Hormones and Reproduction Hormones and Reproduction Hormones and Reproduction Hormones and Reproduction Hormones and Reproduction
of Vertebrates, of Vertebrates, of Vertebrates, of Vertebrates, of Vertebrates,
Volume 1dFishes Volume 2dAmphibians Volume 3dReptiles Volume 4dBirds Volume 5dMammals
Hormones and Reproduction of Vertebrates Volume 3: Reptiles
David O. Norris Department of Integrative Physiology University of Colorado Boulder, Colorado
Kristin H. Lopez Department of Integrative Physiology University of Colorado Boulder, Colorado
AMSTERDAM BOSTON HEIDELBERG LONDON NEW YORK OXFORD PARIS SAN DIEGO SAN FRANCISCO SINGAPORE SYDNEY TOKYO Academic Press is an imprint of Elsevier
Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA First edition 2011 Copyright Ó 2011 Elsevier Inc. All rights reserved Cover images Front cover image: Amphiprion percula, the orange clownfish. Courtesy of iStockphoto: Image 6571184. Back cover image: Atlantic hagfish (Myxine glutinosa) eggs. Courtesy of Stacia A. Sower, University of New Hampshire, Durham, NH, USA and Scott I. Kavanaugh, University of Colorado, Boulder, CO, USA. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email:
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(Set) (Volume (Volume (Volume (Volume (Volume
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Dedication
Richard Evan Jones
This series of five volumes on the hormones and reproduction of vertebrates is appropriately dedicated to our friend and colleague of many years, Professor Emeritus Richard Evan Jones, who inspired us to undertake this project. Dick spent his professional life as a truly comparative reproductive endocrinologist who published many papers on hormones and reproduction in fishes, amphibians, reptiles, birds, and mammals. Additionally, he published a number of important books including The Ovary (Jones, 1975, Plenum Press), Hormones and Reproduction in Fishes, Amphibians, and Reptiles (Norris and Jones, 1987, Plenum Press), and a textbook, Human Reproductive Biology (Jones & Lopez, 3rd edition 2006, Academic Press). Throughout his productive career he consistently stressed the importance of an evolutionary perspective to understanding reproduction and reproductive endocrinology. His enthusiasm for these subjects inspired all with whom he interacted, especially the many graduate students he fostered, including a number of those who have contributed to these volumes.
v
Preface
Hormones and Reproduction of Vertebrates Preface to the Series Every aspect of our physiology and behavior is either regulated directly by hormones or modified by their actions, as exemplified by the essential and diverse roles of hormones in reproductive processes. Central to the evolutionary success of all vertebrates are the regulatory chemicals secreted by cells that control sexual determination, sexual differentiation, sexual maturation, reproductive physiology, and reproductive behavior. To understand these processes and their evolution in vertebrates, it is necessary to employ an integrated approach that combines our knowledge of endocrine systems, genetics and evolution, and environmental factors in a comparative manner. In addition to providing insight into the evolution and physiology of vertebrates, the study of comparative vertebrate reproduction has had a considerable impact on the biomedical sciences and has provided a useful array of model systems for investigations that are of fundamental importance to human health. The purpose of this series on the hormones and reproduction of vertebrates is to bring together our current knowledge of comparative reproductive endocrinology in one place as a resource for scientists involved in reproductive endocrinology and for students who are just becoming interested in this field. In this series of five volumes, we have selected authors with broad perspectives on reproductive endocrinology from a dozen countries. These authors are especially knowledgeable in their specific areas of interest and are familiar with both the historical aspects of their fields and the cutting edge of today’s research. We have intentionally included many younger scientists in an effort to bring in fresh viewpoints. Topics in each volume include sex determination, neuroendocrine regulation of the hypothalamuse pituitaryegonadal (HPG) axis, separate discussions of testicular and ovarian functions and control, stress and reproductive function, hormones and reproductive behaviors, and comparisons of reproductive patterns. Emphasis on the use of model species is balanced throughout the series with comparative treatments of reproductive cycles in major taxa.
Chemical pollution and climate change pose serious challenges to the conservation and reproductive health of wildlife populations and humans in the twenty-first century, and these issues must be part of our modern perspective on reproduction. Consequently, we have included chapters that specifically deal with the accumulation of endocrinedisrupting chemicals (EDCs) in the environment at very low concentrations that mimic or block the critical functions of our reproductive hormones. Many authors throughout the series also have provided information connecting reproductive endocrinology to species conservation. The series consists of five volumes, each of which deals with a major traditional grouping of vertebrates: in volume order, fishes, amphibians, reptiles, birds, and mammals. Each volume is organized in a similar manner so that themes can be easily followed across volumes. Terminology and abbreviations have been standardized by the editors to reflect the more common usage by scientists working with this diverse assembly of organisms we identify as vertebrates. Additionally, we have provided indices that allow readers to locate terms of interest, chemicals of interest, and particular species. A glossary of abbreviations used is provided with each chapter. Finally, we must thank the many contributors to this work for their willingness to share their expertise, for their timely and thoughtful submissions, and for their patience with our interventions and requests for revisions. Their chapters cite the work of innumerable reproductive biologists and endocrinologists whose efforts have contributed to this rich and rewarding literature. And, of course, our special thanks go to our editor, Patricia Gonzalez of Academic Press, for her help with keeping us all on track and overseeing the incorporation of these valuable contributions into the work. David O. Norris Kristin H. Lopez
xiii
Preface
Preface to Volume 3 Reptiles This volume provides a background in the development and function of the reproductive system of reptilian vertebrates with an emphasis on the roles of bioregulators (hormones, pheromones, etc.) in directing the formation and activities of the hypothalamus–pituitary–gonadal (HPG) axis. Where possible, the topics have been arranged similarly to the other volumes in this series to facilitate the efforts of readers looking for comparative data on reproductive processes. Viviparity has been selected as a special topic due to its evolutionary importance for reptiles and because its multiple origins in this taxon provide many opportunities to examine how viviparity evolved. The role of pheromones is another topic of special interest for reptilian
reproduction. Furthermore, the role of stress hormones and other factors affecting HPG axis functions and reproductive behavior are emphasized in several chapters. Reproductive cycles also are described for the major reptilian groups (turtles, crocodilians, lizards, and snakes). Finally, there is a chapter dealing with the impacts of environmental chemicals that function as endocrine disruptors of reproduction in reptiles. This chapter is useful for conservationists, ecologists, toxicologists, and physiologists who are concerned about how organisms interact with and adapt to their environments and how chemical pollution at minute but environmentally relevant levels may affect individual species or reptiles as a group.
xv
Contributors
Daniel A. Warner, Iowa State University, Ames, IA, USA Michele A. Johnson and Juli Wade, Michigan State University, East Lansing, MI, USA
Sunil Kumar, Brototi Roy, and Umesh Rai, University of Delhi, Delhi, India
Susan M. Jones, University of Tasmania, Hobart, TAS, Australia Daniel H. Gist, University of Cincinnati, Cincinnati, OH, USA Jose´ Martı´n and Pilar Lo´pez, Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain
Richard R. Tokarz, University of Miami, Coral Gables, FL, USA Cliff H. Summers, University of South Dakota, Vermillion, SD, USA
Barry Sinervo, University of California, Santa Cruz, CA, USA Donald B. Miles, Ohio University, Athens, OH, USA
Lori C. Albergotti and Louis J. Guillette, Jr., University of Florida, Gainesville, FL, USA
Gae¨lle Blanvillain and David Wm. Owens, College of Charleston, Charleston, SC, USA
Gerald Kuchling, The University of Western Australia, Crawley, WA, Australia
Matthew R. Milnes, Zoological Society of San Diego, Escondido, CA, USA
Matthew B. Lovern, Oklahoma State University, Stillwater, OK, USA Emily N. Taylor, California Polytechnic State University, San Luis Obispo, CA, USA
Dale F. DeNardo, Arizona State University, Tempe, AZ, USA Ashley S.P. Boggs, Nicole L. Botteri, Heather J. Hamlin and Louis J. Guillette, Jr., University of Florida, Gainesville, FL, USA
xvii
Chapter 1
Sex Determination in Reptiles Daniel A. Warner Iowa State University, Ames, IA, USA
SUMMARY The sex-determining mechanisms (SDMs) of reptiles are remarkably diverse, ranging from systems that are under complete genetic control to those that are highly dependent upon temperatures that embryos experience during development. Because reptiles exhibit a remarkable diversity of SDMs, this group provides excellent models for addressing critical questions about the proximate mechanisms of sex determination, as well as its ecology and evolution. The goal of this chapter is to integrate studies from different disciplines (e.g., ecology, evolution, physiology, molecular biology) to broadly summarize the current understanding of reptilian sex determination. This chapter is divided into six topics that cover the (1) diversity of reptilian SDMs, (2) taxonomic distribution of SDMs, (3) molecular and physiological mechanisms underlying sex determination, (4) timing of sex determination during embryogenesis, (5) ecology and evolution of reptilian SDMs, and (6) current gaps in our understanding of this field and where future research should be directed.
1. INTRODUCTION In sexually reproducing species, the sex of an individual is arguably one of the most important aspects of its phenotype. Whether an embryo develops as a male or a female will have profound consequences on its life history, behavior, physiology, morphology, and ultimately its fitness. Indeed, differences between the sexes are among the most spectacular sources of phenotypic variation within populations. It is not surprising, then, that factors governing sexual development are of primary interest to scientists and have been debated for over three millennia. Over this expanse of time, our understanding of sex determination has transformed from mythological speculations to explanations based on hard-won scientific evidence (reviewed in Mittwoch, 2000). The end of the 19th century marked a critical point at which advances in cytogenetics enabled the discovery of sex chromosomes and their relationship to sex determination (reviewed in Brown, 2003). After this discovery, it was widely accepted that the sexual phenotype
Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
of most organisms is determined by genetic factors located on sex-specific chromosomes. Research has since revealed far greater diversity in sexdetermining mechanisms (SDMs). Karyological studies have revealed a variety of sex-specific chromosomal arrangements (e.g., male vs. female heterogamety (Mittwoch, 1996)). In many species, heteromorphic sex chromosomes do not exist, but instead sex-determining factors lie on autosomes. In other organisms, sex is determined by the ratio of X chromosomes to autosomes or the ploidy level of the zygote (Cook, 2002). During the latter half of the 20th century, the ubiquity of these genetic systems was challenged by studies that demonstrate a role of environmental factors in the sex-determination process. Indeed, in many species, environmental conditions experienced during embryogenesis (rather than genotypic factors) trigger the developmental cascade that eventually leads to the male or female phenotype. This environmental sex determination (ESD) has since been shown to exhibit remarkable diversity in itself (Bull, 1983). It is now well established that SDMs range from those under complete genetic control to those that are highly dependent upon environmental parameters. The diversity in SDMs is intriguing since the division of the sexes is so similar throughout most animals. Indeed, without knowledge of such diversity, one might expect that a single mechanism would have been stabilized at an early stage of evolution. Why, then, have so many different mechanisms evolved as a means to produce males and females? How do the proximate mechanisms of different SDMs vary? Which SDMs are ancestral and how have new mechanisms arisen? Answers to these and many related questions are continuously sought after by biologists in many disciplines. The ideal approach for tackling such questions requires an integrative examination of closely related organisms that vary in their SDMs. The class Reptilia is a group that satisfies this requirement. Reptiles show spectacular diversity in SDMs, ranging from systems that have a strong genetic basis to those that are under almost complete environmental influence
1
2
(Bull, 1980). In many cases, closely related species differ in fundamental aspects of their SDMs, making these taxa well-suited for comparative analyses. Many characteristics of reptiles make them especially amenable to experimental manipulation, which facilitates tests of theoretical predictions about the evolution of alternative SDMs (Janzen & Paukstis, 1991a). Not surprisingly, reptiles have received considerable research attention, and have provided critical insights toward our understanding of sex determination. Our understanding, however, is far from complete, and reptiles will undoubtedly continue to serve as excellent models for addressing fundamental issues in this field. The primary objective of this chapter is to provide an overview of our current understanding of sex determination in reptiles. Given that the vast literature on this topic spans several decades, specific details on all aspects of such a broad topic cannot be covered in a single review. The author’s hope is that this review will provide a sense of the current state of the field and a framework that will guide research in specific directions that warrant further study. An additional goal of this review is to provide an awareness of different perspectives on reptilian sex determination (e.g., viewing SDMs as a dichotomy vs. a continuum), as well as to identify gaps in our knowledge and finally to provide suggestions for future research. This chapter is divided into six major sections that focus on the ‘what,’ ‘who,’ ‘how,’ ‘when,’ ‘why,’ and ‘where’ questions of reptilian sex determination. The first section begins by introducing the diversity of SDMs (the ‘what’ question) found within reptiles by detailing the specifics of genotypic sex determination (GSD) and the variations in reaction norms that describe temperaturedependent sex determination (TSD). Secondly, a brief overview of the taxonomic and phylogenetic distribution of alternative SDMs (the ‘who’ question) is provided, and the evolutionary transitions between systems discussed. The third section addresses the proximate mechanisms involved in reptilian sex determination (the ‘how’ question). This section discusses the molecular and hormonal aspects of sex determination and evaluates commonalities and differences among mechanisms. The fourth section explores the timing of sexual lability during embryonic development (the ‘when’ question), and compares different methods for addressing this issue. The fifth section explores the ecology and evolution of reptile SDMs (the ‘why’ question). This section establishes the existence of TSD in nature and evaluates hypotheses of adaptive significance and micro-evolutionary potential of SDMs. The final section briefly points out emergent themes and critical gaps in this field, and suggests avenues of future research that warrant further investigation (the ‘where’ question).
Hormones and Reproduction of Vertebrates
2. WHAT MECHANISMS OCCUR IN REPTILES? DIVERSITY OF SEX-DETERMINING MECHANISMS (SDMs) AND PATTERNS Reptilian SDMs are traditionally placed into two main categories: one in which sex is determined solely by genetic factors (i.e., GSD) and the other a form of ESD in which sex is determined primarily by the temperature that embryos experience during development (i.e., TSD). Intriguingly, within both of these sex-determining systems, we see remarkable diversity in patterns. For example, under GSD, both male and female heterogamety evolved independently multiple times within reptiles. Under TSD, shapes of reaction norms that describe the relationship between developmental temperature and sex determination vary considerably among taxa (Figure 1.1). Moreover, evidence is accumulating that elements of both TSD and GSD may co-occur in some species, even within single populations (e.g., Shine, Elphick, & Donnellan, 2002). In this section, the current knowledge on the diversity of sexdetermining patterns of reptiles is reviewed. To ease discussion, GSD and TSD are treated independently in many parts of this review, but recent arguments suggest that this dichotomy may oversimplify the complexity of SDMs and that TSD and GSD may not be alternative mechanisms but instead represent endpoints of a continuum (Sarre, Georges, & Quinn, 2004).
2.1. Patterns of Genotypic Sex Determination Genotypic sex determination is a system in which offspring sex is irreversibly fixed by its own (or its parent’s) genotype (Bull, 1980; Janzen & Paukstis, 1991a). That is, genetic factors inherited from the parents determine the sex of the offspring. These genetic factors may reside on sex chromosomes, which differ from autosomes in their size, number, and gene content, and are elements of the genome that segregate during meiosis. Importantly, sex-determining genes located on sex chromosomes direct the pathways that lead to male or female development. The two most common types of GSD are male and female heterogamety. Under male heterogamety, offspring that inherit the Y chromosome from the father develop into males (XY) and those that inherit the father’s X chromosome develop into females (XX). In many species, this system is reversed and females are the heterogametic sex; sex chromosomes in this system are referred to as Z and W (i.e., males are ZZ, and females ZW). Although GSD is common in reptiles, many species that have been karyotyped show no evidence of heteromorphic sex chromosomes (Table 1.1), presumably because homomorphic chromosomes have been retained, or have changed very little, from the ancestral state (Ohno, 1967).
Chapter | 1
Sex Determination in Reptiles
3
FIGURE 1.1 Diversity of sex-determining patterns in reptiles (modified from Warner & Janzen, 2010). All graphs show sex ratio (% male) as a function of increasing egg incubation temperature (x-axes). The three major patterns of sex determination with respect to incubation temperature are shown to the left of the arrows. Patterns to the right of the arrows are variants of those patterns. (a) Pattern of temperature-dependent sex determination (TSD) in which females are produced at both temperature extremes, and males at intermediate temperatures (FMF pattern). In some species, intermediate temperatures produce mixed sex ratios, and other species show geographic variation in the shapes of reaction norms (e.g., the lines illustrate population-specific reaction norms in the common snapping turtle, Chelydra serpentina (Ewert, Lang, & Nelson 2005)). (b) Patterns of TSD in which males and females are produced at one or the other temperature extreme (FM and MF patterns). Pivotal temperature (i.e., temperature that produces 1 : 1 sex ratio) varies considerably among species, populations, and embryos produced by different females within populations. Additionally, considerable diversity occurs in the transitional range of temperatures (i.e., range of temperatures that yields mixed sex ratios) among and within species. (c) Sex ratio is not influenced by incubation temperature (genotypic sex determination). Recent studies demonstrate that extreme incubation temperatures reverse genotypic females to phenotypic males (Radder, Quinn, Georges, Sarre, & Shine, 2008) and vice versa (Quinn et al., 2007) in certain lizard species.
Nevertheless, reptiles exhibit remarkable variation in the degree of sex chromosome differentiation (Solari, 1994), which may reflect different stages in the evolutionary transitions between homomorphic and heteromorphic systems (Marshall-Graves & Shetty, 2001; Charlesworth, D., Charlesworth, B., & Marais, 2005). To detect GSD in species with apparently homomorphic chromosomes, experimental approaches are needed to verify that sex determination is unresponsive to environmental parameters (e.g., temperature) (Valenzuela, Adams, & Janzen, 2003). Indeed, incubation temperature does not influence primary sex ratios in many turtle and squamate species that lack differentiated chromosomes (e.g., Bull & Vogt, 1979;
Uller, Mott, Odierna, & Olsson, 2006; Uller, Odierna, & Olsson, 2008). A caveat, however, is that most cytological studies define sex chromosomes only when they are morphologically distinguishable and often do not attempt to detect differences in gene content. Unfortunately, cytological techniques used for karyotyping (e.g., C-banding, reverse fluorescent staining) vary in their ability to detect sex chromosomes that are not highly differentiated. As a result, seemingly homomorphic chromosomes may actually exhibit some differentiation, which can be detected by more advanced techniques. For example, early work on the lizard Pogona vitticeps suggested homomorphic sex chromosomes (Witten, 1983), but more advanced techniques
4
TABLE 1.1 Summary of sex-determining mechanisms (SDMs) found in reptilian families. Family classifications and number of extant species correspond to Pough et al. (2004). XY, XXY are forms of male heterogamety; ZW, ZZW, ZWW are forms of female heterogamety. Experimental evidence for a thermally sensitive SDM comes from laboratory-based egg incubation studies. Under the MF pattern, males are produced at cool incubation temperatures and females at warm temperatures; this pattern is reversed under the FM pattern. Under the FMF pattern, females are produced at extreme incubation temperatures and males at intermediate temperatures Karyological evidence for morphologically differentiated sex chromosomes
Experimental evidence for a thermally sensitive SDM
Number of extant species
Number shown/ examined
Heterogametic system(s)
Referencea
Number shown/ examined
TSD pattern(s)
Referencea
Podocnemidae
8
0/8
-
15
3/3
MF
8,22
Pelomedusidae
18
0/5
-
15
2/2
FMF
8,22
Chelidae
50
3/23
XY
6,13,14,15
0/9
-
8,22
Carettochelyidae
1
0/1
-
15
1/1
MF
8,22
Trionychidae
27
1/8
ZW
15
0/3
-
8,22
Kinosternidae
22
2/16
XY
15
15/18
MF, FMF
8,22
Dermatemydidae
1
0/1
-
15
1/1
MF
8,22
Dermochelyidae
1
0/1
-
15
1/1
MF
8,22
Cheloniidae
6
0/5
-
15
6/6
MF
8,22
Chelydridae
3
0/3
-
15
2/2
FMF
8,22
Emydidae
40
0/30
-
15
23/24
MF
8,22
Testudinidae
40
0/15
-
15
6/6
MF
8,22
Bataguridae
65
2/33
XY, ZW
15
8/8
MF, FMF
8,22
Alligatoridae
8
0/8
-
15
6/6
FMF
8,22
Crocodylidae
13
0/13
-
15
6/6
FMF
8,22
Gavialidae
2
0/1
-
15
1/1
FMF
22
Order Family Chelonia
Rhynchocephalia
Hormones and Reproduction of Vertebrates
Crocodilia
Chapter | 1
2
0/2
-
15
2/2
FM
22
Gekkonidae
1050
13/144
XY, XXY, ZW
15
20/27b,c
FMF
2,7,22,23
Dibamidae
10
1/1
XY
15
0/0
-
Squamata (Sauria)
7,12,19,22
0/0
-
-
15
0/0
-
-
-
15
0/0
-
-
1/61
XY
15
0/3
-
22,23
5/22
XY, XXY
15
0/0
1260
6/118
XY
15,19
4/6
Xantusiidae
18
0/12
-
15
Gerrhosauridae
35
0/12
-
Cordylidae
42
0/11
Teiidae
125
Gymnophthalmidae
150þ 250
42/103
ZW, ZZW
15
40
4/22
ZW
15
0/2
Lanthanotidae
1
0/0
-
-
0/0
Shinisauridae
1
0/0
-
-
0/0
Anguidae
110
0/13
-
15
0/1
Helodermatidae
2
0/1
-
15
0/0
Xenosauridae
6
0/1
-
15
0/0
Agamidae Iguanidae
130 380þ
1/50 2/89
ZW ZW
15 5,15,20,21
-
-
c,d
-
4,7,22,23
c
-
7,23
-
-
-
-
-
7,22
-
-
-
-
1/5
Varanidae
Chamaeleonidae
d
MF, FM
Scincidae
Lacertidae
b
0/2
c
c
13/25
915þ
76/250
XY, XXY
15
0/5
135
0/25
-
15
0/0
c
b
FM, FMF
Sex Determination in Reptiles
Sphenodontidae
1,7,23 d
7,21,22
-
7,22,23
-
-
Squamata (Amphisbaenia) Amphisbaenidae
(Continued)
5
6
TABLE 1.1 Summary of sex-determining mechanisms (SDMs) found in reptilian families. Family classifications and number of extant species correspond to Pough et al. (2004). XY, XXY are forms of male heterogamety; ZW, ZZW, ZWW are forms of female heterogamety. Experimental evidence for a thermally sensitive SDM comes from laboratory-based egg incubation studies. Under the MF pattern, males are produced at cool incubation temperatures and females at warm temperatures; this pattern is reversed under the FM pattern. Under the FMF pattern, females are produced at extreme incubation temperatures and males at intermediate temperaturesdcont’d Karyological evidence for morphologically differentiated sex chromosomes
Experimental evidence for a thermally sensitive SDM
Number of extant species
Number shown/ examined
Heterogametic system(s)
Referencea
Number shown/ examined
TSD pattern(s)
Referencea
Rhineuridae
1
0/1
-
15
0/0
-
-
Trogonophidae
6
0/2
-
15
0/0
-
-
Bipedidae
3
1/3
ZW
15
0/0
-
-
Anomochilidae
2
0/0
-
-
0/0
-
-
Aniliidae
1
0/0
-
-
0/0
-
-
Xenophidiidae
2
0/0
-
-
0/0
-
-
Tropidophiidae
31
0/1
-
15
0/0
-
-
Bolyeriidae
2
0/0
-
-
0/0
-
-
Acrochordidae
3
0/2
-
15
0/0
-
-
Boidae
74
6/29
ZW
15
0/1
-
18
Uropeltidae
55
0/0
-
-
0/0
-
-
Xenopeltidae
2
0/1
-
15
0/0
-
-
Loxocemidae
1
0/1
-
15
0/0
-
-
Order Family
Squamata (Serpentes)
Hormones and Reproduction of Vertebrates
90
0/1
-
15
0/0
-
-
Anomalepididae
15
0/0
-
-
0/0
-
-
Typhlopidae
200
0/6
-
15
0/1
-
17
Colubridae
1800þ
57/140
ZW
15
0/7
-
3,9,11,23,24
Atractaspididae
18
0/0
-
-
0/0
-
-
Elapidae
300
77/110
ZW, ZZW, ZWW
15
0/2
-
10,16
Viperidae
228
38/61
ZW
15
0/0
-
-
7803D
338/1466
Total
121/186
Note: Thermal effects on sex determination may have been evaluated in more squamate species than reported in this table, but offspring sex ratios are seldom reported. a To reduce the number of citations, most references refer to literature reviews rather than original publications. b Some species contain both heteromorphic chromosomes and thermal-sensitivity of sex determination. c Thermal effects on sex determination are reported in some species, but evidence for TSD is unconvincing (see Viets, Ewert, Talent, & Nelson, 1994; Harlow, 2004). d Sex ratios of some species are 1 : 1 at one extreme temperature but sex-biased at the other extreme. 1 Andrews (2005); 2 Blumberg, Lewis, and Skoloff (2002); 3 Du and Ji (2002); 4 Du and Ji (2006); 5 Ezaz et al. (2005); 6 Ezaz et al. (2006); 7 Harlow (2004); 8 Janzen and Paukstis (1991a); 9 Ji and Du (2001a); 10 Ji and Du (2001b); 11 Ji, Du, and Xu (2001); 12 Ji, Du, and Xu (2006); 13 Martinez, Ezaz, Valenzuela, Georges, and Marshall-Graves (2008); 14 McBee, Bickham, Rhodin, and Mittermeier (1985); 15 Olmo and Signorino (2005); 16 Shine (1989); 17 Shine and Webb (1990); 18 Shine, Madsen, Elphick, and Harlow (1997); 19 Shine, Elphick, and Donnellan (2002); 20 Uller, Mott, Odierna, and Olsson (2006); 21 Uller, Odierna, and Olsson (2008); 22 Valenzuela (2004b); 23 Viets, Ewert, Talent, and Nelson (1994); 24 Webb, Brown, and Shine (2001).
Sex Determination in Reptiles
c
Chapter | 1
Leptotyphlopidae
7
8
(i.e., comparative genomic hybridization) reveal a ZZ/ZW sex microchromosome system in this species (Ezaz et al., 2005). This reclassification of chromosomal systems is also illustrated in two turtles with GSD (Ezaz et al., 2006; Martinez, et al., 2008). Considering that early studies have not identified heteromorphic chromosomes in many GSD reptiles (Table 1.1), advanced techniques may reveal more chromosomal differentiation than was detected in past studies. Some squamate species exhibit multiple sex chromosomes and more than one chromosome sort according to sex, resulting in different diploid numbers for males and females (Solari, 1994). For example, male heterogamety has been identified in the lizard Sceloporus poinsettia, but diploid numbers differ between the sexes (male 2n ¼ 31, female 2n ¼ 32; a system referred to as X1X2Y _/X1X1X2X2 \ (Cole, Lowe, & Wright, 1967)). Similar sex differences in diploid numbers occur in several other squamate reptiles (Olmo, 1986; Olmo & Signorino, 2005), including species with female heterogamety (e.g., in some lizards of the genus Lacerta, females differ from males in diploid number (Odierna et al., 1996)). These multiple sex chromosome systems may have resulted from the fusion of an autosome with a sex chromosome (Solari, 1994), and there is no evidence that they cause any fundamental change in the SDM (Bull, 1980). Therefore, these multiple
Hormones and Reproduction of Vertebrates
sex chromosome systems will be treated as either XX/XY or ZZ/ZW systems accordingly.
2.2. Patterns of Temperature-dependent Sex Determination Under TSD, offspring sex is irreversibly determined by the temperature embryos encounter during development. All of the extant reptilian orders contain some members with TSD, but its prevalence varies among the major groups (Figure 1.2). The presence of TSD is identified experimentally when eggs are incubated under a variety of constant temperatures in the laboratory. The resultant offspring are then sexed and the effect of incubation temperature on the primary sex ratio is evaluated. Experiments must rule out the possibility of sex biases in embryonic mortality, which can cause skews in secondary sex ratios and give the impression of TSD. Such differential embryonic mortality has been described in snakes (Burger & Zappalorti, 1988) and birds (Eiby, Wilmer, & Booth, 2008). Primary sex ratios of TSD species vary dramatically depending upon incubation regimens. Typically, a narrow range of temperatures (sometimes just 1–2 C) produces mixed sex ratios, and temperatures above or below this range yield all of one or the other sex; this range
FIGURE 1.2 Phylogenetic distribution of genotypic (XY and ZW) and temperature-dependent sex determination (TSD) in vertebrates, with particular attention given to reptiles. Temperature-dependent sex determination in Amphibia has never been demonstrated in nature, but thermal effects on offspring sex are well documented and are often referred to as a thermal sex reversal (see Dournon, Houillon, & Pieau, 1990; Chardard, Penrad-Mobayed, Chesnel, Pieau, & Dournon, 2004).
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Sex Determination in Reptiles
is called the transitional range of temperatures (TRT) and is an important parameter that describes the shape of the TSD reaction norm. The constant incubation temperature that produces a population-wide 1 : 1 sex ratio is called the pivotal temperature; this temperature expressed at the individual level is called the threshold temperature (Mrosovsky & Pieau, 1991). Perhaps the most spectacular aspect of reptilian sex determination is the diversity in TSD patterns (Figure 1.1). Each pattern contains at least one pivotal temperature and at least one TRT, but these parameters can vary considerably among taxa and populations (threshold temperatures can even vary among individuals within populations) (Ewert, Jackson, & Nelson, 2004; Ewert, et al., 2005; Warner, Lovern, & Shine, 2008). Accordingly, evidence suggests that the pivotal temperature has a heritable genetic basis (Bull, Vogt, & Bulmer, 1982a; Janzen, 1992; Rhen & Lang, 1998; Janes & Wayne, 2006). Given these features, pivotal temperatures have the potential to evolve in response to selective pressures, which may explain the diversity of TSD patterns. Most descriptions of TSD discuss three types of reaction norms that describe the relationship between constant-temperature incubation and sex ratio (Head, May, & Pendleton, 1987; Pieau, 1996; Kraak & Pen, 2002), but in reality these reaction norms are broad generalizations of a variety of patterns (Figure 1.1). Under one pattern, males are produced at low incubation temperatures and females at high temperatures (male– female or ‘MF’ pattern); this is characteristic of many turtle and lizard species (Ewert, Etchberger, & Nelson, 1994; Harlow, 2004). Although rare, the reverse pattern is found in some species (female–male or ‘FM’ pattern), whereby low incubation temperatures produce females and high temperatures produce males (i.e., tuatara and some lizards (Harlow, 2004; Mitchell et al., 2006)). Another pattern yields female offspring at extreme incubation temperatures and males at intermediate temperatures (female–male–female or ‘FMF’ pattern). This pattern contains two pivotal temperatures and, hence, has two TRTs. The FMF pattern is found in all crocodilians and in several lizard and turtle species. The literature often refers to these TSD patterns as type Ia, type Ib, and type II, respectively, but here the less ambiguous letters are adopted to symbolize the sex produced at low or high temperatures. Even within these three patterns, we see remarkable diversity in the shapes of the reaction norms (Figure 1.1). The pivotal temperature varies considerably among species, such that incubation temperatures that produce predominantly males in one species will produce predominantly females in another species. In some North American turtles, variation in pivotal temperatures is explained by latitude (Bull, Vogt, & McCoy, 1982b; Ewert et al., 1994; 2004; 2005). For example, in Chelydra
9
serpentina, a species with an FMF pattern of TSD, the upper pivotal temperature is greater in northern populations than in southern populations (Ewert et al., 2005). Threshold temperatures and the slope of the sex-determining reaction norm (determined by the width of the TRT) can vary substantially among clutches within the same population (Rhen & Lang, 1998; St. Juliana, Bowden, & Jansen, 2004; Warner et al., 2008). In the alligator snapping turtle (Macrochelys temminkii), for example, the sex-determining response of embryos from some clutches has a steep relationship with incubation temperature, whereas embryos from other clutches are relatively unresponsive to temperature, resembling a pattern expected under GSD (Ewert et al., 1994). Considerable among-clutch variation has also been shown among populations of painted turtles (Chrysemys picta) and snapping turtles (Chelydra serpentina) (Ewert et al., 2004; 2005). To add to this diversity, some species exhibit a pattern in which no incubation temperature produces exclusively male offspring. For example, in the Australian jacky dragon (Amphibolurus muricatus), extreme incubation temperatures produce all females but intermediate temperatures yield about 1 : 1 sex ratios, and often these sex ratios are slightly female-biased (Harlow & Taylor, 2000; Warner, Lovern, & Shine, 2007); hence, the pivotal temperature is not readily identifiable under this pattern. Moreover, family effects on sex ratios in A. muricatus can vary irrespective of developmental temperature; that is, some clutches have balanced sex ratios whereas others are extremely male- or female-biased when incubated at temperatures expected to produce 1 : 1 sex ratios (Warner et al., 2008). Additionally, sex ratios of many kinosternid turtles and crocodilians never achieve 100% males across any constant-temperature incubation regime (Lang & Andrews 1994; Ewert et al., 2004). Intuition would suggest that such patterns would yield highly female-biased primary sex ratios in nature, possibly leading to unstable population demographics (Girondot et al., 2004). However, field data supporting this idea are scarce and it is possible that more males are produced in nature than one would expect from the results of artificial incubation experiments (see Section 6, on ecology and evolution). The evolution of this diversity in TSD patterns is not entirely clear, but it has been proposed that the FMF pattern was the ancestral state from which the other patterns evolved (Deeming & Ferguson, 1988; Pieau, 1996). Perhaps selection favored shifts in the sex-determining response along the temperature range until lethal extremes precluded sex determination (Pieau, 1996). Such changes in thermal sensitivities could result in the FM or MF patterns depending on the direction of the shift. Given the evidence for variable and heritable pivotal temperatures (Bull, Vogt, & Bulmer, 1982a; Janzen, 1992; Rhen & Lang,
10
1998; Dodd, Murdock, & Wibbels, 2006), such a scenario is possible. Additional information on species-specific patterns of TSD, as well as resolved phylogenies to which these patterns can be mapped, will enable robust tests of this hypothesis.
2.3. Sex-determining Mechanisms as a Dichotomy or a Continuum? Traditional classification schemes place reptilian SDMs into one of two discrete categories: GSD or TSD. This dichotomous perspective implies that these two mechanisms are fundamentally different from each other in terms of process and are mutually exclusive (Bull, 1983). Indeed, the inheritance of sex and sex-linked traits is fundamentally different under TSD systems, which lack sex chromosomes, vs. GSD systems, with sex chromosomes. However, evidence is accumulating that the underlying mechanisms that shape the sexual phenotype of offspring in GSD and TSD systems are similar (Place & Lance, 2004), and that these mechanisms may represent a continuum (Sarre et al., 2004; Barske & Capel, 2008). One end point of the continuum is pure GSD, where sex is determined by genetic factors; pure TSD is at the other endpoint, where sex is determined by developmental temperature. At intermediate points along the continuum, sex is determined by variable levels of genetic and environmental contributions. Much discussion of reptile SDMs now treats these two perspectives (dichotomous vs. continuous) as opposing schools of thought that have very different impacts on our understanding of the proximate mechanisms and evolution of SDMs. The goal here is to briefly discuss the basis for both perspectives and illustrate that the source of conflict between ideas may involve confusion about the level at which SDMs are viewed (i.e., evolutionary genetic level vs. developmental level) (Figure 1.3). It is important to first establish what is meant by GSD and TSD at both the evolutionary genetic level and the developmental level (Valenzuela, 2008a). At an evolutionary genetic level, GSD is defined by the presence of sex-specific heritable factors (e.g., heteromorphic or homomorphic sex chromosomes), whereas TSD is defined by the absence of consistent heritable genetic differences between the sexes (as in Valenzuela et al., 2003). Under TSD, male and female genomes are identical, but sex differences are induced by a thermal trigger that influences sex-specific gene expression (see Section 4 on proximate mechanisms). Given these definitions, the distinction between GSD and TSD is whether or not one sex contains a single copy of a sex-determining gene(s). If sex chromosomes are present, the genetic mechanism by which sex is determined is transmitted between generations in a sexdependent manner (i.e., one of the parents can contribute
Hormones and Reproduction of Vertebrates
two different types of chromosome). Clearly, the presence (GSD) or absence (TSD) of sex chromosomes will have fundamentally different consequences on population genetics, sex-ratio evolution, sex-linked traits, and autosomal traits (Bull, 1983; Reeve & Pfennig, 2002). Thus, when SDMs are viewed this way, a dichotomous classification appears appropriate. Importantly, however, thermosensitivity of sexual differentiation can also be inherited (Bull, et al.,1982a), which may occur in many species with sex chromosomes (e.g., Radder, Quinn, Georges, Sarre, & Shine, 2008; Luchenback, Borski, Daniels, & Godwin, 2009; Nakamura, 2009). Another consideration is that sex chromosomes have not been identified in many GSD species; if sex chromosomes do not occur in these taxa, then inheritance of sex chromosomes may not always exist in GSD taxa. Clearly, more research is urgently needed to evaluate the co-occurrence, stability, and genetic inheritance of both TSD and GSD mechanisms within single populations under natural developmental conditions (e.g., Radder et al., 2008; Bull, 2008). From a developmental perspective, biologists are interested primarily in the proximate developmental mechanisms involved in sexual differentiation (as in Sarre et al., 2004). Under pure GSD, the embryo’s genotype triggers the developmental pathway that leads to the male or female phenotype. Under pure TSD, this pathway is triggered by developmental temperature. Under these definitions, the boundary between GSD and TSD is blurred because elements of both sex-determining systems cooccur within many species (Barske & Capel, 2008). For example, we see remarkable genetic variation among and within species in their sensitivity to incubation temperature, suggesting that many species do not exhibit pure TSD or pure GSD. Instead, different degrees of thermal sensitivity of sex determination occur in the background of GSD. Support for this idea is widespread; thermally sensitive SDMs coexist with differentiated sex chromosomes in many reptilian species (Shine et al., 2002; Sarre et al., 2004; Quinn et al., 2007). Evidence also illustrates that the molecular pathways involved in TSD and GSD are quite similar (Bull, Hillis, & O’Steen, 1988a; Servan, Zaborski, Dorizzi, & Pieau, 1989; Sarre et al., 2004; Valenzuela, 2007), indicating that the processes involved are not discrete. Intriguingly, chromosomal sex determination in some invertebrate (Cook, 2002), fish (Baroiller, Chourrout, Fostier, & Jalabert, 1995; Kraak & Pen, 2002; Luckenback et al., 2009), and amphibian (Dournon et al., 1990; Kraak & Pen, 2002) populations shows sensitivities to temperature, indicating that this is not just a reptilian phenomenon. Overall, the evidence suggests that there is a common underlying mechanism that guides sexual differentiation in ‘GSD’ and ‘TSD’ reptiles, and that categorizing mechanisms may simplify the complexity of these systems when viewed from a developmental perspective.
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Sex Determination in Reptiles
11
FIGURE 1.3 Two contrasting views about reptile sex-determining mechanisms (SDMs). The traditional perspective is that genotypic (GSD) and temperature-dependent sex determination (TSD) are fundamentally different and that the mechanisms are mutually exclusive. This viewpoint is derived from an evolutionary genetics level, where sex-determining factors (e.g., sex chromosomes) are inherited among generations. Species that exhibit thermally sensitive SDMs and contain sex chromosomes represent an intermediate state; in this case environment affects sex, but the genetic variation in sex determination is still inherited on sex chromosomes (dichotomous view to the right) (Valenzuela, N., Adams, D. C., & Janzen, F. J., 2003). Importantly, however, under this dichotomous perspective, cases 2 and 3 are traditionally classified as GSD, but if no sex chromosomes exist then this poses classification problems when using definitions at the level of inheritance (indicated by question marks). A more recent perspective from a developmental standpoint posits that the distinction between GSD and TSD is not clear-cut, and these two mechanisms represent endpoints of a continuum (continuous view to the left) (Sarre, S. D., Georges, A., & Quinn, A., 2004). A continuous perspective provides a different understanding of how elements of both GSD and TSD coexist to determine the sexual phenotype of offspring.
In summary, GSD and TSD are traditionally classified as discrete mechanisms when limiting our view to the level of inheritance, but these SDMs represent a continuum when extending our view to the developmental level. Valenzuela et al. (2003) make clear distinctions between systems at the level of inheritance, but still recognize that a combination of SDMs lies at intermediate states along a continuum (Figure 1.3). For example, in organisms containing both heteromorphic sex chromosomes and thermally sensitive
sex determination (Baroiller et al., 1995; Shine et al., 2002; Kozielska, Pen, Beukeboom, & Weissing, 2006), there is a genetic component that determines sex differences under some circumstances, and this component is inherited on a sex chromosome (Valenzuela et al., 2003). However, at the developmental level, both mechanisms coexist and affect the sexual phenotype of offspring, which places this system at an intermediate state in the GSD to TSD continuum. This state is termed GSD þ environmental
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effect by Valenzuela et al. (2003) and lies between the endpoints of the GSD to TSD continuum according to Sarre et al. (2004). Regardless of whether these intermediate states are stable or transient (Bull, 2008), recognition of a continuum will broaden our understanding of the evolutionary transitions among SDMs.
3. WHO EXHIBITS GENOTYPIC SEX DETERMINATION (GSD) AND TEMPERATURE-DEPENDENT SEX DETERMINATION (TSD)? TAXONOMIC AND PHYLOGENETIC DISTRIBUTION Despite the extensive literature on SDMs in reptiles, we have only scratched the surface in terms of our understanding of their taxonomic distribution. To date, sexdetermining mechanisms have been identified in less than 6% of the extant reptile species (Table 1.1). This statistic reveals our ignorance in many respects, but it also signifies that much exciting and fruitful research is yet to come. Nevertheless, much has been learned to date, and we have gained a detailed understanding of many aspects of the distribution of alternative SDMs in reptiles. For example, we know that SDMs are non-randomly distributed throughout reptiles. Recent evidence suggests that GSD evolved independently in three lineages of extinct Mesozoic marine reptiles (e.g., Sauropterygia, Mosasauroidea, Ichthyosauria), which may have been partially responsible for radiations within these clades (Organ, Janes, Meade, & Pagel, 2009). In extant reptiles, both species of tuatara exhibit the FM pattern of TSD (Mitchell et al., 2006) and all crocodilians that have been studied (13 of 23 species) exhibit the FMF pattern (Lang & Andrews, 1994; Deeming, 2004). The ZZ/ZW chromosomal system is ubiquitous in avian reptiles (sister group to Crocodilia). Because SDMs are conserved within these lineages, insights into their evolution are limited based on studies of these groups. Much more can be learned about the evolution of SDMs by focusing on taxa that exhibit considerable diversity, such as the Chelonia and Squamata. Indeed, both of these groups contain species that exhibit XX/XY and ZZ/ZW chromosomal systems, as well as TSD (Figure 1.2). Hence, this section will focus on these two lineages and follow with a brief overview of evolutionary transition between SDMs.
3.1. Chelonian and Squamate Sex Determination Comparative analyses demonstrate that TSD is the ancestral state for turtles (Janzen & Krenz, 2004; Organ & Janes, 2008) (Figure 1.4a). Of the 149 turtle species that
Hormones and Reproduction of Vertebrates
have been karyotyped, sex chromosomes have been identified in only eight species (Table 1.1). Egg incubation experiments showing an absence of temperature effects on sex have further identified GSD in several species that lack differentiated sex chromosomes (e.g., Bull & Vogt, 1979; Bull, Legler, & Vogt, 1985). In most species examined (81%; see Table 1.1), however, incubation experiments found evidence for TSD with both the FMF and MF patterns, suggesting that TSD is the most prevalent SDM within turtles. Based on our current knowledge of SDMs and turtle phylogeny, comparative analyses suggest that GSD has arisen at least six times in this group (Janzen & Krenz, 2004). Independent origins of sex chromosomes occur twice (XY and ZW systems) in the Bataguridae (Carr & Bickham, 1981; Olmo & Signorino, 2005) and once in Kinosternidae (Sites, Bickham, & Haiduk, 1979). Based on experimental evidence from egg incubation studies, GSD arose once in the Emydidae (Glyptemys insculpta) and is likely ubiquitous in the Chelidae and Trionychidae. Sex chromosomes have been identified in three members of the Chelidae (Ezaz et al., 2006; Martinez et al., 2008) and one member of the Trionychidae (Olmo & Signorino, 2005). As in turtles, comparative analyses suggest that TSD is likely the ancestral condition in squamates (Janzen & Krenz, 2004; Pokorna´ & Kratochvı´l, 2009; but see Organ & Janes, 2008), from which both XY and ZW chromosomal systems have evolved independently multiple times (Figure 1.4b). Of the squamate species that have been karyotyped, less than 26% exhibit differentiated sex chromosomes, most of which are snakes or belong to the lizard genera Anolis, Sceloporus (Iguanidae), or Lacerta (Lacertidae) (but see Table 1.1 for other examples). Incubation experiments provide additional support for GSD in many taxa that lack differentiated sex chromosomes (e.g., Uller et al., 2006; 2008). In contrast, many species have retained (or independently evolved) the ancestral state of TSD, and all three patterns (FM, MF, and FMF) occur in lizards. Based on reliable evidence, rather than anecdotal observations that are occasionally reported (see Harlow, 2004), TSD appears to be confined to only two or three lineages (Agamidae, Gekkota, and probably Scincidae). Importantly, however, thermal effects on sex determination have not been studied in the vast majority of squamate species (Table 1.1). Phylogenetic reconstructions of the evolution of lizard SDMs are relatively unclear, as well-resolved trees and extensive taxon sampling are currently lacking. At present, at least three phylogenetic hypotheses for relationships of the major lizard families have been proposed (Estes, de Queiroz, & Gauthier, 1988; Townsend, Larson, Louis, & Macey, 2004; Vidal & Hedges, 2005), and the evolution of SDMs varies depending on which phylogeny is used (Pokorna´ & Kratochvı´l, 2009). Adding to this
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Sex Determination in Reptiles
13
FIGURE 1.4 Phylogenetic distribution of sex-determining mechanisms (SDM) in extant families of (a) Chelonia and (b) Lepidosauria (Squamata þ Rhynchocephalia). Family classifications correspond to Pough et al. (2004). The chelonian phylogenetic hypothesis is based on nuclear DNA (Krenz, Naylor, Shaffer, & Janzen, 2005) and the squamate phylogenetic hypothesis is based on nuclear and mitochondrial DNA (Townsend et al., 2004). Temperature-dependent sex determination (TSD) is ancestral in turtles, from which genotypic sex determination (GSD) evolved at least six times. The evolutionary reconstruction of squamate SDMs is not resolved due to extremely low taxon sampling both among and within families. Nevertheless, evidence suggests that TSD is ancestral for this clade (Janzen & Krenz, 2004; Pokorna´ and Kratochvı´l, 2009) and may have evolved independently in the Agamidae. Both male and female heterogamety have multiple independent origins in squamates. Because the MacClade phylogenetic anaylsis program used here (Maddison & Maddison, 2001) produces the most parsimonious topology, the phylogeny indicates that ancestors of some squamate families exhibit GSD (white branches); however, this conclusion is premature because of insufficient data on squamate SDMs. Additional details on SDMs within families are given in Table 1.1. See Organ and Janes (2008) and Pokorna´ and Kratochvı´l (2009) for more detailed phylogenetic analyses of reptile SDMs.
uncertainty, phylogenetic relationships among species within families are currently not resolved (e.g., Han, Zhou, & Bauer, 2004; Smith, Sadlier, Bauer, Austin, & Jackman, 2007; Hugall, Foster, Hutchinson, & Lee, 2008). Another problem is the lack of research on SDMs of lizards (SDMs are known in < 5% of the species), as well as questionable reports of TSD in several species. Indeed, anecdotal observations of sex-ratio skews in response to incubation temperatures have been reported in many families (e.g., Chameleonidae, Iguanidae, Lacertidae, and Varanidae (Viets et al., 1994)), but the evidence for TSD in these groups is unconvincing due to low sample sizes or inconclusive evidence (see review by Harlow, 2004). Because of these issues, the ‘phylogenetic reconstruction’ of squamate SDMs presented in Figure 1.4b excludes
these reports and is extremely generalized, but it none the less provides information on some important aspects of the evolution of squamate SDMs. For example, (1) both male heterogamety (e.g., iguanids, gekkonids, scincids) and female heterogamety (e.g., snakes, gekkonids, lacertids) have arisen multiple independent times; (2) SDMs are evolutionarily labile within the Agamidae and Gekkonidae; and (3) thermally sensitive SDMs may have been retained (or independently arisen) in the Scincidae despite the presence of sex chromosomes in this lineage. Perhaps the most compelling evidence of TSD in squamates occurs in the family Agamidae (Harlow, 2004). Both TSD and GSD have evolved independently multiple times within this group (Pokorna´ & Kratochvı´l,
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Hormones and Reproduction of Vertebrates
FIGURE 1.5 Phylogenetic reconstruction of sex-determining mechanisms (SDMs) in the lizard family Agamidae. The agamid phylogeny is based on hypotheses proposed by Schulte, Melville, & Larsan, (2003) and Hugall et al. (2008). Parsimony analysis was performed with MacClade Software (Maddison and Maddison, 2001). The phylogeny suggests that genotypic sex determination (GSD) was the ancestral state in this family. Temperaturedependent sex determination (TSD) evolved two independent times, accompanied by at least three independent reversals back to GSD. This analysis is treated as preliminary given that SDMs are known for only 26 of the 380þ extant species of Agamidae.
2009) (Figure 1.5). In some cases, sister taxa differ in SDMs, suggesting a recent evolution of either GSD or TSD (e.g., Amphibolurus and Ctenophorus). Most species exhibit the FMF pattern, but some have the FM pattern (e.g., Lophognathus temporalis (Harlow, 2004)). Because of this diversity within a single family, the Agamidae provides excellent model systems for exploring ecological and evolutionary aspects of vertebrate SDMs (Warner & Shine, 2005; Uller & Olsson, 2006; Quinn et al., 2007; Warner, Uller, & Shine, 2009). However, as SDMs have only been identified in 26 of the 380þ agamid species worldwide, much exciting work is yet to be done. The Gekkonidae is another family that exhibits considerable diversity in SDMs (Pokorna´ and Kratochvı´l, 2009). Temperature-dependent sex determination occurs in three lineages of this family (all FMF pattern: Diplodactylinae, Eublepharinae, and Gekkoninae) (Viets et al.,
1994), from which GSD has evolved independently at least three times (Janzen & Krenz, 2004; Pokorna´ and Kratochvı´l, 2009). Intriguingly, comparative analyses indicate that male heterogamety evolved multiple times in some gecko lineages, whereas female heterogamety evolved in others (Pokorna´ and Kratochvı´l, 2009). Considering the current diversity of SDMs that has been described in such a small fraction of the extant gekkonids (SDMs are known in only about 33 of 1050þ species), it would not be surprising if far more origins of alternative SDMs occur in this family than in others, and that both SDMs may occur within a single species. For example, strongly differentiated sex chromosomes occur in Gekko japonicus, but sex determination is also highly sensitive to incubation temperature in this species (Yoshida & Itoh, 1974; Tokunaga, 1985). Recent laboratory experiments and correlative data from the field provide convincing evidence of thermal
Chapter | 1
Sex Determination in Reptiles
sensitivity of sexual differentiation in scincid lizards. For example, the Australian skink, Bassiana duperreyi, exhibits male heterogamety, but eggs incubated at cool temperatures that mimic thermal regimens of nests overproduce males, whereas warmer temperatures produce a 1 : 1 sex ratio. Maternal factors, such as yolk allocation (i.e., egg size), can also interact with incubation temperature to affect offspring sex ratios in this species (Shine et al., 2002; Radder, Pike, Quinn, & Shine, 2009). Sex is also sensitive to temperature in three distantly related viviparous scincid lizards (Eulamprus tympanum, Niveoscincus ocellatus, and Sphenomorphus indicus). Experimental manipulations of basking conditions, and hence maternal thermoregulation, have profound effects on offspring sex ratios. In E. tympanum, warm gestation temperatures overproduce male offspring and cool temperatures produce 1 : 1 sex ratios (Robert & Thompson, 2001), but this pattern is reversed in the viviparous skink N. ocellatus (Wapstra et al., 2004; 2009). Skewing of sex ratios in response to gestation temperature are more substantial in the viviparous skink S. indicus (Ji et al., 2006) than that reported in E. tympanum or N. ocellatus.
3.2. Transitions between Sex-determining Mechanisms (SDMs) The evolution of reptilian SDMs involves multiple transitions between TSD systems and GSD systems with welldifferentiated sex chromosomes. It is generally accepted that sex chromosomes arise from an autosomal ancestor when a gene involved in ESD acquires a mutation that consistently directs either male (under XY systems) or female (ZW systems) development (Ohno, 1967). After this primordial sex-determining locus emerges, the pair of autosomes that contain that locus become sex chromosomes. During early stages of chromosome evolution, sex chromosomes are essentially identical except at the sexdetermining locus, where they differ only in gene content and not morphology (Matsuda et al., 2007). This sexdetermining locus resides only on the Y or W chromosomes, and recombination with its homolog becomes increasingly limited (Charlesworth, 1991). As a result, the sex-linked differential segment is free to diverge from the homologous region, causing sex chromosomes to become progressively differentiated from each other (Rice, 1996; Vallender & Lahn, 2004; 2006). The homogametic chromosome (X or Z) maintains its size and gene content, while the heterogametic chromosome (Y or W) loses these features (Vallender & Lahn, 2004; Charlesworth et al., 2005). Intriguingly, the complete range of chromosome differentiation occurs in snakes (Modi & Crews, 2005;
15
Matsubara et al., 2006). Sex chromosomes are only slightly differentiated in basal species (Boidae) and fully differentiated in the more derived viperid snakes. Members of the family Colubridae exhibit intermediate levels of chromosome differentiation. Given this diversity within snakes, this group provides an excellent model for comparative studies of sex chromosome evolution. In addition, comparative gene mapping demonstrates that the sex chromosomes of snakes, mammals, and birds were derived independently from different autosomal ancestors (Marshall-Graves & Shetty, 2001; Matsubara et al., 2006). Given the evolutionary lability of GSD, it is possible that this process has also occurred multiple times within reptiles, as has already been shown in turtles (Sites et al., 1979; Carr & Bickham, 1981). What about transitions in the opposite direction, from GSD to TSD? Some authors argue that highly differentiated sex chromosomes place constraints on reversals back to homomorphic chromosomes; that is, once chromosomal differentiation has begun, the probability of subsequent change in the SDM is greatly diminished (Marı´n & Baker, 1998). In support of this model, a recent phylogenetic reconstruction of the evolution of SDMs in squamates suggests that the transition from TSD to GSD is unidirectional and that sex chromosomes are an ‘evolutionary trap’ preventing subsequent evolution back to TSD (Pokorna´ & Kratochvı´l, 2009). Whether this is truly the case remains to be seen, as sampling of more taxa and better resolution of phylogenies are needed (TSD may have arisen from GSD in agamid lizards (Figure 1.5)). Indeed, one phylogenetic reconstruction of reptilian SDMs suggests that transitions between XY systems and TSD occur in both directions, but a ZW system is less likely to give rise to TSD, perhaps because of a greater instability of XY systems (Organ & Janes, 2008). Importantly, transitions back and forth between TSD and GSD may involve very few changes that could occur at different locations in the sex-determining pathway, which in turn can affect master thermal switches (Sarre et al., 2004; Barske & Capel, 2008; Valenzuela, 2008b). Transitions from heteromorphic to homomorphic systems can occur by a translocation of a piece of an autosome onto the Y (or W) chromosome (Rice, 1996), particularly if little chromosome differentiation has occurred. Additionally, sex chromosomes could become extinct at the end of chromosome degeneration (Charlesworth & Charlesworth, 2000; Charlesworth et al., 2005), resulting in homomorphic chromosomes, which could lead to a thermally sensitive SDM. Moreover, Bull (1981) provides a theoretical framework illustrating how ESD can evolve from male-heterogametic systems. Overall, we cannot ignore the possibility that transitions between TSD and GSD occur in both directions, which may explain the impressive diversity and distribution of SDMs in reptiles.
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4. HOW IS SEX DETERMINED? PROXIMATE MECHANISMS Our understanding of the molecular and physiological underpinnings of reptilian sex determination is far from complete. Research on this topic has focused primarily on species with TSD to understand how interactions among genes, steroid hormones, and temperature affect sex determination and gonad differentiation. Since the gene regulatory networks and physiological pathways that influence key events during gonadal differentiation are highly conserved and have common elements among all vertebrates (Place & Lance, 2004), our in-depth understanding of mammalian systems offers some critical insights into GSD and TSD in reptiles. As many reviews have already covered this material in depth (Crews et al., 1994; Crews, 1996; Lance, 1997; Pieau, Dorizzi, & Richard-Mercier, 1999; Morrish & Sinclair, 2002; Elf, 2004; Pieau & Dorizzi, 2004; Place & Lance, 2004), this section will provide only a brief overview of gonadal differentiation and its molecular and physiological basis under GSD and TSD.
4.1. Gonadal Differentiation and Gene Expression To understand proximate mechanisms, a distinction between sex determination and sexual differentiation is useful. Sex determination is the process that guides the undifferentiated gonads to develop into testes or ovaries. This process can be initiated by a master switch, which is either under genetic control (under pure GSD), environmental control (under pure TSD), or has elements of both mechanisms. Sexual differentiation, on the other hand, refers to the development of specialized sex organs (i.e., testes or ovaries). Under these definitions, the process of sex determination guides sexual differentiation and ends when gonadal development is irreversibly committed to becoming either a testis or an ovary. Although the specific details of gonadal development (i.e., gonadogenesis) vary among vertebrate taxa, many general patterns are conserved (see Place & Lance, 2004). Prior to gonadogenesis in mammals, male and female embryos develop similarly and the rudiments of the testes and ovaries (genital ridges) are at first indistinguishable. At this early stage, embryos contain two sets of ducts: the Wolffian ducts (male reproductive tract) and the Mu¨llerian ducts (female reproductive tract). In embryos that develop into males, the genital ridges begin to develop into seminiferous tubules, which indicate testis development. The embryonic testes produce two important hormones that influence sexual development of males. The first is anti-Mu¨llerian hormone (AMH) (also known
Hormones and Reproduction of Vertebrates
as Mu¨llerian-inhibiting substance (MIS)), which causes the Mu¨llerian ducts to degenerate. The second hormone, testosterone, stabilizes the Wolffian ducts, which eventually develop into seminal vesicles, epididymis and vas deferens. In the absence of these two hormones, the Wolffian ducts degenerate and the Mu¨llerian ducts develop into oviducts and the uterus (Mittwoch, 1996; Place & Lance, 2004). Each stage of gonadal differentiation is governed by numerous genes that perform different roles and interact with each other in complex ways (Table 1.2). In mammals, a major sex-determining switch (Sry gene located on the Y chromosome) sets the testicular developmental pathway in motion (Sinclair et al., 1990). The Sry gene triggers expression of the downstream gene Sox9, which is expressed in the Sertoli cells (but not in the ovary) and is the key regulator of the Amh gene in mammals (Arango, Lovell-Badge, & Behringer, 1999). Other genes (e.g., Wt1 and Sf1) also contribute to testis formation by activating the transcription of the Amh gene, the product (AMH) of which initiates the degeneration of the Mu¨llerian duct. In contrast, ovaries develop in the absence of the Sry gene. The gene Dax1 is expressed in ovaries, which suppresses the activation of Wt1 and Sf1, enabling the retention of the Mu¨llerian ducts (Nachtigal et al., 1998). Thus, expression of this gene will lead to female development. Another gene that is likely involved in testis formation is Dmrt1. This gene is expressed in the bipotential gonads of both sexes, but at later stages it is only expressed in the testes. Recent research using RNA interference to knock down Dmrt1 shows convincing evidence that this gene is required for testis determination in the chicken (Smith et al., 2009), but its role in sex determination is largely unknown in non-avian reptiles. Indeed, no upstream or downstream targets of Dmrt1 have been identified. The roles of the genes discussed above are described primarily from a mammalian perspective, but many of these genes have homologs in reptiles with GSD and TSD (Bull et al., 1988a; Smith, McClive, Western, Reed, & Sinclair, 1999; Pieau & Dorizzi, 2004; Place & Lance, 2004). At present, a master sex-determining switch (e.g., Sry in mammals) has not been identified in reptiles with GSD or TSD. Instead, expression of many of the genes involved in gonadal differentiation is temperature-sensitive in TSD species. For example, in American alligators (Alligator mississippiensis) and leopard geckos (Eublepharis macularius), Sox9 expression is upregulated in gonads of embryos exposed to male-producing temperatures, but this expression occurs after the initiation of male gonadal differentiation (Western, Harry, Graves, & Sinclair, 1999a; Valleley, Carwright, Croft, Markham, & Coletta, 2001). Interestingly, Amh expression precedes Sox9 expression in alligators, suggesting that, unlike the
Chapter | 1
Sex-determining factor
Full name
Definition or function for sexual development
Sry
Sex-determining region Y
Major testis-determining factor located on the Y chromosome. Found only in placental mammals
Sox9
SRY-like HMG box
Regulates transcription of the anti-Mu¨llerian hormone gene in mammals. Involved in testes differentiation
Sf1
Steroidogenic factor 1
Involved in the formation of the primary steroidogenic organs (adrenal glands and gonads). Plays a role in testis development and regulation of Amh
Wt1
Wilms’ tumor suppressor gene
Transcription factor involved in urogenital development. Necessary for maintenance of seminiferous tubules and Sertoli cells in mammals
Amh or Mis
Anti-Mu¨llerian hormone or Mu¨llerian-inhibiting substance
Regulates anti-Mu¨llerian hormone, which inhibits development of the Mu¨llerian ducts and hence is important in testis development
Dmrt1
Doublesex- and mab-3-related transcription factor
Transcription factor involved in gonadal differentiation, but its role is poorly understood
Dax1
Dosage-sensitive sex reversal
May be antagonist of Sry function in mammals. Represses Wt1 and Sf1 expression (i.e., suppresses testis formation) and important in ovarian differentiation
DHT
Dihydrotestosterone
Non-aromatizable androgen involved in testis development
T
Testosterone
Androgen involved in testis development
E2
Estradiol
Estrogenic steroid hormone involved in ovarian development
Aromatase
-
Enzyme that converts androgens to estrogens
Reductase
-
Enzyme that converts testosterone to dihydrotestosterone
Gene
Sex Determination in Reptiles
TABLE 1.2 Genes, steroids, and enzymes involved in sex determination in vertebrates. With the exception of the Sry gene, all factors listed are found in all vertebrate taxa, and serve similar functions under temperature-dependent sex determination (TSD) and genotypic sex determination (GSD)
Steroid hormones and steroidogenic enzymes
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regulation of Amh by Sox9 in mammals, other genes may regulate Amh in alligators (Western, Harry, Graves, & Sinclair, 1999b). In contrast, Sox9 expression precedes Amh expression in the TSD turtle Trachemys scripta (Shoemaker, Ramsey, Queen, & Crews, 2007). Expression of Wt1 occurs during the early stages of gonadogenesis and at both male- and female-producing temperatures in alligators (Western, Harry, Marshall-Graves, & Sinclair, 2000). However, levels of expression of Wt1 are greater at malethan at female-producing temperatures in turtles (T. scripta and C. picta) that exhibit TSD (Spotila, Spotila, & Hall, 1998; Valenzuela, 2007). Moreover, Wt1 has been shown to regulate Sf1 in mammals (Wilhelm & Englert, 2002)danother gene involved in gonad formation. The expression of Sf1 also varies depending upon incubation temperature in turtles (Crews, Fleming, Willingham, Baldwin, & Skipper, 2001). Indeed, both Wt1 and Sf1 are implicated as candidates for master thermal switches in TSD turtles (Valenzuela, LeClere, & Shikano, 2006; Valenzuela, 2007; 2008b). Dax1 and Dmrt1 expression occur in both sexes prior to gonadal differentiation in alligators and turtles, but their expression is temperaturesensitive during the embryonic stages that coincide with sexual differentiation (Smith et al., 1999; Kettlewell, Raymond, & Zarkower, 2000; Shoemaker et al., 2007; Valenzuela, 2008b). Nevertheless, the exact roles of these genes and their position in the regulatory network require more investigation to fully understand the molecular basis for GSD and TSD. Excellent reviews by Shoemaker and Crews (2009) and Ramsey and Crews (2009) provide more details of the regulatory network. Thermal sensitivity of gene expression has recently been demonstrated in a turtle with GSD. In the softshell turtle Apalone mutica, expression of the Wt1 gene is sensitive to incubation temperature, similar to that shown in TSD species (Valenzuela, 2007). Expression of Dax1 is also sensitive to temperature in A. mutica, presumably because it is regulated by Wt1 (Valenzuela, 2008b). Despite this thermal sensitivity, sex ratios remain unaffected by incubation temperature in this species. A possible reason for this involves the thermal insensitivity of the Sf1 gene, which lies directly downstream from Wt1 and Dax1 (Valenzuela et al., 2006; Valenzuela, 2008b). Because Sf1 is not sensitive to these effects, thermally sensitive expression of Wt1 and Dax1 is not functional in sex differentiation, indicating that there may be no selection operating on the thermal sensitivity of gene expression in GSD taxa. This thermal sensitivity of gene expression in A. mutica is likely a characteristic that is retained from a TSD ancestor, suggesting that this species may exhibit an intermediate state in the transition to or from GSD (Figure 1.3). It has been proposed that molecular switches that affect the thermal sensitivity of SDMs may occur at many different
Hormones and Reproduction of Vertebrates
levels in the regulatory network, and the molecular pathways for TSD (and possibly GSD as well) could differ among species (Sarre et al., 2004; Valenzuela, 2008b). Thus, even within TSD (or GSD), multiple processes may reveal a similar pattern, further illustrating that all TSD and all GSD systems are not comprised of single mechanisms. From this perspective, SDMs cannot be grouped into dichotomous categories.
4.2. Physiological Mechanisms under Temperature-dependent Sex Determination (TSD) It is generally accepted that temperature exerts its effect on sex determination in TSD species by acting on genetic mechanisms that govern steroid production, steroidogenic enzymes, or steroid hormone receptors (see Table 1.2 for list of hormones and enzymes). Such an effect will change the hormonal environment of the embryo, thereby directing development in a male or female direction. Indeed, levels of steroid hormones in reptilian egg yolks decline dramatically during development and the degree of this decline can depend on incubation temperatures (Conley et al., 1997; Elf, 2004); such effects may direct gonadal development. This notion is supported by experiments that manipulate the hormonal environment of the developing embryo. For example, administration of the estrogen 17b-estradiol (E2) to eggs of many reptile species induces female development even at maleproducing temperatures (Bull, Gutzke, & Crews, 1988; Crews, 1996; Freedberg, Bowden, Ewert, Sengelaub, & Nelson, 2006). The ability of E2 to counteract the effects of temperature is greatest during the developmental window that corresponds with gonadogenesis (Gutzke & Chymiy, 1988; Wibbels, Bull, & Crews, 1991) (see also Section 5). Perhaps this effect occurs via an influence on gene expression, as exogenous E2 application to eggs of T. scripta results in downregulation of Sf1 and Dmrt1 expression (potentially repressing testis formation) at male-producing temperatures (Fleming & Crews, 2001; Murdock & Wibbels, 2006). Moreover, E2 has an increasingly potent effect on ovarian development as incubation temperatures move from strictly maleproducing towards the threshold temperature (Crews & Bergeron, 1994). Administration of E2 to eggs can also impact sexual differentiation in species with GSD (Bull et al., 1988b). Despite male heterogamety in the turtles Staurotypus triporcatus and Staurotypus salvinii, E2 exposure during development permanently induces female development (Freedberg et al., 2006). In both TSD and GSD reptiles, these hormonal manipulations lead to fully functional females similar to those from unmanipulated eggs (Crews et al., 1994). Clearly, E2 is the primary
Chapter | 1
Sex Determination in Reptiles
steroid that is required for ovarian differentiation in reptiles (Pieau & Dorizzi, 2004). Another key player involved in directing sexual differentiation of the gonad is aromatase. Aromatase is an enzyme that is responsible for converting androgens into estrogens in both TSD and GSD species (Crews et al., 1994; Pieau & Dorizzi, 2004). Aromatase activity or aromatase gene expression in the gonad is greatest at femaleproducing incubation temperatures during the period of gonadal differentiation in several TSD species such as Emys orbicularis and Malaclemys terrapin (Desvages & Pieau, 1992; Jeyasuria & Place, 1998), but not in T. scripta or C. picta (Murdock & Wibbels, 2003; Valenzuela et al., 2006). The initial trigger of aromatase activity has not been identified, but it may involve thermal sensitivity of genes that lie upstream in the regulatory network (Lance, 1997; Valenzuela & Shikano, 2007). At the onset of gonadal differentiation, aromatase activity is orders of magnitude greater in female gonads than in male gonads (Lance, 1997), indicating that the aromatization of androgens provides an important source of estrogens needed for ovarian development. Indeed, the application of testosterone (T)da precursor to E2dto eggs has feminizing effects on some developing embryos of T. scripta when exposed to female-producing temperatures; this non-intuitive result is presumably due to the aromatization of T to E2 (Crews & Bergeron, 1994; Crews, 1996). Another line of evidence that illustrates the importance of aromatase is provided by studies that block E2 synthesis with aromatase inhibitors. By applying aromatase inhibitors to eggs, embryos develop into males when incubated at female-producing temperatures. This has been demonstrated in numerous reptilian species that exhibit TSD (e.g., Crews et al., 1994; Rhen & Lang, 1994; Warner & Shine, 2005). Moreover, aromatase inhibitors can induce male development in eggs of all-female parthenogenetic lizards (i.e., Aspidoscelis uniparens) (Wibbels & Crews, 1994). These manipulations result in phenotypic males with similar gonadal morphology and similar behaviors to naturally produced males; they are also capable of spermatogenesis as adults (Elbrecht & Smith, 1992; Wennstrom & Crews, 1995; Shine, Warner, & Radder, 2007; Warner & Shine 2008a). Many other chemicals (e.g., hormones, enzyme inhibitors, receptor antagonists) applied to eggs have varying effects on sexual development (reviewed in Crews et al., 1994; Crews, 1996). Unlike E2 application at maleproducing temperatures, androgens cannot overcome the effects of female-producing incubation temperatures. For example, dihydrotestosterone (DHT), a non-aromatizable androgen, only induces male development at temperatures near the threshold temperature for sex determination. Nevertheless, manipulations of 5a-reductase (the enzyme that converts T to DHT) provide evidence that androgens
19
are important for male development. For example, by blocking DHT synthesis using 5a-reductase inhibitors, male development is also blocked, suggesting that DHT is important for male development (Crews & Bergeron, 1994). Moreover, simultaneous administration of T and a 5a-reductase inhibitor to eggs results in a greater number of female hatchlings than control treatments in the redeared slider turtle (T. scripta) (Crews & Bergeron, 1994), whereas simultaneous application of both T and an aromatase inhibitor results in nearly 100% male offspring. Overall, these results illustrate that steroidogenic or steroid-metabolizing enzymes (aromatase and 5a-reductase) play a critical role in sex determination and differentiation. The manipulative studies described above clearly demonstrate critical roles for steroid hormones in sex determination, but what about hormone levels that occur naturally prior to gonadogenesis? Indeed, embryonic production of steroids during initial stages of gonadal development is negligible in the reptiles examined (White & Thomas, 1992). Yet, both E2 and T are plentiful in egg yolks at the time of oviposition, long before embryos are capable of producing these steroids (Bowden, Ewert, & Nelson, 2000; Bowden, Ewert, Freedberg, & Nelson, 2002; Elf, Fivizzani, & Lang, 2002; Lovern & Wade, 2003). Recent research demonstrates that these steroid hormones are maternally derived and deposited into the yolks of eggs during vitellogenesis (reviewed in Elf, 2004). Moreover, variation in yolk steroids is largely explained by amongclutch differences (i.e., clutch effects (Elf, 2004; Warner et al., 2008)), and this variation often reflects levels of circulating hormones in females (Callard, Lance, Salhanick, & Barad, 1978; Janzen, Wilson, Tucker, & Ford, 2002). Given these patterns, maternal condition could influence the hormonal environment of developing embryos (Kratochvı´l, Kubicka, & Landova´, 2006; Warner et al., 2007; Lovern & Adams, 2008), thereby influencing sexual differentiation. As expected, offspring sex of several reptilian species is associated with maternally derived E2 or T when eggs are incubated at pivotal temperatures (reviewed in Radder, 2007). Indeed, eggs from clutches with relatively high levels of E2 tend to produce female offspring in a turtle with TSD (C. picta) (Bowden et al., 2000), and eggs with relatively high levels of T tend to produce male offspring in a lizard with GSD (Anolis carolinensis) (Lovern & Wade, 2003). Maternally derived corticosterone also influences offspring sex ratios in the lizard Amphibolurus muricatus (Warner et al., 2007), but experimental work suggests that elevated levels of corticosterone in eggs in this species yields female-biased secondary sex ratios via differential embryonic mortality (Warner, Radder, & Shine, 2009a). Despite these important maternal effects, in some species an association between maternally derived steroids and offspring sex is not
20
detected (C. serpentina (St. Juliana et al., 2004); B. duperreyi (Radder, Ali, & Shine, 2007); A. muricatus (Warner et al., 2007)). In these cases, perhaps the levels of maternally derived steroids are too low to have a significant effect on sex determination. Nevertheless, steroids deposited into yolk represent an important maternal contribution, and their role in modulating sex determination requires further investigation.
5. WHEN IS SEX DETERMINED? TIMING OF EMBRYONIC SEXUAL LABILITY Under pure GSD, only the genetic makeup of the embryo determines the direction of gonadal development, and accordingly natural external factors should have no influence on sexual differentiation. Hence, the genetic sex of the zygote is determined at fertilization under this system, but the process of gonadal sex differentiation begins when sex-determining genes are expressed. In most cases of GSD, the genetic constitution of the zygote will determine offspring sex. However, in some GSD taxa, external factors (e.g., temperature, hormones) can irreversibly direct gonadal differentiation at some point after fertilization (Crews, 1996; Shine et al., 2002; Freedberg et al., 2006; Quinn et al., 2007). If the role of sex chromosomes in sex determination is overridden by an environmental factor, then phenotypic sex (i.e., the gonad) is not determined solely by the genetic constitution of the embryo but by an interaction with the environment it experiences during the critical time of development. The effect of environmental factors in many GSD species is dependent upon the dosage of that factor (e.g., hormones, temperature) and has a permanent effect on gonadal sex (Freedberg et al., 2006; Radder et al., 2008). Thus, the distinction between TSD and GSD becomes blurred from a developmental perspective. Under pure TSD, sex is determined strictly by environmental parameters regardless of genotype. In this system, embryonic sex is not determined by genetic factors inherited at fertilization, but instead temperature triggers the developmental cascade of events that direct gonadal differentiation during a critical window of development. During this window, embryonic sex remains labile and can be modified by environmental cues, namely temperature. This interval is commonly referred to as the thermosensitive period (TSP) (Mrosovsky & Pieau, 1991). Although the TSP occurs at roughly the middle third of development in TSD reptiles, it has not been evaluated in most TSD species. This section evaluates the different methods used to identify the TSP and explores the ecological significance of the variation in TSPs that occurs among reptiles.
Hormones and Reproduction of Vertebrates
5.1. Methods for Establishing the Thermosensitive Period (TSP) Most studies that quantify the onset and duration of the TSP in TSD reptiles have relied upon temperature-shift experiments (Bull & Vogt, 1981; Pieau & Dorizzi, 1981; Bull, 1987; Lang & Andrews, 1994; Valenzuela, 2001). Under these experiments, researchers incubate eggs at a constant temperature that produces some known sex ratio, typically 100% of one sex. Eggs are then moved during specified developmental intervals to thermal conditions that are expected to generate the other sex. Investigators can then manipulate the timing and duration of the temperature shifts among treatments to identify the timing and size of the developmental window in which sex determination processes are responsive to temperature. These experiments involve either a one-step or a two-step temperature shift design to identify the onset and duration of the TSP. Although shift experiments have been instructive in identifying the TSP in many species, such designs are not without complications. First, switching temperature modifies developmental rates, rendering it difficult to quantify the percentage of the developmental period during which sex is responsive to external cues. Secondly, shift experiments may fail to elicit sex reversal even if the embryo is potentially able to respond, because the stimulus for change may not always be sufficiently strong. Related to this, because results from shift experiments depend on the duration and magnitude of the shifts (Lang & Andrews, 1994), prior knowledge of the appropriate temperatures and length of exposure for inducing a response is needed. To avoid these problems, recent work by Shine et al. (2007) used chemical manipulation of eggs to identify the window of embryonic sexual lability. Specifically, the researchers applied an aromatase inhibitor to eggs of A. muricatus and B. duperreyi at specified times during embryonic development. By inhibiting aromatase activity during development, the conversion of T to E2 is blocked, thereby inducing male development at female-producing temperatures (Crews et al., 1994). Chemically manipulating eggs at different times of embryogenesis and identifying the sex of the resultant offspring provides information on when embryonic sex is responsive to external cues without affecting developmental temperature and, thus, developmental rates. Do temperature-shift experiments and chemicalapplication experiments (e.g., application of aromatase inhibitors or E2) yield similar conclusions? Studies in which exogenous estrogens were applied to eggs at different stages of development suggest that embryo sensitivity to these chemicals coincides with the TSP (Gutzke & Chymiy, 1988; Wibbels et al., 1991). However, caution is needed because embryo responses to
Chapter | 1
Sex Determination in Reptiles
developmental temperatures and chemical applications depend on temperature magnitude and chemical dosage, respectively, and these two approaches may provide differing results. Moreover, chemical manipulations may reverse embryo sex at times of development when normal thermal cues are unable to do so. Another concern is that chemicals may remain in the egg during times when embryos are insensitive to such cues, thereby rendering it difficult to determine the exact timing of the response. Thus, although chemical-application experiments may identify the window of sexual lability, this may not always equate to the TSP. Indeed, results of a preliminary temperature-shift experiment on eggs of the jacky dragon (A. muricatus) indicate that onset of the TSP may be later in development than that suggested by chemical-application experiments (application of an aromatase inhibitor; Figure 1.6). These contrasting results may be due to (1) differing ‘dosage effects’ of the aromatase inhibitor vs. incubation temperature or (2) the aromatase inhibitor applied early in development may not take effect until a later period that corresponds with the actual critical sexdetermining period. Both types of experiment supply useful information about windows of sexual lability during development, but only temperature-shift experiments can accurately identify the TSP.
5.2. Variation in the Thermosensitive Period (TSP) and its Ecological Implications The developmental timing of the TSP is relatively similar among reptiles with TSD, but some variation exists (Figure 1.7). To date, most of our knowledge of the TSP has been derived from studies of turtles. For instance, in the European turtle Emy orbicularis, the TSP begins at a stage just prior to the onset of gonadal differentiation (stage 16), and the duration of the TSP, in terms of developmental stages, depends on incubation temperature (Pieau & Dorizzi, 1981). As a consequence, male differentiation occurs slightly earlier and may last longer than female differentiation. Similar patterns occur in the North American turtles Graptemys ouachitensis and C. picta (Bull & Vogt, 1981). In general, the TSP extends throughout much of the middle third of development coinciding with gonadal differentiation (Bull and Vogt, 1981; Pieau & Dorizzi, 1981). Similar patterns occur in other turtle families (Yntema, 1979; Yntema & Mrosovsky, 1982; Valenzuela, 2001). For crocodilians, the TSP begins at a later period in development compared to that of turtles, but still coincides with gonadal differentiation (Deeming & Ferguson, 1988). Detailed shift experiments on A. mississippiensis indicate that sex ratios are labile from embryonic stages 21 to 24 (Lang & Andrews, 1994), which encompass roughly the third quarter of embryonic development. Differences
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among eggs from different clutches also explain a significant amount of the variation in the TSP, hinting at a genetic component to the timing of sexual differentiation. Similar experiments on eggs of the leopard gecko show that sex is determined within stages 32–37, which occurs shortly after oviposition and extends to about half of the incubation period. For comparison, embryonic morphology is similar at the onset of the TSP for geckos, turtles, and crocodilians (Bull, 1987). The TSP in tuatara is likely to be similar to that in other TSD reptiles. Although embryos were not staged, the estimated TSP for tuatara occurs between 25% and 55% of the total incubation period, which likely corresponds closely with the period of gonadogensis (Mitchell et al., 2006). The major reptilian lineages vary considerably in the degree of embryonic development that occurs in utero prior to oviposition (Andrews, 2004) (Figure 1.7). Crocodilians, turtles, and tuatara oviposit during very early stages of development prior to neurulation (e.g., gastrula or neurula stages), whereas most oviparous squamates retain developing embryos for about 30% of the total period of development (Andrews & Mathies, 2000). Thus, comparisons of the squamate TSP with that of other reptilian lineages can vary depending on whether the TSP is expressed in terms of total developmental period or total incubation period subsequent to oviposition (Shine et al., 2007). The timing of the TSP relative to oviposition has important consequences on maternal control of the sex ratio in TSD species. Because squamates typically oviposit at advanced stages of embryonic development, this may enable reproductive females to predict the thermal conditions of potential nest sites during the TSP of their embryos. Hence, squamates may have considerable control over offspring sex ratios via active nest-site selection (Warner & Shine, 2008b). Intriguingly, viviparity will enable even more maternal control over the sex ratio via active thermoregulatory behaviors (Robert & Thompson, 2001; Wapstra et al., 2004; Ji et al., 2006). This type of control in many viviparous squamates may enable mothers to facultatively adjust sex ratios in adaptive directions (Robert, Thompson, & Seebacher, 2003; but see Allsop, Warner, Langkilde, Du, & Shine, 2006). Due to the greater temporal separation of the TSP and oviposition in turtles, crocodilians, and tuatara, similar maternal control is unlikely in these species, as predictive ability of future nest conditions is low. The timing of thermal effects on fitness-relevant phenotypes (other than sex) of offspring has important consequences for the evolution of the TSP. In squamates, for example, incubation temperature soon after oviposition (i.e., near the time of the TSP) has significantly greater impacts on offspring phenotypes than do temperatures later in development (Shine & Elphick, 2001; Andrews, 2004). Since incubation temperatures
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Hormones and Reproduction of Vertebrates
FIGURE 1.6 Comparison of two methods for identifying the onset of the thermosensitive period (TSP) of sex determination in embryos of the lizard Amphibolurus muricatus. (a) Experimental design for a one-step temperature-shift experiment. Dotted lines represent two treatments where eggs were incubated at a constant 23 C (female-producing temperature) or 28 C (1 : 1 sex ratio temperature). The dark line represents a third treatment where eggs were shifted from 23 C to 28 C on day 20 after oviposition. (b) Sex ratios produced from each treatment (c2¼7.5, P¼0.006). As expected, predominately females were produced under a constant 23 C, but the shift treatment yielded 1 : 1 sex ratios similar to that of the constant 28 C treatment, suggesting that the TSP begins at some point after the first 20 days of incubation (from an unpublished dataset obtained by D. Warner, R. Radder, and R. Shine). (c) Results from aromatase-inhibitor applications given to eggs at day 0 and day 20 after oviposition (graph derived from data in Shine, R., Warner, D. A., & Radder, R., 2007). By manipulating the hormonal environment of embryos, sex determination is affected at very early stages of development (before or at day 20 of incubation). Hence, temperature-shift and chemical-application experiments provide different conclusions about the onset of the TSP.
Chapter | 1
Sex Determination in Reptiles
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FIGURE 1.7 Thermosensitive periods (TSP) of sex determination in reptiles (modified from Shine, R., Warner, D. A., & Radder, R., 2007). In tuatara, turtles, and crocodilians, oviposition occurs at very early stages of embryonic development (Andrews, 2004), but in lizards oviposition occurs later in development at a stage close to the onset of the TSP. The TSPs for the top four graphs were estimated using temperature-shift experiments. However, the bottom graph for the dragon and skink was derived from studies using a chemical-application experiment (Shine et al., 2007). Interestingly, temperatureshift experiments suggest that the TSP may begin at some point after the dashed line (see Figure 1.6) rather than immediately after oviposition for the dragon, Amphibolurus muricatus.
during this early developmental phase are important in shaping offspring phenotypes (e.g., size, locomotor performance), we might expect selection to move the TSP to coincide with this important phase of incubation (Bull, 1987). Indeed, an overlap of the TSP with the phase of
incubation that affects fitness-relevant phenotypes would be critical in how incubation temperature shapes sexspecific phenotypes, a condition that would support models for the adaptive significance of TSD (Shine, 1999) (more details in Section 6).
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6. WHY DO REPTILES EXHIBIT A DIVERSITY OF SEX-DETERMINING MECHANISMS (SDMS)? ECOLOGY AND EVOLUTION Theoretical and empirical studies have provided an important foundation for understanding the selective pressures operating on SDMs (Charnov & Bull, 1977; Bulmer & Bull, 1982; Bull & Charnov, 1989; Warner & Shine, 2008a). These studies have revealed that the evolution of SDMs can be highly dynamic, can be facilitated or constrained by pre-existing features of an organism’s biology, and that selection can operate at multiple levels (Uller, Pen, Wapstra, Beukeboom, & Komdeur, 2007). To accurately understand the evolution of reptilian SDMs, it is important to first establish that TSD is not just a laboratory phenomenon. This section will begin by evaluating the evidence that TSD exists in nature and subsequently discuss models that explain when selection would favor TSD over GSD (and vice versa) and argue that different SDMs evolved in different reptilian lineages under different selective pressures. Lastly, the micro-evolutionary potential of TSD in response to changing environments will be examined.
6.1. Ecological Relevance: Does Temperature-dependent Sex Determination (TSD) Occur in Nature? Temperature-dependent sex determination is usually, if not always, determined via laboratory experimentation that fails to mimic the complexities of natural thermal regimens. Additionally, sex-determining reaction norms are rarely evaluated from natural nests. This raises two important questions concerning the presence of TSD in nature. First, do the complex thermal regimens of natural nests have predicable impacts on sex ratios in TSD species? Secondly, are all locations along the laboratory-generated reaction norm ecologically relevant? In nature, eggs that experience mean daily temperatures above the laboratory-established pivotal temperature are expected to yield one sex, whereas eggs exposed to mean daily temperatures below the pivotal temperatures should yield the other sex. Although many field studies are in broad agreement with these expectations (Bull & Vogt, 1979; Janzen, 1994a), sex ratios are not always related to the mean temperature within nests (Warner & Shine, 2009). For example, a study on painted turtles (C. picta) in Canada shows that sex ratios are not related to mean temperatures in natural nests (Schwarzkopf & Brooks, 1985). In the European pond turtle (Emys orbicularis), mean daily temperatures above the pivotal temperature produce the opposite sex from that predicted from laboratory studies (Pieau, 1982). Research on map turtles (genus Graptemys)
Hormones and Reproduction of Vertebrates
demonstrates that both the mean and variance of nest temperatures are important in predicting sex ratios (Bull, 1985). In the jacky dragon (A. muricatus), the average of the daily temperature range throughout incubation is the best predictor of offspring sex ratios (Warner, 2007). These examples demonstrate that the mean nest temperature is often inadequate in fully explaining variation in sex ratios produced under natural conditions. What, then, are the appropriate thermal parameters that we need to measure to accurately predict offspring sex ratios? To accurately predict offspring sex ratios from natural thermal regimens, several characteristics of the nests’ thermal patterns and the embryos’ developmental patterns must be considered (Shine & Harlow, 1996; Georges, Doody, Beggs, & Young, 2004). First, because warm temperatures accelerate embryonic development (Andrews, 2004), more development will occur when daily temperatures fluctuate above, rather than below, the pivotal temperature (Shine & Harlow, 1996). Consequently, sexual differentiation will depend on the relative proportion of development taking place above or below the pivotal temperature. Temperatures below the ‘developmental zero’ will have no impact on sexual differentiation because development is arrested when thermal fluctuations drop this low. Secondly, only temperatures during the TSP should be considered, as temperatures before or after this critical window have no impact on sex determination (Mrosovsky & Pieau, 1991). Because daily fluctuations and seasonal trends in nest temperatures prevent a constant rate of embryonic development, identifying the onset and end of the TSP in nature is not straightforward. To address these issues, models have been developed for evaluating the influence of natural thermal regimens of nests on offspring sex ratios (Georges, 1989; Georges et al., 2004; Georges, Beggs, Young, & Doody, 2005). These models propose that, if similar amounts of development (during the critical sex-determining stages) occur above the pivotal temperature as below it, then both sexes will be produced. However, if more than half of development is spent above the pivotal temperature during each day of incubation, then males (for FM pattern) or females (for MF pattern) will be produced. That is, the key predictor of sex ratios from natural nests is not the mean temperature or its variance, but instead the temperature above and below which half of development occurs, calculated on a daily basis. This predictor statistic is referred to as the constant temperature equivalent (CTE), and produces temperature values that are equivalent to a constant incubation temperature in an incubator. This CTE approach has received strong support in several studies of turtles and tuatara because the CTE calculated from natural nest temperatures accurately predicts the sex ratios expected under constant temperature incubation in the laboratory
Chapter | 1
Sex Determination in Reptiles
(Georges, 1989; Georges, Limpus, & Stoutjeskijk, 1994; Demuth, 2001; Mitchell et al., 2006). However, a disadvantage of this approach is that it only applies to species that have a single pivotal temperature (i.e., those with either the MF or FM pattern of TSD). Predicting sex ratios from natural nests in species with the FMF pattern of TSD has been problematic for two reasons. First, the CTE models cannot be applied to species with this pattern because two pivotal temperatures exist (Georges et al., 2004). Although warm and cool temperatures produce females, embryonic sensitivity of the sex-determining response to these two extremes will differ dramatically (i.e., more development occurs at temperatures above the upper pivotal temperature than at temperatures below the lower pivotal temperature). Moreover, the proximate mechanisms involved in TSD may differ between embryos experiencing high vs. low incubation temperatures, even though these extremes produce similar sex ratios. Indeed, the amount of developmental time spent at female-producing temperatures (high and low temperatures combined) does not predict offspring sex ratio in A. muricatus, a lizard with the FMF pattern of TSD (Warner & Shine, 2009). A second problem involves the functional significance of one of the pivotal temperatures in the FMF pattern. Field data from nests of the snapping turtle (Chelydra serpentina) and Amazonian river turtle (Podocnemis expansa) (both with FMF pattern) illustrate that temperatures towards the lower pivotal temperature are rarely experienced in nature, suggesting that only the MF pattern is realized in natural nests (Valenzuela, 2001; Janzen, 2008). Similarly, mean nest temperatures beyond the upper pivotal temperatures may not occur in the lizard A. muricatus (compare Harlow and Taylor (2000) with Warner and Shine (2008b)), yet daily extremes still fluctuate well beyond the upper pivotal temperature. These issues are not accounted for in current CTE models, and thus the development of sophisticated models is needed to fully understand the existence of the FMF pattern in nature. Overall, the existence of TSD in nature is well supported for turtles (Bull, 1985), crocodilians (Ferguson & Joanen, 1982), and tuatara (Mitchell et al., 2006). For lizards, however, few field data exist, but there is no evidence against its existence in this group (see Doody et al., 2006; Warner and Shine, 2008b; Wapstra et al., 2009). In reptiles generally, field data clearly demonstrate that warm and cool nests produce substantially different sex ratios (Bull & Vogt, 1979; Janzen 1994a; 1994b) and that daily and seasonal thermal fluctuations contribute to this variation (Bull, 1985; Georges et al., 2004). Moreover, laboratory experiments that mimic the thermal fluctuations of natural nests also illustrate the importance of temperature variance, not only on offspring sex (Valenzuela, 2001; Les, Paitz, & Bowden, 2007) but also on a variety of
25
fitness-relevant phenotypes (Du & Ji, 2006; Mullins & Janzen, 2006).
6.2. Adaptive Significance of Sex-determining Mechanisms (SDMs) Theoretical models of sex ratio evolution predict that reproductive females invest equally into each sex when the cost of producing a son equals that of a daughter (Fisher, 1930). Indeed, balanced primary sex ratios are considered evolutionarily stable because frequency-dependent selection will favor the rarer sex if population sex ratios are perturbed from unity. Consequently, selection should favor SDMs that ensure equal numbers of sons and daughters. In many reptiles, and most other animals, GSD provides a convenient mechanism by which 1:1 sex ratios are produced due to random segregation of chromosomes during meiosis. Hence, selection will favor GSD when the fitness returns of producing a son are equal to that of a daughter. In many situations, however, one sex may provide greater fitness returns than the other, thereby shifting the selective pressures that operate on SDMs. In such cases, maternal control over the sex ratio will enhance parental fitness by enabling overproduction of the sex that benefits most from prevailing conditions (Trivers & Willard, 1973; Cockburn, Legge, & Double, 2002). For example, many female adders (Vipera berus) in a Swedish population produce only one litter in their lifetimes, and that litter consists of equal numbers of sons and daughters. Because of intense competition among males, mothers that survive to produce a second litter reduce competition between their already-produced sons and their current offspring by overproducing daughters in their second litter (Madsen & Shine, 1992). Although sex-biased investment has been demonstrated in numerous reptiles that exhibit GSD (Madsen & Shine, 1992; Lovern & Wade, 2003; Calsbeek & Sinervo, 2004; Calsbeek & Bonneaud, 2008), the precise mechanisms that enable these shifts are not well understood. Theoretical and empirical research suggests that mechanisms for sex-ratio adjustment likely occur at multiple levels (Uller et al., 2007) and through multiple pathways (Pike & Petrie, 2003; Uller & Badyaev, 2009). Unfortunately, reptiles have received relatively little attention as models for empirical studies of sex allocation theory (Wapstra et al., 2007; Wapstra & Warner 2010). Undoubtedly, more research in this area will provide new insights into the selective forces responsible for diversity of SDMs in reptiles. Sex allocation theory (recently reviewed in West, 2009) provides a useful framework for explaining the adaptive significance of ESD, particularly for TSD in reptiles. As described above, models of sex allocation theory propose that if some conditions are more
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conducive towards one sex than the other, maternal fitness would be enhanced if the sex best-suited for the given conditions was over-produced (Trivers & Willard, 1973; Charnov, 1982). These ideas have been extended by Charnov and Bull (1977) to explain when selection will favor ESD over GSD. In terms of TSD, their model proposes that, if male and female offspring have different optimal developmental temperatures, selection will favor a SDM that enables each sex to be produced at their respective optimal developmental temperature. Indeed, experimental studies on a variety of reptilian species demonstrate that egg incubation temperatures affect offspring phenotypes in ways that could influence their fitness (e.g., Andrews, Mathies, & Warner, 2000; Van Damme, Bauwens, Bran˜a, & Verheyen, 1992), and some of these effects are long-lasting (Freedberg, Stumpf, Ewert, & Nelson, 2004; Elphick & Shine, 1998). If those traits differentially impact the fitness of sons vs. daughters (e.g., Gutzke & Crews, 1988; Warner & Shine, 2005), selection will favor an SDM that is sensitive to temperature so that each sex develops at its optimal temperature. Thus, under these situations, TSD would be favored and maintained by selection because it would confer higher parental fitness than GSD (Bull, 1983). The matching of offspring sex with developmental temperature via selection can occur through multiple pathways. Hence, the Charnov–Bull model comprises several differential-fitness hypotheses that link incubation temperature with sex-specific fitness to explain the adaptive significance of TSD. Several of these hypotheses are outlined in Table 1.3, and since previous reviews have covered these at length (Shine, 1999; Valenzuela, 2004a; Janzen & Phillips, 2006), they will not be detailed here. Despite numerous hypotheses for the adaptive value of TSD, however, satisfactory empirical support has been elusive and TSD has remained an enigma in reptiles (Bull, 1983). Most attempts to empirically test the Charnov–Bull model have been impeded by the following factors: (1) Most reptiles with TSD are poorly suited for lifetime fitness studies. The vast majority of research on this topic has been conducted on turtles and crocodilians (Joanen, McNease, & Ferguson, 1987; Janzen, 1995), and measuring reproductive success (fitness) of these late-maturing, long-lived species is logistically difficult. (2) Offspring sex is often difficult to identify without sacrificing animals, which would preclude any measurement of fitness. (3) The effects of incubation temperature and offspring sex are naturally confounded. In order to evaluate the sex-specific effect of incubation temperature on offspring fitness, both sexes need to be produced across a broad range of incubation temperaturesdan obvious problem with most TSD species. (4) As discussed above, incubation temperature may differentially affect fitness in males vs. females via multiple
Hormones and Reproduction of Vertebrates
complex pathways (shown in Table 1.3). Thus, any attempt to test the Charnov–Bull model must examine a wide range of variables, thereby posing a substantial challenge to comprehensive empirical analysis. Recent work on an Australian lizard (A. muricatus) with TSD has overcome these obstacles to provide the first substantial support for the Charnov–Bull model in reptiles (Warner & Shine, 2005; 2008a). In this study, the authors incubated eggs across a range of temperatures that naturally occur in the field and applied an aromatase inhibitor to a subset of eggs in each incubation treatment. This manipulation blocked the conversion of T to E2 to produce male offspring at female-producing temperatures, thereby decoupling the confounded effects of sex and incubation temperature. After eggs hatched, the offspring were harmlessly sexed by manual eversion of hemipenes on males and then raised under semi-natural conditions among six replicated field enclosures for four years. Because A. muricatus matures within one year of hatching and has a short life span (likely three to four years) relative to other TSD reptiles, the authors were able to measure near lifetime reproductive success of the offspring that were produced under the controlled incubation conditions. With the use of microsatellite DNA markers, parentage of all second generation offspring was assigned over three reproductive seasons, enabling a direct measure of reproductive fitness (i.e., lifetime number of offspring produced). Remarkably, the results provide strong support for the theoretical predictions of the Charnov–Bull model. Males that hatched from naturally male-producing temperatures sired more offspring than did sex-reversed males from female-producing temperatures. The reverse was true for females; temperatures that exclusively produce daughters were optimal for females (Warner & Shine, 2008a). Although this pattern supports the Charnov–Bull predictions, the mechanism(s) by which incubation temperature differentially affects the fitness of sons and daughters remain unclear. Hence, subsequent studies that address the alternative hypotheses outlined in Table 1.3 are needed. Although all hypotheses have not been tested, the current data on A. muricatus provide support for hypothesis 4, which proposes that TSD enables each sex to hatch at its own optimal time of the season. Indeed, developmental temperature strongly affects the timing of hatching, which in turn has long-lasting impacts on fitness (Warner & Shine, 2005; 2007). Because early hatching likely benefits one sex more than the other, perhaps TSD evolved to create an adaptive match between the timing of hatching and the appropriate sex (Warner et al., 2009b); this pattern is similar to the scenario seen in the Atlantic silverside fish whereby earlier hatching benefits females more than males (Conover, 1984; Warner et al., 2009b). Nevertheless, in A. muricatus, the interactive effect of sex and incubation temperature on offspring fitness remains
Chapter | 1
Hypothesis
Role of incubation temperature
Prediction if adaptive
References
1. Different optimal egg size for sons vs. daughters
Enables mother to adjust clutch sex ratios via nest-site selection
Sons and daughters produced from different sized eggs, and sex-specific relationship between egg size and fitness
14, but see 11
2. Different phenotypic optima for sons vs. daughters
Induces changes to phenotype independent of sex
Incubation temperature affects phenotypes, and the phenotypic determinants of fitness are sex-specific
6,10,13, but see 20
3. Different norms of reaction for sons vs. daughters
Influences phenotypes and/or fitness of hatchlings, but differently in sons and daughters
Significant interaction between incubation temperature and sex on fitness-related phenotypes
4,15,16,19
4. Different optimal hatching times for sons vs. daughters
Induces variation in time of hatching, and seasonal variation in sex ratio
Seasonal variation in offspring sex ratio production, and sex-specific relationship between time of hatching and fitness
2,7,18
5. Natal homing and nestsite philopatry
Induces variation in thermal quality of nesting sites
Females return to natal nest sites for oviposition; fitness of daughters is enhanced when hatching at natal site but male fitness is unaffected
12, but see 17
6. Sexual size dimorphism
Induces variation in growth rate
Sex with larger adult body size is produced at temperatures that induce rapid growth, thereby generating sexual size dimorphism
5,8, but see 9
7. Differential mortality
Induces embryonic mortality, but differently in male vs. female embryos
Female and male embryos have different sensitivities to incubation temperature
1,3
Sex Determination in Reptiles
TABLE 1.3 Summary of seven differential-fitness hypotheses that fall within the Charnov–Bull (1977) framework. All hypotheses propose adaptive values or mechanisms for the maintenance of temperature-dependent sex determination (TSD)
1 Burger and Zappalorti (1988); 2 Conover (1984); 3 Eiby, Wilmer, and Booth (2008); 4 Elphick and Shine (1999); 5 Ewert and Nelson (1991); 6 Gutzke and Crews (1988); 7 Harlow and Taylor (2000); 8 Head, May, and Pendleton (1987); 9 Janzen and Paukstis (1991b); 10 Langkilde and Shine (2005); 11 Morjan and Janzen (2003); 12 Reinhold (1998); 13 Rhen and Lang (1995); 14 Roosenburg (1996); 15 Shine, Elphick, and Harlow (1995); 16 Shine, Elphick, and Harlow (1997); 17 Valenzuela and Janzen (2001); 18 Warner and Shine (2005); 19 Warner and Shine (2008a); 20 Warner, Woo, Van Dyk, Evans, and Shine (2010).
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significant whether or not analyses are corrected for the timing of hatching (Warner & Shine, 2008a), which suggests that another mechanism could be involved. For example, perhaps continuous selection for males at certain temperatures and females at others results in developmental processes being optimized at different temperatures for each sex regardless of the timing of hatching. Additional work that evaluates the level at which temperature optimizes development is needed to address this hypothesis. A single adaptive explanation for TSD in reptiles is unlikely to exist (Ewert & Nelson, 1991; Valenzuela, 2004a; Warner et al., 2009b). Given the diversity in life histories, mating systems, demographics, and developmental patterns of TSD reptiles, it is more likely that TSD and GSD evolved in different taxa for different reasons. For example, thermally sensitive SDMs in viviparous taxa may have evolved to enable mothers to facultatively adjust offspring sex ratios (via active thermoregulation) in adaptive directions (Robert et al., 2003; Wapstra et al., 2004). Alternatively, TSD may have evolved to create an adaptive match between the timing of hatching and sex in short-lived species, as described above (Conover, 1984; Harlow & Taylor, 2000; Warner et al., 2009b); such a match, however, is less likely to be important in longer-lived reptiles with TSD. In long-lived species, long-term effects of developmental temperature on adult reproduction are likely to be swamped out over time, suggesting that TSD may have evolved via different mechanisms in these taxa (Ewert & Nelson, 1991). In fact, models suggest that TSD may be maintained in long-lived species despite negative impacts of extreme sex-ratio fluctuations, which is a demographic feature associated with this SDM (Bull & Bulmer, 1989; Freedberg & Taylor, 2007). Other explanations concerning the adaptive value and maintenance of TSD relate to inbreeding avoidance, group selection (Ewert & Nelson, 1991; but see Burke, 1993), and cultural inheritance of nest sites (Freedberg & Wade, 2001); neutral hypotheses also have been proposed for different taxa (see Valenzuela, 2004a).
6.3. Evolutionary Potential of Temperaturedependent Sex Determination (TSD) After TSD is established in populations, how will natural selection maintain viable offspring sex ratios in a changing environment? That is, what is the microevolutionary potential of TSD, and what factors might constrain sexratio evolution? Theoretical models predict that microevolution of the sex ratio can occur via selection on either (1) the thermal sensitivity of embryonic sex determination or (2) maternal choice of the nest site (Bulmer & Bull, 1982; Morjan, 2003a). Importantly, evolution of these two
Hormones and Reproduction of Vertebrates
key factors depends upon the amount of heritable genetic variation that is present. Although empirical studies have indeed shown that both of these factors have genetic components, genetic and non-genetic maternal factors may alter their evolutionary potential. Variation in pivotal temperatures is often considered an index for variation in the thermal sensitivity of embryonic sex determination. Empirical studies demonstrate that most reptilian species with TSD exhibit remarkable variation in pivotal temperatures at the among-population level, and in threshold temperatures among clutches within populations. For example, pivotal temperatures shift geographically in some North American turtle species, implying that the sexdetermining response of embryos may be adapted to local climates (Ewert et al., 1994; 2005) (Figure 1.1). However, the direction of this trend is opposite from that expected (i.e., pivotal temperatures increase with latitude (Bull et al., 1982b; Ewert et al., 2004)). Additional studies illustrate impressive variation in threshold temperatures among clutches within populations, implying a strong genetic component (Rhen and Lang, 1998; Janes and Wayne, 2006; Warner et al., 2008). Indeed, constant-incubation experiments suggest that the heritability of sex ratios produced at pivotal temperatures can be quite high, but heritability may be substantially muted under field conditions because of environmental noise imposed by natural thermal regimens among nests (Bull et al., 1982a; Janzen, 1992). Non-genetic maternal effects, such as yolk hormones or maternal nutrition (Bowden et al., 2000; Warner et al., 2007), also may place constraints on the evolutionary potential of the pivotal temperature. Maternal nest-site choice also plays an important role in the microevolutionary potential of TSD (Bulmer & Bull, 1982). The availability of suitable nest sites varies substantially both within and among populations, and selection has apparently shaped maternal nesting behaviors so that females choose nest sites with favorable thermal conditions for their developing embryos (Janzen & Morjan, 2001; Warner & Andrews, 2002; Doody et al., 2006). Although laboratory experiments on eublepharine geckos with TSD demonstrate relatively little variation in nest-site selection (Bull, Gutzke, & Bulmer, 1988c; Bragg, Fawcett, Bragg, & Viets, 2000), field studies on other TSD reptiles show substantial within-population variation in thermal characteristics of nests chosen by females (Janzen & Morjan, 2001; St. Juliana et al., 2004; Kamel & Mrosovsky, 2005; Warner & Shine, 2008b). Moreover, field studies of the painted turtle (C. picta) illustrate strong individual repeatability for choice of specific microhabitat characteristics, such as the amount of vegetative cover over nest sites (Janzen & Morjan, 2001). Indeed, vegetation cover, which influences the nest thermal environment, is a strong predictor of clutch sex ratios in C. picta (Janzen, 1994a).
Chapter | 1
Sex Determination in Reptiles
In fact, vegetative cover has consistently been shown to be an important cue driving nest-site selection in many TSD reptiles (Morjan, 2003b; Kolbe & Janzen, 2002; St. Juliana et al., 2004; Warner & Shine, 2008b), but heritability of maternal choice of nest microhabitat has not yet been demonstrated. Perhaps the most convincing evidence for the evolutionary potential of TSD involves the nesting behavior of the water dragon, Physignathus lesueurii (Doody et al., 2006). This species has a broad latitudinal range spanning 19 along the eastern coast of Australia, and hence occupies habitats that vary considerably in microclimate. Extensive work by Doody et al. (2006) clearly demonstrates that these lizards compensate for climatic differences by selecting low-vegetated nest sites (which receive more solar radiation) in cool locations of its geographic range, and vice versa in warm locations. Adjustments in nesting phenology may also be important in the microevolution of TSD. Evidence suggests that the timing of the nesting season may shift in response to spatial or temporal variation in climate (e.g., Ewert et al., 2005; Doody et al., 2006; Schwanz & Janzen, 2008; Tucker, Dolan, Lamer, & Dustman, 2008). Importantly, whether these responses have a heritable genetic basis or are due to behavioral plasticity, shifts in nesting phenology may maintain viable offspring sex ratios in response to changing climates. However, recent analyses suggest that shifts in nesting phenology may not be enough to compensate for the predicted changes in climate (Schwanz & Janzen, 2008; Telemeco, Elphick, & Shine, 2009). Nevertheless, heritable variation in all three of these factors (changes in pivotal temperatures, nesting behavior, and nesting phenology) warrant further investigation, as such research will provide important insights into how TSD reptiles may cope with changes in habitat and global climate (Janzen 1994b; Telemeco et al. 2009).
6.4. Comments on the Coexistence of Temperature-dependent Sex Determination (TSD) and Sex Chromosomes Theoretical models predict that a stable sex ratio will evolve and be maintained regardless of whether sex is determined strictly by genetic mechanisms or by temperature (Bull, 1981; 2008). These models, as well as empirical evidence, suggest that elements of TSD and GSD can coexist within single populations (Lagomarsino & Conover, 1993; Baroiller et al., 1995). However, genetic models suggest that the coexistence of TSD and GSD is selected against only when GSD systems contain differentiated sex chromosomes (Bull, 1980; 1983). For example, consider a system with male heterogamety (XX/XY) and TSD. In
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this system, phenotypic sex of some individuals will not match their chromosomal constitution. That is, developmental temperatures will override genotypic sex determination to produce some XX males and some XY females, whereas others in the population will maintain their usual condition (i.e., XY males and XX females). Consequently, XY females will mate with XY males, and a fourth of their progeny will inherit the YY genotype; this genotype is predicted to be non-viable due to the accumulation of nonfunctional genes on the Y chromosome. Frequencydependent selection will then favor XX females as they will produce more progeny than their XY-female counterparts, thereby selecting against TSD in this system. This argument posits that TSD and GSD represent two distinct peaks in an adaptive landscape and that intermediate states would be selected against. See Bull (1980; 1983; 2008) for more discussion on this topic. Despite this argument against the coexistence of TSD and sex chromosomes, the multiple evolutionary transitions between SDMs in reptiles suggest that some degree of coexistence must occur even if during a brief transient period. Intriguingly, recent discoveries of TSD and sex chromosomes in populations of two Australian lizards have challenged the incompatibility of these systems (Shine et al., 2002; Quinn et al., 2007). In these species, the production of YY (or WW) individuals does not occur because the environmental effect on sex determination occurs only at one temperature extreme. For example, in the male-heterogametic skink Bassiana duperreyi, cool temperatures override GSD so that predominately males are produced, but relatively warm nests produce balanced sex ratios, as expected under GSD. Because this thermal effect on sex determination occurs only under cool temperatures, this creates a mismatch between phenotypic and genotypic sex for some males, but not for females. That is, XX and XY males are produced, but XY females never occur, implying that YY progeny will not be produced in this system. Intriguingly, the thermal effect on sex determination is reversed (i.e., females are produced at high temperatures) in the female-heterogametic agamid Pogona vitticeps (Quinn et al., 2007), hence no WW progeny will be produced in this species. Similar cooccurrences of sex chromosomes and thermal effects on sex determination have been observed in other reptiles, fishes, amphibians, and invertebrates (e.g., Dournon, et al., 1990; Kozielska et al., 2006; Luckenback et al., 2009). Together, these systems provide evidence that TSD and GSD may be more compatible than previously considered and that these systems represent some intermediate states in the TSD to GSD continuum (Figure 1.3). Whether this coexistence is accidental, transient, or adaptive raises additional questions that warrant further empirical and theoretical investigation (see Bull (2008) for discussion).
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7. WHERE TO GO NEXT: FUTURE RESEARCH AND CONCLUSIONS Decades of theoretical and empirical research have provided many critical insights into the diversity of SDMs in reptiles, their phylogenetic distribution, proximate mechanisms, and evolutionary significance. This review has touched on many aspects of these issues and has attempted to provide a general consensus of where the field stands, the emerging directions, and what gaps need to be filled. Here, some of the emergent directions of the field will be reiterated and major gaps of inquiry where future research should be directed will be pointed out. A major component of this review explores two different perspectives on reptilian SDMs: the traditional perspective divides reptilian SDMs into discrete categories, whereas the emerging alternative perspective treats TSD and GSD as endpoints of a continuum (Valenzuela et al., 2003; Sarre et al., 2004). Commonalities in proximate mechanisms and direct evidence of the co-existence of both SDMs within populations suggest greater complexity in sex-determining systems than is embodied in a dichotomous perspective. These two perspectives have fundamentally different consequences for our understanding of reptilian SDMs in terms of proximate mechanisms, ultimate explanations, and evolutionary transitions. Under a dichotomous view, SDMs are divided based on the presence or absence of consistent heritable genetic differences between the sexes, and hence are forced into one or the other category. This view may bias our perception of underlying mechanisms and limit our understanding of evolutionary transitions. On the other hand, a continuous view makes less of a distinction among SDMs, and therefore provides a logical understanding of why commonalities exist in TSD and GSD systems, which may give insights into why evolutionary transitions between SDMs have occurred so frequently in reptiles. More empirical and theoretical research is urgently needed to further understand the frequency and stability of co-existing SDMs in natural populations. A second theme of this review involves our ignorance of the phylogenetic distribution of SDMs. At present, SDMs have been identified in less than 6% of reptile species, and this clearly limits the scope for evaluating the evolutionary history of SDMs. In addition, new technologies will help in identifying cryptic sex chromosomes that have escaped detection in early studies. Both karyological and experimental studies are needed to identify the relative influences of genotype and environment (and their interaction) on sex determination in the vast majority of reptilian species. Coupled with better-resolved phylogenies, these studies will undoubtedly change our current understanding of the phylogenetic distribution of SDMs
Hormones and Reproduction of Vertebrates
and provide insights into the origins of and transitions among SDMs. More exploration into the mechanistic underpinnings of TSD and GSD is needed. We have only a crude understanding of the molecular regulatory networks and hormonal pathways involved in sex determination and sexual differentiation. Comparative work that simultaneously evaluates the gene networks in closely related TSD and GSD taxa will provide critical insights into the evolution or loss of master thermal switches involved in reptilian sex determination. Additionally, research on the thermal sensitivities of gene expression during gonadogenesis under fluctuating temperature regimens will provide a better understanding of how these systems work in nature. The role of maternally derived steroids in egg yolks in modulating sexual differentiation has only recently been given research attention. Hence, much work is needed to fully understand the factors that influence maternal allocation of steroids into yolk, as well as their interaction with incubation temperature and sex-determining genes. Additional research on TSD under natural conditions is urgently needed. At present, almost all studies describe sexdetermining reaction norms based on constant-temperature incubation in the laboratory. Experiments that mimic natural thermal regimens will be critical not only in identifying how these complex conditions affect sex ratios but also in our understanding of how natural thermal conditions affect sex-specific traits that are relevant to fitness. More work is needed to evaluate the long-term effects of natural incubation regimes on fitness (i.e., reproductive success) in nature. Certainly, short-lived reptiles with TSD (e.g., agamid lizards) will facilitate such research, but work is also needed on longer-lived species (e.g., turtles and crocodilians) to evaluate generalities or lack thereof. These types of experiment will provide critical insights into how Charnov– Bull (1977) predictions explain the adaptive significance of TSD in nature. Further, sophisticated CTE models, similar to those developed by Georges et al. (2004; 2005), are needed to successfully predict offspring sex ratios from natural nest temperatures in species with the FMF pattern of TSD. Lastly, in the face of human-induced habitat modifications and global climate change, more research is needed to understand the microevolutionary potential of both GSD and TSD. Experiments designed to evaluate variation in and heritabilities of embryonic sex-determining reaction norms and maternal nest-site selection will provide critical insights into how TSD reptiles will deal with rapid environmental changes. The field of reptilian sex determination has a very dynamic history and its future will undoubtedly continue in this way as more information is gathered (Bull, 2004). To fully understand the functional and evolutionary aspects of SDMs, future research needs to take a comprehensive
Chapter | 1
Sex Determination in Reptiles
integrative approach that utilizes skills from investigators in multiple disciplines. Given the diversity of SDMs within reptiles and their feasibility as model organisms, this group will continue to serve as a model for our general understanding of the proximate mechanisms and evolution of sex-determining mechanisms.
ACKNOWLEDGEMENTS I dedicate this chapter to my colleague and friend, Raju Radder, who passed away during the early stages of this contribution. Raju’s research and our many discussions on reptilian sex determination have influenced many aspects of my understanding and interest in this field; his contributions to this field will be greatly missed. I thank F. Janzen, S. McGaugh, K. Lopez, T. Mitchell, D. Norris, T. Schwartz, R. Shine, T. Uller, and N. Valenzuela for insightful discussions and comments on early drafts of this chapter. Thanks to L. Kratochvı´l for providing access to an unpublished manuscript. The author was supported by National Science Foundation grant DEB-0640932 (to F. Janzen) during the preparation of this chapter.
ABBREVIATIONS AMH CTE Dax1 DHT Dmrt1 E2 ESD FM pattern FMF pattern GSD MF pattern MIS SDM Sf1 Sox9 Sry T TRT TSD TSP Wt1
Anti-Mu¨llerian hormone Constant temperature equivalent Dosage-sensitive sex reversal gene Dihydrotestosterone Doublesex and Mab-3 related transcription factor 17b-estradiol Environmental sex determination Females produced at low temperatures, males at high temperatures Females produced at extreme temperatures, males at intermediate temperatures Genotypic sex determination Males produced at low temperatures, females at high temperatures Mu¨llerian-inhibiting substance Sex-determining mechanism Steroidgenic factor 1 Sry-like HMG box Sex-determining region on the Y chromosome Testosterone Transitional range of temperatures Temperature-dependent sex determination Thermosensitive period Wilm’s tumour suppressor gene
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Webb, J. K., Brown, G. P., & Shine, R. (2001). Body size, locomotor speed and antipredator behaviour in a tropical snake (Tropidonophis mairii, Colubridae): the influence of incubation environments and genetic factors. Funct. Ecol., 15, 561–568. West, S. (2009). Sex Allocation. Princeton: Princeton University Press. Western, P. S., Harry, J. L., Graves, J. A., & Sinclair, A. H. (1999a). Temperature-dependent sex determination: upregulation of SOX9 expression after commitment to male development. Developmental Dynamics, 214, 171–177. Western, P. S., Harry, J. L., Graves, J. A., & Sinclair, A. H. (1999b). Temperature-dependent sex determination in the American alligator: AMH precedes SOX9 expression. Developmental Dynamics, 216, 411–419. Western, P. S., Harry, J. L., Marshall-Graves, J. A., & Sinclair, A. H. (2000). Temperature-dependent sex determination in the American alligator: expression of SF1, WT1 and DAX1 during gonadogenesis. Gene, 241, 223–232. Wennstrom, K. L., & Crews, D. (1995). Making males from females: the effects of aromatase inhibitors on a parthenogenetic species of whiptail lizards. Gen. Comp. Endocrinol., 99, 316–322. White, R. B., & Thomas, P. (1992). Adrenal–kidney and gonadal steroidogenesis during sexual differentiation of a reptile with
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Chapter 2
Neuroendocrinology of Reptilian Reproductive Behavior Michele A. Johnson and Juli Wade Michigan State University, East Lansing, MI, USA
SUMMARY Reptiles provide ideal species in which to address questions of the neuroendocrine influence on reproduction, as they exhibit extensive variation in many reproductive traits. This chapter describes the use of comparisons among and within reptilian species that allow detailed investigations of the relationships between neuroendocrine mechanisms and reproductive behaviors. In particular, we explore comparisons among species with different courtship and copulatory behaviors and different modes of sex determination, between sexes and among alternative reproductive morphs, and across environmental conditions that may facilitate or inhibit breeding. The roles of hormones and their receptors, neural structures, and associated muscular systems in the production of reproductive behaviors are reviewed, along with the effects of developmental and adult plasticity in producing their diversity. This discussion is framed around the lizard Anolis carolinensis, the species for which the most neuroendocrine information is available, and research on other species is reviewed.
1. INTRODUCTION Reptiles provide ideal opportunities for addressing many questions regarding the neuroendocrinology of reproduction, even though mechanisms controlling the display of sexual behaviors have been studied more extensively in mammalian and avian systems to date. A particularly useful strategy for identifying factors critical in reproductive evolution involves comparisons at multiple taxonomic levels, across diverse organisms that exist under a variety of ecological and physiological constraints. At the broadest level, evaluating traits across vertebrate groups facilitates our understanding of which mechanisms are fundamental and which have evolved based on particular features of organisms and their environments. Comparisons within animal classes allow examination of organisms that differ in general features of reproduction, offering more direct ways of testing hypotheses on the function or regulation of those features. Finally, studies within species can be
Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
especially valuable in determining relationships between structure and function and the mechanisms controlling them. These intraspecific investigations might include analyses between males and females, among morphs within a sex, and across seasons within individuals. Reptiles offer a spectacular set of natural experiments. Reptilian species exhibit an impressive degree of variation in many behavioral traits, including foraging modes, antipredation strategies, and thermoregulatory behaviors, but none are more diverse than the reproductive functions exhibited in this group. In particular, courtship and copulatory strategies, modes of sex determination, and types of gestation provide exceptional models for understanding the factors that regulate reproduction. In addition, many squamates, and lizards in particular, can readily be studied in both their natural environments, which allow the full range of natural behavior, and in laboratory settings, which allow more controlled manipulations. Despite all of this diversity, there are relatively few reptilian model systems for which we have extensive information regarding the many factors associated with the neuroendocrine regulation of reproductive behaviors. The reptile with the longest and most extensive history of neuroendocrine study is the green anole (Anolis carolinensis) (Figure 2.1). Publications describing the behavior of this species date as far back as the 1800s (e.g., Monks, 1881), and substantial work on its endocrine control began in the 1940s (Evans & Clapp, 1940; Noble & Greenberg, 1941a; 1941b). Investigations on the neural and muscular systems critical for the display of reproductive behaviors started about 30 years later and continue today, focusing on the integrated field of neuroendocrinology. While more recent studies of neuroendocrine mechanisms in reptiles have encompassed a broader taxonomic range, A. carolinensis remains the species for which the largest quantity of detailed and diverse information is available. Therefore, the information presented in this chapter is framed in the context of this species, with relevant material from other reptiles included. 39
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FIGURE 2.1 Male Anolis carolinesis, Louisiana, USA. Photo by M. Johnson. See color plate section.
This chapter primarily focuses on the features associated with displays of sexual behavior, drawing mostly from research on the species that have been most extensively studied. We consider behavioral displays in their ecological contexts, associated seasonal and hormonal changes, the muscles and portions of the nervous system critical for production of the behaviors, and the development and plasticity of reproductive traits. Finally, we attempt to integrate across the levels and discuss relationships among morphology, behavior, and the endocrine and other mechanisms regulating these traits. Territorial aggression and dominance (and the stress associated with these social behaviors) are also critical components of reproductive success that involve neuroendocrine mechanisms, including
an important role for neurotransmitters (e.g., serotonin and dopamine (recently reviewed in Overli et al., 2007)). This literature is described in detail in Chapter 7, this volume; the focus here is on the sexual behaviors themselves.
2. BEHAVIORAL DISPLAYS IN ECOLOGICAL CONTEXT 2.1. Anolis Lizards and the Ecology of Visual Reproductive Displays Anolis carolinensis is a member of the incredibly diverse and speciose genus Anolis, a group of approximately 400
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Neuroendocrinology of Reptilian Reproductive Behavior
arboreal lizard species whose members occur throughout the southeastern USA, the Caribbean, and Central and South America (Schwartz & Henderson, 1991; Nicholson et al., 2005). In general, species naturally occurring in the Caribbean have been studied more extensively than those on the mainland, with the exception of A. carolinensis, whose native range extends throughout the southeastern USA. Of the ~150 Caribbean species, most can be assigned to one of six groups of habitat specialists, called ecomorphs. These ecomorphs are named by the arboreal microhabitat to which they are morphologically, ecologically, and behaviorally adapted, and include the designations trunk-ground, trunk-crown, crown-giant, twig, trunk, and grass-bush (Williams, 1983). Phylogenetic studies have revealed that each ecomorph has independently evolved multiple times on the four islands of the Greater Antilles (Jackman, Losos, Larson, & de Queiroz, 1997; Losos, Jackman, Larson, de Queiroz, & Rodrı´guez-Schettino, 1998), such that species are likely to be more closely related to species in other ecomorphs on the same island than to species in the same ecomorph on other islands. Among anoles, structures involved in reproduction are highly variable. In particular, the morphology of the dewlap, an extendable throat fan involved in courtship, is remarkably diverse, with most species exhibiting a unique combination of dewlap color, pattern, and size (Nicholson, Harmon, & Losos, 2007). The variation of sexual dimorphism in dewlap morphology is also extensive. While in most species of anole the dewlap is substantially larger in males than females, species also exist in which the two sexes have similarly sized dewlaps (in some cases diminutive in both males and females, while in others the dewlap is sizable in both sexes). The natural experiments offered by the diversity in ecology and morphology across anole species provide a unique opportunity for a wide variety of investigations of numerous factors associated with reproductive behaviors and other functions. Despite the impressive diversity of anoles, comprehensive data on male and female reproductive behaviors and the neural circuits that produce them are available only for A. carolinensis, a species in the trunk-crown ecomorph. These seasonally breeding lizards reproduce in the field from approximately April through July; long days and warm temperatures induce testicular and ovarian growth (Licht, 1967a; 1967b; see also Lovern, Holmes, & Wade, 2004). The gonads become refractory to environmental conditions in late summer, followed by a period of modest testicular (although minimal ovarian) growth during the winter. Outside of the breeding season, individuals aggregate in concealed locations, but may bask on warm days. Growth of the gonads is completed in the early spring, and secretions of these organs activate the display of sexual behaviors. The sequence of social behaviors displayed by male green anoles is stereotyped and easily observed in both field
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and laboratory conditions. Courtship displays consist of headbobs, pushups, and extensions of the bright reddishpink dewlap (Greenberg & Noble, 1944; Crews & Greenberg, 1981; Decourcy & Jenssen, 1994). Males also perform these displays in aggressive encounters with conspecific males, mainly during territory defense. In this latter context, however, the behavior is usually accompanied by the formation of a black spot behind each eye, the raising of a crest along the back, and postural changes (Greenberg, 2003). Following courtship, a male green anole will mount the female, maneuver his tail under hers, evert one of two hemipenes from inside his cloacal vent, intromit, and ejaculate (Crews, 1980). This possession of bilateral, independently controlled hemipenes is typical of squamates, and males often alternate which one is used in consecutive intromissions (Shine, Olsson, LeMaster, Moore, & Mason, 2000). During courtship and copulation, female green anoles may bob their heads in a manner similar to males, but generally without dewlap extensions (Nunez, Jenssen, & Ersland, 1997). Females do not have a characteristic receptive posture but, if a female is receptive, she will remain stationary and allow the male to mount, and she will often bend her neck to facilitate his grasping the skin over the neck between his teeth (Crews, 1980).
2.2. Reproductive Communication in Other Reptilian Taxa 2.2.1. Differences between males and females Courtship behaviors provide key information related to mate selection. The modes of communication used in courtship are species-specific and extremely variable among reptiles, in terms of signal complexity and the sensory systems associated with interpretation of the displays. Most iguanian lizards (including anoles) communicate primarily through visual signals (reviewed in Ord, Blumstein, & Evans, 2002), but many other reptiles use largely chemical and/or auditory signals to identify and attract potential mates and to detract rivals. In red-sided garter snakes (Thamnophis sirtalis parietalis), male courtship is facilitated by a female pheromone. Interestingly, some males produce this pheromone, potentially allowing them to gain access to females in mating aggregations and increase their reproductive competence (Mason & Crews, 1985). The composition of the pheromone differs across individuals, allowing males to discriminate between large and small females (LeMaster & Mason, 2002), as well as females from their own vs. another population (LeMaster & Mason, 2003). Male leopard geckos (Eublepharis macularius) also use chemical signals during courtship. When approaching a female, a male will explore for female pheromones by
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licking the substrate or the air (i.e., performing ‘tongue flicks’), a behavior common among chemical-sensing reptiles. Male leopard geckos can also recognize individual females based on these chemical cues (LaDage & Ferkin, 2006). When females are detected, male geckos use auditory signals as well, generating a buzzing sound by rapidly vibrating their tails (Godwin & Crews, 2002). Individuals of several genera, including Sceloporus, Cnemidophorus, and Urosaurus, transfer sex pheromones from femoral pores to the substrate. These chemicals can send a variety of messages important in mate choice. For example, in female Sceloporus graciosus, the sagebrush lizard, these femoral pore secretions can communicate physiological condition and reproductive status (Martins, Ord, Slaven, Wright, & Housworth, 2006) or receptivity, as females produce more secretions when exposed to a male performing courtship displays than a male performing aggressive displays (Kelso & Martins, 2008). Femoral gland secretions from males of the Iberian rock lizard (Lacerta monticola) (Martin, Moriera, & Lopez, 2007) and the lizard Liolaemus monticola (Labra, 2006) indicate social dominance status. Interestingly, the ability to perceive different types of sensory information in courtship may also be associated with differences in brain morphology and activity, as suggested by the differences exhibited between male and female Podarcis hispanica, a species in which males have larger accessory olfactory bulbs, and in parallel are more responsive to chemical cues, than females (Sampedro, Font, & Desfilis, 2008). Parallel to the diversity in courtship structures, the morphology of male copulatory organs (hemipenes) is strikingly diverse among many reptilian species, particularly in snakes (Dowling & Savage, 1960) and lacertids (e.g., Arnold, 1986), in which the structures may be covered with features such as spines, ridges, and fleshy protuberances. However, even with the diversity of these organs, copulatory behavior across reptiles appears generally conserved. The function of copulationdsperm delivery from the male to the femaledis identical across species, and morphological constraints on this function have likely directed the evolution of copulatory behavior as well.
2.2.2. Variation within sexes: Alternative reproductive strategies The taxa described above provide many opportunities to compare reproductive structure and function between males and females, but reptiles also allow for examinations of the differences among groups within a sex. In particular, males within a species often exhibit generally non-overlapping phenotypic variation (called different ‘morphs’). In such species, one morph frequently displays the full range of male-typical morphological and behavioral traits and
Hormones and Reproduction of Vertebrates
another is more female-like in some of these traits (reviewed in Knapp, 2003). Generally, both male types are able to produce functional sperm, but, as described below, they use different behavioral tactics to achieve reproduction (Knapp, 2003). Two species of iguanid lizard in particular have become model systems in which to examine relationships between reproductive behavior and neuroendocrine traits in these alternative morphsdUrosaurus ornatus, the tree lizard, and Uta stansburiana, the side-blotched lizarddalthough studies of several other species have made important contributions as well (see references below). U. ornatus male morphs are distinguished by their dewlap coloration. While up to nine variations exist, most populations of this species have two general types: males with orange-blue dewlaps, which aggressively defend territories that typically overlap the territories of multiple females, and males with orange dewlaps, which are less aggressive and may switch between sedentary satellite and nomadic behavior (Moore, Hews, & Knapp, 1998). Results from a common garden experiment indicate that morph type is genetically based (Thompson, Moore, I., & Moore, M., 1993), and behavioral and morphological differences between morphs appear very early in male development (Moore et al., 1998). Male U. ornatus exhibit behavioral courtship displays similar to those of Anolis lizards, including dewlap extensions, pushups, and lateral compression, the lattermost of which serves to present the throat coloration and bright blue ventrolateral (belly) patches. Independent of morph, the size of this patch predicts bite force, an important indication of fighting ability (Meyers, Irschick, Vanhooydonck, & Herrel, 2006), and larger belly patches are preferred by female U. ornatus (Hamilton & Sullivan, 2005). U. stansburiana males exhibit three alternative morphs, also distinguished by dewlap color: orange, blue, and yellow-throated males, which differ in reproductive and aggressive behaviors. Orange males are aggressive and defend territories encompassing multiple females; less aggressive blue males diligently guard females on their territories; and yellow males are non-territorial sneakers, or deceptive female mimics that ‘steal’ copulations when the other males are inattentive (Sinervo & Lively, 1996; Sinervo, Miles, Frankino, Klukowski, & DeNardo, 2000; Sinervo & Clobert, 2003). These morphs are genetically determined, and multiple lines of evidence indicate that this variation is based on a single Mendelian gene (Sinervo, Bleay, & Adamopoulou, 2001). The three morphs are maintained because of frequency-dependent selection that favors the rarest morph in a classic rock-paper-scissors ‘game’ in which each morph can be reproductively outcompeted by another (Zamudio & Sinervo, 2000). The same locus that determines male morphs in U. stansburiana also determines female reproductive strategies that differ in resource allocation to offspring.
Chapter | 2
Neuroendocrinology of Reptilian Reproductive Behavior
Orange-throated females have been described as rstrategists, laying larger clutches of smaller eggs, while yellow-throated females are K-strategists, laying smaller clutches of larger eggs (Sinervo & Licht, 1991; Sinervo, Svensson, & Comendant, 2000). Males of the lacertid lizard Podarcis melisellensis may exhibit reproductive morphs based on throat (and abdomen) color as well. White, yellow, and orange males differ from one another in body size and performance (but not in activity budgets), and these differences may underlie different reproductive strategies (Huyghe, Vanhooydonck, Herrel, Tadic, & Van Damme, 2007), although further work is needed to address this hypothesis. Galapagos marine iguanas (Amblyrhynchus cristatus) also exhibit multiple male morphs, but they are determined largely by male age and size; as a male grows, he adopts a different reproductive strategy (Wikelski, Carbone, & Trillmich, 1996; Wikelski, Carbone, Bednekoff, Choudhury, & Tebbich, 2001). The smallest (and youngest) male iguanas look very similar to females, and because they cannot be distinguished from females by larger males, these small males live on territories of other males and use a sneaking strategy to mate with unattended females. As sneakers grow larger, they adopt a satellite strategy, living around other males’ territories and attempting to mate with females as resident males leave these territories. The largest, oldest males in the population defend territories in which females live and they court these females using headbobbing displays. Similarly, collared lizard (Crotophytus collaris) males exhibit developmentally plastic reproductive strategies as they mature from smaller, younger, non-territorial males to larger, dominant, territory-holding males (Baird & Hews, 2007).
2.3. Summary The diversity of reproductive behaviors exhibited by reptiles provides many interspecific and intraspecific comparisons of courtship and copulatory structure and function. Because all behaviors are performed in an ecological context, an appreciation of the social and physical environments in which reproductive behaviors have evolved is necessary for informed investigations of the mechanisms underlying these behaviors. Having described these general features of reproductive behaviors in reptiles, the relevant mechanisms associated with their control will now be considered.
3. HORMONAL CONTROL OF REPRODUCTIVE BEHAVIORS IN ADULTHOOD Reproductive behaviors in many species of vertebrates are influenced by sex steroid hormones. Testosterone (T),
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primarily produced by the testes, and its metabolites are particularly important for males across many animal taxa. For example, increases in T in males within a species can often be associated with larger territories, more sexual and aggressive behavior, higher dominance, larger or more colorful ornaments, and greater endurance (reviewed in Miles, Sinervo, Hazard, Svensson, & Costa, 2007). In parallel, female behaviors such as sexual receptivity are routinely facilitated by ovarian hormones such as estradiol (E2) and progesterone (P4) (Crews & Silver, 1985; Blaustein & Erskine, 2002).
3.1. Hormonal Control of Anole Reproduction Consistent with the general patterns described above, T activates reproductive behaviors in male green anoles. Castration diminishes courtship and copulation, and treatment with T prevents and/or reverses that effect (Crews, Traina, Wetzel, & Muller, 1978; Adkins & Schlesinger, 1979; Winkler & Wade, 1998; Rosen, O’Bryant, Matthews, Zacharewski, & Wade, 2002). It is common among mammalian and avian species that, for an animal to produce male reproductive behaviors, T must be metabolized into E2 or 5a-dihydrotestosterone (DHT) within the brain (Ball & Balthazart, 2002; Hull, Meisel, & Sachs, 2002). However, in green anoles, T itself is likely the primary factor responsible for the activation of male sexual behaviors, as systemic treatment with E2 and inhibition of E2 synthesis have little or no effect on male sexual behavior (Mason & Adkins, 1976; Crews et al., 1978; Winkler & Wade, 1998). 5a-dihydrotestosterone is also not sufficient to activate the display of male reproductive behaviors, but metabolism of T into DHT is required for maximal behavioral performance (Rosen & Wade, 2000). Interestingly, the effects of these hormones depend on the season, as the ability of exogenous T to activate male sexual behavior is increased in the breeding season (BS) compared to the non-breeding season (NBS) (Neal & Wade, 2007a). In Anolis sagrei, E2 is also not effective in activating sexual behaviors in castrated males to the levels seen in intact males (Tokarz, 1986), and treatment of anoles with antiandrogens produces variable results: cyproterone acetate (which prevents androgens from binding to their receptors and suppresses luteinizing hormone (LH)) inhibits aspects of male sexual behavior, but flutamide (which also blocks androgen receptors (ARs)) does not (Tokarz, 1987). The most detailed description of the reproductive cycle in female green anoles indicates that individuals ovulate a single egg about every 14 days alternately from each of the two ovaries (Jones, Guillette, Summers, Tokarz, & Crews, 1983). More recent laboratory work suggests that eggs can be laid as frequently as every seven days (Lovern et al., 2004a). Regardless, this pattern results in females carrying
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a single egg in the oviduct through much of their cycle, but a period of a few days exists in which a shelled egg is soon to be laid and another recently ovulated one is in the early stage of development in the other oviduct. Removal of one ovary in green anoles causes compensatory follicular development on the other side and influences hypothalamic catecholamine asymmetries, likely through both neural and hormonal signals (Jones et al., 1997). When an egg is present in each oviduct, adult female plasma levels of E2 and P4 are both around 2.2 ng/ml whereas, in the one-egg condition, P4 levels are slightly higher and E2 is substantially lower (~600 pg/ml) (Jones et al., 1983). On average, across the stages of the ovulatory cycle, yolking follicles contain roughly 800 pg/ml of E2 while the level in the plasma of females carrying these follicles is less than half of that (Lovern & Wade, 2001). Levels of T are somewhat higher in yolking follicles (~6 ng/ml) but far lower in the plasma of these females (less than 200 pg/ml) (Lovern & Wade, 2001). Experimental manipulations confirm that both E2 and T play an important role in activating female receptivity in green anoles. For example, E2 alone can facilitate female sexual behaviors (Winkler & Wade, 1998) and subthreshold doses can prime P4 to activate the behavior (Wu, Whittier, & Crews, 1985). Parallel to the effects of T on male sexual behavior, this latter regime is more effective in the BS than the NBS (Wu et al., 1985). Administration of the anti-estrogen CI-628 can inhibit receptivity activated by E2 treatment in green anole females (Tokarz & Crews, 1980). Testosterone also facilitates receptivity in green anole females, a function at least in part due to aromatization (Winkler & Wade, 1998). Parallel to patterns observed in males, circulating T is increased in reproductively active compared to inactive females; further, among breeding females, those with a large yolking follicle have increased T compared to those with one or more eggs in the oviduct (Lovern & Wade, 2001). Thus, T is maximal when females are most likely to display sexual behavior. Prostaglandin F2a can inhibit receptivity in estrogentreated female anoles (Tokarz & Crews, 1981). In contrast, luteinizing hormone-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) facilitate the display of sexual behaviors in E2-primed individuals (Alderete, Tokarz, & Crews, 1980). Although specific mechanisms are unclear, these data suggest that the reproductive behaviors of green anole females are directly or indirectly modulated by a variety of extra-gonadal factors.
3.2. Hormonal Control in Species with Alternative Reproductive Strategies In U. ornatus, T and P4 also play important roles in the activation of male reproductive behaviors (Weiss & Moore, 2004). Although the two male morphs differ in adult
Hormones and Reproduction of Vertebrates
behavior, territorial orange-blue males and non-territorial orange males do not differ in their levels of circulating T (Moore et al., 1998). The variability in behavior may be the consequence of differential sensitivity to hormones resulting from early organizational effects (Weiss & Moore, 2004; see Section 5.2). In addition, a general relationship exists between T and behaviors related to territoriality, such that animals with higher levels of T exhibit higher frequency and intensity of displays (Kabelik, Weiss, & Moore, 2006), and these male behaviors are activated by both T and P4 (Weiss & Moore, 2004). Collared lizard males exhibit age- and size-related morphs (territorial vs. non-territorial) that also do not differ in T, DHT, or the stress hormone corticosterone (CORT) (Baird & Hews, 2007). Although courtship behaviors are more frequent in territorial males, there is no clear activational effect of steroid hormones on these behaviors; in fact, non-territorial males who move into a recently vacated territory increase their display rates, but this experience decreases T and CORT (Baird & Hews, 2007). Unlike tree and collared lizards, side-blotched lizard male morphs differ in sex steroid hormone levels; territorial orange males have higher levels of T than blue and yellow males (Sinervo et al., 2000a). Further, experimentally increasing T in yellow and blue males causes an increase in orange-male typical behaviors, including higher endurance, general activity, and control over access to females (Sinervo et al., 2000a). These effects of T may be influenced by the gonadotropins (GTHs) LH and folliclestimulating hormone (FSH), as these hormones regulate the function of the gonads. Both LH and FSH increase T secretion in this species, and both hormones influence many physiological and behavioral traits independently of the presumed function of FSH in sperm production (Mills et al., 2008). Hormones may also affect female reproductive strategies in side-blotched lizards. Treatment with FSH increases clutch size, but at the expense of offspring size (Sinervo & Licht, 1991), demonstrating the classic r (fewer, larger offspring) vs. K (more, smaller offspring) strategy tradeoff. These results indicate that clutch size is influenced by circulating plasma GTHs (Sinervo & Licht, 1991). Further, in crowded social conditions during the BS, both yellow (K-strategist) and orange (r-strategist) females have higher levels of CORT, but yellow females have higher levels than do orange females (Comendant, Sinervo, Svensson, & Wingfield, 2003). If this rise in CORT enhances the females’ competitive abilities, yellow females should experience the greater benefit. However, CORT appears to provide strategy-specific benefits to both morphs, such that the hormone improves offspring quality in K-strategist females and increases reproductive rates in r-strategist females (Lancaster, Hazard, Clobert, & Sinervo, 2007).
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Neuroendocrinology of Reptilian Reproductive Behavior
3.3. Hormonal Control of Reproduction in Other Reptilian Taxa A substantial body of work on the role of hormones in reproduction has been conducted in whiptail lizards, focusing mainly on two species, Cnemidophorus inornatus and Cnemidophorus uniparens. The former is gonochoristic (consists of both males and females); the latter is a direct descendent of C. inornatus and is composed only of females that reproduce by parthenogenesis (Crews, 1989). Control of masculine sexual behaviors in whiptail lizards is similar to green anoles. 5a-dihydrotestosterone, and particularly T, activate sexual behaviors in male C. inornatus. Interestingly, even months after castration, female C. inornatus and parthenogenetic C. uniparens appear sensitive to the ability of these hormones to activate malelike behaviors in response to female stimulus animals (Wade, Huang, & Crews, 1993). Progesterone can also activate the display of sexual behaviors in approximately one-third of male C. inornatus (Lindzey & Crews, 1992). This effect can be facilitated by low doses of DHT, and this androgen is more effective in activating the behaviors in P4-sensitive compared to P4-insensitive males (Lindzey & Crews, 1992), suggesting the potential for interactions between androgens and progestins in the regulation of masculine behaviors in this species. This ability of P4 to facilitate reproductive behaviors in some C. inornatus is particularly intriguing because the all-female C. uniparens also display male-like sexual behaviors (including mounting and assuming a stereotypical donut-shaped copulatory posture), and they do so predominantly during the post-ovulatory phase of their cycles when circulating P4 is high (Moore, Whittier, & Crews, 1985a; Moore, Whittier, Billy, & Crews, 1985b). In contrast, they tend to display female-typical receptivity prior to ovulation, when they have large yolking follicles and relatively high levels of E2 (Crews, 1980). The pattern of receptivity is comparable in females of the gonochoristic species, C. inornatus, and E2 treatment of gonadectomized females of both species activates receptivity (e.g., Wade et al., 1993). Further, in leopard geckos, like other reptiles, E2 activates sexual receptivity in females (Rhen & Crews, 2000). Ovariectomy in adulthood causes females to be significantly less attractive to potential mates, and treatment of ovariectomized females with T decreases their attractiveness further, to the point where many T-treated females are attacked by males (Flores & Crews, 1995; Rhen, Ross, & Crews, 1999). These results suggest that female pheromones (the major component of attractiveness) are affected by circulating hormones. Further, unmanipulated females never perform tail vibrations, but treatment with T causes about half of females to exhibit this typical male courtship behavior (Flores & Crews, 1995).
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The role of steroid hormones in female nesting behavior has also been a focus of neuroendocrine work in turtles, as these behaviors can directly affect the sex of offspring. In addition to the role of estrogens, androgens are an important factor in female reproduction, as indicated by the presence of AR in the turtle oviduct (Selcer, Smith, Clemens, & Palmer, 2005). An examination of hormone levels in green sea turtles (Chelonia mydas) comparing females who successfully oviposited to those who built nests but did not oviposit found that P4 is higher in successful than unsuccessful females, but that T, E2, and CORT did not differ between these groups (Al-Habsi et al., 2006). Further, while nesting female green sea turtles experience clear metabolic changes, hormones generally involved in metabolism (epinephrine, norepinephrine, and CORT) did not change across nesting stages, a finding that may indicate that the actions of these metabolic hormones are modified to facilitate reproduction (Jessop & Hamann, 2004). In contrast to many other reptiles (and other vertebrates), red-sided garter snakes (T. s. parietalis) exhibit a dissociated breeding pattern in which the display of sexual behaviors occurs at a time when gonadal hormones are low. These behaviors are restricted to a few weeks following emergence from winter hibernation, a time when the gonads are not yet recrudesced and circulating steroid levels are reduced. Male courtship behavior is not activated by gonadal androgens but rather by the increase in temperature following hibernation (Crews et al., 1984). This behavior appears to be ‘organized’ by testicular function the previous summer (Crews, 1991). Steroid hormones are also low in females at the time of mating. In these animals, mating stimulates a neuroendocrine reflex that causes a rise in prostaglandins (Whittier & Crews, 1989) and then a surge in E2 (Mendonc¸a & Crews, 1990). Unlike males, females normally will not exhibit sexual behavior on emergence from winter dormancy if they have been gonadectomized, but treatment with E2 just prior to emergence will reinstate receptivity (Mendonc¸a & Crews, 1996). Thus, similar to many other vertebrates, including a variety of lizard species, T is important for male sexual behaviors and E2 activates receptivity in females. However, the timing of gonadal development and steroid hormone production is asynchronous with the actual behavioral displays.
3.4. Summary Reptilian species provide particularly valuable models for the study of the role of sex steroid hormones in reproduction for at least two reasons. Investigations of hormones in reptiles support the general importance of T for male sexual behavior and E2 plus P4 for female sexual behavior, a pattern observed in many vertebrate taxa. However,
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details such as the role of particular metabolites can differ across species, allowing for exploration of their role in the evolution of diversity in hormonal mechanisms critical for the display of reproductive behaviors. These steroid hormones act on neural tissues to influence reproductive behavior. Critical regions of the brain are considered next.
4. NEURAL CONTROL OF REPRODUCTIVE BEHAVIORS Regions of the brain are highly conserved across vertebrate groups, particularly those regions of the forebrain involved in producing social and reproductive behaviors, (the ‘social behavior network’) (Newman, 1999; Goodson, 2005). Included in this network are the ventromedial hypothalamus (VMH), preoptic area (POA) and amygdala (AMG), regions with clear homologies in mammals, birds, and reptiles (Goodson, 2005). Whereas regions outside the forebrain are also involved in reproductive behavior, the forebrain is by far the most studied region of the brain in reptiles.
4.1. Neural and Muscular Control of Anole Reproduction In green anoles, far more is known about the neural control of male than female sexual behaviors. In the forebrain, lesion studies have shown that the anterior hypothalamuspreoptic area (AH-POA) (Wheeler & Crews, 1978) and ventromedial nucleus of the AMG (Greenberg, Scott, & Crews, 1984), equivalent to the posterior portion of the medial AMG (Bruce & Neary, 1995), are important for masculine courtship and copulation. Implants of DHT into the AH-POA can facilitate male courtship, and although systemic treatment with E2 in gonadectomized male green anoles has no effect on their sexual behavior, implants of E2 directly into the AH-POA can facilitate courtship (Crews & Morgantaler, 1979). Manipulations with this degree of specificity have not been conducted in female green anoles, but destruction of the basal hypothalamus impairs receptivity (Farragher & Crews, 1979). A variety of sex differences in the morphology of these regions exist in green anoles, but they largely depend on the animals’ endocrine environment and are therefore discussed below (see Section 6, on adult plasticity). The mechanics of dewlap extension have been studied in A. carolinensis and Anolis equestris. The simplicity of this system makes it particularly amenable to investigations of relationships between structure and function, as well as the mechanisms involved in regulating them. Key structures involve three pieces of bilateral cartilage in the throat and just under the skin overlaying the chest (reviewed in Wade, 2005) (Figure 2.2). Movement of the
Hormones and Reproduction of Vertebrates
cartilage is controlled by the paired ceratohyoid muscles, which lie in the throat; when these muscles contract, a lever-like action is induced among the cartilage components and the second ceratobranchials (which normally lie flat, running from under the chin to mid-chest) bow out, extending the colorful fan of skin; i.e., the dewlap (Bels, 1990; Font & Rome, 1990). Motoneurons innervate the ceratohyoid muscles ipsilaterally through the combined IXþX cranial nerve. These motoneurons are located in the caudal brainstem in the vagal component of the nucleus ambiguus (AmbX) and the region containing the glossopharyngeal portion of the nucleus ambiguus and the ventral motor nucleus of the facial nerve (AmbIX/ VIImv) (Font, 1991; Wade, 1998). Dewlap structure and function are sexually dimorphic in anoles. The dewlaps of males are generally three to seven times the size of those in females (Jenssen, Orrell, & Lovern, 2000), although the degree of this sexual dimorphism differs across species (see Section 2.1). Green anole males extend these structures more often than females do, even when administered equivalent amounts of T after castration (Winkler & Wade, 1998; but also see Mason & Adkins, 1976; Adkins & Schlesinger, 1979). In parallel, the length of the second ceratobranchial cartilage and crosssectional areas of motoneuron somas and the nerve containing their axons are larger in males (Wade, 1998; O’Bryant & Wade, 1999). However, dendritic arborization of dewlap motoneurons does not differ between breeding males and females (O’Bryant & Wade, 2000). The mass of the ceratohyoid muscles is greater in males than females, which is at least partially due to increased size of individual fibers (O’Bryant & Wade, 1999); neuromuscular junctions are also larger in males than females (O’Bryant & Wade, 1999; 2002a). In males, the size of these muscle fibers is correlated with the frequency with which dewlaps are extended in a sexual context (Neal & Wade, 2007b). The composition of fiber types in the ceratohyoid muscle is similar between the sexes, with a majority of fast-twitch fibers (both glycolytic and oxidative-glycolytic). However, the percentage of tonic fibers is increased in males, whereas slow-oxidative fibers are enhanced in females (Rosen, O’Bryant, Swender, & Wade, 2004). These results are consistent with the fact that the basic features of dewlap extension are similar in the two sexes (Jenssen et al., 2000); however, more tonic fibers may help males to stabilize and/ or maintain the extension of their much larger dewlaps. In green anoles, sex differences in the neuromuscular system controlling hemipenis function during copulation are even more dramatic than in the dewlap system (Ruiz & Wade, 2002). Movement of each hemipenis is largely controlled by a set of ipsilateral muscles (reviewed in Wade, 2005) (Figure 2.3). The transversus penis (TPN) muscle wraps around the ventral surface of the hemipenis in a medial to lateral orientation; its contraction causes the
Chapter | 2
Neuroendocrinology of Reptilian Reproductive Behavior
organ to evert through the cloacal vent. The retractor penis magnus (RPM) muscle is attached to the caudal end of the hemipenis as it lies in the tail and controls its retraction following copulation (Arnold, 1984). Transversus penis and RPM motoneurons are located in the last trunk and first sacral segments of the spinal cord (T17-S1; these trunk segments are equivalent to the combined thoracic and lumbar regions of mammals). Within this region of the cord, just over half of the motoneurons project to the TPN and RPM, while most of the rest innervate the caudofemoralis (a muscle that lies mostly in the tail and controls leg movement) and the sphincter cloacae (SC). Only males possess hemipenes and the associated TPN and RPM muscles (Ruiz & Wade, 2002), and T17-S1 motoneurons are larger and more numerous in males than females (Ruiz & Wade, 2002; Holmes and Wade, 2004a). The other two muscles are present in females as well as males (Ruiz & Wade, 2002; Holmes & Wade, 2004a). Details on the copulatory motoneurons have rarely been described in reptilian species, but the general pattern of hemipenis and muscle anatomy is
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reasonably well conserved across squamates. Additional details and exceptions are described in Arnold (1984). Androgen receptors are present in green anoles in the same general regions of the brain as in other vertebrates (e.g., high levels in limbic regions, such as the POA and AMG, and in some motor nuclei), as well as in one of the two motor nuclei for dewlap extension, AmbX (Rosen et al., 2002). Androgen receptors are also expressed in copulatory motoneurons and both the dewlap and copulatory muscles, as well as the target structures for both systems (cartilages and hemipenes) (Holmes & Wade, 2005a). Expression of the AR protein does not appear to vary across seasons, and in none of the reproductive tissues (brain regions, spinal cord, or muscles) does the number of nuclei expressing the AR protein relate to the frequency of behavioral displays in unmanipulated male green anoles (Neal & Wade, 2007b). However, T treatment increases the percentage of nuclei in the copulatory muscles that express AR, an effect that is not detected in the ceratohyoid muscle or in either copulatory or dewlap motoneurons (Holmes &
(a)
(b)
(c)
(d)
FIGURE 2.2 Components of dewlap extension in the green anole. (a) and (b) show lateral views of a male before and during dewlap extension, respectively. (c) shows a dorsal view of the general location within the brain of the motoneurons that extend the dewlap. These cells exist in two regions: the vagal component of the nucleus ambiguus (AmbX) and the region containing the glossopharyngeal portion of the nucleus ambiguus and the ventral motor nucleus of the facial nerve (AmbIX/VIImv). (d) provides a ventral view of the muscles and cartilage that control dewlap extension. M, ceratohyoid muscle; C1, first ceratobranchial cartilage; CH, ceratohyal cartilage. Reprinted with permission from Hormone and Behavior, copyright 2005 (Figure 1; Wade, 2005).
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Hormones and Reproduction of Vertebrates
(a)
(b)
(c)
FIGURE 2.3 Copulatory system anatomy in the green anole. (a) Vertebral morphology with relation to the body. (b) Enlargement of the boxed region in (a). Trunk 15 through caudal 1 spinal segments, the sacral ribs, ilium, and the femur are identified on the left side, while the spinal cord and nerves are shown on the right. (c) Ventral view of the rostral tail muscles. On the left side, the hemipene (H) and associated transversus penis (TPN) and retractor penis magnus (RPM) muscles are depicted, structures which are absent in females. The (more dorsal) muscles present in both sexes are identified on the right side, including a leg muscle called the caudifemoralis (CF), which is located primarily in the tail with the ischiocaudalis (IC) and the iliocaudalis (ILC) tail muscles. In the left portion of (c), the IC and ILC are depicted as slightly pulled apart to expose the RPM for the drawing. The sphincter cloacae (SC) muscle lies along the cloacal vent (V). Reprinted with permission from Hormones and Behavior, copyright 2005 (Figure 2; Wade, 2005).
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Wade, 2005a). Additionally, expression of the AR protein in the POA, AMG, and dewlap and copulatory muscles and motoneurons is not related to the frequency of courtship or copulatory displays (Neal & Wade, 2007b). Estrogen receptor-a (ERa) is also expressed in the green anole in regions comparable to other vertebrates (Martinez-Vargas, Keefer, & Stumpf, 1978; Morrell, Crews, Ballin, Morgentaler, & Pfaff, 1979; Beck & Wade, 2009a; 2009b). In both sexes, ERa mRNA shows a two- to three-fold increase in the VMH (which is likely important for estrogen-dependent female receptivity) compared to the POA and AMG (which are critical for the display of male sexual behaviors). In unmanipulated animals, expression of ERa mRNA is sexually dimorphic in the POA and VMH; levels are higher in females than in males, but do not differ between the BS and NBS (Beck & Wade, 2009a). In gonadectomized individuals, E2 treatment downregulates estrogen receptor mRNA in males but not females. In the VMH, females express greater levels of ERa mRNA compared to males (Beck & Wade, 2009b). The patterns of neural enzyme expression in adult green anoles is similar to other vertebrates (Balthazart, 1997; Lephart, 1996). In particular, the estrogen synthetic enzyme aromatase is relatively high in the hypothalamus and POA and, despite not being critical for masculine sexual behavior, the activity of the enzyme in the brain is increased in males compared to females and is greater in breeding than in non-breeding males (Rosen & Wade, 2001). In contrast, 5a-reductase activity, which seems important for full production of masculine sexual behaviors is relatively high in the brainstem, which contains the dewlap motoneurons (Wade, 1997).
4.2. Neural Control of Whiptail Lizard Reproduction Consistent with the results from green anoles, lesion studies have documented the importance of the AH-POA in the control of masculine sexual behavior in whiptail lizards, in both male C. inornatus and the parthenogenetic C. uniparens (Kingston & Crews, 1994). Implantation of DHT in this region facilitates the display of masculine reproductive behaviors in both whiptail species (Mayo & Crews, 1987; Rozendaal & Crews, 1989). Lesions of the VMH inhibit the display of receptivity and local implants of E2 activate it (Wade & Crews, 1991; Kendrick, Rand, & Crews, 1995). During the BS, the AH-POA of C. inornatus is larger in volume in males and the VMH is larger in females (Crews, Wade, & Wilczynski, 1990). These sex differences parallel their roles in the facilitation of sexual behaviors. The most extensive work to date on the regulation of steroid receptor expression (mRNA for estrogen, androgen,
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and P4 receptors) in reptiles has been conducted in whiptail lizards. In general, these studies have indicated that steroid receptors are present in regions similar to green anoles and other vertebrates, including the POA, VMH, and AMG (reviewed in Godwin & Crews, 2002), which are important for the control of reproductive behaviors. Overall, the parthenogen C. uniparens appears to express increased levels of steroid receptors compared to C. inornatus, perhaps because they are triploid rather than diploid like the gonochoristic species. A variety of effects of sex, season, and hormone manipulation have been detected (reviewed in Godwin & Crews, 2002). For example, consistent with its role in the control of sexual behavior, male C. inornatus express more AR mRNA in the medial POA than females of either species. These AR levels decrease with T treatment in males, but not in females. In contrast, this androgen (T) causes increases in estrogen and P4 receptor (PR) mRNA in the VMH similarly in the two sexes. Aromatization of T to E2 might be important in some cases. Progesterone receptor mRNA in the periventricular POA is increased in both males and females by T, but not by the non-aromatizable androgen DHT. In C. uniparens, E2 selectively increases the expression of estrogen receptors in brain regions including the VMH while decreasing it in other areas, such as the lateral septum. The hormone also increases PRs in the VMH and POA of females of both species, but it is more effective in C. uniparens. In males (C. inornatus), these increases are not induced one week after castration, but after six weeks the response is similar to that of females. Recent research has also confirmed the importance of neurotransmitters in the regulation of reproductive behaviors in whiptails. In the parthenogenetic C. uniparens, serotonin inhibits the display of male-like pseudosexual behaviors (Dias & Crews, 2006). In fact, it inhibits both male-typical and female-typical reproductive behaviors. Specifically, injecting serotonin into the POA of animals that have been ovariectomized and treated with T decreases mounting, whereas injection into the VMH of gonadectomized, E2-treated C. uniparens suppresses receptivity (Dias & Crews, 2008). The level of serotonin is lower in post-ovulatory and ovariectomized, T-treated C. uniparens (both of which are likely to display masculine sexual behaviors) compared to those that were preovulatory or ovariectomized and administered E2 (likely to be receptive) (Dias & Crews, 2008). Masculine and feminine behaviors in these parthenogens may be regulated via different serotonin receptors; systemic stimulation of the 1a receptor inhibits mounting, whereas activating the 2a receptor facilitates receptivity (Dias & Crews, 2008). Dopamine may also be involved in this regulation, as treatment with a dopamine receptor agonist (D1) facilitates male-like pseudosexual behaviors in C. uniparens as well as sexual behaviors in P-sensitive male C. inornatus.
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Interestingly, a lower dose was active in the parthenogen, suggesting increased sensitivity (Woolley, Sakata, Gupta, & Crews, 2001).
4.3. Neural Control of Reproduction in Other Reptiles In red-sided garter snakes, lesioning the POA abolishes courtship behavior in males (Friedman & Crews, 1985, Krohmer & Crews, 1987a). In contrast, destruction of the nucleus sphericus (NS) (a homolog of a portion of the AMG that receives vomeronasal input) (Martinez-Marcos, Lanuza, & Halpern, 1999) facilitates courtship (Krohmer & Crews, 1987b), suggesting that the POA might be under inhibitory control by the NS. Studies of neurotransmitters in this species have shown that melatonin and CORT decrease courtship behavior in males and that their effects appear to be additive and may be modulated by the serotonin system (Lutterschmidt, LeMaster, & Mason, 2004). Removal of the pineal gland in males blocks the onset of courtship following hibernation (Nelson, Mason, Krohmer, & Crews, 1987). Several regions of the forebrain also have been shown to play an important role in the social and sexual behavior of U. ornatus, and these nuclei are influenced by sex steroid hormones. The volume of brain nuclei, including the POA, AMG, and VMH, are greater in males of this species than females (Kabelik et al., 2006). However, orange-blue and orange males were not found to differ in the volume of these brain regions (Kabelik et al., 2006) or in neural activity in the VMH (Kabelik, Crombie, & Moore, 2008a). The size of the POA and VMH also vary in leopard geckos. However, the differences are due to incubation temperature rather than gonadal sex (Coomber, Crews, & Gonzalez-Lima, 1997). Thus, the manner in which they are organized appears to differ from vertebrates with sex chromosomes, in which gonadal hormones frequently play a major role in the organization of both brain and behavior (Adkins-Regan, 1981; De Vries & Simerly, 2002). In leopard geckos, all adults have hemipenes and copulatory muscles, although these structures are much smaller in females than in males (Holmes, Putz, Crews, & Wade, 2005). In addition, sex differences do not appear to exist in the size or number of motoneurons in the region of the caudal spinal cord containing the associated motoneurons. Research on leopard gecko brain anatomy has also revealed that changes in plasma T concentrations combined with expression of AR in the female braindincluding the POA, several regions of the hypothalamus, the AMG, and the lateral septumdare associated with changes in the female reproductive cycle (Rhen, Sakata, Woolley, Porter, & Crews, 2003).
Hormones and Reproduction of Vertebrates
4.4. Summary Steroid hormones act at receptors within particular regions of the brain to regulate the display of sexual behaviors. In many species, these brain areas are sexually dimorphic. In a number of cases, sex differences in neural and other structures associated with reproduction arise during ontogeny. This property is covered in the following section.
5. DEVELOPMENT OF REPRODUCTIVE TRAITS 5.1. Development of Sex Differences in Anoles and Whiptail Lizards Sexual differentiation of the dewlap structures in green anoles occurs when animals are juveniles. The second ceratobranchial cartilage becomes longer in males than females between 30 and 60 days after hatching, and the muscle fibers are significantly larger in males by day 75. It is not clear when the sexual dimorphism of motoneuron soma size first appears, but it is some time between 90 days of age and adulthood (O’Bryant & Wade, 2001). While the ideas need to be tested, it is possible that the large increase in T associated with their first spring BS masculinizes the dewlap structures, or they increase in size due to greater use by adult males than females. Masculinization of muscle and cartilage is controlled at least in part by T in juveniles 30 to 90 days after hatching (Lovern, Holmes, Fuller, & Wade, 2004b); a period when plasma T is elevated in males compared to females (Lovern, McNabb, & Jenssen, 2001). Testosterone implants in females at this time significantly increase both the length of the second ceratobranchials and the size of ceratohyoid muscle fibers, although it does not affect soma size of motoneurons that project to that muscle. In parallel, castration of males on post-hatching day 30 decreases cartilage length. This manipulation does not modify muscle fiber size, but it is possible that conducting this surgery at a younger age would be more effective or that adrenal androgens, which were not eliminated, play some role. Sexual differentiation of the peripheral copulatory structures in green anoles occurs embryonically. Hemipenes and primordial RPM fibers are initially present in both sexes, but they regress completely in females prior to hatching (Holmes & Wade, 2005b). This pattern is typical of many reptiles (Raynaud & Pieau, 1985). Interestingly, both androgens and estrogens appear to play active roles in sexual differentiation of these copulatory structures. Administration of T or DHT rescues male structures in females, whereas E2 treatment feminizes males (Holmes & Wade, 2005b). Further, at embryonic day 13, when the tissues are still present in both sexes, males express higher levels of AR mRNA in their hemipenes and copulatory
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muscles than females, while females tend to express increased ERa mRNA (Beck & Wade, 2008). In contrast to the peripheral structures, the number of T17-S1 motoneurons is equivalent in males and females at hatching (Holmes & Wade, 2005b), suggesting a dissociation between muscle and motoneuron development similar to the dewlap system. Patterns of yolk steroids are consistent with at least the possibility that T masculinizes copulatory structures. For example, T is higher in male than in female eggs sampled while still in the oviduct (Lovern & Wade, 2003a), as well as on the day the eggs are laid (Lovern et al., 2001). Intriguingly, these data suggest a maternal source for the sex difference, as well as an early time period when T may influence sexual differentiation. Maternal transfer to yolk is not passive, however. Plasma T in adult females is approximately 30% that of E2, whereas T in yolking follicles is over 600% that of E2 (Lovern & Wade, 2003b). Embryos appear to start producing T, but perhaps not E2, in the latter half of incubation (Lovern & Wade, 2003b). The sex difference in yolk T is no longer detected one- to two-thirds of the way between laying and hatching (Lovern & Wade, 2001), but we do not know whether embryonic hormone levels differ in males and females. Advances in our understanding of the specific roles of steroids during this pre-hatching developmental period will be greatly facilitated when we can determine the sex of these animals using molecular techniques. Only then will we be able to determine, for example, relative levels of the production of androgens and estrogens by embryonic gonads. The recent sequencing of the green anole genome provides hope that this will become possible relatively soon. Details regarding the development of sex differences in the forebrain of green anoles remain unclear. One study has assessed morphological traits in the POA, VMH, and AMG around the time of hatching (which occurs after approximately 34 days of incubation). A variety of changes occurred between embryonic day 26 and post-hatching day 5, including decreases in soma size, particularly in females, and increases in the density of cells in regions associated with masculine behaviors (POA and AMG). However, only transient sex differences existed in any of these regions, suggesting that they arise at a later developmental stage (Beck & Wade, 2009c). As in adults, ERa expression in both sexes is higher in the VMH than the other two brain regions, which is consistent with the role E2 plays in the display of female sexual behaviors (Beck & Wade, 2009c). Sex differences do not exist in the volume of the AH-POA or VMH in C. inornatus on the day of hatching. Treatment for approximately two weeks with T does not affect the sizes of these brain regions, but ovariectomy on the day of hatching decreases the size of the AH-POA in parthenogenetic C. uniparens (Wade et al., 1993).
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5.2. Development in Reptiles with Alternative Reproductive Morphs In U. ornatus, T and P4 are important factors in the organization of differences between the two male morphs (Hews, Knapp, & Moore, 1994; Hews & Moore, 1996; Moore et al., 1998). This conclusion is based on both measurements of endogenous hormones and studies in which hormones were manipulated. Castration of juvenile males produced more orange males than a control group, while castrated juvenile males given T implants were more likely to be orange-blue than control males (Hews et al., 1994). This study also found the same pattern of results when 30-day-old intact males were given T implants or blank capsules, indicating that the critical period for the role of T or its metabolites in morph differentiation may continue beyond 30 days. Further work determined that T does not influence dewlap color after post-hatching day 60 (Hews & Moore, 1996), suggesting that the critical period for this trait has ended by this developmental stage. Treatments with T or its metabolite DHT are equally effective for the development of male sexual traits, including dewlap color, waxy femoral pore secretions, and hemipene-related tissues, while DHT is required for full expression of the blue belly patches (Hews & Moore, 1995). Progesterone also plays an important role in the organization of morph phenotypes. Manipulations of P4 or T at hatching result in differing morphs (Moore et al., 1998). Progesterone may operate independently of T or, alternatively, it may be the initial trigger of differentiation, with T operating later in development (Moore et al., 1998). Invitro incubation demonstrated that, during the period between embryonic day 30 through hatching, P4 is released by the adrenal glands and T from the testes (Jennings, Painter, & Moore, 2004). This early production of T from U. ornatus gonads contrasts with results from turtles (Pieau, Mignot, Dorizzi, & Guichard, 1982; White & Thomas, 1992a) and crocodiles (Smith & Joss, 1994), in which levels of T are very low in early post-hatching periods. Galapagos marine iguanas provide an example of a species in which developmental and environmental conditions affect reproductive behavior via changes in circulating hormones. In this species, males can be successful territory holders only after achieving a certain body size, but this threshold is affected by climate conditions and population density (Wikelski & Trillmich, 1997) such that, when territories are available, smaller males may adopt territorial behavior. The ontogenetic switches between sneaker and satellite, and satellite and territorial strategies can be imitated via manipulation of T. When exposed to increased T, satellite males begin to defend territories and sneaker males begin to openly court females (Wikelski, Steiger, Gall, & Nelson, 2005), behaviors more typical of larger males. These changes do not increase the
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reproductive success of manipulated males, however, as they experience more frequent fights with other, larger males and expend time and energy performing courtship displays that are largely ignored by females (Wikelski et al., 2005). Further, when T is blocked, territorial males decrease their defensive and courtship behaviors (Wikelski et al., 2005).
5.3. Development in Reptiles with Temperature-dependent Sex Determination (TSD) In addition to the diverse patterns of the neuroendocrine control of behavior described above, reptiles also offer a unique opportunity to compare taxa exhibiting different modes of sex determination. Sex is determined by genotype in many reptilian species; both XX/XY (in which males are the heterogametic sex) and ZZ/ZW (in which females are the heterogametic sex) chromosome systems exist. In other reptiles, sex is environmentally determined, as the temperature of egg incubation can determine sex in many crocodilians, chelonians, and some squamates (reviewed in Wibbels, Bull, & Crews, 1994; see also Chapter 1, this volume). One of the best-studied examples of temperaturedependent sex determination (TSD) is the leopard gecko (Eublepharis macularius). Individuals of this species have no sex chromosomes, and an individual’s sex is dependent on the temperatures experienced as an egg (Viets, Tousignant, Ewert, Nelson, & Crews, 1993). At an incubation temperature of 26 C, all individuals become females, while at 32.5 C mostly males are produced (Crews, Coomber, Baldwin, Azad, & Gonzalez-Lima, 1996), although these effects can be overridden by application of exogenous E2 (Flores, Tousignant, & Crews, 1994). Temperature appears to direct sex development in TSD species at least in part by influencing genes that code for steroidogenic enzymes and steroid hormone receptors (Crews et al., 1994). In an experiment designed to determine whether incubation temperature and gonadal sex hormones produce irreversible effects on behavior in leopard geckos, Rhen and Crews (1999) found that some effects of temperature on male behaviors are permanently organized during development. When gonadectomized and then treated with androgens, adult males from male-biased incubation temperatures perform more scent-marking behaviors than males from female-biased temperatures, but males from female-biased temperatures perform more copulatory (mounting) behavior. In addition, they found that even when treated with androgens, adult females perform very little courtship or mounting behavior. Incubation temperature has also been found to affect the influence of T on aggressive behavior, suggesting that incubation temperature may have long-term effects on sensitivities to
Hormones and Reproduction of Vertebrates
hormones (Flores et al. 1994; Flores & Crews, 1995; Rhen & Crews, 1999). Incubation temperature also influences the volume and metabolic capacity of several brain nuclei in leopard geckos. Experimental manipulations indicate that changes in androgen and estrogen levels do not affect POA or VMH volumes in females from all-female temperatures, or neural activity in the anterior hypothalamus, AMG, dorsal ventricular ridge, or septum; however, hormones do alter the volume of these brain areas in individuals from both sexes who were incubated at male-biased temperatures (Crews et al., 1996). These manipulations showed that incubation temperature itself organizes the brain, and not simply the gonadal hormones produced once sex is determined (Crews et al., 1996). Hormonal changes during development also affect leopard gecko reproductive morphology. Androgen (but not estrogen) levels increase during the course of male ontogeny, and hemipenis development tracks these changes. In particular, at 10 weeks of age, signs of hemipenes appear as androgens gradually increase, and, at 25 weeks of age when androgen levels increase, hemipenes are fully developed (Rhen, Sakata, & Crews, 2005). In females, plasma levels of E2 and DHT (but not T) vary by incubation temperature and change over the course of development (Rhen et al., 2005). Most turtle embryos also experience a thermosensitive period (TSP) (reviewed in Pieau & Dorizzi, 2004), when gonadal sex is influenced by both temperature and hormones (Wibbels et al., 1994). The synergistic relationship between temperature and hormones is exemplified in studies of red-eared slider turtles (Trachemys scripta elegans), in which treatments manipulating temperature and E2 independently have the same effect on the sex of incubating eggs and appear to act through the same developmental pathways (Wibbels, Bull, & Crews, 1991). The gonads are a main site of aromatase activity and estrogen synthesis during the TSP, as well as a site of steroid action. One study that examined the relationship between ER and AR in the gonads during the TSP in the red-eared slider found that ERa and AR levels dramatically increase at female-producing temperatures during (but not before) this sensitive period and that expression of these receptors was reduced at male-producing temperatures (Ramsey & Crews, 2007). Also, treatment with E2 at maleproducing temperatures caused feminization of the gonads, showing that temperature and hormonal conditions can both influence the sex of the individual, although these effects can occur through differing steroid signaling pathways (Ramsey & Crews, 2007). Other work in red-eared sliders showed that P4 secreted from adrenal and kidney tissues (White & Thomas, 1992a) also appears to be a factor in sex determination. During the temperaturesensitive embryonic period, plasma levels of P4 are
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Neuroendocrinology of Reptilian Reproductive Behavior
significantly higher in males than females (White & Thomas, 1992b).
5.4. Summary The development of many morphological structures necessary for successful courtship and copulation depends on exposure to hormones (and/or temperature) during embryogenesis or after hatching, and these organizational effects generally occur during a particular critical period. While many adult reproductive traits are directly influenced by social and/or neuroendocrine traits during an animal’s development, in a wide diversity of reptilian taxa, variation in reproductive behavior may alternatively be the result of adult plasticity due to changing external or internal environmental factors, as described below.
6. ADULT PLASTICITY 6.1. Effects of Season and Hormone Manipulation in Anoles In the green anole, dewlap and copulatory system neuromuscular morphology differ in their responsiveness to adult T. Morphology of the dewlap neuromuscular system appears not to be plastic in adulthood. For example, despite large seasonal changes in circulating T and the fact that castration removes the main source of the hormone, muscle fiber and motoneuron components are generally stable in size across the BS and NBS, and with castration and T replacement (O’Bryant & Wade, 1999; Neal & Wade, 2007a). However, functionally, the dewlap muscle does appear to undergo changes in adulthood. Males have a higher percentage of fast oxidative-glycolytic fibers during the BS compared to the NBS, and T treatment increases the percentage of fast oxidative-glycolytic dewlap fibers. Thus, routine changes in this hormone may mediate fiber type in gonadally intact males (Holmes, Bartrem, & Wade, 2007). The pattern of hormone responsiveness for the copulatory system in green anoles is quite different, as the morphology of these structures is plastic in adulthood (Holmes & Wade, 2004b). Whereas the cross-sectional areas of hemipenes and RPM fibers do not differ between intact animals under breeding and non-breeding environmental conditions, they increase dramatically in T-treated compared to control castrated males. Yet, these structures are not induced in females with adult T treatment (Holmes et al., 2007). The effect of T in males is greater in those housed with long days and warm temperatures (i.e., breeding conditions), suggesting that sensitivity to T in peripheral structures decreases as a result of non-breeding environmental conditions (Neal & Wade, 2007a). It is possible that this change exists to prevent a decline in
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morphology due to a seasonal decrease in T. However, as males captured in the field after the end of the BS still have ~10 ng/ml of T (compared to ~20 ng/ml during the BS) (Lovern et al., 2001), they may have sufficient hormone available to maintain the structures. In addition, when males are gonadectomized, RPM fibers, and especially hemipenes, appear smaller than in intact animals. Thus, it seems more likely that the copulatory system of males shows an increased dynamic range in its sensitivity to T during breeding compared to non-breeding environmental conditions. This process might facilitate the response of the copulatory system to even relatively small T fluctuations that may occur with particular experiences, possibly exposure to females. In contrast to effects on gross morphology, seasonal environmental conditions and T manipulation have little to no effect on copulatory muscle fiber type (Holmes et al., 2007). Thus, this feature is not the primary mediator for seasonal changes in male copulatory behaviors. Finally, it is possible that a small decrease in T17-S1 motoneuron soma size occurs in the NBS. However, this effect is probably not specific to cells in that region of the spinal cord, and it is not mediated by plasma T levels (Holmes & Wade, 2004b). In the forebrain of unmanipulated adult green anoles, volumes of the POA and VMH (but not AMG) are greater during the BS than in the NBS in both sexes (Beck, O’Bryant, & Wade, 2008). However, mechanisms regulating the seasonal change in volume are not clear, as increased volume during the BS is not detected in the POA or VMH in gonadectomized animals, treated with E2 or not (Beck & Wade, 2009b). Thus, the sexual dimorphism is present across diverse endocrine conditions and is probably not maintained by adult steroid hormones. The seasonal difference detected in intact animals could therefore involve gonadal secretions during adulthood other than E2; perhaps T or non-gonadal factors such as melatonin. Testosterone treatment of gonadectomized adult green anoles increases soma size in the POA and AMG, and in the AMG it does so to a greater extent in the BS than the NBS (Neal & Wade, 2007a). While intact anoles show no sex difference in the volume of the AMG, gonadectomized males have larger AMGs than gonadectomized females (Beck & Wade, 2009b). These results fit with the larger soma sizes in gonadectomized males than gonadectomized females detected in this region during the NBS, regardless of whether the animals are treated with T (O’Bryant & Wade, 2002b). Estradiol treatment enhances the sexual dimorphism in AMG volume, suggesting it may support change in one or more of the characteristics that contribute to overall AMG volume other than total cell number (which is unaffected by this hormone), such as soma size or dendritic arborization. This issue has not been investigated, but T treatment does increase soma size in the AMG of males,
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and the same dose is more effective in the BS than NBS (Neal & Wade, 2007a). Perhaps even the relatively low levels of T in female anoles (~100 pg/ml in the NBS and ~750 pg/ml in the BS) (Lovern & Wade, 2001) are sufficient to maximally increase brain region volume, thus masking an underlying sex difference in AMG size. The same may be true for the VMH, which is larger in gonadectomized males than females (regardless of whether they are treated with E2), but not in gonad-intact individuals (Beck & Wade, 2009b). Female green anoles also have a greater number of cells in the VMH and AMG than males (at least than gonadectomized males), suggesting that they may be smaller and/or more densely packed than in males (Beck & Wade, 2009b). A greater total number of cells exists in the AMG during the NBS than BS, in both unmanipulated and gonadectomized animals, regardless of whether they were administered E2 (Beck et al., 2008; Beck & Wade, 2009b). These results suggest that some feature(s) of non-breeding environmental conditions facilitate the addition to or survival of cells in this region. Data on the estimated total cell number in the VMH are a little more complicated, as an increase was detected in the NBS compared to the BS in gonadectomized animals, some of which received E2 treatment, but not in intact green anoles (Beck et al., 2008; Beck & Wade, 2009b). It will be important in the future to determine the functional relevance of the increased number of cells in the AMG and VMH during the NBS, as well as whether they are mediated by differences in cell birth, incorporation, or survival.
6.2. Effects of Season and Hormone Manipulation in Other Reptiles In male whiptail lizards, volumes of the AH-POA and VMH are plastic. During the NBS, they become femalelike in size in male C. inornatus, an effect that is regulated by T. In contrast, the volume of these two brain regions appears to be stable in adult female C. inornatus and C. uniparens (Wade & Crews, 1991; Wade et al., 1993). In tree lizards, the POA and AMG are greater in breeding than NBS males (Kabelik et al., 2006). The relationship between neural anatomy, hormone levels, and behavior in this species is still somewhat unclear. While one study indicates that T and P4 both cause an increase in the volume of the VMH and AMG (Weiss & Moore, 2004), another found that while T and P4 increase aggression, only increased T affected brain nuclei volumes (Kabelik, Weiss, & Moore, 2008b). Further, neural activity in the dorsolateral VMH, or VMHdl (as measured indirectly using the phosphorylated form of cyclicAMP response element binding protein (pCREB)), increases with greater aggressive behavior but is not affected by T (Kabelik et al.,
Hormones and Reproduction of Vertebrates
2008a). Therefore, while T influences both brain anatomy and aggressive behavior, these appear to be independent effects of the hormone (Kabelik et al., 2006; 2008b), as no direct relationship between forebrain nuclei and aggression has been identified. In leopard geckos, treating females with T dramatically increased the size of the hemipenes and the fiber size of the muscles that control hemipenis movement, but these structures do not reach the sizes of those in control males (Holmes & Wade, 2005a). Leopard gecko hemipenes contain AR (Endo & Park, 2003; Rhen & Crews, 2001) and so T likely acts directly on the hemipenes to cause their growth, but we do not yet know whether copulatory muscles contain AR. Compared to green anoles, it is clear that copulatory structures in adult leopard geckos are much more sensitive to hormonal changes. While far more work must be done to address the idea, it would be fascinating if the lack of sex chromosomes in leopard geckos in some way lessens the degree to which these structures differentiate. Sex and seasonal differences in the limbic forebrain were investigated in one study in red-sided garter snakes, and relatively few sexual dimorphisms were found. They included a larger POA in males compared to females only during hibernation and in the NS in animals not subjected to hibernation. The reproductive forebrain appears not to exhibit dramatic changes in morphology in males, but in females the POA and VMH were both smaller during hibernation than under other environmental conditions (Crews, Robker, & Mendonc¸a, 1993). In side-blotched lizards (U. stansburiana), some males may change morphs during the reproductive season. In particular, late in the BS some yellow-throated males can begin to exhibit partial blue-throated morphology, with correlated increases in T levels and endurance (Sinervo et al., 2000a). In general, T appears to be a major factor in the physiological changes that all three color morphs undergo during development (Sinervo et al., 2000a). In tree lizards (U. ornatus), although male morph type is established early in development, reproductive tactics within the orange morph are determined by activational effects of hormones (Figure 2.4) (Moore et al., 1998). Territorial orange-blue males are always territorial, but the non-territorial orange morph adopts a satellite tactic in some years and a nomadic tactic in others. This behavioral variation is correlated with climatic conditions, such that in drier years orange males are nomads while they exhibit the more sedentary, satellite behavior in years with more rainfall (Knapp & Moore, 1997; Knapp, Hews, Thompson, Ray, & Moore, 2003). This difference appears to be driven by variation in sensitivity to T and CORT (Knapp & Moore, 1997). In harsher, dry conditions, males of both morphs have higher CORT levels, but territorial orange-blue males are much less sensitive to the effect of CORT on the
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FIGURE 2.4 Dewlap polymorphism in the tree lizard (Urosaurus ornatus). This figure depicts a summary of the proposed endocrine mechanisms influencing dewlap coloration and territorial strategies in tree lizards. The permanent differences between orange and orange-blue males appear to result from organizational influences of progesterone and testosterone. Low levels of these hormones during early development result in orange males, while high levels produce orange-blue males. However, orange males may switch between satellite and nomad tactics, a shift that appears to result from a twostep activational process that involves the stress increase in corticosterone and the consequent decrease in testosterone levels. Reprinted with permission from American Zoologist, copyright 1998 (Figure 12; Moore, Hews, & Knapp, 1998).
suppression of T (Knapp & Moore, 1997; Knapp et al., 2003). These differences in CORT sensitivity may be due to differences between the morphs in the binding capacity of androgen-glucocorticoid-binding globulin (AGBG), which lead to higher levels of circulating CORT in nonterritorial orange males than territorial orange-blue males, particularly when CORT is increased in response to stress (Jennings, Moore, Knapp, Matthews, & Orchinik, 2000). Therefore, in harsher environmental conditions, only orange males have lower T, which seems to drive plasticity in reproductive tactics.
6.3. Summary In seasonally breeding animals, reproductive morphology often differs dramatically between breeding and nonbreeding seasons, in a number of cases (although not always) in a manner that mirrors changes in the display of sexual behaviors. Further, adult neuroendocrine traits may vary as a function of the social environment, which may also produce plastic behaviors in adulthood.
7. CONCLUSIONS AND FUTURE DIRECTIONS Work with the species described in this chapter has greatly informed our understanding of the relationships involving the neural and endocrine influences on reproductive behavior. However, our knowledge to this point is based on relatively few species, with the longest history of research in this area on the green anole. Increasing the
diversity of study organisms, including more research with non-squamate reptiles such as crocodilians and turtles, should yield valuable insights into critical mechanisms across life stages. In addition, it will be particularly useful to explore individual species in more depth, as the tools for this work become available. The recent (2008) sequencing of the green anole genome is a critical first step in this process. These data, and similar information from other reptiles as it becomes available, will greatly facilitate investigations of the molecular mechanisms regulating reproduction, including those in neural and muscular structures that influence or are influenced by hormones.
ABBREVIATIONS AGBG AH-POA AMG AR BS CI-628 CORT D1 DHT E2 ERa FSH GnRH GTH LH LHRH NBS NS
Androgen-glucocorticoid-binding globulin Anterior hypothalamus-preoptic area Amygdala Androgen receptor Breeding season an anti-estrogen Corticosterone Dopamine receptor agonist 5a-dihydrotestosterone Estradiol Estrogen receptor-a Follicle-stimulating hormone Gonadotropin-releasing hormone Gonadotropin Luteinizing hormone see GnRH Non-breeding season Nucleus sphericus
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P4 pCREB POA PR RPM SC T TPN TRH TSD TSP VMH VMHdl
Hormones and Reproduction of Vertebrates
Progesterone Phosphorylated cyclic AMP response element binding protein Preoptic area Progesterone receptors Retractor penis magnus Sphincter cloacae Testosterone Transversus penis muscle Thyrotropin-releasing hormone Temperature-dependent sex determination Thermosensitive period Ventromedial hypothalamus Dorsolateral ventromedial hypothalamus
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Martı´nez-Marcos, A., Lanuza, E., & Halpern, M. (1999). Organization of the ophidian amygdala: chemosensory pathways to the hypothalamus. J. Comp. Neurol., 412, 51–68. Martinez-Vargas, M. C., Keefer, D. A., & Stumpf, W. E. (1978). Estrogen localization in the brain of the lizard, Anolis carolinensis. J. Exp. Zool., 205, 141–147. Martins, E. P., Ord, T. J., Slaven, J., Wright, J. L., & Housworth, E. A. (2006). Individual, sexual, seasonal, and temporal variation in the amount of sagebrush lizard scent marks. J. Chem. Ecol., 32, 881–893. Mason, P., & Adkins, E. K. (1976). Hormones and social behavior in the lizard, Anolis carolinensis. Horm. Behav., 7, 75–86. Mason, R. T., & Crews, D. (1985). Female mimicry in garter snakes. Nature, 316, 59–60. Mayo, M. L., & Crews, D. (1987). Neural control of male-like pseudocopulatory behavior in the all-female lizard, Cnemidophorus uniparens: Effects of intracranial implantation of dihydrotestosterone. Horm. Behav., 21, 181–192. Mendonc¸a, M. T., & Crews, D. (1990). Mating-induced ovarian recrudescence in the red-sided garter snake. J. Comp. Physiol. Sensory Neural Behav. Physiol., 166, 629–632. Mendonc¸a, M. T., & Crews, D. (1996). Effects of ovariectomy and estrogen replacement on attractivity and receptivity in the red-sided garter snake (Thamnophis sirtalis parietalis). J. Comp. Physiol. Sensory Neural Behav. Physiol., 178, 373–381. Meyers, J. J., Irschick, D. J., Vanhooydonck, B., & Herrel, A. (2006). Divergent roles for multiple sexual signals in a polygynous lizard. Funct. Ecol., 20, 709–716. Miles, D. B., Sinervo, B., Hazard, L. C., Svensson, E. I., & Costa, D. (2007). Relating endocrinology, physiology, and behaviour using species with alternative mating strategies. Funct. Ecol., 21, 653–665. Mills, S. C., Hazard, L., Lancaster, L., Mappes, T., Miles, D., Oksanen, T. A., & Sinervo, B. (2008). Gonadotropin hormone modulation of testosterone, immune function, performance, and behavioral trade-offs among male morphs of the lizard Uta stansburiana. Am. Nat., 171, 339–357. Monks, S. P. (1881). A partial biography of the green lizard. Am. Nat., 15, 96–99. Moore, M. C., Whittier, J. M., & Crews, D. (1985a). Sex steroidhormones during the ovarian cycle of an all-female, parthenogenetic lizard and their correlation with pseudosexual behavior. Gen. Comp. Endocrinol., 60, 144–153. Moore, M. C., Whittier, J. M., Billy, A. J., & Crews, D. (1985b). Malelike behavior in an all-female lizarddrelationship to ovarian cycle. Anim. Behav., 33, 284–289. Moore, M. C., Hews, D. K., & Knapp, R. (1998). Hormonal control and evolution of alternative male phenotypes: Generalizations of models for sexual differentiation. Am. Zool., 38, 133–151. Morrell, J. I., Crews, D., Ballin, A., Morgentaler, A., & Pfaff, D. W. (1979). 3H-estradiol, 3H-testosterone and 3H-dihydrotestosterone localization in the brain of the lizard Anolis carolinensis: An autoradiographic study. J. Comp. Neurol., 188, 201–224. Neal, J. K., & Wade, J. (2007a). Courtship and copulation in the adult male green anole: Effects of season, hormone and female contact on reproductive behavior and morphology. Behav. Brain Res., 177, 177–185. Neal, J. K., & Wade, J. (2007b). Androgen receptor expression and morphology of forebrain and neuromuscular systems in male green
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Chapter 3
Hormonal Regulation of Testicular Functions in Reptiles Sunil Kumar, Brototi Roy, and Umesh Rai University of Delhi, Delhi, India
SUMMARY Testicular functions are controlled by multifactor, environmentally important, hypothalamo–hypophysial, and testicular cells-secreted paracrine factors. The existence of different gonadotropins and their role in testicular functions are interesting in reptiles. In chelonians and crocodilians, two distinct pituitary gonadotropins similar to mammalian follicle-stimulating hormone (FSH) and luteinizing hormone (LH) regulate two different functions: spermatogenesis and steroidogenesis, respectively. Conversely, the purification and characterization of pituitary gonadotropin from different families of snake reveal the existence of a single gonadotropin that controls both testicular functions in squamates. To date, cDNA has been cloned for either FSH or LH but not for both FSH and LH from a single squamate. With respect to sex steroids, androgens differentially regulate spermatogenesis, depending on reproductive phases, while estrogens are implicated in post-spermatogenic testicular regression. In addition, several uncharacterized paracrine factors secreted by Leydig and Sertoli cells, macrophages, and mast cells play critical roles in the regulation of spermatogenesis, steroidogenesis, and testicular immune responses.
1. INTRODUCTION Reptiles represent an important phylogenic link between the anamniotes and amniote vertebrates. Despite the fact that all extant reptiles are ectotherms like fishes and amphibians, they are grouped with birds and mammals due to the presence of an amniotic membrane during development. Moreover, they are ancestral to both birds and mammals. The living reptiles can be classified into four different orders: lizards and snakes in Squamata; Sphenodon punctatus (living fossils) in Sphenodonta; turtles and tortoises in Chelonia; and alligators and crocodiles in Crocodilia. The basic components of the testes of reptiles are quite similar to those of mammals, implying conserved development and cellular differentiation. The genital ridge develops as a thickening of the coelomic epithelium on the ventromedial surface of the
Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
mesonephric kidney. Initially, gonads are bi-potential and indistinguishable in either sex. A detailed study of gonadal development has been carried out in turtles (Pieau, Dorizzi, & Richard-Mercier, 1999), in which germ cells migrate from the posterior germinal crescent to the genital ridge at the end of gastrulation. This migration is assumed to occur through the vascular system as in avian species, since germ cells lie at the surface of the coelomic epithelium in both groups. During the course of development of gonadal primordia, the medullary region of the developing gonad is invaded by epithelial cells from (1) the external epithelium of Bowman’s capsule, (2) the coelomic epithelium bordering the mesonephric kidneys on the lateral side of each gonad, and (3) the germinal epithelium of the gonad. Cells from the Malpighian corpuscles and lateral coelomic epithelium become organized in the dorsal part of the gonadal primordium to form the rete cords. Germinal epithelial cells give rise to thin sex cords that penetrate into the underlying initial mesenchyme (Figure 3.1). Temperature plays the most important role in differentiation of the bipotential gonad into a testis (see Chapter 1, this volume). At the male-producing temperature, sex cords envelop the germ cells and differentiate into typical testis cords; otherwise, they degenerate (Figure 3.2) (Yao & Capel, 2005). Many conserved genes are involved in sex differentiation and gonadal development in reptiles (Figure 3.3). Among these, steroidogenic factor 1 (Sf1) and Wilms’ tumor 1 (Wt1) genes are important for the initial formation of the bi-potential gonad. Double sex and mab-3-related transcription factor 1 (Dmrt1) is an important testis-determining gene as its expression increases during the temperature-sensitive testisdetermining period (TDP) and remains high during testicular development. The action of Dmrt1 in testes development is downstream to temperature and upstream to another important gene, anti-Mu¨llerian hormone (Amh) (AMH). The upregulation of AMH in reptiles is not 63
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FIGURE 3.1 Development and differentiation of gonads in reptiles before, during, and after the thermosensitive period (TSP). a, albuginea; BcMc, Bowman’s capsule of Malpighian corpuscle; bv, blood vessel; c, cortex; ca, cortex anlage; ce, coelomic epithelium; dm, dorsal mesentery; gc, germ cells; ge, germinal epithelium; l, lacunae; Lc, Leydig cells; m, medulla; mc, medullary cords; mm, mesonephric mesenchyme; mt, mesonephric tube; oo, oocyte; pf, primordial follicle; rc, rete cord; rca, rete cord anlage; sca, seminiferous cord anlagen. Courtesy: Pieau, Dorizzi, & Richard-Mercier (1999).
regulated by Sox 9, as in mammals. Rather, Sox 9 expression is evidenced later than AMH expression (Figure 3.3) (Yao et al., 2005). Downstream molecular events in testis differentiation need to be explored in a large number of reptiles in order to develop conceptual knowledge of the molecular regulation of sex differentiation.
There are two important functions of the testis: the production of male gametes (spermatogenesis) and the synthesis of sex steroids (steroidogenesis). These functions are regulated by the complex interaction of extra- and intratesticular factors. However, the purification and characterization of intra-testicular factors regulating testicular functions in a paracrine manner in reptiles are yet to be
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FIGURE 3.2 Temperature-dependent cellular reorganization in the bipotential gonad of a turtle, Trachemys scripta. In the bipotential gonad, at stage 17 primordial germ cells (PGCs) (red) are associated with the coelomic epithelium. By stage 23, temperature-dependent sex differentiation occurs. At 26 C temperature, PGCs become enclosed within the testis cord, while at 31 C sex cords degenerate (DSC) and PGCs remains in the periphery. The primordial germ cells are highlighted by an anti-VASA antibody (red) and sex cords (SC) and testis cords (TC) are outlined by an anti-laminin antibody (green). Modified from Yao and Capel (2005). See color plate section.
done. In addition to endocrine and paracrine factors, environmental cues such as photoperiod, temperature, and rainfall are implicated in the control of reproductive activity by influencing the release of hypothalamic hormones, particularly gonadotropin-releasing hormone (GnRH). This in turn stimulates secretion of gonadotropins (GTHs) and consequently regulates testicular functions.
2. TESTICULAR STRUCTURE The reptilian testis differs from the testes of anamniotes, which are characterized by a cystic form of spermatogenesis. However, like other vertebrates, the two important testicular functions occur in two distinct compartments of the testis; i.e., the tubular and interstitial compartments.
FIGURE 3.3 Temporal expression of genes that play critical roles in sex determination and differentiation in the male red-eared slider turtle (Trachemys scripta). The shaded area represents the temperature-sensitive testis-determining period. Amh, anti-Mu¨llerian hormone; Dmrt1, Drosophila Doublesex and C. elegans Mab-3 related transcription factor 1; Sox9, Sry-like HMG-box protein 9; Sf1, steroidogenic factor 1; Wt1, Wilms’ tumor gene 1. Modified from Yao and Capel (2005).
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2.1. Tubular Compartment of the Testis The tubular compartment is the site of spermatogenesis. It consists of seminiferous tubules. Each tubule is lined with a non-cellular basement membrane and cellular peritubular (fibroblast/myoid-like) cells. These cells make their first phyletic appearance in the reptilian testis (Unsicker, 1975), since they are absent in anamniotes. Depending on the reproductive state, peritubular cells change from being fibroblast-like in the non-breeding phase to myoid-like in the spermatogenetically active phase (Unsicker, 1975). The peritubular cells are arranged in multilayers during the quiescent phase in the Indian wall lizard, Hemidactylus flaviviridis, whereas during the breeding phase they are arranged in a single layer (Figure 3.4) (Khan & Rai, 2004a). The peritubular myoid cells in reptiles, like their mammalian counterparts, share several ultrastructural characteristics with smooth muscle cells. They contain ˚ thick intra-cytoplasmic filaments, plasmalemmal 50–70 A
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and intracellular dense attachment sites for thin filaments, and smooth membrane inpocketing. The presence of desmosomes and tight junctions between adjacent myoid cells suggests the existence of a permeability barrier even at the level of the basement membrane. The myoid-like appearance of peritubular cells during the spermatogenetically active phase suggests their involvement in the contraction of seminiferous tubules, thereby aiding sperm transport. The transformation of peritubular cells from fibroblast to myoid-like is closely associated with the increase of steroidogenic activity of Leydig cells. In addition to peritubular cells, the Sertoli cells and germ cells constitute the tubular compartment of the testis. Sertoli cells are irregular in shape and extend from base to lumen of the tubule. Their irregular-shaped nucleus occupies the basal position. The presence of numerous spherical and elongated mitochondria along with smooth endoplasmic reticulum (SER) and Golgi bodies suggests the active involvement of Sertoli cells in steroid and protein
(a)
(b) FIGURE 3.4 Electron micrograph of basal lamina of seminiferous tubules during (a) breeding ( 7,950) and (b) regressed phase ( 6,150) in the Indian wall lizard, Hemidactylus flaviviridis. The peritubular cells change from myoid-like, single layer in the breeding phase to fibroblast-like multilayer during the regressed phase. MY, myoid cells; BM, basement membrane; F, fibroblast-like peritubular cells; D, desmosomes-like junctions; arrow heads, nuclear indentations.
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biosynthesis (Figure 3.5). Further, Sertoli cells provide physical support to developing germ cells, as evident by the presence of various adhering junctional complexes between Sertoli and germ cells (Figure 3.6) (Khan & Rai, 2004a). Another important function of the Sertoli cell is to create a protected microenvironment for developing germ cells by forming the blood–testis barrier. This is formed by occluding junctional complexes between adjacent Sertoli cells (Figure 3.7). The blood–testis barrier demarcates the seminiferous tubule into two distinct subcompartments. The basal subcompartment faces the interstitial space, houses the spermatogonia, and has direct access to the serum components. The adluminal subcompartment includes primary and secondary spermatocytes, and spermatids. Germ cells of the adluminal subcompartment derive their nutrients from the Sertoli cells. Lactate, a Sertoli cell metabolite, is utilized by germ cells as an
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energy substrate (Nirmal & Rai, 1999). In addition to the nursing function, Sertoli cells play an important role in spermiation, as evident by the presence of residual bodies in their cytoplasm. The residual bodies are a membraneenclosed cytoplasmic matrix containing non-functional cell organelles such as Golgi complex, SER, and ribosomes shed from the elongated spermatids at the time of their release from the Sertoli cells (Figure 3.8). The phagocytosed, degenerated germ cells and residual bodies are eventually disposed of by the Sertoli cells (Dubois, Pudney, & Callard, 1988; Khan & Rai, 2004a).
2.2. Interstitial Compartment The interstitial compartment is the extra-tubular space between the seminiferous tubules. This compartment consists of blood and lymphatic vessels, nerve fibers,
FIGURE 3.5 Electron micrograph of a regressed testis showing the basal portion of Sertoli cells with a horseshoe-shaped nucleus (N) and basement membrane invaginations. Numerous spherical mitochondria (M) can be seen. 7,950.
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FIGURE 3.6 Basal portion of the seminiferous epithelium of a regressed testis in the Indian wall lizard Hemidactylus flaviviridis showing different types of junctional interactions between the testicular cells: desmosome-like junctions between Sertoli cells (D), Sertoli cell-germ cell (arrow), and germ cellgerm cell (rectangle). S, septate-like junctions between germ cells; spg, spermatogonia; Sc, Sertoli cell. 4,650.
a large population of steroid-secreting Leydig cells, and immune cells (macrophages and a few mast cells). Leydig cells, the primary site for steroid biosynthesis, are characterized by the presence of elongated mitochondria with tubular cristae, SER, lipid droplets, and 3b-hydroxysteroid dehydrogenase (3b-HSD) enzyme activity. Since reptiles are seasonal breeders, marked variations in Leydig cell ultrastructural features are observed during different phases of the reproductive cycle (Khan & Rai, 2004a). The cytoplasm of Leydig cells during the regressed phase contains numerous large lipid droplets, irregular-shaped nuclei, sparsely distributed SER, and tubular mitochondria. Very weak steroidogenic activity of Leydig cells is evidenced by weak 3b-HSD activity. During recrudescence, Leydig cells resume their steroidogenic activity with a marked decrease in the size and number of lipid droplets. Leydig cells in the spermatogenetically active phase are characterized by abundant SER and a large number of tubular mitochondria
(Figure 3.9) (Dubois et al., 1988; Khan & Rai, 2004a; Khan & Rai, 2005). Regarding other testicular interstitial cells, studies are limited and confined to lacertilians. Macrophages have been isolated from the testes of Indian wall lizards and identified by their morphological and functional characteristics (Figures 3.10 and 3.11) (Khan & Rai, 2007). They constitute the second largest population of the testicular interstitium. Corresponding to Leydig cells, the number of testicular macrophages varies over different phases of the reproductive cycle. With the onset of recrudescence, macrophages increase in number and reach a maximum in the breeding phase. Interestingly, the ratio of macrophages to Leydig cells remains constant throughout the reproductive cycle, implying a close relationship between these two cell populations. The presence of mast cells has been demonstrated in the testis of Podarcis sicula sicula using electron microscopy. The cell number varies during the
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Hormonal Regulation of Testicular Functions in Reptiles
69
FIGURE 3.7 Electron micrograph of the blood–testis barrier between the Sertoli cells of the Indian wall lizard. Occluding junctions including tight junctions (arrow heads) and gap junctions (arrows) between Sertoli cells (Sc). BM, basement membrane. 6,150.
year, with peaks in spring and winter (Minucci, Vitiello, Marmorino, Di Matteo, & Baccari, 1995). Unlike that of macrophages, mast cell number has an inverse relationship with Leydig cell number. Administration of a Leydig cellspecific cytotoxin, ethane dimethane sulfonate (EDS), increases mast cell number.
3. TESTICULAR FUNCTIONS 3.1. Spermatogenesis Spermatogenesis is an elaborate cytological process by which spermatogonia give rise to sperm. In anamniotes, temporal spermatogenesis occurs within a cyst in which a cohort of germ cells progress through the phases of spermatogenesis. All the germ cells present in a cyst are in the same stage of spermatogenesis. When spermatogenesis is completed, the cysts rupture and sperm are released as a single spermiation event. Although reptiles resemble
other amniotes in the possession of seminiferous tubules lined with seminiferous epithelium, spermatogenesis in temperate species is generally aligned with anamniotes. Instead of spatial germ cell development as in mammals and birds, a temporal germ cell development strategy has been described in detail for three temperate species representing different orders of reptiles (Table 3.1). In the slider turtle, Trachemys scripta, the initiation of spermatogenesis is characterized by the proliferation of spermatogonia near the basement membrane of the seminiferous epithelium in June and early July. Three types of spermatogonia are seen: resting, A, and B. The resting spermatogonia located towards the base of the tubules are small in size and do not undergo mitosis. It is the type A spermatogonia that undergo mitosis to form type B spermatogonia. With the initiation of meiosis, primary spermatocytes are formed from type B spermatogonia. They give rise to spermatids after the completion of the second meiotic division. These haploid germ cells are transformed
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Hormones and Reproduction of Vertebrates
FIGURE 3.8 Degenerated residual body (rb) deep within Sertoli cell cytoplasm during spermatogenetically active phase in Hemidactylus flaviviridis. Elongated spermatids (std) embedded in apical portion of Sertoli cell cytoplasm can be seen. Some empty recesses (r) after spermiation are also visible. 4,350. Reproduced from Khan and Rai (2004a).
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Hormonal Regulation of Testicular Functions in Reptiles
(a)
(b)
(c)
(d)
FIGURE 3.9 Ultrastructural features of Leydig cells (Lc) during different phases of the reproductive cycle of the Indian wall lizard. (a) In the regressed testis, abundant lipid droplets (l) are seen in the Leydig cell lying adjacent to a blood capillary, c; 3,450. (b) In the recrudescent phase, a few elongated mitochondria (M) with a decreased number of lipid droplets are observed; 10 000. (c) The voluminous cytoplasm of Leydig cells during the spermatogenetically active phase includes an increased number of mitochondria with tubular cristae and an increased amount of smooth endoplasmic reticulum (SER). Lipids droplets (l) are rarely seen. The chromatin is evenly distributed in the nucleus (N); 12 600. (d) Leydig cells showing D5-3b-hydroxysteroid dehydrogenase enzyme activity after incubation with ovine follicle-stimulating hormone (oFSH) for 24 hours (800). Reproduced from Khan and Rai (2004a).
to sperm by the process referred to as spermiogenesis, which is completed by late October to early November. Based on acrosomal development, nuclear condensation, cytoplasmic elimination, and flagellum formation, spermiogenesis in the slider turtle is divided into eight recognizable steps (Gribbins, Gist, & Congdon, 2003). The male European wall lizard Podarcis muralis differs from T. scripta in having a spermatogenically active seminiferous epithelium throughout the year with a very short quiescent phase. Spermatogenesis slows down
during the quiescent phase instead of ceasing completely. Only two types of spermatogonia, namely A and B, are present within the seminiferous epithelium, and spermiogenesis is divided into seven recognizable steps (Table 3.1) (Gribbins & Gist, 2003). Similarly to the European wall lizard, a prenuptial pattern of sperm development (described below) occurs in the American alligator, Alligator mississippiensis, though spermatogenesis is confined to a short period from February to June (Gribbins, Elsey, & Gist, 2006). Since both these species have
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FIGURE 3.10 Electron micrograph of lizard testicular macrophage with ruffled border, indented nucleus (N), and the presence of numerous phagolysosomes.
FIGURE 3.11 The testicular macrophage (M) of the Indian wall lizard, showing phagocytosis of a heat-killed yeast cell (arrow). Numerous phagocytic processes can be seen; 480. See color plate section.
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Hormonal Regulation of Testicular Functions in Reptiles
TABLE 3.1 A comparison of testicular cycles with an emphasis on the pattern of spermatogenesis and the various steps of spermiogenesis in different orders of living reptiles.
SPERMATOGONIA TYPES
RESTING A
A
B
B
B
SPERMATOGENICALLY ACTIVE PHASE
JUNE – NOVEMBER
ENTIRE YEAR
FEB – JUNE
SPERMIOGENESIS
8 - STEPS
7- STEPS
8 - STEPS
SPERMATOGENESIS PATTERN
POSTNUPTIAL
PRENUPTIAL
PRENUPTIAL
QUISCENT PHASE
VERY LONG
VERY SHORT
VERY SHORT
REFRACTORY PERIOD
PRESENT
ABSENT
ABSENT
A
FIGURE 3.12 Schematic representation of two major patterns of spermatogenesis and their correlation with the androgen (testosterone) profile. T, testes; V, vas deferens. Modified from Licht (1984).
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an actively dividing population of spermatogonia A and B throughout the year, favorable temperature as well as GTH and testosterone (T) administration during the quiescent phase can initiate recrudescence. Thus, alligators and European wall lizards lack a refractory period. On the other hand, a long refractory period is observed in slider turtles, in which the testes contain only resting spermatogonia during the quiescent phase and no mitotically active A and B spermatogonia. The resting spermatogonia fail to respond to temperature or hormonal manipulations (Gribbins et al., 2003). Based on the seasonal timing of spermatogenesis and mating, two major patterns for testicular cycles are recognized in reptiles: prenuptial and postnuptial (Figure 3.12) (Saint Girons, 1963). In the prenuptial species, spermatogenesis takes place before or during the mating season, and a single annual peak of testosterone is generally seen corresponding to the culmination of spermatogenesis. On the
Hormones and Reproduction of Vertebrates
other hand, timing of spermatogenesis does not coincide with mating in postnuptial species. In these species, spermatogenesis takes place after the mating season and sperm are stored in the male or female ducts for long periods to be used in the next mating season. Peak plasma androgen levels and sexual behaviors occur when the seminiferous tubules are regressed. In general, lizards exhibit a prenuptial type of spermatogenesis, while chelonians are characteristically postnuptial. Snakes are less consistent and may fall into either category (Licht, 1984).
3.2. Steroidogenesis The synthesis of androgens begins with the mobilization of cholesterol from cholesterol depots present in the form of conspicuous lipid droplets within the cytoplasm. Cholesterol is transported to the inner-mitochondrial membrane, where it is converted to pregnenolone by C27 cytochrome
FIGURE 3.13 The biosynthetic pathway of sex steroids. The conversion of cholesterol to androgens is carried out by three primary enzyme systems, namely C27 side-chain cleavage P450, C21 side-chain cleavage P450, and D5-3b-hydroxysteroid dehydrogenase and D5-4-isomerase. The C27 side-chain cleavage P450 enzymes are located in the mitochondria, whereas the other two enzyme systems are in the smooth endoplasmic reticulum. See color plate section.
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Hormonal Regulation of Testicular Functions in Reptiles
P450 side-chain cleavage enzyme complex. Thereafter, pregnenolone is carried to the microsomal compartment where the membrane-bound enzyme 17a-hydroxylase and C17,20-lyase converts it into dehydroepiandrosterone (DHEA). 3b-hydroxysteroid dehydrogenase, along with D5-4-isomerase, converts DHEA into androstenedione (AND), the immediate precursor of T. The synthesis of T is regulated by 17b-HSD (Figure 3.13). Based on 3b-HSD activity and ultrastructural features (Callard, I., Callard, G., Lance, Bolaffi & Rosset, 1978), both Leydig and Sertoli cells are considered to be sites of steroid biosynthesis in reptiles. Although the importance of Sertoli cell-secreted steroids is not very clear, they may contribute meaningfully to maintaining the high intratubular androgen concentrations required for spermatogenesis, or in maintaining accessory sex organs during the mating season, when the testes are regressed in postnuptial species. On the other hand, Leydig cell-secreted androgens enter the peripheral circulation and control development of
accessory sex organs and sexual behavior. In most reptiles, T is the principle androgen and shows pronounced seasonal variation (see Table 3.2). Additionally, estrogens are synthesized by the testes of reptiles. Recently, aromatase, the rate-limiting enzyme regulating estrogen biosynthesis, was immunocytochemically demonstrated in Sertoli and Leydig cells of the turtle T. scripta. Aromatase immunoreactivity shows an inverse relationship with the testicular cycle. A strong immunochemical reaction for aromatase is observed during the quiescent phase, but only a weak reaction is detected when the germ cells undergo meiosis (Gist, Bradshaw, Morrow, Congdon, & Hess, 2007). However, no such relationship is reported in crocodiles and alligators, probably due to very low levels of estrogens, detectable only by using a highly sensitive assay system. The plasma estrogen levels in male alligators range from 0.23 to 3.14 pg/ml. Based on much higher mRNA expression of aromatase in the brain than in the testes, this extragonadal site is suggested to contribute majorly to estrogen
TABLE 3.2 Peak plasma androgen level in certain prenuptial and postnuptial reptilian species. Generally, two androgen peaks are observed in postnuptial species, whereas a single peak parallel to spermatogenesis is seen in prenuptial species. SPECIES
PEAK(S)
CORRELATION
POST NUPTIAL TYPE Number of peaks Large Peak
Small Peak
Chrysemys picta (Callard et al. 1976; Licht et al., 1985)
Double
10–16 ng/ml ~ 5 ng/ml
Neither peak coincides with spermatogenesis
Chelydra serpentina (Mahmoud et al., 1985)
Double
50 ng/ml
Neither peak coincides with spermatogenesis
Chelonia mydas (Licht, et al., 1985)
Double
27.39 ng/ml 3 ng/ml
20 ng/ml
Exceptionally large peak of androgens coincides with spermatogenesis
PRENUPTIAL TYPE Geochelone nigra (Schramm et al., 1999)
Single
28.94 6.38 ng/ml
With spermatogenesis
Podarcis s. sicula (Ando et al., 1990)
Single
174.8 ng/ml
With spermatogenesis
Calotes versicolor (Radder et al., 2001)
Single
w13 ng/ml
With spermatogenesis
Sternotherus odoratus (McPherson et al., 1982; Mendonca and Licht, 1986)
Single
90–125 ng/ml
With spermatogenesis
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biosynthesis (Lance, Conley, Mapes, Steven, & Place, 2003). However, Milnes, Woodward, Rooney, and Guillette (2002) reported plasma estradiol (E2) levels of 10–60 pg/ml in juvenile alligators from the Florida wetlands. The discrepancies in estrogen levels in the different studies are attributed in part to differences in the sensitivity of the assay system used, and also to the presence of endocrine disruptors in the Florida wetlands. In contrast, the male lizard Podarcis sicula had very high plasma estrogen levels (> 1000 pg/ml in males has been recorded (Ando` et al., 1992)).
4. REGULATION OF TESTICULAR FUNCTIONS 4.1. Environmental Factors Studies on the role of environmental factors in the control of testicular functions are few and scattered. Temperature acts as the main cue for testicular recrudescence in temperate species. For example, in Anolis carolinensis, the responsiveness of testes to mammalian/chelonian GTHs is reported to be maximal at 30 C compared to 20 C (Licht, 1975). Similarly, androgen production is temperaturedependent in the lizard Tiliqua rugosa (Bourne, Raylor, & Watson, 1986) and the turtle T. scripta (Licht, Denver, & Pavgi, 1989). However, temperature is ineffective in influencing the in-vitro effect of GnRH on LH secretion from pituitary cells in T. scripta (Licht et al., 1989). On the other hand, in tropical species, rainfall/moisture regulates gonadal recrudescence and breeding because temperature remains relatively constant throughout the year (Licht, 1984; Lofts, 1987; Whittier & Crews, 1987). The largest testicular size in four tropical gecko species from Brazil is observed during the rainy season (Vitt, 1986). It is of interest to note that the Indian wall lizard Hemidactylus flaviviridis inhabiting the temperate zone of India (north India) shows distinct phases of the testicular cycle from regressed to spermatogenetically active, whereas testes in H. brooki, which inhabits the tropical region of India (south India), are reported to be spermatogenetically active throughout the year (Shanbhag, Karegouder, & Saidapur, 2000). In some reptiles, photoperiod is an important regulator of testicular functions. An increase in photoperiod results in testicular recrudescence and active spermatogenesis in the red-eared turtle, T. scripta elegans (Burger, 1937); the desert night lizard, Xantusia vigilis (Bartholomew 1950; 1953); and A. carolinensis (Underwood & Hall, 1982). Further, the pineal hormone, melatonin (MEL), is implicated in mediating photoperiod effects on testes, since pinealectomy in A. carolinensis induced testicular development during the fall when testes normally are regressed. This effect is reversed after MEL
Hormones and Reproduction of Vertebrates
replacement therapy (Underwood, 1995). The antigonadal role of MEL is also reported in the lizard Calotes versicolor. Administration of MEL to intact animals resulted in inhibition of gonadal activity, whereas an increase in gonadal weight and accessory sex organs occurs in pinealectomized lizards (Haldar-Misra & Thapliyal, 1981a).
4.2. Extratesticular Factors 4.2.1. Hypothalamic hormones Gonadotropin-releasing hormone was first identified by its stimulatory effect on synthesis and the release of pituitary GTHs, which in turn regulate reproductive functions (Matsuo, Baba, Nair, Arimura, & Schally, 1971; Burgus et al., 1972). The lack of GnRH expression or mutation in the gene encoding for GnRH receptors produces sterility in all vertebrates, including reptiles (Licht, Porter, & Millar, 1987; Tsai & Licht, 1993b; Somoza, Miranda, Strobl-Mazzulla, & Guilgur, 2002). To date, 25 GnRH variants have been isolated and characterized from different vertebrate and invertebrate nervous tissues (Kavanaugh, Nozaki, & Stacia, 2008). Although varying forms of GnRH are encoded by different genes, they are grouped into one family since all are decapeptides with conserved amino acids at one, four, nine, and ten positions. Among the different structural variants, chicken GnRH-II (cGnRH-II) is universally present in all vertebrates without any sequence alterations. In fact, cGnRH-II appears to have remained unchanged during the evolution of jawed vertebrates. In reptiles, many different forms of GnRH have been demonstrated using various techniques. For example, two forms of GnRH, cGnRH-I and cGnRH-II, are present in A. mississippiensis (Lovejoy et al., 1991); crocodiles, Crocodylus niloticus (Millar & King, 1994); and turtles, T. scripta (Sherwood & Whittier, 1988; Tsai & Licht, 1993b). In the lacertilians, the girdle-tailed lizard, Cordylis nigra, and the ruin lizard, P. sicula, cGnRH-II and salmon GnRH (sGnRH) are present (Powell, King, & Millar, 1985; Powell, Ciarcia, Lance, Millar, & King, 1986). Many unidentified GnRH-like forms are also reported in P. sicula (Powell et al., 1985; 1986), the striped skink, Mabuya capensis; and a tortoise, Chersine angulata (King & Millar, 1979; 1980). However, the physiological significance of varying forms of GnRH is still obscure. Synthetic cGnRHII stimulates pituitary GTH secretion, but its anatomical location in the mid-brain and hind-brain (Tsai & Licht, 1993b) raises doubts about its availability to reach the pituitary gonadotropes through portal vessels. On the other hand, cGnRH-I, the axons of which terminate on the hypophysial portal vessels that perfuse the pituitary, does not always stimulate LH secretion in vivo (Licht & Porter, 1985; Tsai & Licht, 1993c). None the less, cGnRH-I is
Chapter | 3
Hormonal Regulation of Testicular Functions in Reptiles
shown as a potent GTH releaser in-vitro (Licht & Porter, 1985; Licht et al., 1987). With increasing knowledge of multiple GnRH variants in the brain, a search for distinct GnRH receptors (GnRH-Rs) in target tissues began. The first complete study demonstrating the presence of distinct GnRH-R subtypes was performed in goldfish (Illing et al., 1999). Phylogenetic analysis classifies the vertebrate GnRH-Rs into four groups: I/III, 2/non-mammalian I, 3/II, and 4/mammalian I (Ikemoto & Park, 2005). In reptiles, the first GnRH-R was cloned and characterized from the leopard gecko Eublepharis macularius (Ikemoto, Enomoto, & Park, 2004). Molecular phylogenetic analysis revealed that the cloned GnRH-R belongs to type-2/non-mammalian I. As its expression levels were quite low in the pituitary gland of reproductively active leopard geckos, the possibility of some other type of GnRH-R being responsible for GTH secretion in reptiles was suggested. In 2007, three GnRHRs that respond respectively to GnRH ligand, cGnRH-I, and cGnRH-II were demonstrated in the lizard E. macularius. It is noteworthy that two of the three GnRH-R subtypes (GnRH-R 2 and GnRH-R 3) could not be detected in the anterior pituitary; rather, these are present in the posterior or intermediate lobe (Ikemoto & Park, 2007). Further studies are required to explain the structure, expression pattern, and functions of GnRH-Rs in reptiles. Studies on the role of GnRH in the regulation of testicular functions are limited and exhibit inconsistencies. Mammalian GnRH (mGnRH) or its agonist does not affect plasma LH and gonadal steroids in the male turtles Chelonia mydas, Sternotherus odoratus, and Lepidochelys olivacia (Licht, Owen, Cliffton, & Pessaflores, 1982; Licht et al., 1984). Similarly, cGnRH-I is ineffective in S. odoratus. On the other hand, mGnRH stimulates plasma T secretion in male alligators (Lance, Villet, & Bolaffi, 1985). The inconsistency in the results might be due to GnRH variants, dose, or frequency of treatment, as diverse effects of GnRH based on these factors are reported in P. s. sicula. In this species, a single dose of mGnRH, cGnRH-II, or sGnRH increases plasma T levels, though mGnRH and cGnRH-II are more potent than sGnRH. In contrast to a single injection, prolonged GnRH treatment inhibits testicular and epididymal activity, possibly due to desensitization of pituitary receptors (Ciarcia, Paolutti, & Botte, 1989). The concept of GnRH-induced desensitization of pituitary receptors was reinforced by in-vitro observations in female T. scripta in which prolonged cGnRH-I or cGNRH-II treatment attenuated LH release from pituitary cells (Tsai & Licht, 1993a). In addition to GnRH, a novel hypothalamic dodecapeptide called gonadotropin-inhibitory hormone (GnIH) has been identified in the avian pituitary that may play an important role in the regulation of GTH secretion (Tsutsui et al., 2000). Gonadotropin-inhibitory hormone is localized
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in the neurons of the avian paraventricular nucleus (PVN), ventral paleostriatum, septal area, preoptic area (POA), and other areas of the hypothalamus as well as in the optic tectum, median eminence, and the dorsal motor nucleus of the vagus in the medulla oblongata. Gonadotropininhibitory hormone fibers are observed in close proximity to GnRH neurons in the avian POA. Moreover, GnIH significantly inhibits GTH release and consequently testicular development and functions in birds (Tsutsui & Osugi, 2009). Although homolog peptides of GnIH sharing a common C-terminal LPXRFamide motif have been identified in the hypothalamus of non-avian vertebrates including reptiles (unpublished data, cited in Tsutsui & Osugi, 2009), their role in testicular function has not been explored in reptiles.
4.2.2. Pituitary hormones In Chelonia and Crocodilia, two distinct gonadotropins similar to mammalian FSH and LH have been isolated and characterized (Licht, Farmer, & Papkoff, 1976; Papkoff, Farmer, & Licht, 1976; Licht & Papkoff, 1985). This was further confirmed by the cloning of a partial cDNA sequence for an FSH b-subunit and a full length cDNA sequence for a LH b-subunit from the pituitary of Reeves’ turtle, Geoclemys reevesii (Aizawa & Ishii, 2003). These two GTHs, FSH and LH, are non-covalently linked heterodimeric glycoproteins consisting of a- and b-subunits encoded by distinct genes. The a-subunit of both GTHs is identical and interchangeable, while the b-subunit is hormone-specific as well as species-specific and confers biological action through signal transduction. Both subunits are synthesized as precursor proteins that are posttranslationally glycosylated, assembled, and secreted into the circulation. In testes, FSH normally regulates Sertoli cell functions, and Leydig cell androgen synthesis is controlled by LH. However, the precise role of FSH and LH in the regulation of testicular functions in Chelonia and Crocodilia is still not clear. Mammalian FSH (mFSH) is reported to be more potent in regulating steroidogenesis in turtles and alligators as compared to mammalian LH (mLH) (Callard & Ryan, 1977; Tsui & Licht, 1977; Lance & Vliet, 1987). However, altogether different results are reported with homologous GTHs in alligators. The in-vitro incubation of interstitial cells with alligator LH, not FSH, results in an increase in androgen production (Tsui & Licht, 1977). The confusion regarding two GTHs and two testicular functions is further exacerbated in the squamates. The biochemical analysis of pituitary GTH from five species of snake belonging to three different families does not establish clear evidence for two separate types of gonadotropin (Licht, Farmer, Gallo, & Papkoff, 1979). Rather, a single GTH is suggested to be present that is
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structurally similar to neither FSH nor LH, but shares biological actions of both FSH and LH, depending on the animal model in which it is tested. The snake GTH showed a high degree of species specificity. It had high binding affinity and biological activity with respect to androgen production in homologous or heterologous species of snakes, had relatively less potency in lizards, but was totally devoid of activity in non-squamate reptiles or other vertebrates. Further, other studies using mammalian GTHs report that a single FSH-like molecule regulates both the testicular functions in squamates (Licht & Pearson, 1969; Reddy & Prasad, 1970; Licht & Hartree, 1971; Licht & Papkoff, 1971; Eyeson, 1971; Licht, 1972; Licht, Gallo, Hartree, & Shownkeen, 1977; Rai & Haider, 1986; 1995). A high affinity binding site for only mFSH is present in both the tubular and interstitial compartments of the testes of the whiptail lizard, Cnemidophorus tigris (Licht & Midgley, 1977). Recently, in-vitro experiments in the Indian wall lizard have provided direct evidence of ovine FSH (oFSH) action in the control of Sertoli as well as Leydig cell activities (Khan & Rai, 2005). However, instead of only an FSH-like GTH, the single GTH present in the Japanese grass lizard Takydromus tachydromoides seems to be LH-like, as a cDNA encoding only for the LH bsubunit has been cloned in this species (Aizawa & Ishii, 2003). The concept of the existence of a single GTH, however, is contradicted in some of the squamates. In the lizard P. sicula campestris, two GTHs are immunocytochemically demonstrated in the pars distalis using hFSHb and hLHb antibodies (Desantis, Labata, & Corriero, 1998). In another study, Southern blot analysis shows that the P. sicula genome contains a nucleotide sequence encoding b-subunits of both FSH and LH. However, it is not clear whether these nucleotide sequences are translated into two different functional GTH b-subunits or a single b-subunit that is similar to mammalian FSHb as well as LHb (Borrelli, Bovenzi, & Filosa, 1997). The two GTHs/two testicular functions hypothesis is validated in the lizard Agama agama. In hypophysectomized A. agama, mFSH maintains spermatogenesis, whereas mLH regulates interstitial (Leydig) cell function (Eyeson, 1971). Nevertheless, to date only a cDNA for FSH receptor has been cloned from the gonads of squamates (Borrelli, De Stasio, Parisi, & Filosa, 2001; Bluhm et al., 2004), strengthening the one GTH-two functions hypothesis.
Hormones and Reproduction of Vertebrates
Naulleau, Fleury, & Boissin, 1987; Bermudez et al., 2005). An inverse correlation between plasma thyroid hormones and androgens is reported in the Chinese cobra, Naja Naja, and six-lined racerunner, Cnemidophorus sexlineatus (Bona-Gallo, Licht, MacKensie, & Lofts, 1980; Sellers, Wit, Ganjean, Etheridge, & Ragland, 1982). Experimental studies show a direct role for thyroid hormones in spermatogenesis. Either hypothyroidism induced by thyroidectomy in the variegated lizard, Coleonyx variegatus (Plowman & Lynn, 1973) and the garden lizard C. versicolor (Haldar-Misra & Thapliyal, 1981b), or hyperthyroidism caused by administration of thyroid hormones to intact Calotes (Haldar-Misra & Thapliyal, 1981b), leads to impairment of testicular functions, indicating that abnormalities in thyroid functions adversely affect spermatogenesis. The direct involvement of thyroid hormone was evidenced with the demonstration of its a receptor in the testis of P. sicula (Cardone, Angelini, Esposito, Comitato, & Varriale, 2000). Recently, thyroid hormones were reported to increase the expression of AR mRNA levels and, thus, enhance the androgen responsiveness of testicular cells during spermatogenesis (Cardone et al., 2000). Data on testicular sensitivity to thyroid hormones are lacking for chelonians and crocodilians. Glucocorticoids are also known to influence testicular functions in reptiles (see also Chapter 7, this volume). Stress induces an increase in plasma corticosterone (CORT), with a parallel decrease in sex steroid levels (Lance & Elsey, 1986; Greenberg & Wingfield, 1987; Guillette, Cree, & Rooney, 1995). Administration of CORT decreases plasma androgen levels in the lizards Uta stansburiana (DeNardo & Licht, 1993) and Anolis sagrei (Tokarz, 1987a). In addition to steroidogenesis, exogenous CORT adversely affects spermatogenesis in A. sagrei (Tokarz, 1987a) and Mabuya carinata (Yajurvedi & Nijagal, 2000). It has been postulated that CORT affects testicular functions by acting directly on the gonads in M. carinata, as CORT interferes with spermatogenesis even in the presence of GTHs (Yajurvedi & Nijagal, 2000).
4.3. Intratesticular Factors Sertoli cells, Leydig cells, and testicular immune cells in mammals are reported to secrete steroidal and non-steroidal factors that regulate testicular functions in a paracrine manner. As far as reptiles are concerned, studies are meager and very rudimentary.
4.2.3. Other hormones The importance of the thyroid gland in the regulation of testicular activities was recognized long ago in reptiles, based on seasonal variation of plasma T3 (Kar & ChandolaSaklani, 1985) and T4 (Kar & Chandola-Saklani, 1985;
4.3.1. Androgens Earlier studies in reptiles present a confusing picture pertaining to the role of androgens in the regulation of spermatogenesis. For example, androgen administration in
Chapter | 3
Hormonal Regulation of Testicular Functions in Reptiles
juvenile terrapins stimulates spermatogenesis (Lofts & Chiu, 1968), whereas this treatment is ineffective in alligators (Forbes, 1939) as well as in the lizard Sceloporus occidentalis (Gorbman, 1939). An inhibitory effect of androgens is observed in some species such as Eumeces fasciatus (Reynolds, 1943), Uromastix hardwickii (Ramaswami & Jacob, 1963), and A. agama (Eyeson, 1971). This androgen-induced inhibition of spermatogenesis is attributed to the decrease of basal GTH level by negative feedback (Eyeson, 1970). However, in these studies, the reproductive state of animals was not considered. In the Indian wall lizard, depending on its reproductive state, different effects of androgens on spermatogenesis are demonstrated. During the breeding phase, the administration of AR antagonists such as cyproterone acetate (CA) and flutamide inhibits spermatogenesis (Haider & Rai, 1986). This provides a clue as to why a direct correlation exists between Leydig cell steroidogenic activity and spermatogenesis in prenuptial species. In the non-breeding or recrudescent phase, androgen administration leads to a decrease of spermatogonial cells in the Indian wall lizard (Rai & Haider, 1986; 1995). Recently, in-vitro experiments in wall lizards have resolved these discrepancies. Sertoli cells were collected from regressed testes and subjected to androgen treatment in the presence or absence of FSH. Dihydrotestosterone (DHT) alone inhibited lactate production from inactive Sertoli cells while enhancing metabolic activity of the Sertoli cells pretreated with FSH or simultaneously incubated with FSH and DHT. This suggests that the observed discrepancies of androgens acting on spermatogenesis are dependent on the inactivated/GTH-activated state of the Sertoli cells (Khan & Rai, 2004b). In P. sicula, androgens upregulate the expression of the AR (Cardone, Angelini, & Varriale, 1998). However, the precise location of AR within the testis has not been demonstrated; hence, it is difficult to explain precisely the autocrine/paracrine role of androgens on testicular functions at this time.
4.3.2. Estrogens The de novo biosynthesis of estrogens is reported in the testes of reptiles. In the turtle T. scripta, aromatase, the ratelimiting enzyme involved in estrogen biosynthesis, is localized immunocytochemically in Leydig as well as Sertoli cells (Gist et al., 2007), and plasma levels of estrogens have been assessed in the lizards P. s. sicula (Ciarcia, Angelini, Polzonetti, Zeroni, & Botte, 1986) and Chalcides ocellatus (Angelini, Ciarcia, Zerani, Polzonetti, & Botte, 1987). The involvement of estrogens in the control of testicular functions is implicated by the presence of estrogen receptors (ERs) in the testes. Estrogen receptora is localized in Leydig cells, while ERb is found in
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spermatogonia, Sertoli cells, and epithelial cells of excurrent ducts in the turtle (Gist et al., 2007). Further, the presence of ERb in germ cells is reported in P. s. sicula (Chieffi & Varriale, 2004). In this lizard, estrogens are implicated in post-reproductive refractoriness as administration of E2 induces regression of testes and epididymis during the reproductively active phase (Angelini, Ciarcia, Picariello, Botte, & Pagano, 1986). This is corroborated by induction of the precocious resumption of spermatogenesis and sperm release in the autumnal early recrudescent phase using the aromatase inhibitor, fadrazole (Cardone, Comitato, Bellini, & Angelini, 2002). Further, estrogens decrease AR mRNA expression in androgen-primed testicular cells in the European wall lizard (Cardone et al., 1998) and are hypothesized to cause testicular regression by either inhibiting the hypothalamo–hypophysial–gonadal (HPG) axis (Angelini et al., 1986), and/or by directly downregulating AR gene expression. In Indian wall lizards, E2 decreases the metabolic activity of Sertoli cells (Khan & Rai, 2004b), suggesting the inhibitory role of estrogens on spermatogenesis by decreasing the nursing function of Sertoli cells. In parallel to lacertilians, estrogens may maintain testes in the quiescent state in chelonians. An intense immunoreactivity for aromatase along with higher expression of ERa and b is reported in the quiescent testes of the turtle T. scripta (Gist et al., 2007). However, a key role for estrogens in early phases of spermatogenesis is suggested in P. s. sicula, based on the observations that estrogens induce proliferation of type A spermatogonia through activation of ERK1/2 and that pre-treatment with an antiestrogen ICI 182-780 inhibits spermatogonial mitotic divisions (Chieffi, D’Amato, Guarino, & Salvatore, 2002). The role of estrogens in spermatogenesis has been questioned in alligators because estrogen levels as well as aromatase expression are very low in males (Lance et al., 2003).
4.3.3. Other paracrine factors Cell–cell interaction plays a key role in the regulation of testicular functions. In reptiles, the literature is scanty and confined to the Indian wall lizard. In-vitro experiments have been performed in which the effect of conditioned medium of one cell type on the function of another cell type is studied. These studies suggest that some uncharacterized non-steroidal paracrine factors may be involved in the control of testicular cells. 4.3.3.1. Sertoli cell-secreted factors Sertoli cell-conditioned medium (SCCM) is shown to regulate the activities of Leydig cells collected from regressed testes. The spent culture medium collected from oFSH-preactivated or non-activated Sertoli cells increases
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Leydig cell steroidogenesis, although oFSH-preactivated SCCM is more potent than non-activated SCCM. Interestingly, preactivation of Leydig cells with oFSH increases their responsiveness to SCCM. Perhaps the Sertoli cells secrete a steroidogenic factor that regulates basal as well as FSH-induced T production by Leydig cells. In addition to this hypothesized steroidogenic factor, FSH-activated Sertoli cells secrete an unknown mitogenic factor that induces proliferation of Leydig cells, but only when they are in an activated state in response to oFSH (Khan & Rai 2005). 4.3.3.2. Testicular macrophage-secreted factor Macrophages lie in close proximity to the Leydig cells in the testicular interstitium. As discussed in Section 2.2, there is a direct relationship between the Leydig cell number and the size of the testicular macrophage population. The ratio of macrophages to Leydig cells (i.e., 1 : 3–4) remains constant throughout the reproductive cycle, though their total numbers increase from the regressed to the spermatogenetically active phase. This reflects a close communication between these two cell populations. In-vitro experiments substantiate the role played by macrophages in the regulation of Leydig cell activities. The conditioned medium of testicular macrophages (TMCM) enhances T production by Leydig cells collected from regressed testes of lizards. The steroidogenic effect of TMCM is more pronounced when testicular macrophages are pre-activated with FSH. This suggests that macrophages secrete a steroidogenic factor that regulates basal as well as FSHinduced T production by Leydig cells. Further, FSH has a stimulatory effect on the secretion of this presumed steroidogenic factor from testicular macrophages. Also, testicular macrophages in response to FSH are reported to secrete a mitogenic factor that induces proliferation of Leydig cells only when the latter are activated with GTH (e.g., oFSH). Khan and Rai (2008a) examined the role of testicular macrophages in the regulation of Leydig cell activities under inflammatory conditions. The testicular macrophages were treated with a bacterial lipopolysaccharide (LPS) and the spent medium was collected. Lipopolysaccharide-treated TMCM inhibited Leydig cell steroidogenesis as well as proliferation. Thus, it was concluded that testicular macrophages differentially regulate Leydig cell functions under normal physiological conditions and during inflammation. 4.3.3.3. Histamine The mast-cell secreted factor, histamine, is ineffective in influencing basal T production by Leydig cells. However, histamine affects FSH-induced Leydig cell steroidogenesis in a biphasic manner, depending on its concentration. It increases T production at low concentrations but decreases
Hormones and Reproduction of Vertebrates
it at high concentrations (Khan & Rai, 2007). The biphasic effects of histamine apparently are mediated by two different histaminergic receptors. The inhibition of steroidogenesis at the high concentration of histamine is mediated by H1 receptors, while the stimulation at low concentration works through H2 receptors. However, histamine does not influence Leydig cell proliferation and therefore it appears that the increased T production in response to histamine is due to altered steroidogenic enzyme levels. 4.3.3.4. Leydig cell-secreted factors Leydig cell-secreted paracrine factors appear to regulate the nursing function of Sertoli cells. Follicle-stimulating hormone-activated Leydig cell-conditioned medium (LCCM) that contains FSH as well as FSH-induced Leydig cell-secreted factor increases lactate production by Sertoli cells. On the other hand, FSH-preactivated LCCM that contains FSH-induced Leydig cell-secreted factor but not FSH inhibits metabolic activity in Sertoli cell (Khan & Rai, unpublished data). In a separate study, DHT is reported to exert diverse effects on the metabolic activity of Sertoli cells, depending on their state of activation; i.e., inhibitory on non-activated and stimulatory on FSH-activated cells (Khan & Rai, 2004b). Perhaps the Leydig cell-secreted factor is an androgen that differentially affects lactate production by Sertoli cells.
5. REGULATION OF TESTICULAR IMMUNE FUNCTIONS The immune cells in the interstitial compartment ensure an infection-free microenvironment for the developing germ cells. Although the presence of lymphocytes in the testicular interstitium has not been reported in reptiles, macrophages lying in close proximity to Leydig cells immunonologically regulate the testis microenvironment by phagocytosis and release of cytotoxic substances (Figure 3.14). The functions of testicular macrophages are regulated by pituitary GTH as well as by paracrine factors secreted from neighboring cells. A study in the Indian wall lizard demonstrates the stimulatory role of oFSH in the regulation of macrophage-based immune responses (Khan & Rai, 2008b). Leydig cell-secreted factor also affects the phagocytic and respiratory burst activities of testicular macrophages (Khan & Rai, 2008b). Androgens differentially can regulate phagocytosis. In the presence of FSH, DHT enhances phagocytosis, but it downregulates phagocytosis in the absence of oFSH. In contrast, DHT suppresses the respiratory burst activity of non-activated or even FSH-activated testicular macrophages. Intriguingly, FSH-activated/-preactivated LCCM increases superoxide production and hence implicates the involvement of an
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FIGURE 3.14 Schematic representation of the regulation of testicular immune responses in the Indian wall lizard Hemidactylus flaviviridis. Red lines denote inhibition whereas green arrows indicate stimulation of a particular response. ROS, reactive oxygen species. See color plate section.
FSH-induced Leydig cell-secreted proteinaceous factor. This has been corroborated using heat-inactivated FSHactivated LCCM, which dramatically decreased superoxide production. Since FSH and DHT have distinct signaling pathways, exploring the crosstalk between these hormones in terms of control of testicular functions would be an interesting area of research. The mast cell-secreted product histamine is also important in the regulation of testicular immune responses. Histamine differentially regulates phagocytic and respiratory burst activities, depending on concentrations. Both phagocytosis and superoxide production are inhibited by high concentrations of histamine. On the other hand, superoxide production increases at a low concentration of histamine, and phagocytosis remains unaltered (Figure 3.14).
6. MAINTENANCE OF MALE ACCESSORY SEX ORGANS AND COURTSHIP BEHAVIOR 6.1. Accessory Sex Organs The renal sexual segment (RSS) is an accessory sex organ found exclusively in male squamates. It is a hypertrophied region of the nephron and consists of a single layer of columnar epithelial cells with basal nuclei. The sexual segment associated with each kidney undergoes seasonal changes corresponding to the testicular cycle (Shivanandappa, 1978; Thippareddy, 1979). The RSS is barely visible during the quiescent phase but is highly developed
with large secretory granules in the breeding season. Bilateral castration leads to atrophy of the androgendependent RSS, and exogenous T restores its growth and secretory activity (Bishop, 1959; Pandha & Thapliyal, 1964). Further, RSS hypertrophy with secretory granule formation can be achieved in immature animals by T administration (Kro¨hmer, Martinez, & Mason, 2004). The excurrent duct system of each testis includes an epididymis and a vas deferens. Phylogenically, reptiles are the first group in which a functional epididymis is present. The anterior, middle, and posterior segments of the epididymis of wall lizards H. flaviviridis are histologically differentiated and comparable to the caput, corpus, and cauda of the mammalian epididymis. The epithelial cell height decreases while lumen diameter with sperm content increases from anterior to posterior segments (Haider & Rai, 1987). The anterior and middle portions are thought to be involved in sperm maturation. Mature sperm are stored in the posterior portion of the epididymis. Similarly, the vas deferens is differentiated into proximal, middle, and distal regions based on epithelial cell height (Aranha, Bhagya, Yajurvedi, & Sagar, 2004). The androgenic regulation of the excurrent duct system is well established. In prenuptial species, development and secretory activity of the duct coincides with spermatogenesis and increased Leydig cell activity (Arslan, Jalali, Nasreen, & Qazi, 1986; Rai & Haider, 1991). Further, castration or administration of the antiandrogen flutamide leads to regression of these ducts (Shivanandappa & Sarkar, 1987; Rai & Haider, 1991).
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(a)
(b)
FIGURE 3.15 Femoral pores. (a) Ventral view of male lizard Amevia amevia showing rows of femoral pores (arrows) on the thigh region. (b) Magnified view of femoral pores in which opening of the glandular ducts (arrows) can be seen in the center of a row of modified scales resembling a rosette. Courtesy: Imparato, Anteomiazzi, Rodrigues, and Jarred (2007).
Femoral glands are present on the ventral position of each thigh in both sexes (Imparato, Anteomiazzi, Rodrigues, & Jarred, 2007). These are made up of branching tubes and tubules that open outside via femoral pores (Figure 3.15). After hatching, the gland in males increases in size and shows seasonal variation with maximal complexity during the breeding season. The pore size and secretory activity of the femoral gland is positively correlated with plasma T levels in lacertilians (Alberts, Pratt, & Phillips, 1992). Unlike males, the glands remain rudimentary in females.
6.2. Courtship Behavior Based on the relationships between androgen levels, spermatogenesis, and mating behavior, two distinct types of reproductive pattern, associated and dissociated, are observed in reptiles (Crews, 1984). In the associated pattern, the mating behavior coincides with peak androgen levels and spermatogenesis. This type of reproductive pattern is a characteristic of prenuptial species in which castration eliminates while T administration restores the courtship behavior (Woolley, Sakata, & Crews, 2004). The role of androgens in the regulation of courtship behavior is also corroborated using antiandrogens (cyproterone acetate, flutamide) in A. sagrei males (Tokarz, 1987b). On the other hand, in the case of the dissociated pattern of reproduction, spermatogenesis, androgen levels, and mating behavior are not temporally synchronized. The redsided garter snake Thamnophis sirtalis parietalis, is the best-studied reptilian model for the dissociated pattern of reproduction (Kro¨hmer, 2004). Mating in this snake occurs immediately after emergence from hibernation and apparently is independent of androgens as castration prior to or after coming out from hibernation does not affect courtship behavior. However, courtship behavior slowly declines over the years in castrated males. Long-term androgen replacement therapy during summer, when intact males usually experience a surge in androgens, results in an
increase in courtship behavior in the next spring following hibernation, suggesting that androgenic stimulation is required in reorganizing the neural events of mating behavior. Similar organizing effects of androgens occur during sexual displays in other adult snakes. The implication of neurosteroids synthesized de novo in the brain in organizing courtship behavior in such species should be examined. In addition to androgens, the hypothalamic neuropeptide arginine vasotocin (AVT) is implicated in the regulation of sexual behavior in reptiles (Woolley et al., 2004). Expression of AVT is greater in males than females and is androgen-dependent. Administration of T increases expression of AVT in the POA and anterior hypothalamus in Cnemidophorus uniparens and Cnemidophorus inornatus (Hillsman, Sanderson, & Crews, 2007). Temperature may be an important environmental cue to regulate AVT expression in the wall lizard Lacerta muralis (Bons, 1983).
7. CONCLUSIONS Reptiles are the first phylogenetic group in which spermatogenesis occurs in seminiferous tubules lined by peritubular cells. Based on the timing of spermatogenesis, Leydig cell steroidogenic activity, and mating, spermatogenesis can be categorized into two major types: prenuptial and postnuptial. Testicular functions are regulated by a complex interplay of endocrine and paracrine factors. Several variants of GnRH occur in the brains of reptiles, cGnRH-II being the most widespread although GnRH1 is probably the most important for releasing GTHs. The presence and role of another potential hypothalamic hormone, GnIH, need to be determined for reptiles. Gonadotropin-releasing factor stimulates GTH release from the anterior pituitary. Two distinct GTHs regulating different testicular functions are established in Chelonia and Crocodilia. However, in squamate reptiles, even after four decades of research, it is ironic that our knowledge about GTHs is rudimentary. The concept of
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FIGURE 3.16 Model proposed for endocrine and paracrine regulation of testicular functions in squamates. Red lines denote inhibition/negative feedback whereas the green arrows represent stimulation of a particular response. LPS, lipopolysaccharide; ROS, reactive oxygen species. See color plate section.
a single GTH resembling mammalian FSH, which regulates all testicular functions, is supported in lizards and snakes. Two separate GTHs have not been cloned successfully or characterized for any squamate species. Sex steroids, androgens, and estrogens play important roles in the regulation of testicular functions. Androgens differentially regulate spermatogenesis, depending on the reproductive state of the testis. Stimulation of spermatogenesis by androgens is seen during the breeding phase, but no effect is observed during the non-breeding phase. Surprisingly, no efforts have been made to investigate the exact location of ARs in testicular cells. Hence, it is not clear whether androgens regulate spermatogenesis by acting directly on the germ cells or by acting indirectly through Sertoli cells/peritubular myoid cells, or both. In
recent years, ERa as well as ERb have been localized in the reptilian testis. Also, aromatase enzyme has been immunocytochemically demonstrated in Leydig and Sertoli cells. Estrogens are implicated in inducing testicular regression by inhibiting the HPG axis and/or by downregulating AR expression. The inhibitory effect of estrogens on the nursing function of Sertoli cells might be instrumental in decreasing spermatogenesis. Studies on cell–cell interaction in the testis are very limited and confined to a single species, the Indian wall lizard H. flaviviridis (Figure 3.16). Hence, it is difficult to generalize about the paracrine regulation of testicular functions in reptiles. None the less, factors secreted from Sertoli cells, testicular macrophages, and mast cells appear to regulate the Leydig cell functions. Steroidal as well as
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non-steroidal factors from Leydig cells are implicated in control of the nursing function of Sertoli cells. Additionally, Leydig cell-secreted factors contribute to maintaining immune responses in the testis. Unfortunately, these factors are still unidentified and have not been characterized. We have proposed a model to explain the endocrine and paracrine control of testicular functions in squamates (Figure 3.16). However, in order to reach a logical conclusion on phylogenetic relationships with respect to GTH specificity and testicular functions, cloning and expression of pituitary GTHs and their biological activities in homologous systems as well as isolation and characterization of testicular paracrine factors and their functions need to be investigated in a large number of species across the reptilian orders.
ABBREVIATIONS 3b-HSD Amh AND AR AVT CA cGnRH-II CORT DHEA DHT Dmrt1 EDS ER FSH GnIH GnRH GnRH-R GTH HPG LCCM LH LPS MEL mFSH mGnRH mLH oFSH POA PVN RSS SCCM SER Sf1 sGnRH T TDP TMCM TSP
3b-hydroxysteroid dehydrogenase Anti-Mu¨llerian hormone Androstenedione Androgen receptor Arginine vasotocin Cyproterone acetate Chicken-II gonadotropin-releasing hormone Corticosterone Dehydroepiandrosterone 5a-dihydrotestosterone Drosophila doublesex and C. elegans Mab-3 related transcription factor Ethane dimethane sulfonate Estrogen receptor Follicle-stimulating hormone Gonadotropin-inhibitory hormone Gonadotropin-releasing hormone Gonadotropin-releasing hormone receptor Gonadotropin Hypothalamo–hypophysial–gonadal Leydig cell-conditioned medium Luteinizing hormone Lipopolysaccharide Melatonin Mammalian follicle-stimulating hormone Mammalian gonadotropin-releasing hormone Mammalian luteinizing hormone Ovine follicle-stimulating hormone Preoptic area Paraventricular nucleus Renal sexual segment Sertoli cell-conditioned medium Smooth endoplasmic reticulum Steroidgenic factor 1 Salmon gonadotropin-releasing hormone Testosterone Testis-determining period Testicular macrophage-conditioned medium Temperature-dependent period
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Chapter | 3
Hormonal Regulation of Testicular Functions in Reptiles
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Chapter 4
Hormonal Regulation of Ovarian Function in Reptiles Susan M. Jones University of Tasmania, Hobart, TAS, Australia
SUMMARY The reptilian ovary has two primary functions: the production of gametes and the secretion of hormones. Ovarian differentiation begins during embryonic development and, in many species, primordial follicles are present in the ovary by the time of hatching. The ovary produces steroid hormones (estrogens, androgens, and progestins) that circulate in association with plasma-binding proteins and bind to three complementary classes of specific tissue receptors. Metabolism of the primary steroids by peripheral tissues may involve either derivatization or conjugation. Ovarian activity is regulated via the hypothalamo– pituitary–gonadal (HPG) axis. Follicular maturation typically involves the deposition of large amounts of yolk during vitellogenesis, which is stimulated by estrogens secreted by the granulosa cells. In both oviparous and viviparous reptiles, the postovulatory follicles are transformed into secretory corpora lutea, which produce progesterone for species-specific periods. Luteolysis is stimulated by prostaglandins. Further research is particularly required into non-steroidal hormones produced by, or influencing, the reptilian ovary.
1. INTRODUCTION The reptilian ovary has two primary functions: the production of gametes and the secretion of hormones with regulatory effects on the female reproductive system. As in all vertebrates, endocrine control of ovarian development and physiology primarily involves the hypothalamo–pituitary–gonadal (HPG) axis. However, our knowledge of HPG axis functioning in reptiles is still sketchy compared with our current understanding of the mammalian system.
Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
1.1. Ovarian Structure In reptiles, the ovaries are paired, hollow, oval (or, in snakes, elongate) structures attached to the dorsal body wall by a mesovarium (Jones, 1978). Each ovary is covered by a thin epithelium and contains follicles and corpora lutea embedded in a stroma of connective tissue. Small regions of the dorsal surface of the ovary represent the germinal beds, where multiplication of oogonia and formation of primordial follicles occur (Guraya, 1989). In reptiles, unlike birds and mammals, the ovarian stroma is sparse, and there is little interstitial tissue.
1.2. Ovarian Development Ovarian differentiation begins at about stage 24–30 (sensu Dufaure & Hubert, 1961) of embryonic development, corresponding roughly to the time of oviposition in oviparous squamates. However, ovarian development is not always complete at the time of hatching or birth: in alligators, for example, ovarian development continues during the first few months of life (Moore, Uribe-Aranza´bal, Boggs, & Guillette, 2008). Studies of gonadal differentiation in reptiles have largely focused on oviparous species with temperaturedependent sex determination (TSD) due to interest in the underlying mechanisms of TSD (see Chapter 1, this volume). Detailed stage-by-stage descriptions of the differentiation and development of the ovary are available for the oviparous lizards Sceloporus undulatus (Austin, 1988) and Calotes versicolor (Doddamani, 1994) and the viviparous lizards Niveoscincus ocellatus (Neaves, Wapstra, Birch, Girling, & Joss, 2006) and Lacerta vivipara (Dufaure, 1966, cited in Merchant-Larios, 1978), and
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a major review of the embryonic development of the genital system in both female and male reptiles is available (Raynaud & Pieau, 1985). The following description of ovarian development in squamates is derived from these sources. Six stages of gonadal development can be identified in embryonic lizards (Table 4.1). At embryonic stage 24–30 (Dufaure & Hubert, 1961), the gonad first develops as a thickening of undifferentiated mesenchymal tissue underlying the coelomic epithelium, extending down the ventromedial surface of each mesonephros. This forms the genital ridge, which grows and bulges into the coelom. At this early stage, the gonad is indifferent, or bipotential, and contains both medullary and cortical elements. The bipotential stage in lizards lasts for a much shorter period than in turtles and crocodilians (see Section 1.2.1). Primordial germ cells migrate into the developing gonad and multiply by mitosis; rete cords form as single rows of cells extending from the mesonephros into the medulla of the gonad. Differentiation of the ovary begins as the primordial germ cells congregate in the cortex and begin to differentiate into oogonia at stages 31–32. By stages 33–35, gonadal sex can be recognized; the ovary contains a prominent cortex with one or two layers of oogonia, the tunica albuginea begins to develop, and medullary oogonia become scarce. At this stage, oocytes begin to appear in the rete and cortex, particularly in the anteriodorsal region of the gonads: these regions may later form the germinal beds (Austin, 1988).
During the final stages of embryonic development (stages 36–40), the ovarian cortex becomes well-developed, containing six to ten layers of oogonia and oocytes; the medulla degrades and becomes filled with stromal tissue; and the rete cords are absent or degenerating. Folliculogenesis begins after hatching or birth. The ovaries of neonatal lizards contain clearly defined germinal beds, and some primordial follicles, consisting of an oocyte surrounded by a single layer of monomorphic granulosa cells, are apparent about 10 days after birth. In C. versicolor, follicles with a polymorphic granulosa including pyriform cells (see Section 3.2) are present in two-month-old hatchlings (Doddamani, 1994). The development of the ovary follows a similar pattern in other reptilian taxa. The main morphological stages are similar in turtles and alligators: in many, but not all, species, primordial follicles are present in the ovary by the time of hatching (Pieau, Dorizzi, & Richard-Mercier, 1999). In alligators, three broad stages can be recognized: genital ridge formation, the period of bipotentiality, and, finally, differentiation of the gonad. Differentiation takes place during the last third or quarter of embryonic life, as the gonads enter a rapid development phase (Joss, 1989). Ovarian development is incomplete at hatching although the gonad is clearly differentiated. Moore et al. (2008) provide a thorough description of ovarian development in neonatal American alligators, Alligator mississippiensis (to three months post-hatching), and review comparative
TABLE 4.1 Stages of ovarian development in lizards (compiled from Austin, 1988; Doddamani, 1994; Neaves, Wapstra, Birch, Girling, & Joss, 2006). Embryo stages are sensu Dufaure and Hubert (1961) Stage
Embryonic stage
Gonadal component
Description
1
24–30
Genital ridge
Genital ridge first appears on either side of the dorsal mesentery; some primordial germ cells present
2
28–32
Undifferentiated, bipotential gonad
Ridge develops several layers of cells, forms ovoid mass, and bulges into coelom
3
31–32
Undifferentiated gonad
Rete present; outer cortex and inner medulla develop; germ cells in both cortex and medulla; primordial germ cells begin to differentiate into oogonia
4
33–35
Ovary
Cortex prominent and 2–3 cells thick; oogonia mainly in cortex and rete; oocytes in meiotic prophase appear
5
36–38
Ovary
Cortex 3–5 cells thick; vascularity of ovary increases
6
37–40
Ovary
Cortex is 6–10 cells thick; rete degenerating or absent; cortex filled with oogonia and oocytes
6þ
Neonate
Ovary
Germinal beds apparent; primordial follicles appear some time after birth
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literature from birds. The ovary develops rapidly during the first few months post-hatching: cortical production of oocytes continues, folliculogenesis is initiated, and the medullary region undergoes considerable re-structuring into the complex of vascularized lacunae that is a distinctive feature of crocodile, turtle, and bird ovaries.
1.2.1. Hormonal control of ovarian development Differentiation of the bipotential embryonic gonad into an ovary, rather than a testis, is dependent upon the endocrine milieu during the organizational period. Estrogens provide the proximate stimulus critical to ovarian development, which is mediated via the estrogen receptor (Crews, 1996). There are, however, two ultimate mechanisms of sex determination in reptiles. In many species (but almost exclusively squamates), sex is determined by genetics; i.e., genes carried on heteromorphic sex chromosomes trigger a genetic cascade that results in the bipotential gonad developing into either an ovary or a testis. In contrast, many reptilian species exhibit environmental sex determination (ESD). The particular form of ESD characteristic of reptiles is temperature-dependent sex determination (TSD): the sex of an individual embryo is determined by the environmental temperature it experiences during a critical window of development (Bowden, Ewert, & Nelson, 2000). In species with TSD, temperature is transduced into an endocrine signal. That signal directs the sex-determination process and establishes the sex of the individual embryo by influencing endogenous steroid production during embryogenesis. Females result from the activation of an ‘ovary-determining cascade and simultaneous inhibition of a testis-determining cascade’ (Crews, 1994, p.5) during the thermosensitive period. Estrogen levels remain low in testes, but rapidly increase and stimulate development of the cortex as the ovaries differentiate (Pieau et al., 1999). The embryonic stage at which sex is labile varies between taxa, and higher temperatures may produce predominantly either males or females, depending on the species. In crocodilian embryos, for example, incubation temperatures of 30 C produce females, and the steroidogenic enzymes 3b-hydroxysteroid dehydrogenase (3b-HSD) and aromatase, which catalyzes the conversion of aromatizable androgens to estrogens, are active in the developing gonads of embryos incubated at this temperature. A marked increase in aromatase activity occurs coincident with germ cell proliferation and development of the ovarian cortex (Smith & Joss, 1994). Experimentally, TSD may be overridden by application of exogenous hormones, hormone agonists, or aromatase inhibitors such as fadrozole, which blocks the conversion of androgens to estrogens,
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implicating temperature-dependent regulation of aromatase activity as a primary mechanism of TSD. (For further discussion, see Chapter 1, this volume.)
1.3. Major Ovarian Hormones Reproductive activity in female reptiles is associated with dramatic increases in circulating concentrations of the primary sex steroids, 17b-estradiol (E2), testosterone (T) and progesterone (P4) (e.g., Edwards & Jones, 2001; Taylor, DeNardo, & Jennings, 2004). The ovary is the endocrine gland primarily responsible for secretion of the sex steroids in female reptiles, although P4 may be synthesized by other tissues such as the adrenal gland (Highfill & Mead, 1975) or the uterus and placenta in viviparous reptiles (Painter, Jennings, & Moore, 2002; Girling & Jones, 2003). These hormones belong, respectively, to the C18 (estrane), C19 (androstane), and C21 (pregnane) series of steroids. The two classes of steroids most closely associated with female reproduction are the estrogens and the progestins. 17b-estradiol is generally assumed to be the primary gonadal estrogen in reptiles, as it is in many other vertebrates (Kime, 1987; Norris, 2007), and plasma concentrations of this steroid are routinely assayed in studies of female reproduction. However, this assumption may not always hold true. The very low plasma concentrations, even during vitellogenesis, of E2 in the lizard Pogona barbata (Amey & Whittier, 2000) led the authors to speculate that a different estrogen may be more important physiologically in P. barbata, with E2 simply being a short-lived metabolite of that hormone. A possible alternative to E2 has also been postulated for the blotched blue-tongued lizard, Tiliqua nigrolutea (Edwards, Jones, & Davies, 2002) in which an unidentified, highly polar estrogen (rather than E2) is the end product of steroid biosynthesis from pregnenolone. Similarly, the red-sided garter snake Thamnophis sirtalis parietalis produces a six-substituted E2 that represents 30– 60% of the immunoreactive estrogens in the plasma of this species (Whittier & Hess, 1992). This species may also produce an alternative progestin (Whittier, Mason, & Crews, 1987). However, P4 is generally assumed to be the primary progestin in reptiles, as in other vertebrates (Callard et al., 1992). The ovary also produces a third class of steroids: the androgens. Androgens such as T fulfill an important function as precursors of estrogens, but the presence of tissue androgen receptors and significant circulating levels is good evidence that androgens have a wider biological significance in female as well as male vertebrates (Staub & De Beer, 1997). Testosterone is certainly synthesized by the reptilian ovary (Edwards, Jones, & Davies, 2003;
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Gobbetti, Zerani, Di Fiore, & Botte, 1993b), and plasma concentrations of T vary significantly through the female reproductive cycle (e.g., Callard, Lance, Salhanick, & Barad, 1978; Edwards & Jones, 2001), providing circumstantial evidence for a physiological role for this hormone. Some non-steroidal hormones are also produced by the reptilian ovary. Both ovarian follicles and corpora lutea have the capacity to synthesize prostaglandins (Guillette, Herman, & Dickey, 1988; Guillette, Dubois, & Cree, 1991; Gobbetti, Zerani, Di Fiore, & Botte, 1993a), which may have roles in promoting vitellogenesis and in ovulation, as well as influencing the expression of reproductive behaviors (Whittier & Crews, 1986; Whittier & Tokarz, 1992). In addition, gonadotropin-releasing hormone (GnRH-1) and bradykinin, plus their respective receptors, have been identified in ovaries of a number of non-mammalian vertebrates including the lizard C. versicolor (Singh, Krishna, & Sridaran, 2007), and may have autocrine or paracrine actions within the ovary.
1.4. Hypophysial Regulation of Ovarian Function In vertebrates, ovarian secretion of steroids is regulated by the HPG axis. Gonadotropin (GTH) release is regulated by hypothalamic GnRH. In females, production of gonadal P4 and estrogens is stimulated by GTHs. These steroids may exert feedback control on GTH secretion at the level of the hypothalamus (Callard, Chan, & Potts, 1972). In reptiles, as in other vertebrates, multiple molecular forms of GnRH are present in the brain of a single individual (Montaner, Gonzalez, Paz, Affanni, & Somoza, 2000). Implantation of osmotic pumps containing chicken II (cGnRH-II) analog or mammalian GnRH I induced a five-fold increase in plasma estrogens and elicited receptive behaviors in female iguanas (Phillips et al., 1987): this chicken hormone was about twice as potent as the mammalian form. In recent years, a gonadotropin release-inhibiting hormone (GRIH) has also been identified, but studies are still restricted to birds and mammals (Ubuka, McGuire, Calisi, Perfito, & Bentley, 2008). Sexual maturation in reptiles is dependent upon maturation of the HPG axis: at puberty (defined as the attainment of fertility), the ovaries become receptive to gonadotropic stimulation (Masson & Guillette, 1985). In mammals, increased secretion of GnRH at puberty activates the pituitary–gonadal axis (Ebling, 2005). However, we know very little of the neuroendocrine initiation of puberty in reptiles. A study of testicular responses to injections of ovine follicle-stimulating hormone (FSH) suggested that puberty may be extended over several years in large reptiles such as alligators (Edwards, Gunderson,
Hormones and Reproduction of Vertebrates
Milnes, & Guillette, 2004). In female reptiles, puberty is often associated with the attainment of a threshold body size or mass. For example, in the green iguana (Iguana iguana), males attain puberty at 15–17 months of age and 170–200 mm snout-to-vent length. Females exhibit a marked increase in plasma concentrations of E2 at 15–17 months of age but do not mature for at least another year; they do not ovulate unless they reach the critical snout-tovent length of 250 mm (Pratt, Phillips, Alberts, & Bolda, 1994). The estrogen surge in juveniles at 15–17 months occurs concurrently with reproductive activity of mature females, and may serve to synchronize the juvenile iguana females prior to their first reproductive season. In eutherian and marsupial mammals, the separate roles of the two pituitary GTHs in regulating ovarian function are well-established. Follicle-stimulating hormone regulates oogonial proliferation, oocyte growth, and differentiation, while luteinizing hormone (LH) stimulates ovulation and steroidogenesis. However, the picture in reptiles is less clear and, for many taxa, it is still uncertain whether one or two GTHs are present. Fractionation studies showed that turtles and alligators have two distinct GTHs that appear to be homologous to the mammalian LH and FSH (Licht, Farmer, & Papkoff, 1976; Papkoff, Farmer, & Licht, 1976), but that snakes have only a single GTH (Licht, Farmer, Gallo, & Papkoff, 1979). Reviewing (then) current knowledge of pituitary GTHs in reptiles, Licht (1980) concluded that most tetrapod vertebrates do have two distinct GTHs but that squamate reptiles deviate from this pattern in having a single pituitary GTH that lacks FSH/LH specificity. More recent work has shown that the lizard genome contains nucleotide sequences homologous to those that code for the b subunits of mammalian FSH and LH (Borrelli, De Stasio, Bovenzi, Parisi, & Filosa, 1997). However, Bluhm et al. (2004) could not find evidence for an LH receptor in the snake Bothrops jararac; they were able to clone a cDNA for the FSH receptor (FSHR), and FSHR was expressed in the ovary and testis. This provides further evidence that, in possessing only a single GTH that combines FSH- and LH-like activity, squamate reptiles have diverged from the fundamental pattern in tetrapod vertebrates (Licht, 1983).
2. OVARIAN STEROIDS Estrogens, androgens, and progestins are synthesized and secreted by the ovary and carried in the bloodstream to their target tissues, where they exert their biological effects via specific receptors. The dominant end product of steroid synthesis varies among stages of the reproductive cycle. Broadly speaking, the follicular phase is characterized primarily by the production of estrogens by the
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FIGURE 4.1 The D4 and D5 pathways of androgen steroidogenesis from pregnenolone. The D4 pathway is preferred in reptilian ovaries, although both pathways may operate. Estrogens are synthesized from androgens such as testosterone and androstenedione by aromatization. Reproduced from Norris (2007) with permission.
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developing follicles, while P4 is produced by the corpora lutea during the post-ovulatory phase. Steroids secreted by the ovary may be later modified by other tissues through further metabolism, either into biologically active metabolites or into conjugated forms to facilitate excretion.
2.1. Steroid Synthesis Steroids are synthesized from cholesterol, which may be taken up from the bloodstream or synthesized de novo within the endocrine cell. Cholesterol is converted to the C21 steroid pregnenolone, the key intermediate in steroidogenesis, by side-chain hydrolysis. All other steroid hormones are synthesized from pregnenolone via a series of enzymecatalyzed steps, including conversion of pregnenolone to P4 by the enzyme 3b-HSD (Norris, 2007). Kime (1987) and Gore-Langton and Armstrong (1994) provide detailed descriptions of the various pathways of steroid biosynthesis. In particular, there are two alternative pathways through which androgens may be synthesized (Figure 4.1). The 17a-hydroxylase, C-17,20-lyase enzyme complex, can utilize either P4 or pregnenolone as its substrate to form androstenedione or dehydroepiandrosterone (DHEA) via the intermediates 17a-hydroxy-progesterone or 17ahydroxy-pregnenolone, respectively. These pathways are known as the D4 (4-ene-3-oxo) and D5 (5-ene-3b-hydroxy) pathways. As the preferred pathway may vary between taxa, between sexes within taxa, or with reproductive stage, this aspect of steroidogenesis has been of special interest to comparative endocrinologists. Steroidogenic pathways in reptilian ovaries were investigated in a series of early studies (ca. 1965–1972) in which chromatographic techniques were used to identify pathway intermediates synthesized by tissues incubated in-vitro with unlabeled or radioactively labeled steroid precursors (Yaron, 1972). Alternatively, histochemical localization and semi-quantification of key steroidogenic enzymes, particularly 3b-HSD, can be used to infer steroidogenic activity in particular tissues (Norris, 2007). The advent of sensitive assays for plasma hormones in the early 1970s led to a new research emphasis on documenting cycles of plasma steroids (Chieffi & Pierantoni, 1987), and more recent studies of steroidogenic pathways in reptiles are sparse. However, it is clear that the pathways of steroid biosynthesis in reptilian ovaries are basically similar to those in mammals, reflecting their evolutionary conservation amongst higher vertebrates. Progesterone occupies a position early in the steroidogenic pathway and is therefore a key precursor for other steroids as well as being secreted as an active hormone
Hormones and Reproduction of Vertebrates
during the luteal phase. Corpora lutea of the viviparous lizard Chalcides chalcides, for example, produce high levels of P4 during early gestation, and this production is reduced by the addition of trilostane, a potent inhibitor of 3b-HSD activity, to the culture medium (Guarino, Pailesu, Cardone, Bellini, & Ghiara, 1998). In-vitro incubations of ovarian tissue from L. vivipara with 3H-pregnenolone demonstrated that P4 is the primary steroid produced during gestation, with 85% of metabolized pregnenolone appearing as P4 in incubations of ovaries (with corpora lutea) from late-gestating females. In contrast, P4 is rapidly metabolized into 5a- or 5b-reduced steroids at other stages of the reproductive cycle (Xavier, 1982). Hypertrophied (vitellogenic) ovaries of T. nigrolutea also rapidly convert P4 into downstream metabolites such as T and androstenedione (Edwards et al., 2003). The little information available for female reptiles suggests that the D4 pathway of androgen biosynthesis is more active in the ovary, although the D5 pathway may make some contribution to steroid synthesis. Pregnenolone, 17ahydroxy-pregnenolone, DHEA, P4, androstenedione, E2, and estrone (E1) were all detected in extracts of ovaries of the oviparous lizard Lacerta sicula (Lupo Di Prisco, Delrio, & Chieffi, 1968), indicating that both major pathways of steroid biosynthesis were operating. Incubation of snake Naja naja (Lance & Lofts, 1977), turtle, and lizard ovaries (Chan & Callard, 1974) with radioactively labeled pregnenolone precursor indicated that androgens were preferentially synthesized via the D4 pathway, but both pathways appear to operate in ovaries (and testes) of Tiliqua rugosa (Bourne, 1981). Indeed, pathway preference may vary within a species and/or with sex, season and/or reproductive condition. A detailed study of steroid biosynthetic pathways in the lizard T. nigrolutea demonstrated activity of the D4 pathway in both female and male gonads. Intermediates of the D5 pathway were detected only in males, and only when regressed rather than hypertrophied (active) testes were incubated (Edwards et al., 2003). The final steps of the steroidogenic pathway are those through which some androgens are aromatized to estrogens (Figure 4.2). As already noted, the end product of estrogen biosynthesis in reptiles is generally assumed to be E2, but alternative estrogens, including E1 (Chan & Callard, 1974; Lupo Di Prisco et al., 1968), may also be synthesized. In mammals, there is considerable evidence to support the two cell-type/two gonadotropin theory of estrogen biosynthesis. In brief, androgens are produced by cells of the theca interna under LH stimulation and are then transported across the follicular basement membrane to the granulosa, where they are aromatized into estrogens under the influence of FSH (Gore-Langton & Armstrong, 1994). To date, we have no experimental evidence that a similar system operates in reptiles.
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Hormonal Regulation of Ovarian Function in Reptiles
O
HO
3 HSD
Dehydroepiandrosterone (DHEA)
O
O
Androstenedione
P450aro
17 HSD
O
OH
O
HO
Testosterone
P450aro
Estrone
17 HSD
OH
HO
Estradiol
OH OH
HO Estriol
FIGURE 4.2 Synthesis of estrogens from androgens. Reproduced from Norris (2007) with permission.
Finally, steroid synthesis is a dynamic process, and shifts in pathway preference or the activity of specific enzymes may represent proximate mechanisms for regulating ovarian steroid production. At least some of the enzymes of the steroidogenic pathways appear to be temperature-sensitive in reptiles (Bourne, Taylor, & Watson, 1986; Girling & Jones, 2006), suggesting a mechanism whereby environmental temperature may cue seasonal shifts in activity of the HPG axis.
2.2. Tissue Receptors Gonadal steroids exert their physiological effects by binding to specific, high affinity tissue receptors. Steroid receptors were traditionally considered to be nuclear transcription factors: after ligand binding, the dimerized steroid-receptor complexes are translocated to the nucleus where they interact with the genome to alter patterns of mRNA expression (Ho, Mak, Salhanick, Heiserman, & Callard, 1985). These nuclear receptors have six functionally distinct domains (identified as A to F). The E
domain is responsible for ligand-binding, while the C domain binds to the DNA via a zinc finger. More recently, however, a different type of steroid receptor has been identified: these are membrane-linked, extra-nuclear steroid receptors that act via G-protein coupling, causing post-translational modifications or phosphorylation of existing proteins. These receptors initiate rapid signaling actions of steroid hormones (see reviews by Hammes & Levin, 2007; Kohno, Katsu, Iguchi, & Guillette, 2008). Research into this type of receptor has focused on mammals, although membrane-bound P4 receptors have been identified in amphibian oocytes (Revelli, Massobrio, & Tesarik, 1998). There are separate classes of receptor for each major group of sex steroids (estrogens, androgens, and progestins) (Callard & Callard, 1987). Reptilian steroid hormone receptors have a high degree of sequence homology with those of other vertebrates (Young, Lopreato, Horan, & Crews, 1994). Differential expression of sex steroid receptors in different tissues or at different times of the reproductive cycle provides a mechanism for regulating
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sensitivity to their respective hormones. Three steroid hormone receptors have been identified in males of the leopard gecko Eublepharis macularius, and were quantified in different tissues using mRNA expression of each receptor. Receptor expression appeared to vary with environmental conditions (low temperature and short photoperiod upregulate androgen receptors (ARs)), and both ARs and estrogen receptors (ERs) were expressed in a wide variety of tissues (Endo & Park, 2003). Two major forms of the estrogen receptor, ERa and ERb, with differential ligand activation and different tissue distributions, are ubiquitous among vertebrates, although a third type, ERg, is present in fishes (Lange, Hartel, & Meyer, 2003). During the evolutionary sequence from fish to mammals, the affinity of the ER for E2 has increased about 1000-fold, and this is mirrored by a corresponding decrease in endogenous plasma concentrations of E2. Sequence homologies demonstrate that the estradiolbinding pocket has been conserved (Lange et al., 2003). The ERa has been partially or fully sequenced in a range of reptilian species, including full sequences for a caiman (Caiman crocodilus) and a lizard (Cnemidophorus uniparens) (Sumida, Ooe, Saito, & Kaneko, 2001). Both ERa and ERb have been cloned and sequenced for the American alligator (Katsu et al., 2006). Progesterone receptors (PRs) have been identified in tissues of a range of reptilian taxa including the viviparous snake Nerodia sp. (Kleis-San Francisco & Callard, 1986), the lizard Podarcis sicula (Paolucci & Di Cristo, 2002), the turtle Chrysemys picta (Giannoukos & Callard, 1995), and the American alligator (Vonier, Guillette, McLachlan, & Arnold, 1997). Progesterone receptor concentrations in the oviduct and liver of P. sicula vary differentially across the reproductive cycle: in particular, PR is detectable by binding assays and western blotting in the liver during periods when vitellogenin (Vtg) is not being synthesized, and in the oviduct except during the passage of eggs (Paolucci & Di Cristo, 2002). Such observations suggest specific actions of P4 in turtles. Two isoforms of PR, PRA and PRB, have been identified in both the oviduct and liver of C. picta (Giannoukos & Callard, 1995; 1996). Differential expression of these isoforms during the ovarian cycle may provide a mechanism through which P4 inhibits vitellogenesis (see Section 3.3.2). Additionally, expression of PRB in the turtle ovary suggests a role for P4 in the regulation of oocyte maturation in reptiles (Custodia-Lora & Callard, 2002). The physiological roles of androgens in female vertebrates are poorly understood (Staub & De Beer, 1997). Perhaps for this reason, ARs in female reptiles have received little attention. Androgen receptors have, however, been identified in some female reptiles. Patterns of expression of AR-mRNA in the brain and oviduct of the gecko E. macularius vary across the reproductive cycle,
Hormones and Reproduction of Vertebrates
and high levels of AR in the brain appear to be correlated with elevated circulating concentrations of androgens. These results suggest physiological roles for androgens in the regulation of the female reproductive tract as well as female receptivity (Rhen, Sakata, Woolley, Porter, & Crews, 2003), in addition to their function as aromatizable precursors to estrogens. In recent years, there has been a great deal of research into endocrine-disrupting chemicals (EDCs). Many such environmental contaminants exert their effects via their capacity to bind to endogenous steroid receptors. Some endocrine-disrupting chemicals act as receptor agonists by binding to the receptors to induce conformational changes similar to those resulting from binding of the natural ligand, inverse agonists if inappropriate changes occur, or receptor antagonists if they occupy the receptor without leading to conformational change and a physiological response (Rooney & Guillette, 2000; Orchnik & Propper, 2006). Interactions of EDCs with receptors for reproductive steroids may have profound consequences for reptilian reproductive success and sexual development. For further discussion, see Chapter 14, this volume.
2.3. Steroid-binding Proteins in Plasma Non-receptor steroid-binding proteins (S-BPs) are ubiquitous among vertebrates. They are distinguished from steroid receptors by their higher binding capacity (Bmax ¼ 106 to 109) but lower affinity (Kd ¼107 to 109 M) (Callard & Callard, 1987), and by their more limited specificity for particular steroids. Plasma-binding proteins are thought to fulfill several important functions. Steroids bound to binding proteins are protected from excretion, thus reducing the metabolic clearance rate of the hormones (Siiteri et al., 1982; Ho, Lance, & Megaloudis, 1987). Thus, binding proteins are an important means of maintaining a reservoir of steroid in the circulation. Binding proteins also regulate the access of steroids to receptors in target cells because only free steroid is able to enter the cell. Differences in affinity drive the dynamic equilibrium between a hormone, its binding protein, and its receptor, and thus influence the proportion of steroid that is free to bind with tissue receptors (Callard & Callard, 1987). As a generality, two major classes of binding protein are recognized in vertebrates: the first is a corticotropin-binding globulin (CBG, or transcortin) that primarily transports adrenal (C21) steroids, and the second is a sex-hormone binding protein (SHBP) that binds estrogens and androgens (C18 and C19 steroids). In addition, steroids may bind nonspecifically to other plasma proteins, notably albumin. Although albumin has a lower affinity for steroids than do the binding proteins, it has high capacity because it is present
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Hormonal Regulation of Ovarian Function in Reptiles
in relatively high concentrations in the blood; thus, albumin may make an important contribution to steroid transport in the circulation (Callard & Callard, 1987). There are phylogenetic differences among vertebrate taxa in the type and concentration of steroid-binding proteins present and in their degree of specificity (reviewed by Callard & Callard, 1987). In particular, there is considerable variation among species and taxa as to whether either of the major binding proteins binds P4. High levels of a transcortin-type protein are present in the plasma of gestating L. vivipara, in which plasma concentrations of P4 are about 100 basal levels (Xavier, 1982). Sex-hormone binding proteins have been identified in crocodilians (Ho et al., 1987), turtles (Salhanick & Callard, 1980; Ikonomopoulou, Bradley, Whittier, & Ibrahim, 2006) and squamates (Martin & Xavier, 1981; Riley, Kleis-San Francisco, & Callard, 1988; Paolucci, Di Fiore, Ciarcia, & Botte, 1992). The viviparous snake Nerodia sp. appears to have a single medium-high affinity (107 to 108), limited capacity (0.09–0.7 M), and binding protein that binds T, E2, and 5a-dihydrotestosterone (DHT) but also binds corticosterone and P4 (Riley et al., 1988). In contrast, the lizard L. vivipara has two distinct plasma steroid-binding proteins: a steroid hormone-binding globulin (SHBG), with high affinity but limited capacity for E2, and a CBG with high affinity (108 M) for P4. Testosterone showed a similar affinity for both the CBG and SHBG molecules: this high concentration of testosterone-binding proteins may reflect the relatively high plasma concentrations of testosterone, which are five times higher than circulating E2 concentrations in females of this species (Callard et al., 1978). Studies of plasma sex steroids in female reptiles invariably report total (rather than free) concentrations of hormones and can, therefore, give a false picture of hormone activity. Annual variations in plasma concentrations of SHBPs may be very important in regulating the availability of the ovarian steroids to their target tissues, but may have received almost no attention in the literature. In the viviparous Nerodia sp., significantly lower concentrations of SHBG are present during midlate pregnancy although there is little variation at other times of the cycle. During mid-late pregnancy, plasma P4 is at a peak but plasma estrogen concentrations are low: Riley et al. (1988) suggested that the decrease in SHBG may facilitate the supply of P4 for ‘uterine maintenance.’ Comparative studies of viviparous and oviparous species could elucidate the role of SHBPs in maintaining appropriate availability of P4 to tissues during gestation. It is also important to appreciate that binding affinity varies with temperature. For example, Ikonomopoulou et al. (2006) found that, at 4 or 23 C, E2 binds with high affinity to a SHBG in plasma of nesting Chelonia mydas,
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but that binding was abolished at 36 C; thus, at high core body temperatures most of the E2 would become biologically available to the tissues. In contrast, T binding was thermostable, with the binding protein showing similar affinity and capacity at 4, 23, and 36 C. Shifts in binding affinity with body temperature, which would alter the availability of hormones to tissues, may play a role in regulating seasonal patterns of endocrine activity in reptiles, and warrant further investigation.
2.4. Peripheral Metabolism of Steroids Enzymes that catalyze the metabolism of a single functional group on a steroid molecule can either activate or deactivate that steroid. Selective expression of such enzymes, particularly in peripheral tissues, provides an important mechanism for regulating steroid hormone action (Baker, 2004). Peripheral metabolism of steroids may involve either derivatization or conjugation. Even though it is considered a primary steroid, T secreted by the gonad may regularly be converted by peripheral tissues into more biologically active derivatives with greater affinity for the tissue receptors: such derivatives are, in effect, a more active form of the hormone. In particular, T may be derivatized by reduction to DHT via the 5a-reductase enzyme, or aromatized to E2: for example, the epididymis, cloaca, and skin of the male lizard T. nigrolutea readily convert T to DHT (Edwards, Jones, & Davies, 2005). Conjugation to either sulfate or glucuronide moieties deactivates steroids and converts them into water-soluble forms that do not bind to plasma-binding proteins, rendering them readily excretable (Kime, 1987; Norris, 2007). In addition, either metabolites or conjugates may play a physiological role as pheromones. Chemical communication is highly developed in terrestrial reptiles, and steroid metabolism, particularly by cloaca or skin, may be the means through which pheromones are produced. The liver is the most important site of steroid conjugation, although other tissues, including the gonads themselves, can also metabolize steroids. Injection of radioactive P4 in vivo into male or pregnant females of the lizard T. rugosa showed that P4 is rapidly metabolized to free derivatives and conjugated steroids. The major free derivative was 20b-hydroxypregn-4-ene-3-one (20b-HP), which is the major metabolite of P4 in other vertebrates. High levels of radioactivity associated with conjugate fractions from liver and kidney suggested that both organs are routes of steroid excretion (Bourne, 1981). Only one study has directly examined steroid metabolism by peripheral tissues of a reptilian species. In female T. nigrolutea, steroid conjugates are produced by the skin, kidney, cloaca, skeletal muscle, adrenal, oviduct, and ovary, although the liver is the most active (Edwards et al., 2005).
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In this in vitro tissue culture system, the liver conjugated about 75% of the initial substrate, with approximately 10% being converted to highly polar free derivatives. The proportion of E2 metabolized by each tissue in vitro varied with reproductive stage: tissues from gestating females produced the greatest proportion of conjugated steroids, suggesting that this could be a mechanism for protecting tissues from high levels of E2 during gestation. In mammals, steroid sulfates may be reconverted to active steroids via the sulfotransferase/sulfatase pathway, particularly in the fetoplacental unit where the system serves to protect developing embryos from high levels of maternal hormones (Kime, 1987). No-one has yet explored this possibility in viviparous female reptiles. However, it has been suggested that in oviparous reptiles the sulfotransferase/sulfatase pathway may serve to buffer developing embryos from maternal yolk steroids.
3. FOLLICULAR DEVELOPMENT Among vertebrates, an ovarian follicle comprises the oocyte, with its acellular zona pellucida, plus associated surrounding layers of granulosa and thecal cells separated by a membrana propria (Norris, 2007). In reptiles, in contrast to birds and mammals, oogenesis and folliculogenesis continue through the female’s reproductive lifetime (Jones, Swain, Guillette, & Fitgerald, 1982). In most reptiles, follicle growth and vitellogenesis (yolk production) occur seasonally so that ovarian morphology and hormone production vary greatly over the annual cycle, although ovarian activity is continuous in some tropical species (Licht, 1984).
3.1. Recruitment Oogonial proliferation and differentiation begin in the embryonic ovary and continue in adult female reptiles. Mitotic divisions of oogonia in the germinal beds produce groups of naked oocytes that then enter meiotic prophase and become surrounded by a single layer of granulosa cells, forming primordial follicles. Such recruitment often occurs seasonally, as in the oviparous lizard Ctenosaura pectinata (Uribe, Portales, & Guillette, 1996) and the viviparous lizard Sceloporus torquatus torquatus (Uribe, Omana, Quintero, & Guillette, 1995).
3.2. Follicular Development In reptiles, new follicles are produced from oogonia located in germinal beds on the dorsal surface of the ovary: these germinal beds are remnants of the embryonic ovary (Miller, 1963). In lizards, each ovary generally contains two germinal beds, although some species of
Hormones and Reproduction of Vertebrates
lizard possess only one germinal bed per ovary whereas others have several (Jones et al., 1982; Guraya, 1989). In snakes, the germinal epithelium is distributed as scattered irregular patches over the outer border of the ovarian stroma (Lance & Lofts, 1978). Oogonia in the germinal beds proliferate via mitotic activity and then enter meiotic prophase to form oocytes. Such activity often occurs seasonally, particularly in temperate reptilian species (Guraya, 1989). Most reptilian oocytes are telolecithal, and so follicular development is typically associated with extensive vitellogenesis. Some features of follicular development vary among squamate, chelonian, and crocodilian reptiles. In squamates, the follicular epithelium, or granulosa, initially consists of small cuboidal cells, but differentiates during the pre-vitellogenic phase to become multi-layered and polymorphic. At this stage, the epithelium is characterized by the presence of unique flaskshaped pyriform cells, in addition to small and intermediate cells (Uribe et al., 1996) (Figure 4.3(a–b)). These pyriform cells differentiate from small somatic follicular cells early in follicular development via the intermediate cell stage to become ‘nurse cells,’ in direct contact with the developing oocyte (Maurizii, Alibardi, & Taddei, 2004). Differentiation of the small cells into pyriform cells appears to be linked to the progressive appearance of glycoproteins with terminal a-N-acetylgalactosamine residues on the cell surface, which may be involved in fusion between the oocyte and the follicle cell membranes as well as maintenance of the differentiated pyriform cells. The pyriform cells are connected to the oocyte via intercellular bridges containing a cytoskeleton of a-tubulin and cytokeratin microtubules (Maurizii et al., 2004). It has been suggested that both intermediate and pyriform cells synthesize and then transfer RNA, ribosomes, vesicles, and other cellular materials to the oocyte across these bridges (Motta, Castriota Scandenberg, Filosa, & Andreuccetti, 1995); however, at least in the lizard Gerrhonotus coeruleus, only relatively small molecules such as ribosomal RNA are transferred (Klosterman, 1987). After the end of the previtellogenic phase, and as active sequestration of yolk into the ooplasm begins, the intermediate and pyriform cells regress, so that the granulosa is again composed of an homogenous single layer of cells (Uribe et al., 1996)(Figure 4.3(c)). Crocodilians, being archosaurs, share some key features of folliculogenesis with birds: these features include the morphology of yolk sphere development. Uribe and Guillette (2000) provided a detailed description of oogenesis and folliculogenesis in the American alligator, A. mississippiensis, and a recent study of C. crocodilus (Caldero´n, De Pe´rez, & Ramı´rez Pinilla, 2004) showed that these processes are similar in both species. In crocodilians, and in contrast to squamate reptiles, the oocytes are surrounded by a granulosa consisting of a single layer of
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Hormonal Regulation of Ovarian Function in Reptiles
(b)
(a)
(c)
FIGURE 4.3 Micrographs of the follicular wall in the lizard Tiliqua nigrolutea. (a) shows the follicular walls of two adjacent previtellogenic oocytes, with the inner granulosa and outer thecal layers clearly differentiated. The two follicular walls are shown at high power in (b). Note the large pyriform cells (p) that are characteristic of the granulosa layer in early follicles of squamates, but do not appear in follicles from other reptilian taxa. (c) shows a high power view of the follicular wall of a vitellogenic follicle of T. nigrolutea. Note that the granulosa is now composed of a single layer of monomorphic cells. Photographs courtesy of Dr Ashley Edwards.
follicular cells that are squamous during early oogenesis, becoming cuboidal during mid to late stages of oogenesis; a well-defined theca forms during previtellogenesis. As vitellogenesis is initiated, small yolk granules appear in the ooplasm. Later, large, acidophilic yolk platelets accumulate in vacuoles containing numerous yolk spheres of different sizes. Platelets located towards the center of the oocyte contain larger yolk spheres than those towards the periphery. At all stages of follicular development, the ovary exhibits a well-developed system of large lacunae in both cortex and medulla, which may provide expansion space for the large developing follicles. In addition, bundles of smooth muscle present around the lacunae and the theca externa of the follicles appear to support the growing follicles; similar structures are observed in bird ovaries (Schroeder & Talbot, 1985).
3.2.1. Roles of hormones in recruitment and folliculogenesis The hormonal regulation of follicle recruitment and, therefore, oocyte number, appears to involve both GTHs and ovarian factors. In the adult female reptile, follicular recruitment is typically initiated via exogenous environmental cues, presumably acting via neuroendocrine stimulation of the HPG axis. Temperature and/or photoperiod are the environmental factors most likely to be involved. Follicular growth and development in female P. sicula held at different temperatures suggest that ovarian sensitivity is cued by temperature in that species (Borrelli, De Stasio, Bovenzi, Parisi, & Filosa, 2000). Similarly, in garter snakes (T. s. parietalis), ovarian recrudescence appears to require exposure to low temperatures during
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hibernation (Bona-Gallo & Licht, 1983); holding females at warmer temperatures during a simulated hibernation induced ovarian regression and loss of FSH receptors. In seasonally breeding reptiles, there may be a refractory period during which the ovary is unresponsive to environmental cues. Females of Anolis carolinensis become unresponsive to long photoperiods and warm temperatures after the breeding season, and it has been suggested that this ovarian refractory period may be due to the presence of large corpora atretica (Crews & Licht, 1974). In P. sicula, the number of prefollicular oocytes and the sensitivity of the ovary to exogenous heterologous FSH (assessed by the responsiveness of adenylate cyclase), vary with season. There are two peaks of maximal sensitivity to FSH that correspond to peaks of ovarian activity: June, during the reproductive period, and December, when oogonial recruitment resumes despite apparently unfavorable environmental conditions (Angelini, Ciarcia, Picariello, Botte, & Pangano, 1985). Other environmental factors such as stress or social conditions, including sexual composition of the group and male reproductive status, may also influence ovarian recrudescence. In A. carolinensis, for example, the presence of intact males appears to be necessary for GTH secretion (Crews, Rosenblatt, & Lehrman, 1974; DeNardo & Autumn, 2001). It is well known that stress inhibits reproductive function in reptiles, as in other vertebrates (Guillette, Cree, & Rooney, 1995; Chapter 7, this volume). For example, the stress of chronic low humidity inhibits ovarian recrudescence in the lizard A. carolinensis (Summers, 1988). In at least some species, b-endorphin, which is released in response to stress, may have a mediating role in determining the timing of ovarian recrudescence: b-endorphin inhibits both seasonal and FSH-induced recrudescence in Mabuya carinata (Ganesh & Yajurvedi, 2003). Alternatively, postreproductive refractoriness and seasonal regression of the ovaries may be attributed to reduced sensitivity to gonadotropins (Angelini et al., 1985). In other species, mating appears to initiate a neuroendocrine reflex that activates female reproduction. Mating is a facultative and, in some circumstances, obligatory stimulus for vitellogenesis in red-sided garter snakes (Mendonc¸a & Crews, 1990) and blood pythons (DeNardo & Autumn, 2001). Presumably, this system ensures that females commit to mobilizing resources for vitellogenesis only if males are present to ensure fertilization. Autocrine and paracrine factors may also influence folliculogenesis in reptiles. In P. sicula, oocyte numbers decreased in excised germinal beds cultured in-vitro for 48 hours unless FSH or follicles were also present (Sica et al., 2001). The cultured follicles released a recruiting factor that appears to be a low molecular weight (ca. 21 kDa) diffusible protein. The authors hypothesized that this factor, which is not dependent upon FSH, may be
Hormones and Reproduction of Vertebrates
responsible for recruitment of oocytes from the pool of oogonia. During folliculogenesis, follicles not only differentiate and grow but also acquire the capacity to respond to gonadotropins. In mammals, granulosa cells of committed preantral follicles must express FSH receptors to progress to the gonadotropin-dependent stage; otherwise, they would become atretic. Growth factors, particularly activin and inhibin, and cytokines interact to promote and sustain folliculogenesis through paracrine or autocrine regulation of gonadotropin receptors. In mammals, inhibin has local stimulatory effects on LHinduced androgen synthesis by thecal cells, although it probably also acts as a peripheral hormone, regulating the activity of the HPG axis via negative feedback effects on the secretion of pituitary FSH (Findlay, 1993). Immunocytochemical analyses have revealed the presence of an inhibin-like protein in the cytoplasm of pyriform cells in previtellogenic follicles of P. sicula, which may reflect a role for inhibin in the maintenance of a follicular hierarchy (Limatola et al., 2002). However, inhibin has been little studied in reptiles.
3.2.2. Determinants of clutch size In reptiles, clutch size, or the number of follicles ovulating in any given cycle, varies with species. Some gekkonid lizards lay only one egg per clutch, although average clutch size in lizards is usually 2–10 eggs; snakes lay clutches of 6–15 eggs and crocodilians 13–55 eggs, with some sea turtles laying 50–100 eggs at a time (Thompson & Speake, 2004). Radder, Pizzato, and Shine (2008) reviewed the literature on germinal beds in lizards and showed that, among lizards, clutch size is linked to ovarian morphology. Species that produce large clutches have multiple germinal beds in each ovary, whereas species ovulating small clutches have only one germinal bed per ovary. Most species exhibit polyautochronic ovulation, in that several ova are ovulated simultaneously from both ovaries. Gekkonid lizards, on the other hand, ovulate two ova simultaneously, one from each ovary (i.e., monoautochronic ovulation) (Boyd, 1940). A. carolinensis exhibits monoallochronic ovulation in that only one ovum ovulates at a time, with left and right ovaries alternating every two weeks (Jones, Roth, Gerrard, & Kiely, 1973). At the functional level, clutch size is determined by the number of follicles that become vitellogenic and the proportion of these follicles that become atretic before ovulation. Etches and Petitte (1990) provided a review of follicular recruitment and the establishment of ovarian hierarchies in reptiles and birds, but the underlying hormonal mechanisms are still poorly understood. A suite of early papers (e.g., Jones et al., 1973; Jones, Tokarz, LaGreek, & Fitzgerald, 1976) investigated endocrine
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Hormonal Regulation of Ovarian Function in Reptiles
influences on clutch size in A. carolinensis, reflecting the particular opportunities for experimental manipulation afforded by a species exhibiting monoallochronic ovulation. However, due to the highly derived pattern of the anoline ovarian cycle, those results are unlikely to be representative of reptiles in general. More typically, squamate reptiles ovulate larger clutches of variable size. In such species, the number of vitellogenic follicles may be increased by administration of exogenous FSH at appropriate times, although those follicles may not ovulate. Treatment with FSH at doses of 1, 10, or 50 mg stimulated follicular growth, but not ovulation, in the lizard Leiolopisma laterale (Jones et al., 1973) while, in the skink Niveoscincus metallicus, additional follicles were recruited only if exogenous FSH was administered during early vitellogenesis (Jones & Swain, 2000).
3.2.3. Follicular atresia In vertebrate ovaries, a large proportion of follicles undergo atresia to form corpora atretica (CA) before completing their development (Guraya, 1989). Through its influence on the number of eggs produced, atresia may be the primary mechanism through which clutch size is regulated (Me´ndez-de la Cruz, Guillette, & VillagranSanta Cruz, 1993; Cummins, 1995). Atresia may occur at any stage of follicular development. Among lizards, atresia is common during the previtellogenic stages (Fox & Guillette, 1987; Uribe et al., 1995), although in six species of Chilean Liolaemus lizards, follicular atresia rates were highest in gravid females (Leyton & Valencia, 1992). Exogenous FSH induced follicular development in female Uta stanisburiana; however, atresia of large follicles was observed after cessation of FSH treatment, suggesting that GTH stimulation is required to maintain follicular maturation (Ferguson, 1966). High levels of atresia observed during gravidity are positively correlated with elevated plasma P4 concentrations during this reproductive phase (Fox & Guillette, 1987) and may reflect inhibition of GTH secretion by P4, resulting in inhibition of follicular development. There has been some debate about whether atretic follicles are steroidogenic. Guraya (1989, p.210) described the stages of atresia in previtellogenic follicles in squamates and reviewed evidence for the presence of several steroid dehydrogenases in early CA in lizard ovaries, suggesting ‘the possibility of transient luteinization or endocrine activity.’ Histochemical visualization of steroidogenic enzymes suggested that granulosa cells of CA in C. versicolor do have some steroidogenic capacity (Gouder, Nadkarni, & Rao, 1979) although this may simply reflect relict activity persisting before degradation. Guraya (1989) concluded that degenerating yolky follicles have no
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endocrine function but are simply vitellogenic follicles in the process of resorption by follicle cells or phagocytes. Interestingly, the presence of CA suppresses ovarian sensitivity to GTHs or to environmental stimulation in A. carolinensis, and surgical removal of the CA significantly increases the response of the ovary to exogenous GTHs (Crews & Licht, 1974). The authors suggest that the mechanism could involve secretion of a local factor that inhibits vitellogenesis or steroidogenesis. Conversely, the relatively large size of the CA could lead to its appropriating the majority of circulating GTH, depriving other follicles of stimulatory hormone.
3.3. Vitellogenesis In reptiles, follicular growth and maturation typically involve the deposition of large amounts of yolk. The bulk of that yolk is derived from the precursor molecule Vtg, a large molecular weight lipophosphoglycoprotein that is synthesized in the liver (Ho, 1991). Vitellogenesis is the process of hepatic synthesis of Vtg and its transport to the oocytes. This requires the mobilization of significant maternal reserves and is therefore dependent upon a threshold level of body condition (Bonnet, Naulleau, & Mauget, 1994). This requirement precludes annual reproduction in females of some species such as the viviparous vipers (Vipera spp.) (Bonnet et al., 1994) and the bluetongued lizard, T. nigrolutea (Edwards, Jones, & Wapstra, 2002b). Activation of the HPG axis to initiate vitellogenesis in appropriate seasons could involve leptin, a hormone produced primarily by adipocytes, which acts as a metabolic signal to the reproductive system in mammals. Leptin has been little studied in reptiles, but plasma leptin concentrations varied seasonally in S. undulatus (Spanovich, Niewiarowski, & Londraville, 2006) and increased during the previtellogenic period in P. sicula (Paolucci, Rocco, & Varricchio, 2001). Vitellogenin itself has a very long evolutionary history. It is a yolk precursor in a wide range of egg-laying animals including insects, nematodes, and vertebrates (Brandt, Zwaan, Beekman, Westendorp, & Slagboom, 2005). Vitellogenin provides the developing embryo with amino acids, lipids, phosphate, and multivalent metal ions, but its lipid content of 12–15% is not sufficient to fuel the needs of amniote embryos, which must reach an advanced stage of development at hatching. In oviparous amniotes, very-lowdensity lipoprotein (VLDL), also synthesized in the maternal liver, provides an additional source of yolk lipid, while a range of other proteins are also incorporated into the yolk: these include serum albumin, vitamin-binding proteins such as cholecalciferol, and immunoglobulins (IgGs) (Thompson & Speake, 2004).
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The embryos of oviparous reptiles depend solely upon yolk for their nourishment during development. However, among viviparous reptiles, there is a spectrum of different degrees of dependence upon lecithotrophy (yolk nutrition) vs. placentotrophy for embryonic nourishment. Most viviparous squamates do ovulate large yolky eggs, but, at the other end of the spectrum, viviparous species of Chalcides and Mabuya ovulate microlecithal eggs; for example, Mabuya heathi (Vitt & Blackburn, 1983), Mabuya bistriata (Vitt & Blackburn, 1991), and Mabuya brachypoda (Hernandez-Franyutti, Aranzabal, & Guillette, 2005) ovulate eggs only about 1 mm in diameter. In such species, oogenesis and oocyte maturation occur as in other reptiles, but vitellogenesis is suppressed, resulting in relatively small eggs (Hernandez-Franyutti et al., 2005). Callard and Ho (1987) provide a pertinent review of vitellogenesis in the context of the evolution of viviparity in vertebrates. It has been suggested that P4 plays a major role in the suppression of vitellogenesis (Callard, Riley, & Perez, 1990a), but endocrine studies are still needed to elucidate the underlying mechanism(s).
3.3.1. Mechanisms of vitellogenesis Vitellogenesis in vertebrates, and particularly in reptiles, has been the subject of several substantial comparative reviews (Ho, Kleis, McPherson, Heisermann, & Callard, 1982; Callard & Ho, 1987; Callard et al., 1990a; Ho, 1991; Polzonetti-Magni, Mosconi, Soverchia, Kikuyama, & Carnevali, 2004). Among vertebrates, Vtg is synthesized not in the ovary but in the liver, and is transported to the developing oocytes in the bloodstream. Vitellogenesis is accompanied by hypertrophy of the liver, signaling hepatic production of Vtg (Gavaud, 1986) and changes in hepatic protein synthesis in response to estrogens are linked to the transfer of yolk precursors to the developing oocytes (Ho, 1991). Calcium, which is transported to the oviduct in the bloodstream, binds to the Vtg molecule. Plasma calcium concentrations have therefore been used as an indicator of vitellogenesis in reptiles (Rostal, Robeck, Grumbles, & Burchfield, 1998). Vitellogenin is sequestered by the oocytes via receptormediated endocytosis and then stored in membrane-bound compartments (Polzonetti-Magni et al., 2004). Electron microscopic studies in P. sicula demonstrate that exogenous yolk precursors, visualized as electron-dense particles, are taken up by the oocyte by micropinocytosis and accumulate in the yolk globules. Although the chemical composition of these particles was not confirmed, they are presumably Vtg (Limatola & Filosa, 1989). In addition, a Vtg-binding protein has been identified in oocytes of P. sicula. In untreated animals, this protein is present in the plasma membranes of all classes of oocyte during the reproductive period, and treatment of winter animals with
Hormones and Reproduction of Vertebrates
exogenous FSH induced Vtg-binding protein in all oocytes (Romano & Limatola, 2000). In the oocytes, Vtg is enzymatically cleaved into phosvitins and lipovitellin, which form the major yolk proteins that are destined to be utilized as an energy source by the developing embryos (PolzonettiMagni et al., 2004).
3.3.2. Hormones and vitellogenesis The proximate stimulus for the induction of vitellogenesis is the presence of circulating estrogens, produced by the ovary from the maturing follicles. Being estrogendependent, the production of Vtg is normally restricted to females. However, male vertebrates also carry the Vtg gene, so exposure to natural or xenobiotic estrogens can trigger its expression in the liver of males as well as females (Ho, 1991). Indeed, Vtg synthesis by males is a useful indicator of endocrine disruption by ecoestrogens (Kime, Nash, & Scott, 1999; Chapter 14, this volume). Ovarian development and vitellogenesis are, therefore, typically associated with elevated plasma concentrations of estradiol in squamates, turtles, and crocodilians, and upregulation of hepatic ER may signal ‘readiness’ for vitellogenesis (Custodia-Lora, Novillo, & Callard, 2004). For example, high plasma E2 concentrations in vitellogenic females of the snake Vipera aspis are associated with mobilization of maternal reserves to fuel vitellogenesis; this is evidenced by high plasma levels of calcium, phosphorus, phospholipids, cholesterol, triglycerides, and proteins. Insertion of silastic implants containing E2 into non-reproductive females (that typically exhibit low plasma E2) resulted in mobilization of reserves as seen in vitellogenic females (Bonnet et al., 1994). Similarly, exogenous E2 induced hypercalcemia, hyperproteinemia, and liver hypertrophy in ovariectomized lizards (L. vivipara), although several other steroids, including P4 and T, had no such effect (Gavaud, 1986). Vitellogenin appears in the plasma six to eight hours after administration of exogenous estradiol to either sex of C. picta (Ho, Danko, & Callard, 1981), and plasma E2 concentrations rise significantly prior to nesting in loggerhead turtles (Caretta caretta) at a time when the ovaries contain hundreds of vitellogenic follicles, but are minimal by the time of the final nesting for that season (Wibbels et al., 1992). There do seem to be some exceptions to the general pattern of elevated estrogens during vitellogenesis. Circulating estrogen concentrations appear to be low during the presumed period of vitellogenesis in the green turtle C. mydas (Licht, 1979), and the lizard P. barbata exhibits very low plasma concentrations of E2, with no elevation during vitellogenesis (Amey & Whittier, 2000), although the authors speculate that this could be because a different estrogen is more important in this species. In
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Hormonal Regulation of Ovarian Function in Reptiles
the cold-adapted tuatara, Sphenodon punctatus, vitellogenesis is slow and extraordinarily protracted, taking place over a period of three years in a reproductive cycle of four years. Plasma steroids still follow patterns characteristic of other reptiles: plasma E2, T, and P4 are low during early vitellogenesis, rising only during the preovulatory period (Cree, Cockrem, & Guillette, 1992), and plasma P4 and T provide reliable indicators of whether a female will ovulate in any given year (Cree, Guillette, & Cockrem, 1991). In tuatara, plasma Vtg does not appear to be correlated with plasma concentrations of E2 (nor of T or P4) (Brown, Cree, Daugherty, Dawkins, & Chambers, 1994), despite the observation that exogenous E2 does stimulate vitellogenesis. However, estrogens are not the only hormones associated with vitellogenesis. Vitellogenesis is under multihormonal control (Ho et al., 1982; Callard et al., 1990a; PolzonettiMagni et al., 2004), and the vitellogenic action of estrogens is modulated by other ovarian steroids and by pituitary hormones. The most detailed studies of the endocrine interactions associated with vitellogenesis have focused on the freshwater turtle, C. picta (Duggan & Callard, 2003), utilizing the specific radioimmunoassay for plasma Vtg developed for that species. Progesterone is generally considered to inhibit vitellogenesis, probably by acting directly on the liver (Callard, Bayen, & McConnell, 1972a; Callard, Riley, & Perez, 1990b). Both P4 and T inhibit estradiol-induced vitellogenesis in C. picta (Ho et al., 1981), and rising levels of these hormones during the periovulatory phase may summate to terminate vitellogenesis at the end of the cycle (Ho, 1991). Callard et al. (1990a) used evidence from chickens to suggest that the inhibitory action of P4 on vitellogenesis could involve interaction of the PR with a progesterone response element (PRE) close to the estrogen response element (ERE) of the Vtg gene. Rarely, in some species such as the lizard C. versicolor, the key reproductive events of vitellogenesis and gravidity may, on occasion, overlap. In gravid females of C. versicolor with vitellogenic follicles, E2 concentrations rise while plasma P4 remains low (Radder, Shanbhag, & Saidapur, 2001), suggesting that elevated P4 is not required for egg retention during late gravidity. Low P4 at this stage may potentiate the growth of a new crop of follicles. Progesterone may also have pro-vitellogenic effects. Positive and negative effects of P4 could be mediated via differential expression in the liver of the two isoforms of the PR. While PRA, which has been suggested to act as a general transcription inhibitor (Duggan & Callard, 2003), is expressed throughout the annual cycle of C. picta, PRB, a gene-specific positive regulator, is expressed only during follicular growth, in autumn and in spring (Giannoukos & Callard, 1995), and appears to be under estrogenic control. This variation, underpinned by changes in the ratio of circulating P4 to estrogens (P : E ratio), suggests
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a mechanism whereby P4 could modulate the estrogenic induction of vitellogenesis (Custodia-Lora et al., 2004). Pituitary hormones may also be involved in the regulation of vitellogenesis in reptiles (Ho, 1991), although studies remain scarce. At the follicular level, uptake of yolk precursor into developing follicles of the lizard P. sicula was stimulated by exogenous FSH (Limatola & Filosa, 1989). At the hepatic level, hypophysectomy abolished the seasonal increase in hepatic ER concentrations observed in C. picta (Ho, Press, & Yu, 1989). Although the specific pituitary hormone(s) involved in the expression of hepatic ER was not determined, the authors suggested that it is likely to be a growth hormone (GH)-related factor. In their review of vitellogenesis and viviparity, Callard and Ho (1987) suggested that GH has synergistic effects on gonadotropin-stimulated vitellogenesis, while prolactin inhibits the synthesis and/or uptake of Vtg into follicles. However, in view of the paucity of evidence, further work, with a larger variety of species, is necessary to confirm and elucidate the putative roles of these pituitary hormones in the regulation of reptilian vitellogenesis. In addition, detailed studies of the endocrine control of vitellogenesis in reptiles other then turtles are required, in both viviparous and oviparous species. The possibility that ovarian GnRH and bradykinin play regulatory roles in follicular development and vitellogenesis should also be explored. In mammals, GnRH has been hypothesized to play an autocrine/paracrine role in ovarian development and function (Leung, Cheng, & Zhu, 2003). Although GnRH was originally identified as a hypothalamic neurohormone, multiple types of GnRH are now known to be expressed in a wide range of tissues, including the ovary. Two GnRH subtypes (GnRH-I and GnRH-II) and three receptor types (GnRHR1, GnRHR2, and GnRHR3) were detected in the ovary of the leopard gecko, E. macularius (Ikemoto & Park, 2007), and seasonal differences in receptor expression suggest some role for ovarian GnRH in the regulation of seasonality. Bradykinin stimulates GnRH release from the hypothalamus but is also produced in the ovary, where it may be involved locally in follicular development and ovulation (Kihara, Kimura, Moriyama, Ohkubo, & Takahashi, 2000). In C. versicolor, the greatest intensity of both GnRH and bradykinin immunoreactivity was in granulosa cells of previtellogenic or early vitellogenic follicles, suggesting some link with the regulation of follicular development (Singh et al., 2007; Singh, Krishna, Sridaran, & Tsutsui, 2008).
3.3.3. Yolk steroids In reptiles, yolk itself contains steroid hormones that are deposited into the yolk during vitellogenesis. Yolk steroids may play an important role in sex determination, particularly in oviparous reptiles, and influence offspring fitness in both
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oviparous and viviparous reptiles. Concentrations of individual steroids vary between layers of yolk within each egg (Conley, Elf, Corbin, Dubowsky, & Fivizzani, 1997; Bowden, Ewert, Lipar, & Nelson, 2001; Elf, Lang, & Fivizzani, 2002), and change during embryonic development. In alligators, concentrations of E2, androstenedione, and T decline markedly during egg incubation, with the decline most striking at stages 21–23, when gonadal differentiation is occurring (Conley et al., 1997). The authors suggested that androstenedione and T sourced from the yolk may be metabolized to estrogens by the gonads of the developing embryos, and thereby play a key role in sex determination. Steroid concentrations in yolk also vary among eggs, both within and among clutches. Steroid concentrations at the time of ovulation may passively reflect concurrent circulating maternal hormone levels: yolk and maternal plasma steroid concentrations tend to co-vary. For example, both plasma and yolk T concentrations were artificially elevated in female turtles with T implants during follicular development (Janzen, Wilson, Tucker, & Ford, 2002). Seasonal variation in yolk steroids has been correlated with seasonal shifts in sex ratio in clutches of C. picta, a turtle with TSD (Bowden et al., 2000). Conversely, it has been hypothesized that differential allocation of yolk steroids to individual eggs within a clutch is an important mechanism through which mothers can influence offspring development and sex determination (Bowden et al., 2000; Lovern & Wade, 2003). Yolk steroids are not, however, derived entirely from maternal sources. During embryonic development, as the embryonic endocrine organs begin to produce hormones, additional steroids may diffuse across into the yolk (Lovern & Wade, 2003). The CORT content of Urosaurus ornatus eggs, which is non-detectable at oviposition, was significantly increased by day 25 of incubation, strongly suggesting that its source is the fetal adrenals (Weiss, Johnston, & Moore, 2007), while in the viviparous Sceloporus jarrovi, yolk P4 and CORT concentrations more closely mirrored fetal, rather than maternal, plasma concentrations of those steroids (Painter et al., 2002). These and similar observations support the notion of a dynamic interaction between maternal and embryonic endocrine systems in both oviparous and viviparous reptiles. Hormones other than steroids may also be deposited into yolk during vitellogenesis. Plasma concentrations of insulinlike growth factor I (IGF-I) are elevated during gravidity in American alligators, providing some circumstantial evidence that this hormone, which is a growth-promoting substance, may be sequestered into yolk (Guillette, Cox, & Crain, 1996). The potential contributions of such endocrine factors to supporting embryonic development remain to be elucidated, but this could be an additional mechanism through which maternal effects on offspring characteristics may be exerted.
Hormones and Reproduction of Vertebrates
4. OVULATION Ovulation, or release of the oocyte from the ovary, is the culmination of vitellogenesis and follicular maturation. Plasma concentrations of estrogens rise significantly during late vitellogenesis as the follicles mature. Following ovulation, plasma estrogen concentrations decline rapidly to pre-reproductive levels as the post-ovulatory follicle is transformed into a corpus luteum.
4.1. Mechanisms of Ovulation in Reptiles Ovulation involves enzymatic degradation and rupture of the multi-layered follicular wall and the escape of the mature oocyte. The point of follicular rupture is visible externally as a pale region known as the stigma. In reptiles, the stigma is avascular (Uribe & Guillette, 2000). In nonmammalian vertebrates, the stigma ruptures due to ischemia, resulting in necrosis rather than via an inflammatory response as occurs in mammals (Jones, 1987). Histological examination of follicles in FSH-treated A. carolinensis demonstrated a reduction in follicular blood flow immediately before rupture. There were dramatic changes in the stigma, including marked accumulation of extracellular fluid and dissociation of collagen bundles to form a ‘lacework of fibrils,’ but no evidence of inflammatory cells in the interstitial spaces (Jones, Austin, Lopez, Rand, & Summers, 1988). The mechanism of oocyte escape from the follicle has not been studied in reptiles, but the process is believed to involve contractile fibers. Follicles of A. carolinensis undergo spontaneous rhythmic contractions (Jones, Austin, & Summers, 1984) that may be related to this process.
4.2. Hormonal Control of Ovulation From an endocrine perspective, ovulation marks the transition from the estrogen-dominated follicular phase to the P4-dominated luteal phase. In female vertebrates, changing patterns of plasma steroids through the ovarian cycle influence GTH secretion via feedback effects at the level of the pituitary gonadotropes or the hypothalamic GnRH neurons. Peak plasma concentrations of E2 precede ovulation, and the ovulatory surge is stimulated via a positive feedback action of E2 at the level of the hypothalamus (Niswender, Juengel, Silva, Keith Rollyson, & McIntush, 2000). In reptiles, most studies of plasma steroids have examined long-term patterns of variation over the reproductive cycle, and relationships between hormonal changes and physiological effects are usually inferred rather than established. There is, therefore, little direct evidence for feedback control of GTH secretion by steroids in reptiles. Some early studies examined the effects of hypothalamic
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Hormonal Regulation of Ovarian Function in Reptiles
implants of P4 or estrogens in female Sceloporus cyanogenys. Estrogenic implants into the hypothalamus (but not other parts of the brain, nor in subcutaneous locations) inhibited ovulation and depressed ovarian steroidogenesis (Callard et al., 1972a), providing evidence for negative feedback inhibition of GTH release, while P4 implants inhibited ovarian growth (vitellogenesis) but not ovulation. In general, P4 also has a negative feedback effect on GTH secretion in reptiles, so that folliculogenesis is inhibited during gravidity. In contrast, it has been suggested that P4, not estrogens, may be responsible for positive feedback regulation of the ovulatory LH surge in turtles (Etches & Petitte, 1990). Ovulation is also correlated with elevated plasma T concentrations in species including the Galapagos tortoise, Geochelone nigra (Rostal et al., 1998), the bluetongued lizard (Edwards & Jones, 2001), and the American alligator (Lance, 1989), although a causal relationship has not yet been established. In most vertebrates, a marked surge of plasma LH and/or FSH stimulates follicular rupture and also initiates subsequent luteinization of the granulosa cells (Jones, 1987). In reptiles, the precise roles of the GTHs in ovulation remain unclear. The paucity of experimental studies and descriptions of changes in plasma GTHs around the time of ovulation reflects a lack of specific assays for these hormones in reptiles other than turtles. In sea turtles, plasma FSH concentrations are low except for a transient peak associated with the onset of oviposition, whereas plasma LH exhibits a marked ‘ovulatory’ surge associated with nesting (Owens & Morris, 1985). The temporal dissociation between these two hormonal peaks suggests that in turtles FSH may prime the ovary to respond to LH. However, time series samples from individual loggerhead turtles (C. caretta) identified concurrent surges of LH and FSH in the periovulatory period (Wibbels et al., 1992), perhaps reflecting a nonspecific action of GnRH on GTH release by the pituitary. Peak LH levels preceded, or were coincident with, peak P4 concentrations in plasma, providing evidence that LH stimulates ovarian production of P4 by the post-ovulatory follicle. In vertebrates, gonadotropin-induced ovulation is mediated by prostaglandins (Jones, 1987), and inhibitors of prostaglandin (PG) synthesis act at the ovarian level to inhibit ovulation. Thus, in A. carolinensis, indomethacin suppresses total ovarian PGE secretion in-vitro, inhibits preovulatory histological changes in the follicular wall, and significantly delays ovulation in response to exogenous FSH (Jones, Orlicky, Rand, & Lopez, 1990). Both PGF2a and PGE2 are produced by follicles of P. sicula. Preovulatory follicles produced the highest levels of PGF2a, while PGE2 production was highest in early vitellogenic follicles. The effects of PGF2a and PGE2 on sex steroid production in-vitro by follicles at different vitellogenic stages suggest that PGF2a could be involved
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in ovulation, while PGE2 has a stimulatory effect on E2 release by the follicles, indicating that PGE2 is involved in maintaining vitellogenesis (Gobbetti et al., 1993b). More detailed information is still required regarding the roles of steroid hormones and PGs in the induction of ovulation in reptiles.
4.3. Oocyte Maturation The final stages of oocyte maturation involve completion of the first meiotic division; until this is completed, the oocyte is not fertilizable. To date, there have been no studies of the endocrine control of oocyte maturation in reptiles: our knowledge of this process in nonmammalian vertebrates is restricted to fishes and amphibians. It has been suggested that P4 may act as a maturation-inducing substance (Nagahama, 1987), but this hypothesis is based solely upon patterns of plasma P4 levels during the reproductive cycle of various reptiles.
5. THE POST-OVULATORY OVARY: THE CORPUS LUTEUM All reptiles, whether oviparous or viviparous, develop true, secretory, corpora lutea from the postovulatory follicles. These corpora lutea are transitory endocrine structures, formed primarily from the granulosa cells, which hypertrophy after ovulation to form a luteal cell mass surrounded by thecal layers (Guraya, 1989). The luteal cells are steroidogenic and secrete P4. Several major reviews have focused on corpora lutea in reptiles or, more generally, in nonmammalian vertebrates (Saidapur, 1982; Xavier, 1987; Guraya, 1989; Jones & Baxter, 1991; Callard et al., 1992). Jones and Baxter (1991) identify five types of corpora lutea among vertebrates, distinguished by their length of lifespan and type of endocrine regulation. They consider that reptilian corpora lutea fall into type II, having an extended life span and being controlled by pituitary luteotropin(s). They suggest that the life of these corpora lutea is extended because the luteinizing hormone becomes a luteotropin, or another pituitary hormone such as prolactin takes over this function, but these ideas remain to be tested experimentally. The significance of the corpus luteum and its primary steroid product, P4, in the evolution of viviparity has been of particular interest, and is discussed further in Chapter 9, this volume.
5.1. Morphological Changes in the Reptilian Corpus Luteum The lifespan of a typical corpus luteum may be divided into three stages: luteogenesis, luteal maturity, and luteal regression, or luteolysis (Fox & Guillette, 1987). The
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associated morphological changes have been described in great detail in several reviews (e.g., Saidapur, 1982; Fox & Guillette, 1987; Xavier, 1987): a synopsis of those descriptions is given here. After ovulation, the collapsed follicle appears flaccid and sac-like, with a large opening in one side through which the oocyte has escaped. The postovulatory follicle then shrinks in size as its walls become thickened and folded to produce a multilayered granulosa. The granulosa cells now become ‘luteinized:’ they hypertrophy, acquire secretory granules and large lipid droplets in their cytoplasm, and are characterized by a large central nucleus with one to three nucleoli. The ultrastructure of the luteinized cells, with abundant smooth endoplasmic reticulum, is characteristic of steroid-secreting cells. The granulosa involutes and hypertrophies to fill the central cavity of the collapsed follicle, forming the luteal cell mass, while the follicular lumen and the opening through which the oocyte emerged eventually disappear in most species, except crocodilians and tuatara (Guillette & Cree, 1997). The thecal cells also proliferate to form two well-defined layers, the theca externa and the theca interna, around the inner luteal cell mass. The thinner theca externa is a loose tissue containing collagen, fibroblasts, a few hypertrophied round cells, and macrophages, while the theca interna is more cellular. At maturity, the corpus luteum is a compact, solid
Hormones and Reproduction of Vertebrates
structure with three distinct zones: the outermost theca externa, the theca interna, and the luteal cell mass (Guraya, 1989) (Figure 4.4). Reptilian corpora lutea differ from those of mammals in that the cells filling the luteal cavity are, in reptiles, almost exclusively of granulosa origin. However, there are some differences among species in the cellular composition of the corpus luteum. Xavier (1987) distinguished three different morphological types of corpora lutea among reptiles in contrast to the physiological types defined by Jones and Baxter (1991). These are differentiated by the degree to which the cells of the theca interna invade the luteal cell mass. In some species, the granulosa cells alone form the luteal cell mass (type 1). In type 2 corpora lutea, fibroblasts from the theca interna invade to form superficial septae, but no capillaries penetrate among the luteal cells. In other species, the corpus luteum becomes richly vascularized and the theca interna invades the luteal cell mass, forming fibrous dividing septae (type 3). The functional significance of these morphological differences is unknown. However, the corpora lutea remain active and secretory for species-specific periods of time. In oviparous reptiles, the corpora lutea persist during gravidity (egg retention) and regress near the time of oviposition. In tuataras, which have an extraordinarily extended
FIGURE 4.4 Micrograph of a corpus luteum of the viviparous lizard Niveoscincus metallicus during mid-gestation, when the embryos were at stage 29 (sensu Dufaure & Hubert, 1961). Note the well-defined inner luteal cell mass (LCM) and the outer thecal layers (Th).
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Hormonal Regulation of Ovarian Function in Reptiles
reproductive cycle, corpora lutea persist during the eight months of egg retention (Guillette & Cree, 1997). Corpora lutea of viviparous reptiles persist for variable periods, but generally remain active until the end of gestation (Xavier, 1987; Guraya, 1989). The nuclear diameter of the luteal cells decreases gradually during gestation (Bennett & Jones, 2002), presumably reflecting changes in endocrine activity by these cells. Eventually, the corpora lutea degenerate as luteolysis begins. Nuclei of the luteal cells become pycnotic, and vacuoles and sudanophilic lipid droplets appear in their cytoplasm; the theca becomes less vascular, fibrous connective tissue invades, and the thecal layers merge. Luteal cells eventually disintegrate and are replaced by connective tissue. The remaining scar is comparable to the corpus albicans of the mammalian ovary (Guraya, 1989).
5.2. Steroid Production by the Corpus Luteum Corpora lutea are generally considered to be the major source of circulating P4 secreted by the reptilian ovary. This has been confirmed by in-vitro tissue incubations and by histochemical studies. In-vitro studies demonstrate that corpora lutea of oviparous turtles (Klicka & Mahmoud, 1972) and viviparous lizards (Xavier, 1982; Girling & Jones, 2003) can convert pregnenolone to P4, and the rate of P4 secretion by non-luteal ovarian tissues is insignificant compared with its rate of production by corpora lutea (Xavier, 1987; Bennett & Jones, 2002). The steroidogenic enzyme 3b-HSD, which converts pregnenolone to P4, is detectable in luteinized granulosa cells by histochemistry. The intensity of 3b-HSD activity in the corpus luteum has been positively correlated with plasma P4 concentrations in both oviparous and viviparous species (Shanbhag, Radder, & Saidpur, 2001; Martı´nezTorres, Herna´ndez-Caballero, Alvarez-Rodriguez, LuisDı´az, & Ortı´z-Lo´pez, 2003), although in some viviparous species the placentae may take over the primary role in P4 production during later gestation (Guarino et al., 1998). Callard et al. (1992) distinguished two basic patterns of P4 secretion in reptiles. The first is a preovulatory or ovulatory surge of plasma P4 that is characteristic of the oviparous turtles. The second pattern, characteristic of squamates, is a peak of plasma P4 after ovulation, or during mid-late pregnancy in viviparous species. However, at variance with this pattern, the viviparous skink Niveoscincus microlepidotus (Girling, Jones, & Swain, 2002) and viviparous snake Natrix sipedon pictiventris (Chan, Ziegel, & Callard, 1973) exhibit, in addition to peaks during gestation, significant preovulatory rises in plasma P4 concentrations, which may reflect P4 synthesis by the
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preovulatory follicles. Luteolysis (Fox & Guillette, 1987; Fergusson & Bradshaw, 1991), luteectomy (Highfill & Mead, 1975; Guillette & Fox, 1985), or ovariectomy (Fergusson & Bradshaw, 1991) are associated with a marked drop in plasma P4 concentrations as the luteal cells lose their capacity to secrete P4 or the source of that P4 is removed. In some species at least, the corpus luteum may produce androgens and estrogens as well as P4 (Xavier, 1987). Tissue concentrations of T and E2 were higher in corpora lutea than in the follicular tissue of gravid Uromastix hardwicki, although the concentration of P4 in the corpora lutea was 100 times that of E2 (Arslan, Zaidi, Lobo, Zaidi, & Qaza, 1978) and the corpora lutea of the lizard P. s. sicula released E2 as well as P4 when incubated in-vitro (Ciarcia, Paolucci, & Di Fiore, 1993; Gobbetti et al., 1993a). The functional significance of these androgenic and estrogenic steroids is unclear, and it is possible that they simply reflect metabolic catabolism of P4.
5.3. Relaxin The mammalian corpus luteum secretes some nonsteroidal hormones including relaxin, a polypeptide hormone that plays important roles in mammalian pregnancy. Among these, relaxin supports oxytocin-stimulated uterine contractions and widens the pelvic symphysis to facilitate parturition. Jones and Baxter (1991) considered that the ‘fundamental effects of relaxin. should all be present in reptiles,’ but we still have surprisingly little knowledge of relaxin in reptiles, although relaxin-like molecules have been identified in several viviparous sharks (see review by Jones & Baxter, 1991). The possibility of relaxin production by reptilian corpora lutea has not yet been reported, although Koob and Callard (1982) speculated that in viviparous reptiles the corpora lutea may begin secreting relaxin as P4 secretion drops towards the end of gestation.
5.4. Hormones and Luteolysis In mammals, PGF2a elicits luteolysis through a series of actions, including reduction of blood flow to the corpus luteum, downregulation of receptors for luteotropic hormones, and inhibition of P4 synthesis (Niswender et al., 2000). Although the proximate mechanisms of luteolysis of reptilian corpora lutea have not been positively identified, there is good evidence that PGs mediate this process. A single injection of PGF2a induces luteolysis (evidenced by loss of ultrastructural features associated with steroid synthesis and a drop in plasma P4) in A. carolinensis (Guillette, Lavia, Walker, & Roberts, 1984) and Chelydra serpentina (Mahmoud, Cyrus, McAsey, Cady, & Woller,
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1988), and reduces P4 (but not androgens or E2) secretion in-vitro by corpora lutea of the lizard P. s. sicula (Gobbetti et al., 1993a). Whether the initiation of luteolysis is also associated with the withdrawal of a pituitary luteotropin remains to be tested in reptiles.
6. FUTURE DIRECTIONS: APPLICATION OF REPRODUCTIVE ENDOCRINOLOGY TO CONSERVATION This review has highlighted a number of areas in which our knowledge of hormones and the reptilian ovary is still scanty compared with our understanding of mammalian systems, although it must be acknowledged that even for mammals we have extensive data for relatively few species. In particular, research into reptilian endocrine systems has been hampered by a lack of homologous preparations of peptide hormones, both for the development of specific assays and for experimental applications. There is one area in which the ability to apply our fundamental knowledge of reptilian reproduction would be of great benefit. Species are at risk due to habitat loss, climate change, environmental pollution, introduced species, diseases, or unsustainable use and over-collecting. Knowledge of reptilian reproductive endocrinology may become vital to species conservation, especially if ex situ captive breeding programs become necessary. To date, such avenues have not been widely exploited, but some examples can be gleaned from the literature. For example, plasma hormone concentrations can be used as indicators of reproductive condition in species of conservation concern when use of non-invasive techniques such as ultrasound are impractical. Gravid female tuatara can be identified by the criteria of plasma T and P4 concentrations of 2.0 ng/ml and 5.8 ng/ml plasma respectively (Cree et al., 1991). Similarly, measurements of plasma sex steroids can be used to monitor reproductive cycles in Gala´pagos tortoises in captivity and to investigate the proximate cues that regulate seasonality in this species (Rostal et al., 1998). Non-invasive monitoring of sex steroids in feces or urine is now widely utilized in mammalian conservation and captive breeding programs. However, such techniques may be more problematic in reptiles, partly due to the variable rates of excretion in ectotherms, and would need careful validation (Atkins, Jones, & Edwards, 2002). Knowledge of ovarian endocrinology may also be applied to stimulating reproduction by captive reptiles. One group has experimented with GnRH as a means of inducing female receptivity and ovulation in iguanas, with some
Hormones and Reproduction of Vertebrates
limited success (Phillips et al., 1987). Exogenous FSH stimulates ovarian growth and receptivity in captive painted dragons, but the presence of males is required for fertilization (Uller & Olsson, 2005), demonstrating that an understanding of how environmental conditions influence ovarian physiology is essential if captive breeding is to succeed. In particular, the stress of captivity may severely disrupt the reptilian reproductive cycle via inhibitory effects on plasma concentrations of ovarian steroids (Guillette et al., 1995). Captive anoline lizards, for example, frequently exhibit cessation of follicular growth and the appearance of atretic follicles, apparently as a consequence of decreased hepatic expression of Vtg. In captive Anolis pulchellus, Vtg synthesis drops and a marked downregulation of Vtg mRNA levels is observed, but these effects can be alleviated or reversed by treatment with estradiol E2 (Morales & Sanchez, 1996). There were no significant effects of captivity in outdoor, semi-natural enclosures on plasma concentrations of sex steroids in the lizard Sceloporus virgatus compared with free-ranging females of the same species. However, the captive females exhibited a marked delay or inhibition of oviposition, suggesting that such conditions may not be appropriate for long-term breeding programs (Weiss, Jennings, & Moore, 2002). The diversity of reproductive strategies among reptiles means it is not always possible to make generalizations about all aspects of their reproductive endocrinology. However, an understanding of how ovarian activity is regulated by the HPG axis and other hormones, and how the endocrine system is influenced by environmental cues, may eventually underpin the successful management of populations of endangered species, both in situ and ex situ.
ABBREVIATIONS 20b-HP 3b-HSD AR CA CBG cGnRH-II DHEA DHT E1 E2 EDC ER ERE ESD FSH FSHR
20b-hydroxypregn-4-ene-3-one 3b-hydroxysteroid dehydrogenase Androgen receptor Corpus atretica Corticotrophin-binding globulin (transcortin) Chicken II gonadotropin-releasing hormone Dehydroepiandrosterone 5a-dihydrotestosterone Estrone 17b-estradiol Endocrine-disrupting chemical Estrogen receptor Estrogen response element Environmental sex determination Follicle-stimulating hormone Follicle-stimulating hormone receptor
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GH GnRH GRIH GTH HPG IGF-1 IgG LH P4 PG PR PRE S-BP SHBP T TSD VLDL Vtg
Hormonal Regulation of Ovarian Function in Reptiles
Growth hormone Gonadotropin-releasing hormone Gonadotropin release-inhibiting hormone Gonadotropin Hypothalamus–pituitary–gonad Insulin-like growth factor 1 Immunoglobulin G Luteinizing hormone Progesterone Prostaglandin Progesterone receptor Progesterone response element Steroid-binding protein Sex-hormone binding protein Testosterone Temperature-dependent sex determination Very-low-density lipoprotein Vitellogenin
REFERENCES Amey, A. P., & Whittier, J. M. (2000). Seasonal patterns of plasma steroid hormones in males and females of the Bearded Dragon Lizard, Pogona barbata. Gen. Comp. Endocrinol., 117, 335–342. Angelini, F., Ciarcia, G., Picariello, O., Botte, V., & Pangano, M. (1985). Sex steroids and postreproductive refractoriness in the lizard, Podarcis s. sicula. Boll. Zool., 53, 59–62. Arslan, M., Zaidi, P., Lobo, J., Zaidi, A. A., & Qaza, M. H. (1978). Steroid levels in preovulatory and gravid lizards (Uromastix hardwicki). Gen. Comp. Endocrinol., 34, 300–303. Atkins, N., Jones, S. M., & Edwards, A. (2002). Fecal testosterone concentrations may not be useful for monitoring reproductive status in male blue-tongued lizards (Tiliqua nigrolutea: Scincidae). J. Herpetol., 36, 106–109. Austin, H. B. (1988). Differentiation and development of the reproductive system in the iguanid lizard, Sceloporus undulatus. Gen. Comp. Endocrinol., 72, 351–363. Baker, M. E. (2004). Co-evolution of steroidogenic and steroidinactivating enzymes and adrenal and sex steroid receptors. Mol. Cell. Endocrinol., 215, 55–62. Bennett, E. J., & Jones, S. M. (2002). Interrelationships among plasma P4 concentrations, luteal anatomy and function, and placental ontogeny during gestation in a viviparous lizard (Niveoscincus metallicus: Scincidae). Comp. Biochem. Physiol., A, 131, 647–656. Bluhm, A. P. C., Toledo, R. A., Mesquita, F. M., Pimenta, M. T., Fernandes, F. M. C., Ribela, M. T. C. P., & Lazari, M. F. M. (2004). Molecular cloning, sequence analysis and expression of the snake follicle-stimulating hormone receptor. Gen. Comp. Endocrinol., 137, 300–311. Bona-Gallo, A., & Licht, P. (1983). Effects of temperature on sexual receptivity and ovarian recrudescence in the garter snake, Thamnophis sirtalis parietalis. Herpetologica, 39, 173–182. Bonnet, X., Naulleau, G., & Mauget, R. (1994). The influence of body condition on 17-b estradiol levels in relation to vitellogenesis in female Vipera aspis. Gen. Comp. Endocrinol., 93, 424–437.
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Chapter 5
Hormones and the Sex Ducts and Sex Accessory Structures of Reptiles Daniel H. Gist University of Cincinnati, Cincinnati, OH, USA
SUMMARY Reproductive ducts of male and oviparous female reptiles are reviewed. Sperm ducts consist of the rete testis, efferent ductules, epididymis, and vas deferens. An accessory organ, the renal sex segment, is present in squamate reptiles. The entire sperm duct system is under androgen control. Male sex hormones stimulate epididymal secretions that may be involved in sperm maturation. Oviducts may be subdivided into the infundibulum, uterine tube, isthmus, uterus, and vagina. The isthmus and uterine tube regions are absent in squamate reptiles. Female sex hormones stimulate the development of tubuloalveolar glands in the walls of the uterine tube and uterus. Progesterone reduces contractility of the uterus, and AVT, a neurohypophysial hormone, together with PGF2a promote uterine contractions. Oviposition is accomplished by interactions of the above hormones tempered by adrenergic innervation. Sperm may be stored in the epididymis of the male or in the tubuloalveolar glands of the female for long periods of time. Such storage is influenced by estrogenic hormones and has implications for strategies of life history and evolutionary ecology.
being shed to the exterior. Internal fertilization has several implications. One is that it requires a copulatory organ to introduce sperm to the female. Another is that the male and female reproductive cycles must be synchronized so that sperm are present in the female tract when eggs are ripe. Alternatively, in cases in which the male and female cycles are dissociated (Crews, 1984), provisions must be made to preserve sperm, either in the male or female, until the eggs are ripe for fertilization. The third feature is that, once fertilized, eggs remain within the female reproductive tract for short (oviparous) or long (viviparous) periods of time prior to oviposition or parturition. This review concentrates on the sperm ducts of the male and the oviduct of the female, and their anatomy and endocrinology. The issue of viviparity is considered in Chapter 9, this volume; this review will focus on oviparous reptiles. None of these features described above are unique to reptiles, but the reptiles are the first vertebrate group to employ all of these features.
2. OVIPARITY 1. INTRODUCTION As a class, the Reptilia made their appearance in the Paleozoic, flourished during the Mesozoic, and became mostly extinct by the Cenozoic. There remain today only four major reptilian groups, the Testudines, Crocodilia, the Sphenodontia, and the Squamata. Most of the living reptiles are lizards and snakes, members of the Squamata. From a reproductive perspective, the Reptilia possess three features, none of which is unique to them but which together serve to distinguish the group. First, all living and most of the extinct reptiles possess an amniotic egg in which the embryo develops in a fluid-filled sac, the amnion. This is thought by some to have enabled reptiles to reproduce successfully in terrestrial, even arid environments. The second is internal fertilization, in which sperm are introduced directly into the female reproductive tract instead of Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
The oviparous mode of reproduction is characteristic of some fish and amphibians, most reptiles, and all birds. In this mode, ovulated eggs receive investments, including a shell in some species, from the oviduct wall, and the eggs are shed (oviposited) from the female prior to the completion of embryonic development. This mode has several features, one of which is the size of the egg. Since eggs are oviposited with the embryo at a relatively early stage of development, all materials essential for development must be incorporated into the egg prior to oviposition. Reptilian eggs are megalecithal, having an extremely large quantity of yolk, which occupies the largest part of the ovulated egg. Precursors to yolk are synthesized by the liver prior to ovulation (vitellogenesis) and incorporated into the developing follicle, a process controlled by ovarian hormones. Vitellogenesis is considered in Chapter 4, this volume. 117
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Our knowledge of vertebrate reproduction as a whole is scanty. What has emerged, however, is that reproduction is quite species-specific, and many examples of mechanisms existing in some species not being applicable to others can be found in the literature (van Tienhoven, 1983). In reptiles, as in most vertebrate taxa, our knowledge of reproductive mechanisms is based on a few intensely studied species. Despite the acknowledged pitfalls of such an approach, what is known for these few species is typically extended to the group as a whole.
3. REPRODUCTIVE CYCLES AND SEX HORMONES Reproduction in most reptiles is cyclic (Callard & Kleis, 1987) and is associated with favorable times of the year, with temperature, photoperiod, and moisture serving as zeitgebers. As is the case with other vertebrates, environmental influences act via the hypothalamus to coordinate the release of gonadotropins from the pituitary gland to regulate the cyclicity. The gonadotropins follicle-stimulating hormone (FSH) and possibly luteinizing hormone (LH) in turn act on the gonads to regulate the secretion of gonadal sex hormones as well as the growth, maturation, and release of the gametes. It is the gonadal sex hormones that regulate either wholly or in part the function of the sex ducts, the oviduct of the female and the sperm duct of the male, and the secondary sex organs. Two different types of male reproductive cycle have been described for reptiles. The first, the most common, is the associated (Crews, 1984) or prenuptial (Licht, 1984) cycle (see also Chapter 13, this volume). In this type, spermatogenesis and male sex hormone levels are temporally synchronized to ovulation in the female. Crocodilians, the tuatara, and most lizards and snakes possess this type of cycle. In the dissociated, or postnuptial, cycle, male sex hormone secretion and spermatogenesis are not linked to
Hormones and Reproduction of Vertebrates
ovulation in the female, and the two events are separated in time. Some turtles and snakes possess this type of cycle. Species with dissociated cycles typically have a prolonged period of sperm storage, either in the epididymis of the male or in the oviduct of the female. In both sexes, the primary sex ducts as well as the accessory sex organs are secondary sex organs, whose size and functional integrity are dependent on the secretion of sex steroids (androgens, estrogens). Over the past several decades, the identities of the gonadal sex steroids have been determined for a large number of vertebrates (see Kime, 1987). In general, testosterone (T), androstenedione, and dihydrotestosterone (DHT) predominate among the various androgen secretions of the male, and estradiol (E2) and estriol predominate among the estrogenic secretions of females. All reptiles, including males and females, secrete progestins as well, with progesterone (P4) being the one most commonly reported.
4. THE MALE The embryology of the reptilian male reproductive system was reviewed by Fox (1977). Only recently has the detailed anatomy of the connections between the testis, efferent ductules, and ductus epididymis been clarified, and from these there appear to be only minor differences among the representatives of the various reptilian orders. Male reptiles do not possess accessory glands, as is common for mammals. The only exception is the sexual segment of the kidney, found in squamate reptiles. The gross anatomy of the reptilian male reproductive system (Figure 5.1) resembles that of other amniote vertebrates.
4.1. Efferent Ductules The seminiferous tubules of the testis empty into a number of tubules emerging from each testis. The number of these
FIGURE 5.1 Generalized diagram of the reptilian male reproductive system. A, ampulla; C1, caput epididymis; C2, corpus epididymis; C3, cauda epididymis; D, ductus deferens; E, efferent ductules; R, rete testis; S, renal sex segment. Hemipenis not shown. Modified from Jones (1998). Reproduced by permission, Society for Reproduction and Fertility.
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Hormones and the Sex Ducts and Sex Accessory Structures of Reptiles
tubules draining a testis is variable, estimated to be 6–8 in the lizard Sitana ponticeriana (Akbarsha, Kadalmani, & Tamilarasan, 2007), 20–30 in the turtle Chrysemys picta (Holmes & Gist, 2004), and 33 in the snake Tropidonotus natrix (Volsoe, 1944). They are lined by a simple nonciliated squamous to cuboidal epithelium and are surrounded by connective tissue. These tubules resemble the rete testis tubules of the mammal histologically (Robaire & Hermo, 1988), but in older reptilian literature (see Volsoe, 1944) were termed ‘ductuli efferentes.’ Despite the fact that only in the turtle do these tubules form a true rete (exterior to the testis), more recent authors have designated these tubules draining the testis as rete testis tubules (Holmes & Gist, 2004). In most reptiles, the rete testis tubules communicate with the efferent ductules and convey the seminal products to the epididymis (ductus epididymis). The junction between rete testis tubules and efferent ductules is usually abrupt, with the unciliated cuboidal epithelium of the rete testis changing to a columnar form with numerous cilia. Efferent ductules are highly convoluted and run through the loose connective tissue before discharging their contents into the epididymis (ductus epididymis) proper. The junction with the epididymis usually occurs at the proximal portion of the epididymis, but can occur at any level; as in the mammal, blind endings are common. Older authors have identified the efferent ductules as ductuli
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epididymis. Subsequent histological examinations of these ducts reveal that both efferent ductules and ductuli epididymis have the same cell types. It seems prudent to adopt the more modern nomenclature if only to avoid confusion among comparable structures in the vertebrate male duct (Guerrero, Calderon, dePerez, & Minilla, 2004; Holmes & Gist, 2004; Akbarsha et al., 2007). Variations do exist in the efferent ductules from different reptilian taxa, but the basic anatomy of these tubules is the same for all reptiles studied. As they leave the rete testis, efferent ductules range in diameter from 18 to 60 mm and are surrounded by a thin layer of smooth muscle. They possess a simple cuboidal to columnar or pseudostratified epithelium consisting of ciliated and nonciliated cells. The nonciliated cells possess microvilli and outnumber the ciliated cells by 3–4 : 1 (Figure 5.2). Cilia in the efferent ductules are unusually long (estimated to be 40 mm in the turtle). Other variations may exist along the length of the efferent ductules. For example, in the lizard S. ponticeriana and in Sphenodon punctatus, the tubules become less convoluted distally (Gabe & Saint Girons, 1964; Akbarsha et al., 2007). In the caiman (Caiman crocodilus) and turtle (C. picta), the epithelial cells, particularly the nonciliated cells, possess prominent secretory granules ranging in diameter from 0.5–5 mm (Guerrero et al., 2004; Holmes & Gist, 2004). In some species, different zones within the efferent ductules
FIGURE 5.2 Cross section of efferent ductule from the turtle Chrysemys picta. Bar ¼ 50 mm.
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are recognized based on the distribution of granulated cells (Volsoe, 1944; Guerrero et al., 2004; Holmes & Gist, 2004). In addition, both large and small clathrin-lined vesicles have been observed in the efferent ductules of all reptiles examined. In the mammalian testis, the efferent ductules are similar in appearance to those of reptiles and are specialized for secretion and absorption of both liquid and particulate matter (Robaire & Hermo, 1988; Hess 2003). It is probable that the reptilian efferent ductules have similar functions. In addition, spermiophagy within the efferent ductules is reported in S. ponticeriana (Akbarsha et al., 2007) and b-N-acetylglucosaminidase activity, associated with sperm remodeling, is present in the tortoise Testudo hermanni (Kuchling, Skolek-Winnisch, & Bamberg, 1981).
4.2. Ductus epididymis The efferent ductules discharge into a single duct, the ductus epididymis. The reptilian epididymis is similar in many respects to its mammalian counterpart. It lies laterally to the testis and consists of a single highly coiled tube embedded in loose connective tissue. It maintains connections with the testis via the connective tissue and the efferent ductules. Subdivisions resembling the mammalian initial segment, caput, corpus, and cauda epididymis are recognized in a number of reptiles (Haider & Rai, 1987; Robaire & Hermo, 1988; Averal, Manimekalai, & Akbarsha, 1992; Akbarsha & Manimekalai, 1999; Akbarsha, Kadalmani, & Tamilarasan, 2006) but not in others (Guerrero et al., 2004; Holmes & Gist, 2004). For the purposes of this review, the proximal portion of the ductus epididymis will be subdivided into an initial segment and, caudally, the caput epididymis; a middle corpus epididymis; and the distal region, the cauda epididymis (Figure 5.3). Where these recognizable subdivisions exist, as in the lizard S. ponticeriana (Akbarsha, Tamilarasan, & Kadalmani, 2006), the initial segment is a large, thinwalled chamber that receives many of the efferent ductules. The caput, corpus, and cauda epididymis generally consist of an epithelium that is highest in the caput and decreases gradually to be lowest in the cauda. In addition, the diameter of the ductus epididymis tubule increases in size from caput to cauda. The epididymis is the primary sperm storage organ for the male. Following spermatogenesis, the epididymis is engorged with sperm and can exceed the testis in size. During breeding, sperm not transferred to females become concentrated in the cauda epididymis. During the time of testicular rest, the epididymis of some species may be devoid of sperm, but in others some sperm remain in the epididymis throughout the year. The reptilian epididymis is lined by a pseudostratified epithelium and is surrounded by
Hormones and Reproduction of Vertebrates
a layer of 5–7 smooth muscle cells. The organ is wellvascularized. The epithelium lining the epididymis varies greatly among species both with respect to the distribution of ciliated vs. nonciliated cells and with the presence and size of cytoplasmic secretory granules (Dufaure & Saint Girons, 1984). The most abundant cell type in the reptilian epididymis, accounting for > 80% of the epithelium, is variously identified as a principal (Akbarsha et al., 2006a), vesicular (Holmes & Gist, 2004), columnar (Guerrero et al., 2004), or secretory cell (Desantis, Labate, M., Labate, G., & Cirillo, 2002). The shape of principal cells changes from columnar in the caput epididymis to cuboidal distally; they possess microvilli and appear to have tight junctions. Some may be ciliated. In all species examined, the apical cytoplasm of principal cells contain many small (< 1 mm) vesicles, some with coated membranes. More basally, multivesicular bodies and large (> 1 mm) vesicles are numerous. These properties are suggestive of an endocytotic function. In C. picta, spermiophagy by principal cells (Holmes & Gist, 2004) has been reported. In addition, principal cells of most species contain varying quantities of rough endoplasmic reticulum (RER) and apically located secretory vesicles; some have secretory blebs. Most of the variation in epididymal histology that exists among reptiles lies in the presence and nature of secretory granules. Dufaure and Saint Girons (1984) examined the epididymides of 89 reproductively active squamates (lizards and snakes) and classified the secretory granules in the cytoplasm into five categories ranging from no granulation to cells containing several large (10–12 mm) secretory granules. Subsequent studies have documented extensive variation in other groups; for example, no secretory granules have been detected in the caiman (C. crododilus) (Guerreo et al., 2004) or turtle (C. picta) epididymis (Holmes & Gist, 2004). Granules 4–8 mm in diameter are found in the lizard (Lacerta vivipara), and smaller granules are reported in the initial segment and caput regions of another turtle, Lissemys punctata punctata (Akbarsha & Manimekalai, 1999). The second most abundant cell in the reptilian epididymal epithelium is a basal cell, so identified on the basis of its location. As in mammals, this cell is believed to replenish the principal cells. Three other cell types, similar to their mammalian counterparts and collectively accounting for about 10–20% of the epithelium, have been identified in the reptilian epididymis. The first is a narrow cell possessing an elongated nucleus and dense cytoplasm. It is found in the cauda epididymis of C. picta (Holmes & Gist, 2004) and in the initial segment of S. ponticeriana (Akbarsha et al., 2006b). The second is a cell containing numerous mitochondria and relatively few vesicles. In S. ponticeriana it is expanded apically and is restricted to the initial segment, whereas in C. picta it is columnar and
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FIGURE 5.3 Epididymis of the lizard Sitana ponticeriana. ED, efferent ductules; IS, initial segment of epididymis; CA, caput epididymis; CO, corpus epididymis. Cauda epididymis not shown. Bar ¼ 0.17 mm. Reproduced with permission from Akbarsha, Kadalmani, & Tamilarasan (2006).
found in the cauda epididymis. A third cell type, a clear cell, is found in the cauda epididymis of S. ponticeriana; it contains endocytotic vesicles, multivesicular bodies, and lysosomes (Akbarsha & Manimekalai, 1999). The functions of these latter three cell types are unknown.
4.3. Ductus Deferens Caudally, the ductus epididymis continues as the ductus deferens. It terminates in the copulatory organ, where a penile groove (sulcus spermaticus) receives the sperm and transfers them to the female. The ductus deferens is relatively undifferentiated in most reptiles, consisting of a simple epithelium, a thin lamina propria, and smooth muscle of varying thickness. With the exception of the renal sex segment of squamates, there are no accessory glands along the ductus deferens contributing to the seminal fluid. As a result, reptilian semen tends to be highly viscous, with few fluid components. There have been few
detailed studies on the epithelial cells lining the ductus deferens, but most consider the pseudostratified epithelium to consist of cells similar to the principal and basal cells of the epididymis (Volsoe, 1944; Fox, 1977; Sever, 2004; Gist, unpublished observations). However, in a detailed examination of the lizard Mabuya carinata, Aranha and coworkers describe five different cell types in the epithelium lining the ductus deferens, with narrow cells, apical cells, and clear cells (intraepithelial leukocytes) in addition to principal and basal cells (Aranha, Bhagya, Yajurvedi, & Sagar, 2004; Aranha, Bhagya, & Yajurvedi, 2006). In a few squamate reptiles, the terminal portion of the ductus deferens is swollen and structurally differentiated to form an ampulla that is used for sperm storage. In the rattlesnake (Crotalis durissus), the entire ductus deferens functions in sperm storage (Almeida-Santos, LaportaFerreira, Antoniazzi, & Jared, 2004). The morphology of the ampulla varies among species. In the black swamp snake Seminatrix pygaea, the epithelium of the ampulla
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remains undifferentiated as in the ductus deferens, but is highly folded and truncated and sperm are stored in the lumen throughout the year (Sever, 2004). In the lizard Calotes versicolor, the ampulla is differentiated into storage (ductal) and secretory portions, the latter containing epithelial secretory cells that vary with reproductive condition (Akbarsha & Meeran, 1995). Spermiophagy has been observed. Both transport and at least three types of secretory function are suggested by the ultrastructure of the epithelial cells in the secretory portion of the ampulla of the lizard S. ponticeriana (Akbarsha, Tamilarasan, Kadalmani, & Daisy, 2005). In the ampulla of lizards, sperm are absent except during reproductive activity.
4.4. Reproductive Cycles and Hormonal Dependence In most reptilian species, the excurrent canal system varies greatly during the reproductive cycle. The variations are associated with specific phases of the reproductive cycle and are orchestrated by the secretion of androgenic hormones. Androgen dependence of the male reproductive tract has been demonstrated in a number of ways. In reptiles possessing an associated reproductive cycle, hypertrophy of the epididymis is associated with gonadal recrudescence, with peak size in most species being found at the time of highest T levels (e.g., Dufaure, Courty, Depeiges, Mesure, & Chevalier, 1986). Castration of reproductively active lizards results in an atrophy of the hypertrophied epididymis as the result of a diminished diameter of the ductus epididymis and a regression of its epithelium (Morel, Courty, Mesure, & Dufaure, 1987), while the same operation in nonreproductive lizards has little or no effect. Administration of FSH to sexually quiescent lizards results in hypertrophy of the efferent ductules and the ductus epididymis, resulting from increased epithelial height and, in the latter, tubule size (Haider & Rai, 1987). Folliclestimulating hormone- or T-induced increases in epididymal size are blocked by the antiandrogens flutamide or cyproterone acetate (Rai & Haider, 1991; 1995). The hemipenes of lizards became enlarged in response to T (Ananthalakshmi, Sarkar, & Shivabasaviah, 1991). In sum, these data indicate that the male reproductive tract is sensitive to the male sex hormones and undergoes seasonal changes specifically in response to them. In reptiles possessing dissociated reproductive cycles, the hormonal dependence is not as strong. For example, in the turtles Stenotherous odoratus (McPherson & Marion, 1981; McPherson, Boots, MacGregor, & Marion, 1982), C. picta (Callard, I., Callard, G., Lance, & Eccles, 1976; Gist, Dawes, Turner, Sheldon, & Congdon, 2001), and Chelydra serpentina (Mahmoud & Cyrus, 1992; Mahmoud & Licht, 1997), plasma androgen levels do not closely
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follow testicular or epididymal sizes. The same is true for the garter snake, Thamnophis sirtalis (Krohmer, Grassman, & Crews, 1987). However, T administration to immature soft-shelled turtles (L. p. punctata) will stimulate development of the efferent ductules and ductus epididymis (De & Maiti, 1985). The asynchrony between hormone levels and epididymal stimulation may relate to the ability of the epididymis to store sperm from one reproductive season to the next.
4.5. Epididymis The granulations in the principal cells of the lizard epididymis have been the subject of intense study. Through the efforts of Dufaure and his associates, the epididymis of the viviparous lizard (L. vivipara) has emerged as a classic example of steroid effects on secretory cells. The secretory granules of the principal cells in this species at the height of the breeding season are large (5–7 mm) and occupy the apical cytoplasm of the principal cells (Mesure, Chevalier, Depeiges, Faure, & Dufaure, 1991). The granules first make their appearance as the testes are undergoing recrudescence; castration at that time prevents their appearance and replacement therapy with testosterone or other androgens results in their reappearance (Dufaure et al., 1986). They are found in all regions of the epididymis except for the cauda epididymis. The granules themselves are membrane-bound and consist of a central, insoluble protein (protein H) surrounded by a soluble protein (protein L) (Gigon-Depeiges & Dufaure, 1977). While little is known of protein H, the soluble form, protein L, has been shown to be a mixture of preproteins, all of approximately the same size (19 000 daltons) and immunological properties, but differing in their pI (Depeiges, Morel, & Dufaure, 1988). Secreted L proteins, identified as lizard epididymal secretory proteins (LESP), include both glycosylated and phosphorylated forms (Ravet, Depeiges, Morel, & Dufaure, 1991; Morel, Dufaure, & Depeiges, 1993). All are known to be members of the lipocalin family of proteins, a family of low-molecular-weight proteins whose function broadly is to transport small lipophilic molecules. Included among the lipocalin proteins are retinol-binding protein and rat epididymal secretory protein I, an androgen-induced protein that has amino acid and mRNA similarities to LESP (Morel et al., 1993; Morel, Dufaure, & Depeiges, 2000). In L. vivipara at the time of maximal sexual activity, the contents of the secretory granules (containing both insoluble H and soluble LESPs) are discharged into the lumen of the epididymis, where they mix with sperm and bind to sperm heads (Depeiges & Dufaure, 1983). This androgeninduced secretion of proteins that bind to (and potentially influence) sperm closely resembles what occurs in mammals during epididymal sperm maturation (Robaire & Hermo, 1988). Whether this binding confers additional
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properties to the sperm remains undetermined, although sperm have been reported to increase their motility as they traverse from the caput to the corpus epididymis (Depeiges & Dacheux, 1985). The details of the androgen stimulation of epididymal secretory granule formation and the synthesis of its proteins are known. The annual cycle of the epididymis of L. vivipara has been divided into 10 stages (Figure 5.4) on the basis of histological and cytological characteristics of principal cells, and related to tissue and circulating testosterone levels (Dufaure et al., 1986; Faure, Mesure, Tort, & Dufaure, 1987). During the period of sexual inactivity characteristic of mid-summer (stage 1), androgen levels are low but the principal cells begin to divide and hypertrophy. Later on, in the fall, (stage 3) but prior to hibernation, androgen levels remain low but secretory granules make their appearance in the cytoplasm. What stimulates these initial cytological changes remains unknown. Following hibernation (March; stage 4), androgen levels are increased and at the same time increased nucleolar volume, 3H-thymidine incorporation, DNA content per cell, and number of nuclei are noted in the principal cells along with the formation of mature secretory granules. Maximal stimulation (stage 5) occurs in May, followed by release of the secretory granules; subsequently, androgen levels decline, the nuclei cease DNA synthesis, nuclei and nucleoli both become reduced in size, and the cells become necrotic (July; stage 9). In the cytoplasm of principal cells, protein synthesis, evidenced by amino acid incorporation into secretory granule protein (Gigon-Depeiges & Dufaure, 1977; Ravet, Courty, Depeiges, & Dufaure, 1987), is underway by stage 3, along with increased amounts of rough endoplasmic reticulum, Golgi formation, and, later, the condensation of vesicles. This is accompanied by accelerated mRNA synthesis, including mRNA specific for LESP (Courty, Morel, & Dufaure, 1987; Courty, 1991). Castration or the administration of the anti-androgen cyproterone acetate obliterates, and testosterone restores, these changes (Morel et al., 1987). The mature granules, consisting of the insoluble protein H at the core and protein L at the periphery, are released from the apical surface of the principal cell at stage 6 (May) into the lumen of the epididymis. Here they bind to sperm heads (Depeiges & Dufaure, 1983; Dufaure et al., 1986). A different mode of secretory granule formation is reported by Akbarsha et al. (2006a) for the fan-throated lizard, S. ponticeriana. Here, the soluble and insoluble proteins are synthesized in separate vesicles that coalesce within the principal cell to form the mature, secretory vesicle. The mature, secreted product in this species is the soluble form of the protein which, in turn, is replaced in vesicles by degradation of the insoluble protein.
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Since the epididymis is an androgen-dependent organ that in mammals and certain lizards is involved in sperm maturation, any changes in sperm occurring during epididymal transport are presumed to be hormonedependent. Whether this is true awaits experimental verification. The presence of hormone receptors has been reported for a number of species. Androgen receptor (AR) levels of 85 fmol/mg protein were reported in the epididymis of L. vivipara (Courty, 1991) at stage 6, the peak of both sexual activity and plasma T levels, but similar studies in other squamates are lacking. Estrogen receptor (ER) levels of 20 fmol/mg protein are present in epididymal cytosol from the turtle C. picta during the autumn (Dufaure, Mak, & Callard, 1983) when sperm are undergoing maturation, and both ERa and ERb are reported to be present in epithelial nuclei from the same species (Gist, Bradshaw, Morrow, Congdon, & Hess, 2007) following spermatogenesis. The function of estrogens in the reptilian epididymis remains unknown but, in mammals, estrogens, through ERa, promote fluid uptake from the efferent ductules and epididymis (Hess et al., 2003). Sperm acquire motility as they move through the epididymis of L. vivipara (Depeiges & Dacheux, 1985) or Hemidactylus flaviviridis (Nirmal & Rai, 1997), but actual motility is highly variable. Motility values are reported to be in the 70% range (percent of total sperm) in the corpus epididymis of the lizards H. flaviviridis and L. vivipara, approximately 40% in whole epididymides of the turtles T. scripta (Gartska & Gross, 1990) and S. odoratus (Gist, Turner, & Congdon, 2000), and in the 1–10% range in sperm harvested from the epididymides of C. picta or T. scripta (Gist et al., 2000). Phosphodiesterase inhibitors such as caffeine or 3-isobutyl-1-methylxanthine increase motility whenever examined (Depeiges & Dacheux, 1985; Gist et al., 2000), suggesting a cAMP-dependent mechanism for sperm motility. Whether hormones have any direct effect on sperm motility or its acquisition is not known. Other hormone receptors present in the reptilian epididymis include natriuretic peptide and endothelin receptors in the turtle Amyda japonica (Kim, Kang, Lee, & Cho, 2000). This may be relevant since sodium is reported to stimulate sperm motility in the lizard H. flaviviridis (Rai & Nirmal, 2003). In summary, there is abundant evidence that the male sex hormones functionally control epididymal function, at least in lizards. They stimulate secretion of epididymal proteins by principal cells in those species containing prominent secretory granules and may do so in species containing less prominent secretory granules. The function of epididymal secretions, however, remains unknown. Although sperm acquire some motility as they move through the ductus epididymis, there is little evidence of a hormonal effect on the acquisition or maintenance of
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FIGURE 5.4 Stages 1–10 of the annual cycle of principal cells from the epididymis of the lizard Lacerta vivipara. Changes in secretory granule, nuclear, and nucleolar morphology and activity are related to the level of plasma testosterone (dotted line). The time of year is given on the X axis from August (A) to July (J). Reprinted with permission from Faure, Mesure, Tort, & Dufaure (1987).
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sperm motility, a property that in mammals is associated with fertility. The other function of the epididymis is sperm storage. Following spermatogenesis and release from the testis, sperm are stored in the epididymis, vas deferens, or ampulla until mating. This interval can be shortdas in reptiles possessing an associated reproductive cycledor long (overwinter or longer)das in reptiles with a dissociated cycle (Crews, 1984). In the garter snake T. sirtalis, spermatogenesis is completed in late summer (Krohmer et al., 1987). Although mating can occur in the autumn, most individuals mate upon emergence the following spring. Thus, sperm remain in the vas deferens of the male over the winter. In the freshwater turtles C. picta and T. scripta, spermatogenesis is completed in the fall; however, matings can occur at any time throughout the year. In these species, sperm may be found in the epididymis throughout the year (Gist et al., 2001). What maintains the viability/fertility of sperm held in the epididymis is not known. One factor may lie in the gametes themselves. Whereas vertebrate spermatozoa are typically short-lived (Harper, 1982), sperm harvested from turtle (C. picta, T. scripta) epididymides at various times of the year maintained equivalent motility parameters and sensitivity to stimuli for up to 30 days following collection (Gist et al., 2000). In the turtle T. scripta, epithelial cells of the efferent ductules and ductus epididymis possess estrogen receptors (ERa, ERb) (Gist et al., 2007). Estrogens, acting via ERa on the efferent ductules, are essential for fertility in mammals (Hess, 2003).
4.6. Renal Sex Segment The renal sex segment is a structure found only in squamate reptiles. It consists of the terminal portions of the renal tubule and sometimes extends into the collecting ducts of the kidneys and ureters. Cells lining these portions of the tubule contain prominent secretory granules (Figure 5.5). In lizards, comparable regions of the renal tubule in females have some granulation and can respond to androgen stimulation, but not to the same extent as males (Del Conte & Tamayo, 1973; Krohmer, 2004; Sever & Hopkins, 2005). Renal sex segment secretions are released into the ducts and ureter, where they mix with seminal products from the testes. Since the squamate male reproductive tract contains no other sex accessory glands contributing to seminal fluid, the secretion of the renal sex segment represents a major component of the semen that is transferred to the female at copulation. The renal sex segment varies in size and granulation during the reproductive cycle of the male, being greatest at the time of spermiation and less so during other phases of the cycle (see Fox, 1977). It is clearly androgen-dependent (Prasad &
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Reddy, 1972; Khromer, 2004), as castration causes regression and androgen therapy results in hypertrophy. The secretory granules of the renal sex segment cells have been studied histochemically and shown to contain carbohydrate, lipid, and protein components (Weil, 1984; Sever & Hopkins, 2005) that change in proportion during the annual cycle. Studies of changes in renal sex segment morphology and those comparing renal sex segment development to circulating androgen levels reveal that granules are synthesized under androgen stimulation at the level of the RER, are packaged, and undergo maturation in condensing vacuoles into electron-dense, mature granules (Figure 5.5) that occupy progressively greater portions of the cytoplasm (Weil, 1984; Sever, Stevens, Ryan, & Hamlett; Khromer, 2004; Sever & Hopkins, 2005; Sever et al., 2008). Mature secretory granules are homogeneous in some species and are secreted from the cell in an apocrine manner. In other species, the homogeneous granule undergoes vesiculation prior to being released from the cell in a merocrine manner; many squamate reptiles possess both types of secretion. Renal sex segment secretions are released at mating, a time of declining androgen secretion. Following mating, granules of the renal sex segment cells in lizards disappear (Sanyal & Prasad, 1966; Sever & Hopkins, 2005), whereas those of snakes merely become reduced in number (Sever et al. 2002; 2008). Khromer (2004) attributes the retention of granulation in renal sex segment cells of nonreproductive snakes to adrenal androgens. Surprisingly, little is known regarding the function of renal sex segment secretions. It is clear that, in some snakes, renal sex segment secretions form a copulatory plug that effectively blocks the oviduct of the female from subsequent inseminations. In the garter snake, Thamnophis sp., renal sex segment secretions are ejaculated as a bolus following the seminal products. These secretions harden to form a copulatory plug, preventing both subsequent matings and sperm loss (Devine, 1975). In the adder (Vipera berus), the renal sex segment secretions do not form a plug, but induce muscular contractions in the uterus so as to physically prevent sperm entrance (Nilson & Andre´n, 1982). In addition to forming a physical barrier for inseminations, the copulatory plug serves a pheromonal function in that its presence inhibits male courtship behaviors (Devine, 1977; Ross & Crews, 1977; Nilson & Andre´n, 1982). Even less is known regarding the function of renal sex segment secretions in lizards. Earlier suggestions that renal sex segment secretions maintained sperm within the oviduct are consistent with more recent observations of sperm storage within the squamate oviduct (Gist & Jones, 1987), but experimental data are absent. Cuellar, Roth, Fawcett, & Jones (1972) observed a higher motility of epididymal sperm from the lizard Anolis carolinensis incubated in the presence of kidney extracts,
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(a)
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(b)
FIGURE 5.5 Light (a) and electron (b) micrographs of the renal sex segment of the snake Agkistrodon piscivorous. g, sex segment secretory granule; l, lumen of sex segment tubule; rt, renal tubule; s, renal sex segment tubule; v, vacuole in renal sex segment cell. Bar ¼ 50 mm (a) and 5 mm (b). Micrographs courtesy of David Sever.
suggesting an activating effect. However, the study by Connor and Crews (1980) of sperm transfer in this same species made no mention of renal sex segment secretions.
4.7. Femoral Glands Femoral glands are epidermal structures present on the hindlimbs of certain, primarily iguanid, lizards. Found in both sexes, the femoral glands of males tend to be larger and vary to a greater extent with reproductive condition than those of females, despite the fact that femoral glands of females are sensitive to androgens (Fergusson, Bradshaw, & Cannon, 1985). Earlier literature on femoral glands and their function were reviewed by Cole (1966). Femoral glands are exocrine, secreting in a holocrine manner a waxy mixture of lipids and protein to the exterior that may be smeared on vegetation or substrate (Alberts, Sharp, Werner, & Weldon, 1992; Imparato, Antoniazzi, Rodrigues, & Jared, 2007). Femoral gland secretions have a pheromonal function. The tongue flick response (number of tongue flicks recorded in response to exposure to femoral gland secretions or extracts) has been used as a behavioral assay of femoral gland function. Exposure of lacertid lizards (Podarcis hispanica) to femoral gland secretions indicates that these secretions can serve to identify sex and reproductive condition since males were able to distinguish nongravid females from males or gravid females (Cooper & Pe`rez-Mellado, 2002). Further, females of this species react to cholesta-5,7-diene-3-ol, a component of male femoral
gland secretions, in a dose-dependent manner, suggesting a role in mate selection (Martin & Lopez, 2006). While reports suggest a complex role(s) for femoral gland secretions, it seems clear that sex recognition is among them. Femoral glands are under androgen control. Castration causes atrophy of the femoral glands in the male lizard Ampibolurus ornatus and a cessation of secretion, and replacement therapy with either T or DHT reverses the degeneration (Fergusson et al., 1985). The size, secretory ability, and quantity of lipid in the secretion from femoral glands of the iguana (Iguana iguana) as well as the degree of social dominance are all correlated with circulating T levels (Alberts, Pratt, & Phillips, 1992).
4.8. Unresolved Questions Despite the well-documented action of T in stimulating epididymal protein synthesis in lizards, our knowledge of reptilian sperm maturation and capacitation is in its infancy. Based on the variability in epididymal anatomy and function, one may anticipate that different mechanisms of sperm maturation may exist among different reptilian groups as well as within a given group. Many reptilian species are becoming increasingly endangered, partly because of human predation, which targets larger, reproductively active individuals. To facilitate their recovery, it may be necessary to utilize assisted reproductive techniques such as sperm preservation and artificial insemination coupled with captive breeding programs. As they exist
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today, these techniques are based on a sound understanding of the endocrine and other influences on sperm maturation and/or fertility; therefore, their use in reptiles awaits further research in this area.
5. THE FEMALE The reptilian oviduct receives eggs ovulated from the ovary and conveys them to the cloaca, where they are expelled to the environment. The oviducts are derived from the mesonephric ridge (Fox, 1977). In adults, they are typically paired and open rostrally to the coelom in the vicinity of the ovaries and caudally to the cloaca. In snakes, the oviducts fuse into a single uterus that in turn communicates with the cloaca. In other squamates, the left oviduct is reduced or absent. The basic oviduct structure consists of a simple epithelium lining the lumen and an underlying lamina propria. Together, these two elements are referred to as the mucosa, and the compound glands lying within the lamina propria as mucosal glands. The lamina propria in turn is surrounded by an inner circular and outer longitudinal layer of smooth muscle. The oviduct is covered on the exterior by the serosa. Ovulatory patterns in reptiles are diverse, ranging from monoallochronic, in which the ovaries alternate in ovulating one egg as in A. carolinensis, to polyautochronic, in which many eggs are ovulated from both ovaries simultaneously, as in sea turtles. Fertilization of ova is thought to occur at the level of the ovary or at the infundibulum of the oviduct, although some have suggested alternative
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locations within the oviduct. Following ovulation, additional materials such as egg white proteins may be added prior to shell formation. These additional materials, as well as eggshell membranes and mineral components, if any, are secreted by the wall of the oviduct. Unlike the male sperm duct, in which a summary of recent information is lacking, the reptilian oviduct is the subject of two excellent reviews (Blackburn, 1998; Girling, 2002) covering recent findings and concentrating on squamates. Because of this, the present review will emphasize findings from non-squamate reptiles, the oviduct of which differs in some respects. Nomenclature of the various regions of the reptilian oviduct has led to confusion largely because of species differences (Figure 5.6). Girling (2002) has addressed these problems and proposed terminology that will be used in this review.
5.1. Infundibulum The infundibulum is found at the anterior end of the oviduct and receives the ovulated egg. The infundibulum is variable in length, ranging from 12% of the oviduct length in the snake to 20% in the turtle and 25% in the lizard (Gist & Jones, 1987; Perkins & Palmer, 1996; Blackburn, 1998). It is thin and highly convoluted, and opens via the ostium to the coelom in the vicinity of the ovary. Near the time of ovulation, the ostium moves to cover the ovarian surface and receive ovulated eggs (Cuellar, 1970; Alkindi, Mahmoud, Woller, & Plude, 2006). The infundibulum is devoid of glands in the lamina propria and because of this the wall of the infundibulum is thin. The simple epithelium
FIGURE 5.6 Vitellogenic ovaries and oviducts from the turtle Trachemys scripta. f, infundibulum; i, isthmus; p, vitellogenic follicle; ut, uterine tube; u, uterus; v, vagina. Scale ¼ 1 mm.
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consists of cuboidal to columnar ciliated and nonciliated cells, the proportion of which varies with species and reproductive condition. Epithelial cells at the ostium are mostly ciliated, and the proportion declines caudally (Motz & Callard, 1991; Girling, Cree, & Guillette, 1998; Alkindi et al., 2006). Nonciliated cells react with Periodic Acid Schiff reagent, can contain secretory blebs, and possess other ultrastructural features of secretory cells (Palmer, Demaco, & Guillette 1993; Girling et al., 1998); the secretion is most likely mucous, and commences at the time of ovulation. The infundibular epithelium may be thrown into folds; these folds are more numerous caudally and prior to ovulation. In some squamates, the cavities and secondary
Hormones and Reproduction of Vertebrates
tubules in between folds can act as sperm receptacles (see Gist & Jones, 1987; Sever & Hamlett, 2002). The smooth muscle layer of the infundibulum is typically reduced.
5.2. Uterine Tube Caudal to the infundibulum is the uterine tube. This portion of the oviduct is absent in squamate reptiles, in which the infundibulum connects directly to the uterus. In crocodilians and turtles, the uterine tube contains glands that secrete egg white proteins and is homologous to the avian magnum. The uterine tube occupies approximately 40% of the length of the oviduct in both alligators and turtles (Gist
FIGURE 5.7 Transverse section through the uterine tube of the turtle Chrysemys picta, showing longitudinal folds. l, oviduct lumen; t, tubuloalveolar glands in the lamina propria. Bar ¼ 0.1 mm.
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& Jones, 1987; Palmer & Guillette, 1992). The simple epithelium of the uterine tube consists of ciliated and nonciliated columnar cells. The proportion of ciliated cells increases in preovulatory animals (Motz & Callard, 1991; Alkindi et al., 2006) and the nonciliated cells most likely secrete mucus. The oviduct wall in the uterine tube is thrown into a number of longitudinal folds that generally run the length of the uterine tube (Figure 5.7). These are broken by shallow and deep furrows that occasionally contain ducts from tubuloalveolar glands in the lamina propria underlying the epithelium. These glands are extensive and can account for up to 80% of the volume of the oviduct wall in vitellogenic animals (Motz & Callard, 1991). The cells of the glands contain prominent secretory granules (Palmer & Guillette, 1992; Gist & Fischer, 1993) that resemble those in the avian magnum. The glands are believed to be the source of egg white proteins. Mahmoud, Paulson, Dudley, Patzlaff, and Alkindi (2004) identified 11 different proteins present in the epithelium and tubuloalveolar glands of the uterine tube of the turtle C. serpentina that were similar to those found in eggs. Most were present throughout the year. Based on their molecular weights, at least one of these proteins has been identified as an ovalbumin-like protein (Rose, Paxton, & Britton, 1990) and another as transferrin (Ciuraszkiewicz et al., 2007). Avidin is likely to be another since it is present
(a)
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in the ‘tube’ portion of the lizard oviduct (Botte, Segal, & Koide, 1974). In turtles, the glands communicate with the oviduct lumen via ducts and through breaks in the epithelium, whereas in alligators there is a prominent duct system conveying tubuloalveolar secretions to the oviduct lumen (Figure 5.8). At either end of the uterine tube, the glands become less numerous and occupy less of the lamina propria. At the caudal end, ducts connecting the tubuloalveolar glands to the oviduct lumen become both prominent and more numerous. These tubuloalveolar glands with prominent ducts function in sperm storage (Gist & Jones, 1989; Gist, Bagwill, Lance, Sever, & Elsey, 2008). Exterior to the glands are thin but prominent layers of smooth muscle. As reptilian eggs are ovulated, they enter the infundibulum and begin their descent down the oviductal tube. Of the myriad of studies of reptilian reproductive events, only a few have mentioned observing eggs in the infundibulum or uterine tube (Palmer et al., 1993). Thus, the conclusion must be made that eggs traverse these regions of the oviduct rapidly, and spend most of their time in the uterine portion of the oviduct, where they receive eggshell investments. There is little doubt that the megalecithal egg descending though the uterine tube produces a distension of the wall. Such distension is thought to account for secretion of the egg white proteins from the glands (see Palmer &
(b)
FIGURE 5.8 Micrographs of the uterine tube of (a) the turtle Chrysemys picta and (b) Alligator mississippiensis, showing the organization of the tubuloalveolar secretory ducts. Arrows, secretory ducts; s, secretory cells. Bars ¼ 10 mm.
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Guillette, 1991). However, in crocodilians, these same glands are connected to the lumen via well-formed ducts, suggesting a more complicated mechanism of secretion (Figure 5.8). Questions that remain today regarding this region of the oviduct include whether species without a uterine tube secrete egg white proteins, how egg white proteins are secreted, and how egg white proteins are allocated among the many eggs comprising an egg clutch.
5.3. Isthmus Located between the uterine tube and the uterus proper is an aglandular region known as the isthmus. Since squamate reptiles lack a uterine tube, some authors have questioned whether an isthmus exists in these taxa. This issue is addressed by Blackburn (1998) and will not be dealt with here. The isthmus of turtles and crocodilians is typically short (less than 5% of total oviduct length). The simple epithelium is identical to that of the uterine tube, consisting of columnar ciliated and nonciliated cells. The lamina propria is devoid of glands, and the muscle layers are similar to those of the uterine tube.
5.4. Uterus The uterus represents a major portion of the reptilian oviduct, and accounts for approximately 60% of its length. The uterine epithelium is similar to that in other regions of the oviduct, consisting of varying combinations of ciliated and secretory nonciliated columnar cells. The lamina propria is filled with glands that are variously described as tubuloalveolar to branched acinar (Girling, 2002). The mucosa of the uterus is thrown into randomly arranged mounds separated from each other by furrows. The glands deliver their secretions to the lumen via ducts. The thickness of the uterine epithelium, the number of glands in the lamina propria and the nature of their secretion, and the degree of vascularization are highly variable depending on the parity mode of the reptile (Blackburn, 1998). The wall of the uterus is thicker than the preceding portions of the oviduct because of the enlarged smooth muscle layers. The uterus is the portion of the oviduct involved in eggshell formation. Reptilian eggshells have an inner fibrous protein and an outer calcareous component, each of which is highly variable among species and parity modes (Packard & DeMarco, 1991). The synthesis of these different components of the eggshell differs among crocodilian and non-crocodilian reptiles. The uterus of squamate and testudine reptiles is a single structure in which protein secretion and formation of the calcareous portion of the eggshell are separated in time, whereas in crocodilians the protein and calcareous components are produced in different regions of the uterus (Palmer et al., 1993).
Hormones and Reproduction of Vertebrates
Only a few studies have focused on the cellular origin of these eggshell components. There is little doubt that the glands of the lamina propria secrete the eggshell protein fibers. In the lizard Sceloporus woodi, Palmer et al. (1993) observed proteinaceous fibers emanating from the ducts of uterine glands 12 hours following ovulation. They speculated that these fibers covered the egg as the egg rotated in the uterus. Similar observations have been made in the turtle C. serpentina (Alkindi et al., 2006) and in the anterior portion of the alligator (Alligator mississippiensis) uterus (Palmer & Guillette, 1992). Even less is known of the cellular origins of the calcium that forms the shell. Guillette, Fox, & Palmer (1989) found that postovulatory epithelial cells of the uterus in the lizard Crotaphytus collaris stained more intensely with Alizarin red S, a calcium stain, than other regions of the uterus. Immunofluorescent detection of Caþþ ATPase pumps indicates maximal activity in the apical and basolateral surfaces of uterine epithelial cells in lizards (Lampropholis guichenoti) containing shelled eggs (Thompson, Lindsay, Herbert, & Murphy, 2007). More direct measurements of calcium concentrations by atomic absorption spectrophotometry in uterine epithelial tissue from the turtle C. serpentina (Alkindi et al., 2006) confirm high concentrations of Caþþ in uteri containing unshelled eggs and lower levels in uteri containing shelled eggs. Possible endocrine controls of protein deposition and calcification within the same uterus remain unstudied. In contrast, the uterus of crocodilians is separated into anterior and posterior components and deposition of the two eggshell components is a function of egg location. The two regions of the uterus are similar except for the thicker layer of tubuloalveolar glands and myometrium in the posterior uterus (Palmer & Guillette, 1992). Fiber deposition is thought to occur in the anterior uterus while egg calcification is thought to occur in the posterior uterus. Palmer and Guillette (1992) observed calcification of the eggshell in the posterior uterus of A. mississippiensis. The caudal terminations of the uterine glands in the oviduct of turtles and crocodilians are similar in morphology to the termination of the glands of the uterine tube noted above. The glands are less numerous, have prominent ducts open to the uterine lumen, and can serve as locations for sperm storage (Gist & Congdon, 1998; Gist et al., 2008).
5.5. Vagina The terminal portion of the reptilian oviduct, the vagina, is short, devoid of glands in the lamina propria, and highly muscular. The paired vaginae (except in snakes) serve as a connector to the cloaca and open directly to it, acting like a sphincter. The simple epithelium of the vagina is composed of cuboidal to columnar cells, most of which are
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Hormones and the Sex Ducts and Sex Accessory Structures of Reptiles
ciliated. The mucosa is thrown into high folds running longitudinally. In some squamates, the grooves between the folds can serve as sperm storage structures and in still others the grooves can become pinched off into tubules that serve the same function (Gist & Jones, 1987; Sever & Hamlett, 2002). The vagina is surrounded by thick inner circular and outer longitudinal layers of smooth muscle.
5.6. Reproductive Cycles and Hormonal Dependence The secretion of female sex hormones is linked to events of the ovarian cycle. Estradiol, T, the aromatizable precursor to E2, and P4 are the most commonly measured hormones; in most species, circulating levels of P4 > T > E2. The pattern of annual changes in these hormones is variable depending on the duration of ovarian events, the number of egg clutches, and the latitude inhabited by the female (Arslan, Zaidi, Lobo, Zaidi, & Qazi, 1978; Callard, Lance, Salhanick, & Barad, 1978; Licht, Wood, Owens, & Wood, 1979; Bona-Gallo, Licht, MacKenzie, & Lofts, 1980; McPherson et al., 1982; Cree, Cockrem, & Guillette, 1992; Guillette et al., 1997; Radder, Shanbhag, & Saidapur, 2001; Taylor, DeNardo, & Jennings, 2004; Alkindi et al., 2006; Ganesh & Yajurvedi, 2007). The endocrinology of the reptilian ovary is addressed in Chapter 4, this volume and only an outline will be presented here. Both estrogens and testosterone are secreted by the vitellogenic ovarian follicle. Vitellogenic follicles are those that incorporate the yolk precursor, vitellogenin, into the yolk. Vitellogenesis itself is under estrogenic control; this aspect of estrogenic action is discussed in Chapter 4, this volume. Levels of estrogens in the blood typically become elevated during vitellogenesis, reaching a peak prior to or at the time of ovulation, and then decline as eggs remain in the uterus. In species possessing multiple egg clutches, spikes of estrogens are associated with each ovulatory event. Progesterone levels rise close to the time of ovulation and are short-lived in oviparous species, lasting longer in viviparous reptiles, and decline shortly before oviposition. Vitellogenic follicles typically enlarge rapidly as they accumulate yolk, leading to the formation of a megalecithal egg in which virtually the entire volume of the egg is occupied by yolk. Following ovulation, the follicular wall forms a corpus luteum that persists for varying intervals. The corpora lutea of viviparous reptiles persist longer than those of oviparous forms (Callard et al., 1992). There is little doubt that the corpus luteum can synthesize and secrete P4. Secretion in oviparous forms reaches a peak prior to ovulation whereas, in viviparous forms, with longer-lasting corpora lutea, elevated P4 levels are observed postovulatorily (Callard et al., 1992).
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5.7. Oviduct It is well-established that oviductal functions are influenced by ovarian sex steroids. The reader is referred to Botte (1974) for a review of earlier literature. Nevertheless, there are only a few studies examining specific estrogen or P4 effects on the oviduct. Mead, Eroshenko, and Highfill (1981) examined oviductal histology following administration of E2 or P4 to ovariectomized garter snakes (Thamnophis elegans). Estradiol was partially effective in reversing the regressive changes in the oviduct following ovariectomy, but P4 was not. A combination of the two hormones was no more effective than E2 alone. Administration of E2 to intact painted turtles (C. picta) in the fall stimulated the tubuloalveolar glands of the uterus to levels seen in the summer but had no effect on the uterine tube (Motz & Callard, 1991). Estradiol also stimulates myometrial contractility in this same species (Callard & Hirsch, 1976), whereas P4 suppresses uterine contractility but has no effect on the uterine glands. More recent studies have focused on the location of and changes in steroid hormone receptors in the reptilian oviduct. The reptilian oviduct contains receptors for T, E2, and P4. Both nuclear and cytoplasmic ARs are reported in the oviduct of T. scripta, localized in the glands of the lamina propria but absent from the epithelium lining the oviduct lumen and the myometrium (Selcer, Smith, Clemens, & Palmer, 2005). More information is available for the latter two receptor types, and oviductal ERs are reported in the turtles C. picta (Salhanick, Vito, Fox, & Callard, 1979) and T. scripta (Selcer & Leavitt, 1991) as well as the garter snake T. s. parietalis (Whittier, West, & Brenner, 1991) and the lizard Podarcis sicula (Paolucci, DiFiore, & Ciarcia, 1992). In the alligator, ERs have been characterized from the anterior uterus (Vonier, Guillette, McLachlan, & Arnold, 1997). Progesterone receptors (PRs) are reported using immunocytochemistry in the lumenal epithelium, glands of the lamina propria, and myometrium of the turtle C. picta (Giannoukos, Coho, & Callard, 1995). A PR was demonstrated in the oviduct of the snake Nerodia (Natrix sp.) (Kleis-San Francisco & Callard, 1986), and two forms of the PR were found in the turtle C. picta (Reese & Callard, 1989). Changes in the number of oviductal ERs and PRs over the reproductive cycle or in response to hormonal or surgical manipulation have been examined. Hypophysectomy reduces oviductal ER levels in the turtle C. picta, but replacement therapy with either E2 or P4 is unable to restore them to normal (Giannoukos & Callard, 1996). Hepatic ER levels in the lizard P. sicula rise during spring vitellogenic growth (Paolucci, 1989), while those in the oviduct are highest during winter ovarian quiescence (Paolucci et al., 1992; Paolucci & DiFiore, 1994). Long-term (14 days) administration of E2 to ovariectomized P. sicula during
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quiescence induces an elevation in hepatic ER, but effects on oviductal ER levels are equivocal. On the other hand, in response to a single injection of E2 to intact quiescent P. sicula, oviductal ER expression was elevated 12 hours later. These results suggest a rather complex regulation of ER expression in reptilian reproductive tissues and further studies will be of benefit. More attention has been devoted to the regulation of the PR. Specific oviductal binding of P4 in most species is elevated at the time of ovulation, well in advance of the rise in plasma P4, and this elevation persists for varying periods of time following ovulation (Kleis-San Francisco & Callard, 1986; Paolucci & DiFiore 1994; Giannoukos et al., 1995). Ovariectomy has little or no effect on oviductal PR levels in the lizard P. sicula (Paolucci & DiFiore, 1994), but the same operation increases specific P4 binding by turtle (C. picta) oviducts (Giannoukos & Callard, 1996). In contrast to mammals, administration of E2 to ovariectomized P. sicula has little effect on oviduct PR (Paolucci & DiFiore, 1994), whereas E2 increases oviductal PR levels in the snake Nerodia (Natrix sp.) (Kleis-San Francisco & Callard, 1986). Part of the conundrum may lie in the findings that two forms of the PR have been isolated from the turtle (C. picta) oviduct (Reese & Callard, 1989). One form (PR-A) has a low P4 affinity (2.8 109 M) and is present throughout the reproductive cycle, being elevated at the time of and shortly following ovulation (Reese & Callard, 1989). The other form (PR-B) has a high P4 affinity (28 109 M) and is expressed from the time of ovulation to egg laying, and then again during the autumnal period of ovarian growth (Giannoukos et al., 1995). While the role of estrogens in regulating oviductal PR remains obscure, it seems clear that P4 is influential in regulating oviductal levels of its own receptor. Progesterone injections induce a downregulation of oviductal PR receptors in turtles (Selcer & Leavitt, 1991; Giannoukos & Callard, 1996). Such regulation may play a role in the timing of oviductal secretions.
5.8. Oviposition It has been known for some time that estrogens and P4 have antagonistic actions on the reptilian myometrium. Thus, E2 stimulates and P4 inhibits oviductal contractions in the turtle C. picta (Callard & Hirsch, 1976). Progesterone can reduce the effectiveness of arginine vasotocin (AVT), one of the reptilian neurohypophysial hormones, in stimulating uterine contractions in the same species (Callard et al., 1992) and delays parturition in the viviparous lizard Sceloporus jarrovi (Guillette, DeMarco, & Palmer, 1991). Removal of the corpora lutea reduces the time that eggs are retained in the oviduct, and P4 administration delays oviposition (Roth, Jones, & Gerrard, 1973; Klicka & Mahmoud, 1977; H. S. Cuellar, 1979). Viviparous species,
Hormones and Reproduction of Vertebrates
which retain eggs in the oviduct for longer times than oviparous species, maintain elevated P4 levels for longer periods than oviparous species (Callard et al., 1992). Combined, this evidence suggest that the corpus luteum, by virtue of P4 secretion, prevents premature oviposition. Oviposition and its equivalent in viviparous species, parturition, are under complex neuroendocrine control. This topic has been reviewed most recently by Guillette, Dubois, & Cree (1991). Arginine vasotocin, a potent stimulator of oviductal contractions (Ewert & Legler, 1978; Mahmoud, Cyrus, McAsey, Cady, & Woller, 1988), is most effective if given late in pregnancy (viviparous forms) or during the gravid period (oviparous forms) (Mahmoud et al., 1988; Guillette, DeMarco, Palmer, & Masson, 1992). A similar pattern of responsiveness is seen with the prostaglandin F2a (PGF2a) in the lizard S. jarrovi (Guillette et al., 1992). Further, indomethacin, an inhibitor of prostaglandin synthesis, can delay parturition in this same species (Guillette et al., 1991a). Prostaglandin F2a concentrations in the blood of sea turtles (Guillette et al., 1991c), the tuatara (Guillette et al., 1990a), and the lizard Tiliqua rugosa (Fergusson & Bradshaw, 1991) are all elevated at the time of oviposition. A link between AVT and prostaglandin stimulation is provided by Guillette et al. (1990a), who demonstrated in S. jarrovi that AVT stimulates the oviductal synthesis of PGF2a in vitro and increases plasma PGF2a levels in vivo, a response likewise blocked by indomethacin. Here too, the effectiveness of AVT in inducing uterine contractions was highest near the end of pregnancy and lowest in early pregnancy. The effectiveness of both AVT and PGF2a in stimulating oviductal contractions is greater in vitro than in vivo, leading to speculation that another factor, possibly neural, acts on the myometrium to prevent premature contractions. In the lizard A. carolinensis, injection of AVT will not result in oviposition unless the animal is pretreated with the b-adrenergic antagonist dichloroisoproterenol (Jones, Summers, & Lopez, 1983). Similarly, blockade of b-adrenergic receptors in the oviduct of the gecko (Hoplodactylus maculatus) enhances PGF2a-induced uterine contractions, but not those induced by AVT (Cree & Guillette, 1991). These data have led to the hypothesis that the neuroendocrine mechanisms influencing oviposition are directed to the uterovaginal musculature to regulate egg egress from the oviduct rather than egg expulsion as the result of myometrial activity (Guillette et al., 1991b). Thus, the uterotonic actions of AVT and PGF are inhibited by elevated P4 levels in early pregnancy or gravidity, and are modified by the autonomic nervous system in late pregnancy or gravidity.
5.9. Unresolved Questions Fundamental aspects of oviductal function remain unknown. Coordination of deposition of eggshell protein
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Hormones and the Sex Ducts and Sex Accessory Structures of Reptiles
fibers and calcification by hormones or other mechanisms have yet to be elucidated in squamates or turtles. Our knowledge of the effects of estrogens and P4 on the oviduct are inadequate. The timing and control of expression of both ER and PR in the reptilian oviduct need further study.
6. SPERM STORAGE Reptilian eggs are oviposited either singly or, more typically, in clusters that comprise an egg clutch. Because of the large size of reptilian eggs, multi-egg clutches and multiple egg clutches in a season pose problems with respect to fertilization because eggs may not come into contact with sufficient numbers of sperm without repeated copulations. Some reptiles have addressed this problem by the storage of sperm, either within the oviduct of the female or in the excurrent canals of the male, until ovulation occurs. The reptilian oviduct is capable of storing sperm from matings for extended periods of time. This ability is not unique to reptiles, being common in birds, but reptiles as a group store sperm for longer periods than other vertebrates, up to several years, for example, in turtles (Gist & Jones, 1987). Sperm storage is common among the Reptilia, being found in all families, and is considered an integral
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part of the reproductive process. The locations of sperm storage within the oviduct are highly variable. In squamates, sperm may be found at the appropriate time of year in tubules formed from folds in the vaginal wall or in pouches or tubules located in the infundibulum (Sever & Hamlett, 2002). In Testudinae (Figure 5.9) and Crocodilidae, sperm are stored in ducts of glands located at the caudal terminations of the glandular areas of the uterine tube and uterus (Gist & Jones, 1989; DePerez & Pinilla, 2002; Gist et al., 2008). An infundibular location of sperm storage is found in the soft-shelled turtle L. punctata (Sarkar, Sakar, & Maiti, 2003) and an anterior uterine location has been reported in another soft-shelled turtle, Trionyx sinensis (Han et al., 2008). It has yet to be established whether the host glands or tubules provide sustenance for stored spermatozoa, although Han et al. (2008) reported both sperm maturation and degradation within storage tubules. In some reptiles, stored sperm are found in association with an amorphous carrier matrix (Halpert, Garstka, & Crews, 1982; Kumari, Sarkar, & Shivanandappa, 1990; Sarkar et al., 2003). The epithelial cells of the tubules or glands containing sperm are generally similar to those not containing sperm, and in addition do not differ histochemically. Studies using an electron microscope reveal no contact between stored
FIGURE 5.9 Sperm storage tubule in the uterine tube of the turtle Chrysemys picta. Bar ¼ 25 mm.
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sperm and the surrounding epithelial cells (Bou-Resli, Bishay, & Al-Zaid, 1981; Gist & Fischer, 1993; Sever & Hamlett, 2002). While the possibility that the surrounding cells secrete materials that preserve or maintain sperm cannot be excluded, the available evidence suggests that sperm-storing glands or tubules provide a safe haven for sperm and that survival of sperm is a property of the male gamete, not the female host (Gist et al., 2000; 2001). How sperm move from the storage sites to the location of fertilization, presumably the infundibulum, is likewise unknown. In birds, sperm stored in the vaginal storage tubules evacuate the tubules at a continuous rate (Birkhead & Moller, 1992). Sperm stored in the glands and tubules of the oviduct form a reservoir that can provide sperm for upcoming ovulations. With techniques such as allozyme assay and microsatellite DNA analysis, it has become apparent that most reptilian egg clutches have multiple paternity (Davis, Glenn, Elsey, Dessauer, & Sawyer, 2001; Pearse & Avise, 2001; Laloi, Richard, Lecomte, Massot, & Clobert, 2004; Oppliger, Degen, Bouteiller-Reuter, & John-Alder, 2007; Moore, Nelson, Keall, & Daugherty, 2008). Thus, sperm storage sites may contain sperm from several males and the sperm within them represents an additional level of female mate choice, but at the time of fertilization. In the lizard Ctenophorus pictus, clutches fertilized by stored sperm are male-biased (Olsson, Schwartz, Uller, & Healey, 2008) suggesting differential survival of male and female sperm within the oviduct. The implications of sperm storage have been studied most extensively in turtles. In multiclutched painted turtles (C. picta) and desert tortoises (Gopherus agassizii), fertilization of eggs by sperm stored in the oviductal glands has been demonstrated both across subsequent egg clutches within a single year as well as from one nesting season to the next (Palmer, Rostal, Grumbles, & Mulvey, 1998; Pearse & Avise, 2001; Pearse, Janzen, & Avise, 2002). The former is in support of the conclusion of Gist and Congdon (1998) that insufficient time exists between consecutive clutches for additional matings to occur. In terms of species with only a single annual egg clutch, multiple paternity is reported to occur in the American alligator (Davis et al., 2001) and the turtle C. serpentina (Galbraith, White, Brooks, & Boag, 1993); sperm storage is also reported in these two species (Gist & Jones, 1989; Gist et al., 2008). Single-clutched species tend to have large egg clutches. Clutches of the alligator can contain up to 200 eggs (Lance, 1989) and those of the snapping turtle contain 20–40 eggs. With these large clutches of megalecithal eggs, it is unlikely that sperm residing in the oviduct lumen could fertilize more than the first few eggs descending down the oviduct of these polyautochronic ovulators. Storage of
Hormones and Reproduction of Vertebrates
sperm could account for the high degree of fecundity in these single-clutched species.
6.1. Unresolved Questions The role of hormones in the process of sperm storage is just beginning to be examined. Sarkar et al. (2003) have investigated the movement and storage of sperm within the oviduct of the soft-shelled turtle L. punctata. Sperm had reached the storage areas of the posterior uterine tube 24 hours following mating and thereafter were found in the storage tubules. Estradiol given to quiescent (nonbreeding) females induced a lengthening and widening of the sperm storage tubules. In this species, E2 levels normally peak at or around the time of ovulation (Sarkar, S., Sarkar, N., & Maiti, 1995), and at that time sperm are observed exiting the sperm storage tubules. Thus, by virtue of its stimulatory action on the tubuloalveolar glands of the lamina propria, estradiol may facilitate sperm entrance and egress from storage glands as ovulation approaches. Immunological aspects of sperm storage have yet to be investigated. Further studies will examine more thoroughly the role of ovarian steroids and other hormones in regulating sperm storage. Long-term storage of sperm in the oviducts of females or in the male epididymis poses some interesting problems. Vertebrate gametes are known to be short-lived, maintaining viability and/or fertility outside the male reproductive tract for only minutes to weeks. This contrasts with the finding that turtle sperm can retain their fertility from one year to the next stored in the female oviduct (Pearse & Avise, 2001). Whether this longevity is conferred on the sperm by the local environment (e.g., sperm storage glands, epididymis), is a property of sperm, or is due to oviductal secretions, is unknown. Results of Gist et al. (2000) showing that epididymal sperm from turtles maintain their viability in physiological saline for up to 30 days following isolation suggest that sperm survival may involve intrinsic properties of the sperm cells. Further research into the physiology of reptilian spermatozoa may reveal how male gametes of reptiles as well as non-reptilian species maintain long-term viability.
ABBREVIATIONS AVT E2 ER LESP P4 PG PR RER T
Arginine vasotocin Estradiol Estrogen receptor Lizard epididymal secretory proteins Progesterone Prostaglandin Progesterone receptors Rough endoplasmic reticulum Testosterone
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Chapter | 5
Hormones and the Sex Ducts and Sex Accessory Structures of Reptiles
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D. C. Deeming, & M. J. W. Ferguson (Eds.), ‘‘Egg Incubation: Its Effects on Embrionic Development in Birds and Reptiles’’ (pp. 29–46). Cambridge: Cambridge Univ. Press. Palmer, B. D., & Guillette, L. J., Jr. (1992). Alligators provide evidence for the evolution of an archosaurian mode of oviparity. Biol. Reprod, 46, 39–47. Palmer, B. D., Demaco, V. G., & Guillette, L. J., Jr. (1993). Oviductal morphology and eggshell formation in the lizard, Sceloporus woodi. J. Morphol., 217, 207–217. Palmer, K. S., Rostal, D. C., Grumbles, J. S., & Mulvey, M. (1998). Longterm sperm storage in the desert tortoise (Gopherus agassizii). Copeia, 1998, 702–705. Paolucci, M. (1989). Estradiol receptor in the lizard liver (Podarcis sicula). Seasonal changes and estradiol and growth hormone dependence. Mol. Cell. Endocrinol., 66, 101–108. Paolucci, M., & DiFiore, M. M. (1994). Estrogen and progesterone receptors in lizard Podarcis s. sicula oviduct: seasonal distribution and hormonal dependence. J. Exp. Zool., 269, 432–441. Paolucci, M., & Di Cristo, C. (2002). Progesterone receptor in the liver and oviduct of the lizard Podarcis sicula. Life Sci., 71, 1417–1427. Paolucci, M., DiFiore, M. M., & Ciarcia, G. (1992). Oviduct 17b-estradiol receptor in the female lizard, Podarcis s. sicula, during the sexual cycle: relation to plasma 17b-estradiol concentration and its binding proteins. Zool. Sci., 9, 1025–1035. Pearse, D. E., & Avise, J. C. (2001). Turtle mating systems: behavior, sperm storage, and genetic paternity. J. Heredity, 92, 206–211. Pearse, D. E., Janzen, F. J., & Avise, J. C. (2002). Multiple paternity, sperm storage, and reproductive success of female and male painted turtles (Chrysemys picta) in nature. Behav. Ecol. Sociobiol., 51, 164–171. Perkins, M. J., & Palmer, B. D. (1996). Histology and functional morphology of the oviduct of an oviparous snake, Diadophis punctatus. J. Morphol., 227, 67–79. Prasad, M. R. N., & Reddy, P. R. K. (1972). Physiology of the sexual segment of the kidney in reptiles. Gen. Comp. Endocrinol. Suppl., 3, 649–662. Radder, R. S., Shanbhag, B. A., & Saidapur, S. K. (2001). Pattern of plasma sex steroid hormone levels during reproductive cycles of male and female tropical lizard, Calotes versicolor. Gen. Comp. Endocrinol., 124, 285–292. Rai, U., & Haider, S. (1991). Testis and epididymis of the Indian Wall lizard (Hemidactylus flaviviridis): effects of flutamide on FSH and testosterone influenced spermatogenesis, Leydig cell, and epididymis. J. Morphol., 209, 133–142. Rai, U., & Haider, S. (1995). Effects of cyperoterone acetate on FSH and testosterone influenced spermatogenesis and epididymis in the Indian wall lizard, Hemidactylus flaviviridis (Ruppell). Eur. J. Morphol., 33, 443–455. Rai, U., & Nirmal, B. K. (2003). Significance of regional difference in ion concentrations in lizard, Hemidactylus flaviviridis (Ru¨ppell): assessment of ionic influence on sperm motility in vitro. Indian J. Exp. Biol., 41, 1431–1435. Ravet, V., Courty, Y., Depeiges, A., & Dufaure, J. P. (1987). Changes in epididymal protein synthesis during the sexual cycle of the lizard, Lacerta vivipara. Biol. Reprod., 37, 901–907. Ravet, V., Depeiges, A., Morel, F., & Dufaure, J. P. (1991). Synthesis and post-translational modifications of an epididymal androgen dependent protein family. Gen. Comp. Endocrinol., 84, 104–114.
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Hormones and the Sex Ducts and Sex Accessory Structures of Reptiles
Reese, J. C., & Callard, I. P. (1989). Two progesterone receptors in the oviduct of the freshwater turtle Chrysemys picta: possible homology to mammalian and avian progesterone receptor systems. J. Steroid Biochem., 33, 297–310. Robaire, B., & Hermo, L. (1988). Efferent ducts, epididymis, and vas deferens: structure, functions, and their regulation. In E. Knobil, & J. Niel (Eds.), ‘‘The Physiology of Reproduction’’ (pp. 999–1080). New York: Raven Press. Rose, F. L., Paxton, R., & Britton, C. H., III (1990). Identification of an ovalbumin-like protein in egg white of turtle and alligator. Comp. Biochem. Physiol., 96B, 651–654. Ross, P., & Crews, D. (1977). Influence of the seminal plug on mating behaviour in the garter snake. Nature, 267, 344–345. Roth, J. J., Jones, R. E., & Gerrard, A. M. (1973). Corpora lutea and oviposition in the lizard Sceloporus undulatus. Gen. Comp. Endocrinol., 21, 569–572. Salhanick, A. R., Vito, C. C., Fox, T. O., & Callard, I. P. (1979). Estrogen-binding proteins in the oviduct of the turtle, Chrysemys picta: evidence for a receptor species. Endocrinology, 105, 1388– 1395. Sanyal, M. K., & Prasad, M. (1966). Sexual segment of the kidney of the Indian house lizard, Hemidactylus flaviviridis Ru¨ppell. J. Morphol., 118, 511–528. Sarkar, S., Sarkar, N. K., & Maiti, B. R. (1995). Histological and functional changes of oviductal endometrium during seasonal reproductive cycle of the soft-shelled turtle, Lissemys punctata punctata. J. Morphol., 224, 1–14. Sarkar, S., Sarkar, N. K., & Maiti, B. R. (2003). Oviductal sperm storage structure and their changes during the seasonal (dissociated) reproductive cycle in the soft-shelled turtle Lissemys punctata punctata. J. Exp. Zool., 295A, 83–91. Selcer, K. W., & Leavitt, W. W. (1991). Progesterone downregulates progesterone receptor, but not estrogen receptor, in the estrogenprimed oviduct of a turtle (Trachemys scripta). Gen. Comp. Endocrinol., 83, 316–323. Selcer, K. W., Smith, S., Clemens, J. W., & Palmer, B. D. (2005). Androgen receptor in the oviduct of the turtle, Trachemys scripta. Comp. Biochem. Physiol., 141B, 61–70.
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Sever, D. (2004). Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea). IV. Occurrence of an ampulla ductus deferentis. J. Morphol., 262, 714–730. Sever, D. M., & Hamlett, W. C. (2002). Female sperm storage in reptiles. J. Exp. Zool., 292, 187–199. Sever, D. M., & Hopkins, W. A. (2005). Renal sexual segment of the ground skink, Scincella laterale (Reptilia, Squamata, Scincidae). J. Morphol., 266, 46–59. Sever, D. M., Stevens, R. A., Ryan, T. J., & Hamlett, W. C. (2002). Unltrstructure of the reproductive system of the black swamp snake (Seminatrix pygaea). III. Sexual segment of the male kidney. J. Morphol., 252, 238–254. Sever, D. M., Siegel, D. S., Bagwill, A., Eckstut, M. E., Alexander, L., Camus, A., & Morgan, C. (2008). Renal sexual segment of the cottonmouth snake Agkistrodon piscivorous (Reptilia, Squamata, Viperidae). J. Morphol., 269, 640–653. Taylor, E. N., DeNardo, D. F., & Jennings, D. H. (2004). Seasonal steroid hormone levels and their relation to reproduction in the western diamond-backed rattlesnake, Crotalus atrox (Serpentes: Viperidae). Gen. Comp. Endocrinol., 136, 328–337. Thompson, M. B., Lindsay, L. A., Herbert, J. F., & Murphy, C. R. (2007). Calcium ATPase expression in the oviducts of the skink, Lampropholis guichenoti. Comp. Biochem. Physiol., 147A, 1090–1094. Van Tienhoven, A. (1983). ‘‘Reproductive Physiology of Vertebrates’’ (2nd ed.). Ithaca, NY: Cornell University Press. Vonier, P. M., Guillette, L. J., Jr., McLachlan, J. A., & Arnold., S. F. (1997). Identification and characterization of estrogen and progesterone receptors from the oviduct of the American alligator (Alligator mississippiensis). Biochem. Biophys. Research Comm., 232, 308–312. Volsoe, H. (1944). Structure and seasonal variation of the male reproductive organs of Vipera berus (L.). Spolia Zool. Copenhagen, 5, 9–159. Weil, M. R. (1984). Seasonal histochemistry of the renal sexual segment in male common water snakes, Nerodia sipedon (L.). Can. J. Zool., 62, 1737–1740. Whittier, J. M., West, N. B., & Brenner, R. M. (1991). Immunorecognition of estrogen receptors by monoclonal antibody H222 in reproductive tissues of the red-sided garter snake. Gen. Comp. Endocrinol., 81, 1–6.
Chapter 6
Pheromones and Reproduction in Reptiles Jose´ Martı´n and Pilar Lo´pez Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain
SUMMARY Pheromones are chemicals involved in intraspecific communication in many animals. Reptiles rely more on their chemical senses than any other vertebrate class and many behavioral studies suggest that chemical cues (sex pheromones) are important in the communication and reproduction of many reptiles. However, although there are some descriptions of glandular chemical products that might potentially function as pheromones, there has been little research that has linked these chemicals with their roles in communication and reproductive behavior. Some recent studies, mainly with snakes and lizards, have started to reveal the importance of specific pheromones in reproduction and sexual selection. We review the results of experiments showing that pheromones are involved in sex and individual recognition, territoriality, intrasexual aggression between males, mate choice, and reproductive decisions. Pheromones not only inform on the presence and sex of a conspecific, but also may provide reliable information on the characteristics, quality, and health state of the sender, which other individuals may use in their reproductive decisions. We are also starting to understand how chemical communication systems evolved in reptiles by examining both the diversity of pheromones and the underlying physiological and endocrinological mechanisms involved in their production and expression. These physiological mechanisms, specially the alternative roles of some chemicals in other metabolic functions such as the maintenance of the immune system, may also explain how pheromones have evolved to be used as reliable sexual signals in reproduction.
1. INTRODUCTION 1.1. Pheromones of Vertebrates Pheromones are classically defined as chemicals or semiochemicals produced by one individual that effect a change in the physiology (‘primer’ pheromone) and/or behavior (‘releaser’ pheromone) of conspecifics (Karlson & Lu¨scher, 1959). This definition originated mainly from the study of insects, where very often just one or a pair of chemicals acts as a pheromone attracting the opposite sex. Each insect species seems to have a frequently exclusive pheromone Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
that allows species recognition. In contrast, in vertebrates, pheromones are often a mixture of several chemical compounds with different properties (Mu¨ller-Schwarze, 2006). These multicomponent pheromones may have different functions or intended receivers, but may also act together providing specific or individual ‘odor profiles’, also named ‘gestalts’, ‘patterns’, or ‘mosaics’, (Johnston, 2005). Not only the mixture of chemicals, but also the relative proportions or concentrations of these chemicals, are often needed for them to be biologically active as a pheromone. Moreover, the pattern of compounds in the scent of each individual may signal sex, age, social status, group, seasonality, and condition. Thus, a pheromone in vertebrates may be defined as a group of active compounds in a secretion that supply information to, or change behavior in, another conspecific (Mu¨ller-Schwarze, 2006). There are also important differences between insects and vertebrates in the role of pheromones in reproduction. Chemically mediated mate choice is common in many insects, where pheromones alone can directly control reproductive behavior in many species. However, in vertebrates, due to the multicomponent sensory nature of these animals, reproductive behavior is often mediated by a combination of sensory stimuli (visual, tactile, chemical, etc.) rather than by a single type of stimulus alone. Nevertheless, pheromones are important, and sometimes requisite, for species and sex recognition, mate choice, and effective mating in many species of vertebrate, especially in fish, mammals, some amphibians, and many reptiles (for reviews see Mason, 1992; Wyatt, 2003; Mu¨ller-Schwarze, 2006; see also in this series Volume 1, Chapter 9; Volume 2, Chapters 5 and 8; Volume 5, Chapter 10).
1.2. Chemosensory Abilities of Reptiles: The Vomeronasal System The vomeronasal system is a complex of different structures that forward specific chemical signals (including pheromones) to the central nervous system. Many reptiles have a well-developed vomeronasal system and are able to make 141
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discriminations of many different scents, from prey, conspecifics, or predators, based on chemicals alone (Halpern 1992; Mason, 1992; Cooper, 1994; Schwenk, 1995; Halpern & Martı´nez-Marcos, 2003). In many lizards and snakes, chemoreception works in a hierarchical fashion, with chemicals being received through the nares and processed by the nasal organs, subsequently triggering tongue flick (TF)mediated vomerolfaction (Halpern, 1992; Cooper, 1994; Schwenk, 1995). Tongue flicking allows an individual to obtain information about conspecifics based on pheromonal cues alone (Halpern, 1992; Mason, 1992; Cooper, 1994). In fact, there are studies of lizards reporting that functional (vs. sealed) vomeronasal ducts are necessary for accurate discrimination of prey chemicals and pheromones (Graves & Halpern, 1990; Cooper & Alberts, 1991). The vomeronasal organ (VNO) is important for activating accessory olfactory pathways that are involved in sexually dimorphic reproductive behavior. For example, the VNO of male garter snakes is critically important for detection of, and response to, female sex pheromones (Huang, Zhang, Wang, Mason, & Halpern, 2006). Female pheromone directly affects male, but not female, snake vomeronasal neurons and results in the opening of ion channels, converting the pheromone signal to an electrical signal (Huang et al., 2006). Behavioral tests of chemosensory recognition in lizards and snakes have often been based on the relationship between chemoreception and TF, an easily observable and quantifiable characteristic behavior (Cooper & Burghardt, 1990). The TF rates can be used as a bioassay for measuring chemosensory discrimination because many lizards and snakes respond to different chemical stimuli by changing the rate of tongue extrusions (Cooper & Burghardt, 1990; Cooper, 1994). An elevation of TF rates in response to the presentation of a chemical stimulus above the baseline TF rates under the experimental conditions in response to an odorless control (e.g., deionized water) indicates detection of that chemical stimulus, whereas differences between TF rates in response to different scent stimuli indicates discrimination of the different stimuli (Cooper & Burghardt, 1990; Cooper, 1994). Other similar quantifiable behaviors such as labial licking, chin rubbing, or gular pumping also have been used as indicators of chemosensory recognition. In other cases, differences in the time spent in areas scent-marked with different chemical stimuli are used as indicators of chemosensory discrimination and preferences for a determined scent.
1.3. Pheromones and Reproduction in Reptiles Reproductive behavior of many reptiles was traditionally thought to be predominantly based on conspicuous visual signals. The importance of pheromones on reproductive
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behavior of reptiles was little considered in most studies in spite of the well known and considerable evidence of the chemosensory abilities of most reptiles, and the widespread occurrence of multiple types of glands that secrete chemicals with the potential of being pheromones, especially during the breeding season (for example see Section 5.2). Only when all this information has been considered and incorporated into new observations and experiments, has the great importance of pheromones in the reproduction of many reptiles emerged. Most of the known data refer to signaling (releaser) pheromones, which can inform conspecifics about simple data such as species, sex or individual identity. But pheromones also may inform on individual characteristics of the signaler, such as morphological traits and health condition that can be very useful in reproductive decision-making by the receiver. Priming pheromones, which directly trigger slower endocrine or developmental processes, seem rare, or not yet known, in reptiles. This is, however, a relatively new emergent field of study that will need to consider new situations and new species to get a complete picture. We will review here the results of experiments showing that chemical cues (pheromones) are involved in sex and individual recognition, territoriality, intrasexual aggression between males, mate choice, and reproductive decisions, especially in many turtles, lizards, and snakes.
2. TESTUDINES/TURTLES Many turtles have well-developed olfactory and vomeronasal systems and consequently are capable of chemosensory detection (Halpern, 1992; Hatanaka & Matsuzaki, 1993; Fadool, Wachowiak, & Brann, 2001; Brann & Fadool, 2006). In addition, most turtles have several types of specialized secretory glands. One or more pairs of Rathke’s glands, which secrete through pores on the shell bridge of the axillary or inguinal areas, are present in all turtle species except in the Testudinidae (tortoises) and in some Emydidae (Ehrenfeld & Ehrenfeld, 1973; Solomon, 1984). Mental or chin glands, located in the throat area, are found in many genera of the closely related families Emydidae, Geoemydidae, Platysternidae, and Testudinidae (Winokur & Legler, 1975). Some of these glands are quiescent in juveniles, sexually dimorphic (larger in males), and active during the breeding season only (Rose, Drotman, & Weaver, 1969), which suggest their potential role in reproduction. Mental and Rathke’s glands secrete a variety of chemicals that have been analyzed and described in a few turtle species (reviewed in Weldon, Flachsbarth, & Schulz, 2008). Some of these chemicals might be potential pheromones. Cloacal secretions and feces are also considered as possible sources of pheromones (Mason, 1992), but these chemicals remain undescribed. Many chemicals produced
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by these glands, especially by Rathke’s glands, have been mainly considered as predator repellents or simply products of excretion without any other known function in intraspecific communication (Ehrenfeld & Ehrenfeld, 1973; Eisner et al., 1977; Kool, 1981; Weldon & Tanner, 1990). Nevertheless, secretions from the Rathke’s glands of common musk turtles (Kinosternum odoratum) may serve a dual function, being involved in antipredator mechanisms all the year, whereas, during the mating season, secretions of females may allow sex-recognition during courtship (Mahmoud, 1967; Eisner et al., 1977; Brann & Fadool, 2006). This suggests that the potential role of glandular chemicals in reproduction should be re-examined in other turtle species. Mostly anecdotic behavioral observations suggest that chemical stimuli may play a major role in sex and species identification, and that many turtle behaviors, such as foraging, orientation and homing behavior, aggregation, aggressive interactions between males, and mating behavior, seem to be mediated by pheromones (e.g., Alberts, Rostal, & Vance, 1994; Quinn & Graves, 1998; Poschadel, Meyer-Lucht, & Plath, 2006; reviewed in Mason, 1992; Mu¨ller-Schwarze, 2006). Experimental elimination of olfactory function results in impaired homing ability and a large reduction in reproductive behavior in Hermann’s tortoises (Testudo hermanni) (Chelazzi & Delfino, 1986). Evidence for the role of pheromones in the reproductive behavior of turtles is scarce (see Sections 2.1, 2.2 and 2.3). This is probably due to the paucity of studies that have considered chemical communication in turtles, because recent experiments are increasingly finding indications of the importance of pheromones in the sexual behavior of turtles.
2.1. Testudinidae Terrestrial tortoises (Fam. Testudinidae) seem to have two primary sources of pheromones: the mental or chin glands, which elicit aggression between males, and cloacal glands, which allow species and sex recognition (Mason, 1992). During the mating season, mental glands of male desert tortoises (Gopherus spp.) are larger and have more secretion (Rose et al., 1969; Alberts et al., 1994b). Secretions are mainly composed of fatty acids, triacylglycerols, steroids, and phospholipids, and also proteins, which differ between species and between sexes (Rose et al., 1969). Secretions from the mental glands allow discrimination between familiar and unfamiliar conspecifics in desert tortoises (Gopherus agassizii) (Alberts et al., 1994b). Mental gland secretions, and also the fatty acids from the secretions alone, elicit intrasexual aggressive behavior in male Texas tortoises (Gopherus berlandieri) (Rose, 1970). Cloacal secretions allow, at least, species and sex determination in the sympatric South American red-footed and yellow-
footed tortoises (Geochelone carbonaria and Geochelone denticulata) (Auffenberg, 1965). Males smell the cloacal area of other tortoises, and chemical discrimination of a conspecific female induces mounting attempts, whereas heterospecific females are ignored.
2.2. Emydidae Olfactory anatomy and behavioral experiments suggest that some freshwater turtles (Fam. Emydidae) can detect pheromones from conspecifics in water and that this discrimination may affect their reproductive behavior. Painted turtles (Chrysemys picta) discriminate between chemical cues from home ponds and other ponds. Female C. picta, but not males, preferred to occupy water from home ponds over water from other ponds that contained conspecifics (Quinn & Graves, 1998). During courtship, male musk turtles (Kinosternon spp.) approach females from behind and smell the cloaca, apparently to determine sex. If the turtle is identified as a female, the male moves to her side to sniff the musk (Rathke’s) glands located on the female shell bridge, and then, if the female is receptive, the male attempts mounting (Mahmoud, 1967). In recent laboratory experiments, freshwater turtles were tested in simultaneous binary choice trials. A focal animal was given a choice between water containing pheromones from a conspecific and water containing control clean water (Mun˜oz, 2004; Poschadel et al., 2006; Polo-Cavia, Lo´pez, & Martı´n, 2009). Results show interand intrasexual pheromonal communication in freshwater turtles. Spanish terrapins (Mauremys leprosa), which have mental and Rathke’s glands, prefer water with the scent of conspecifics and avoid water with the scent from a heterospecific competitor aggressive turtle species (Polo-Cavia et al., 2009) (Figure 6.1). During the mating season, male M. leprosa select water with chemicals from conspecific females but avoid water with chemicals from other conspecific males, and prefer water from their home containers over clean water. Conversely, during the mating season, female M. leprosa prefer water with chemicals from other females but do not select water from their home containers or water from males (Mun˜oz, 2004). However, outside the mating season, both male and female M. leprosa avoid water that contains chemicals from conspecifics of the opposite sex (Mun˜oz, 2004). In similar experiments, male European pond turtles (Emys orbicularis) prefer water with the odor of a female over clean water, whereas females do not show any preference, suggesting that males actively search for females using pheromones (Poschadel et al., 2006). Males prefer water with the scent of larger females, probably because female fecundity is positively correlated with female size. In contrast, male E. orbicularis strongly avoid chemicals of large males, but orient towards chemicals of relatively
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FIGURE 6.1 Chemosensory species and sex discrimination by Spanish terrapins (Mauremys leprosa). Relative preferences (mean þ SE) of male and female M. leprosa for water containing chemical stimuli of male (black boxes) or female (open boxes) conspecific or heterospecific (Trachemys scripta) terrapins (i.e., the difference between percentage time spent on an experimental pond with a given chemical stimulus in comparison with time spent on a nearby control pond with clean water) (N. Polo-Cavia, P. Lo´pez, & J. Martı´n, unpublished data).
smaller males. This result reflects that males form dominance hierarchies, where large males aggressively attack smaller ones, suggesting that pheromones are involved in the establishment of social dominance (Poschadel et al., 2006). Red-eared sliders (Trachemys scripta elegans) lack inguinal and mental glands but can be trained to make discriminations of food based on chemical cues (Boycott & Guillery, 1962). Sliders can also detect chemical cues of conspecifics in water and tend to prefer water with their own scent, while males avoid, more often during the mating season, water with conspecific chemicals, probably to avoid potential aggressive encounters (Polo-Cavia et al., 2009). However, sliders do not show a clear discrimination of male and female conspecific chemicals in water, nor of their own scents, which suggests that sex is not discriminated, or that it does not affect their space use (Polo-Cavia et al., 2009).
2.3. Cheloniidae Sea turtles (Cheloniidae) have secretory Rathke’s glands that produce lipids and water-soluble proteins of high molecular weight (Solomon, 1984; Weldon & Tanner, 1990; reviewed in Weldon et al., 2008), but secretions are
often considered as mere excretory products. Sea turtles use chemoreception in homing behavior; this is not based on conspecific pheromones but on environmental chemical cues from their natal beach. However, behavioral observations of green sea turtles in captivity (Chelonia mydas) show that males smell the cloacae of breeding females more often than those of nonbreeding females (CrowellComuzzie & Owens, 1990), which suggests that female pheromones might be involved in reproduction as indicators of female receptivity, or be used by males to locate females.
3. RHYNCHOCEPHALIA The two extant species of tuatara (Sphenodon spp.) have two cloacal glands that produce potential pheromones (Gabe & Saint Girons, 1965). Chemical analyses showed that cloacal gland secretions of adult male and female Sphenodon punctatus contain several triacylglycerols derived from unusual medium chain-length fatty acids, and a major glycoprotein (Weldon et al., 2008). However, it is still unclear whether pheromones are used in the reproductive behavior of tuataras because chemosensory exploration by TF is not observed during interactions between males or during courtship or mating (Gans, Gillingham, & Clark,
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1984). Nevertheless, tuataras frequently bite cotton balls bearing prey chemicals, suggesting that they may be able to detect at least airborne prey chemicals via olfaction (Cooper, Ferguson, & Habegger, 2001).
4. CROCODILIA Studies of crocodiles mainly have analyzed the morphology of their glands and the chemical composition of their gland secretions (Weldon & Wheeler, 2000; Weldon et al., 2008), but the ecology and behavior of these animals remain little-studied, perhaps because of the practical difficulties of working with them. Crocodiles do not have a vomeronasal system, but olfaction and taste seem to be used for chemosensory detection (Weldon, Swenson, Olson, & Brinkmeier, 1990; Halpern, 1992; Hansen, 2007). The gular and paracloacal glands of crocodiles are thought to produce pheromones involved in nesting and mating behaviors, but their specific function is unknown (Weldon & Ferguson, 1993). The gular glands are a pair of evertible glands, located on the ventral part of the lower jaw, which secrete lipids such as fatty acids, squalene, cholesterol, alcohols, and a-tocopherol. Gular secretions show sexual and individual variations and are thought to be used by females to scentmark nest sites (Weldon, Shafagati, & Wheeler, 1987; Weldon & Sampson, 1988; Weldon, Scott, & Tanner, 1990; Weldon & Tanner, 1991; Weldon & Ferguson, 1993; Weldon & Wheeler, 2000). The paracloacal glands are a pair of glands, located in the cloaca, that secrete hydrocarbons, fatty acids, alcohols, triacylglycerols, steroids and their esters, and phospholipids (Weldon, Shafagati, & Wheeler, 1988; Shafagati, Weldon, & Wheeler, 1989; Weldon et al., 1990b; Weldon & Tanner, 1991; Dunn, Weldon, Howard, & McDaniel, 1993; Wheeler, Ibrahim, & Weldon, 1999; Garcı´a-Rubio, Attygalle, Weldon, & Meinwald, 2002). Additionally, occasionally there are secreted unusual novel compounds with an unknown function, such as aromatic ketones and some steroidal esters, which reflects some novel biosynthetic capabilities of crocodiles (Whyte, Yang, Weldon, Eisner, T., and Meinwald 1999; Yang et al., 1999). Paracloacal gland secretions show sexual and individual variation and are thought to be used in mating and nesting behavior (Weldon & Ferguson, 1993; Weldon & Wheeler, 2000). However, the only behavioral evidences of pheromone detection are that prolonged sniffing behavior has been observed in Caiman crocodylus in response to conspecific gland secretions (Huggins, Parsons, & Pen˜a 1968), and that yearling Alligator mississippiensis respond to airborne conspecific scents (Johnsen & Wellington, 1982).
5. SQUAMATA 5.1. Amphisbaenians Amphisbaenians are a group of reptiles morphologically and functionally adapted to fossorial life. Morphological adaptations to burrowing include trunk elongation, head modification, highly reduced vision, and loss of limbs in most species (Gans, 1978). Ecology and reproductive behavior of amphisbaenians are poorly known, but chemoreception should be especially important for these almost blind reptiles. Many amphisbaenians have several precloacal pores connected to precloacal glands that produce copious holocrine secretion, especially during the breeding season (Gabe & Saint Girons, 1965; Whiting, 1967; Antoniazzi, Jared, Pellegrini, & Macha, 1993; Antoniazzi, Jared, & Junqueira, 1994; Jared, Antoniazzi, Silva, & Freymu¨ller, 1999). Morphological and microscopic examination of secretions suggest that, as amphisbaenians move inside tunnels, the secretion plugs are abraded against the substrate, releasing a secretion trail (Jared et al., 1999). This trail might contain pheromones important in intraspecific communication inside tunnels. Pheromonal communication has been examined in only one amphisbaenian species. Male amphisbaenians (Blanus cinereus) detect and discriminate between pheromones of males and females (Cooper, Lo´pez, & Salvador, 1994) (Figure 6.2), and between their own scent and those of other males (Lo´pez, Cooper, & Salvador, 1997). The ability to discriminate between sexes was greater from precloacal gland secretions than from skin chemicals (Cooper et al., 1994). Also, before starting to burrow in unfamiliar substrates, amphisbaenians search by TF for a longer time than in familiar substrates, which suggests that amphisbaenians recognize their own scent-marked burrows (Lo´pez, Martı´n, & Barbosa, 2000). Chemical analyses of precloacal secretions of both male and female B. cinereus found a great diversity of lipids (Lo´pez & Martı´n, 2005b), mainly steroids (87%) and minor quantities of squalene (more in males), fatty acids, waxy esters and a-tocopherol (only in females). Cholesteryl methyl ether and cholesterol are the main lipids. The abundance of steroids and waxy-type esters in secretions may be useful to scent-mark underground tunnels and for individuals to orient themselves or trail conspecifics inside them (Jared et al., 1999; Lo´pez et al., 2000). The potential roles of these chemicals as pheromones remain to be analyzed. However, there are clear intersexual differences in the presence/absence of some compounds and in the relative proportions of some shared compounds (Lo´pez & Martı´n, 2005b), which may explain pheromonal sex discrimination (Cooper et al., 1994). Squalene was much more abundant in males. Male, but not female, B. cinereus respond aggressively to cotton swabs bearing
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FIGURE 6.2 Chemosensory sex discrimination by amphisbaenians. Number (mean SE) of total tongue flicks (TFs) emitted in one minute by male amphisbaenians (Blanus cinereus) in response to the presentation of cotton swabs bearing deionized water (odorless control), or scent from the precloacal pores or the skin of conspecific males and females. Redrawn from Cooper, Lo´pez, & Salvador, 1994.
high concentrations of squalene (Lo´pez & Martı´n, 2009). Thus, squalene may be part of the male sex recognition system of amphisbaenians. Similarly, only male garter snakes present squalene in the skin, and courtship to females is partially inhibited if squalene is experimentally added to female skin (Mason et al., 1989). Also, male amphisbaenians have a greater number and diversity of fatty acids than females (Lo´pez & Martı´n, 2005b), which could explain sex and individual discrimination (Cooper et al., 1994; Lo´pez et al., 1997).
5.2. Lizards 5.2.1. Secretory glands and potential pheromones Lizards have several possible sources of pheromones. Chemicals with the potential function of pheromones are found in the skin and secreted by large specialized holocrine glands (femoral, preanal, or precloacal glands) (Mason, 1992; Weldon et al., 2008). The activity of these glands depends on sex hormones, such that secretion is greater during the mating season. This strongly suggests the role of these glands and the chemicals secreted by them on the reproduction of lizards. Morphological descriptions of these glands (Gabe & Saint Girons, 1965) and characterization of the chemicals secreted (Weldon et al., 2008) are known for some lizard species from diverse taxonomic groups. However, in most cases it is not clear, or has never been tested to determine, what specific chemicals have the activity of pheromones and/or the specific function of such pheromones. 5.2.1.1. Skin The skin of lizards contains lipids that are sequestered in the mesos layer of the epidermis. These lipids have been
analyzed in some species (reviewed in Weldon et al., 2008). The skin of green iguanas (Iguana iguana) contains alcohols, steroids, fatty acids, and waxy esters (Roberts & Lillywhite, 1980). Similarly, analyses of many lizard species from most families found in the skin steroids, fatty acids, phosphatidylcholines, and phosphatidylethanolamines, and hydrocarbons in some species (Weldon & Bagnall, 1987). Lipids protect against water loss of the skin (Roberts & Lillywhite, 1980), but they may also function as pheromones, or at least allow for species and sex chemosensory recognition. Thus, there is taxonomic variation in the skin lipids, which ‘combinations’ are characteristics of each species (Weldon & Bagnall, 1987). Moreover, in leopard geckos (Eublepharis macularius) there are distinct sex differences in lipid fractions of high molecular weight from the skin (Mason & Gutzke, 1990). Fatty acids, hydrocarbons, and steroids are found in both sexes, but some steroids are only found in males, and long chain methyl ketones are characteristic of females. Males court females, but, when females are shedding their skin, males respond aggressively to females, as if they were males. This suggests that sex identification depends on skin-derived lipids (Mason & Gutzke, 1990). 5.2.1.2. Femoral, precloacal, and preanal glands Many studies have shown pheromonal detection in different lizard species based on precloacal, preanal, or femoral gland secretions (e.g., Alberts, 1993; Arago´n, Lo´pez, & Martı´n, 2001b; Labra, Escobar, Aguilar, & Niemeyer, 2002). All these glands are probably homologous structures that differ in terms of relative position on the body (Gabe & Saint Girons, 1965). Glands are formed by an invagination of the stratum germinativum, which forms follicular units that produce copious amounts of holocrine secretion. Glands are connected to epidermal structures called femoral pores (located on the ventral
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surface of the thigh) (Figure 6.3) or precloacal or preanal pores (located on the anterior edge of the cloacae), through which a ‘waxy’ secretion is slowly secreted (Cole, 1966). The secretory activity of the femoral and precloacal glands is greatest in males and in the breeding season, which indicates that gland secretions are under direct androgenic control and vary seasonally with androgen production (Cole, 1966; Van Wyk, 1990; Alberts, 1993). For example, productivity and chemical composition of femoral secretions of green iguanas (I. iguana) vary seasonally in relationship with plasma testosterone (T) levels (Alberts, Pratt, & Phillips, 1992; Alberts, Sharp, Werner, & Weldon, 1992). Further, castration of male lizards causes femoral glands to atrophy, whereas supplementation of T increases secretion rates (e.g., Fergusson, Bradshaw, & Cannon, 1985; Chauhan, 1987; see review in Mason, 1992). Thus, the amount of secretion in scent marks may reflect the physiological condition and reproductive state of individual lizards (Martins, Ord, Slaven, Wright, & Housworth, 2006). The ventral location of femoral and precloacal pores allows chemical secretions to be passively deposited on substrates as lizards move through their home ranges, which may serve to scent-mark territories. On the other hand, some lizards also show rubbing behavior of the cloaca and pores against substrates, suggesting active scent-marking. Seasonal increases in gland production may allow lizards to mark more sites rather than to influence the quality of the signal on a single site (Martins et al., 2006).
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The number of precloacal or femoral pores varies within a limited range that is characteristic of each species, which fact has been used extensively in taxonomic studies. Interspecific variation could be directly related to the rate of secretion needed for effective scent-marking, compensating for different environmental conditions in the optimal quantity of secretions smeared on the substrate (Alberts, 1992b), or may be related to the relative importance of chemical communication for a given species. Thus, the similar number of femoral or precloacal glands in different lizard species living in similar environmental conditions might reflect convergent evolution (Escobar, Labra, & Niemeyer, 2001). In 20 species of South American Liolaemus lizards (Fam. Tropiduridae), the number of precloacal pores correlates positively with altitude and negatively with latitude, suggesting that lizards produce more secretions under harsh environments (Escobar et al., 2001). However, there is also a strong phylogenetic inertia component in the number of pores across species (Pincheira-Donoso, Hodgson, & Tregenza, 2008). Chemical composition of femoral or precloacal gland secretions has been studied in a few lizard species (reviewed in Weldon et al., 2008), showing that secretions are composed of both lipids and proteins. Proteins may be the major components in secretions (Alberts, 1990; 1991; Alberts, Phillips, & Werner, 1993). The patterns of protein composition of different species depend on taxonomic affinities, but also on environmental characteristics
FIGURE 6.3 Femoral glands of male Iberian rock lizards (Iberolacerta cyreni) (shown as a row of femoral pores with ‘waxy’ secretion on the ventral surface of the thighs). Photograph by J. Martin.
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(Alberts, 1991). However, although inter- and intraspecific variations in the protein content of secretions are consistent over time and might be responsible for self and individual recognition (Alberts, 1991; Alberts & Werner, 1993), most recent evidence points to lipids as the main compounds involved in pheromonal communication (Mason, 1992; Martı´n & Lo´pez, 2006b; see Section 5.2.3). Lipids are more volatile and have a high degree of molecular diversity, which increases the potential information content of a pheromone, and are regulated by the general metabolism, such that they could indicate the characteristics and condition of the signaler.
5.2.1.2.1. Iguania Iguanid lizards were considered mainly visually oriented lizards, but many have secretory glands and are capable of chemosensory conspecific recognition. The lipophilic fraction of femoral gland secretions of male green iguanas (I. iguana) contains fatty acids and their esters, and several steroids (Weldon, Dunn, McDaniel, & Werner, 1990; Alberts et al., 1992a). Lipids in secretions do not seem to differ qualitatively between adults and juveniles (Weldon et al., 1990a). However, the seasonal variations in testosterone levels in plasma are related to changes in lipid composition of secretions. The lipid content and the proportion of unsaturated fatty acids increase during the mating season, which may enhance volatility and detectability of secretions (Alberts et al., 1992a; 1992b). The amounts of steroids and fatty acids in secretions vary among individuals. Some steroids are phytosterols, which have to be obtained from the diet (Alberts et al., 1992a) and thus might potentially inform on the ability of an individual to obtain food or on the quality of his territory. Proteins in femoral secretions have been analyzed in desert iguanas (Dipsosaurus dorsalis) and green iguanas (I. iguana) (Alberts, 1990; 1991; Alberts et al., 1993). There are intraspecific variations in protein composition, which may be used in sex and individual recognition (Alberts & Werner, 1993). Lipids might attract conspecifics from long distances but individual identification might be based on non-volatile proteins in substrate scent marks. In the South American lizards (Liolaemus spp.; Fam. Tropiduridae), the precloacal gland secretion of males is considered a potential source of pheromones (Labra et al., 2002) and allows chemosensory conspecific and selfrecognition (Labra, Escobar, & Niemeyer, 1999; Labra, Beltra´n, & Niemeyer, 2001; Labra, Escbar, & Niemeyer, 2001). Chemical analysis of the precloacal secretions of 20 species of Liolaemus revealed many lipids, mainly alkanes, long chain fatty acids, and steroids, with cholesterol and five fatty acids (from C14 to C18) appearing in all species.
Hormones and Reproduction of Vertebrates
Different chemical patterns occur at intra- and interspecific levels, indicating interspecific and intrapopulational differences in the chemical composition of secretions (Escobar et al., 2001). Therefore, it seems that species and individuals can be characterized by a particular chemical profile (Escobar et al., 2001; Labra et al., 2001b). Small differences in chemicals in secretions between two populations of Liolaemus fabiani are explained by different environmental conditions (Escobar, Escobar, Labra, & Niemeyer, 2003). The preanal gland secretion of male Hardwick’s spinytailed lizards (Uromastix hardwickii) (Fam. Agamidae) contains fatty acids, triacylglycerols, waxy esters, steroids, and phospholipids (Chauhan, 1986). Females have only steroids and phospholipids.
5.2.1.2.2. Scleroglossa The femoral secretions of males and females of the South African giant girdled lizard or sungazer (Cordylus giganteus) (Fam. Cordylidae) contain only semi-volatile chemicals, including fatty acids, alcohols, ketones, esters, and steroids (Louw, Burger, Le Roux, & Van Wyk, 2007). Femoral secretion could be important in the social biology of this lizard as it occurs in the related Cape girdled lizards (Cordylus cordylus), which detect and respond differentially to male and female pheromones (Cooper, Van Wyk, & Mouton, 1996). In the Indian house lizard (Hemidactylus flaviviridis) (Fam. Gekkonidae), the preanal gland secretions of males are composed of fatty acids, triacylglycerols, waxy esters, steroids, and phospholipids (Chauhan, 1986). Recent studies have examined the lipophilic fraction of femoral secretions of males of several species of European lacertid (Lo´pez & Martı´n, 2005c; 2005d; 2006; Martı´n & Lo´pez, 2006c; 2006e; Martı´n, Moreira, & Lo´pez, 2007; Gabirot et al., 2008). Secretions are mainly composed of fatty acids and steroids with, usually, minor quantities of alcohols, squalene, ketones, waxy esters, aldehydes, tocopherol, furanones, etc. The chemicals found and the relative proportions of compounds are characteristics of each species. Cholesterol is the main compound, found in high quantities in Iberian rock lizards (Iberolacerta spp.) (Lo´pez & Martı´n, 2005c; Martı´n et al., 2007c), wall lizards (Podarcis spp.) (Martı´n & Lo´pez, 2006e), and Common lizards (Lacerta vivipara) (Gabirot et al., 2008). However, the abundance and ubiquity of this component in secretions is thought to be only useful to constitute an unreactive apolar matrix that delivers the compounds that are the true semiochemicals (Escobar et al., 2003). Thus, the chemicals that have been thought to have properties of signaling pheromones, such as dehydrocholesterol, ergosterol, or hexadecanol (see Section 5.2.3), are compounds found in
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relatively low concentrations in secretions. In other species, other compounds are the main ones, for example campesterol in Psammodromus algirus (Martı´n & Lo´pez, 2006c). In spiny-footed lizards (Acanthodactylus erythrurus), longchain alcohols (hexacosanol and tetracosanol) are the most abundant compounds; they may form waxy esters to give consistency to secretions in the dry habitat of this lizard (Lo´pez & Martı´n, 2005d). Tocopherol is the main compound in green lizards (Lacerta schreiberi, Lacerta viridis, and Lacerta lepida) (Lo´pez & Martı´n, 2006, unpublished data). Tocopherol may protect other compounds in the secretions from oxidation in wet environments. There are interpopulational differences in secreted chemicals that may be related to different environmental conditions. In Iberian wall lizards (Podarcis hispanica), differences in secretions between two nearby populations inhabiting different altitudes with different microclimates presumably would reflect selection for the persistency and efficiency of pheromones in different environments, less volatile compounds with a higher chemical stability being favored in the population inhabiting more humid climatic conditions (Martı´n & Lo´pez, 2006e). Males, but not females, discriminate between the scent of males of these two populations (Martı´n & Lo´pez, 2006e; 2006f). Secretions of adult male common lizards (L. vivipara) from oviparous and viviparous populations do not differ in the numbers and quality of chemicals, but there are differences in relative proportions of some compounds (Gabirot et al., 2008). These differences might be explained by either small genetic differences between populations or by different microclimatic conditions. Within each species there is high interindividual variability in relative proportions of compounds. Age class differences are found in Ps. algirus (Martı´n & Lo´pez, 2006c), where older males have secretions with lower proportions of some fatty acids and higher proportions of some steroids than younger adult males. These differences might allow conspecifics to obtain information on the age of the sender, which may be important in social behavior because older males are territorial while younger males adopt a satellite–sneaker strategy (Lo´pez, Martı´n, & Cuadrado, 2003). In Iberian rock lizards (Iberolacerta cyreni; formerly Lacerta monticola cyreni) some male traits, such as body size, number of lateral blue spots, number of femoral pores and their level of fluctuating asymmetry, and T-cell immune response (a health indicator) are related to the variability in relative proportions of some compounds in secretions. These relationships suggest that conspecifics could obtain reliable information on the producer of a scent mark based on chemicals alone (Lo´pez, Amo, & Martı´n, 2006), a possibility that is supported by
recent experiments (Martı´n & Lo´pez, 2006a; 2006b; 2007; see Section 5.2.3). 5.2.1.3. Cloacal and urodeal glands Urodeal glands are found in the cloaca of female lizards mainly in the Fam. Cordylidae and Scincidae. These are tubular organs that empty into folds of the cloacal ducts via small orifices (Gabe and Saint-Girons, 1965; Trauth, Cooper, Vitt, & Perrill, 1987). Glands are active and have more secretions during the breeding season. In broadheaded skinks (Eumeces laticeps), the neutral lipid fraction of urodeal glands, consisting of steryl and waxy esters and acylglycerols, has properties of a sex pheromone because it seems to elicit courtship by males (Cooper, Garstka, & Vitt, 1986; Cooper & Garstka, 1987; Trauth et al., 1987). A similar pheromone from the urodeal gland is thought to occur in females of the cordylid black-lined plated lizard (Gerrhosaurus nigrolineatus) (Cooper & Trauth, 1992). 5.2.1.4. Feces Chemicals with the function of pheromones are secreted onto the surface of the feces or scats, probably from cloacal glands, as feces are deposited by the lizard. These chemicals may serve for territorial scent-marking in some lizards (Duvall, Graves, & Carpenter, 1987; Carpenter & Duvall, 1995; Lo´pez, Arago´n, & Martı´n, 1998; Bull, Griffin, & Johnston, 1999; Bull, Griffin, & Perkins, 1999; Arago´n, Lo´pez, & Martı´n, 2000; Bull, Griffin, Bonnett, Gardner, & Cooper, 2001). Chemical signals from scats of Australian tree skinks (Egernia striolata) and Stoke’s skinks (Egernia stokesii) (Fam. Scincidae) have properties of pheromones (Bull et al., 1999a; 1999b). Chemical signals of each individual seem to have a unique signature, because lizards respond more strongly to scats of unfamiliar conspecifics than to their own scats (Bull et al., 1999a; 1999b). This is unrelated to diet because there is no difference in the response to scats from unfamiliar lizards fed on the same or different diets from the test lizard. Chemicals with properties of pheromones have not been identified, but they are probably a combination of several lipids as they are contained in scat extracts made with organic solvents (dichloromethane), and fractionation of the scats with different solvents (pentane and methanol) led to loss of the unique signals needed for individual recognition (Bull et al., 1999a). In Iberian rock lizards (I. cyreni) (Lo´pez et al., 1998), western fence lizards (Sceloporus occidentalis) (Duvall et al., 1987), and western banded geckos (Coleonyx variegatus) (Carpenter & Duvall, 1995), feces are deposited on visually conspicuous sites and have an aggregated spatial distribution, such that the visual location of the pellet at
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long distance might further elicit the search for chemical signals from the scat or from nearby scent marks. Fecal chemicals of adult male rock lizards (I. cyreni) allow for self-recognition, discrimination of familiar vs. unfamiliar males, and signaling of male body size, thus suggesting that feces act as a composite signal (visual and chemical) in territorial marking (Lo´pez et al., 1998; Arago´n et al., 2000).
5.2.2. Chemosensory recognition A plethora of studies using the technique of measuring TF rates for different scent stimuli (see Introduction) have examined and found chemosensory discrimination of chemicals from prey and conspecifics in many lizard species from most taxonomic groups. Pheromones allow lizards to recognize conspecifics from closely related heterospecifics (e.g., Cooper & Vitt, 1987; Cooper & Pe´rez-Mellado, 2002; Barbosa, Font, Desfilis, & Carretero, 2006), males from females (Cooper & Vitt, 1984; Cooper & Trauth, 1992; Cooper et al., 1996), the self from other individuals and familiar from unfamiliar individuals (Graves & Halpern, 1991; Alberts, 1992a; Alberts & Werner, 1993; Bull et al., 1999b; Bull, Griffin, Lanham, & Johnston, 2000; Arago´n, Lo´pez, & Martı´n, 2001a; Arago´n et al., 2001b), and kin from nonkin (Werner, Baker, Gonzales, & Sosa, 1987; Bull, Doherty, Schulze, & Pamula, 1994; Main & Bull, 1996; Lena & de Fraipont, 1998; Bull et al., 2001). Few studies, however, have yet documented how these different recognitions are achieved or the chemical basis of the pheromonal recognition. Further, only a few studies have examined whether lizards can discriminate between different chemical compounds found in gland secretions (Cooper & Pe´rez-Mellado, 2001; Cooper, Pe´rez-Mellado, & Vitt, 2002a; 2002b; Martı´n & Lo´pez, 2006b; 2006d, 2008b). The lacertid Lilford’s wall lizard (Podarcis lilfordi) discriminates between lipids, proteins, and carbohydrates, and also between different lipids such as glycerol, cholesterol, and oleic and hexadecanoic acids (Cooper et al., 2002a; 2002b). Female Iberian wall lizards (P. hispanica) and Iberian rock lizards (I. cyreni) discriminate between several steroids that are found in the femoral secretions of males, and also assess changes in their concentrations (Martı´n & Lo´pez, 2006b; 2006d). Intersexual differences in response to different chemicals found together in secretions of males suggest that secretions contain multiple messages (multiple pheromones) with different intended receivers (Martı´n & Lo´pez, 2008b). 5.2.2.1. Sex and individual recognition Most lizards from the different taxonomic groups tested can discriminate between the scents of male and female conspecifics (e.g., Tropiduridae (Labra & Niemeyer, 1999); Scincidae (Cooper & Vitt, 1984); Gekkonidae (Mason &
Hormones and Reproduction of Vertebrates
Gutzke, 1990; Cooper & Steele, 1997); Lacertidae (Lo´pez & Martı´n, 2001a; Cooper & Pe´rez-Mellado, 2002); Cordylidae (Cooper et al., 1996)). Tongue flick rates that differ between male and female scents are considered a clear indication of sexual discrimination (see Section 1). Moreover, very often male and female pheromones elicit different reproductive behaviors. For example, male leopard geckos (E. macularius) perform TFs at lower rates to male than to female scent or a blank control. Males perform aggressive behaviors toward male scent but not female scent, and perform tail vibrations (a courtship behavior) toward female but not male scents (Cooper & Steele, 1997). Pheromonal sex discrimination may be season-dependent, suggesting their active role in reproduction. For example, during the post-reproductive season, tree-dwelling lizards (Liolaemus tenuis) (Fam. Tropiduridae) TF at similar rates in conspecific and control enclosures, while during the mating season enclosures of females elicit more TFs by both sexes and TF rates are higher than in the postreproductive season (Labra & Niemeyer, 1999). Pheromones also allow lizards to obtain additional information. For example, males of the lacertid Iberian wall lizard (P. hispanica) show different responses to scent from males and females, and from gravid and nongravid females (Cooper & Pe´rez-Mellado, 2002). The relative importance of visual and chemical cues to identifying sex may vary between lizard species, with visual identification being more important in species with sexual dichromatism (see Cooper & Greenberg, 1992). However, chemical stimuli are also clearly involved, and many species might rely more on chemoreception. Experimental manipulation of the coloration and scent of male and female Iberian wall lizards (P. hispanica), to create groups with all combinations of coloration and scents of males and females, showed that males react more aggressively to intruders impregnated with male scent, independently of their actual sex and coloration (Lo´pez & Martı´n, 2001a; Lo´pez, Martı´n, & Cuadrado, 2002). Both color and scent are important in eliciting male courtship. However, females painted as females and impregnated with female scent are preferentially courted. Males impregnated with female scent do not elicit aggressive responses but are often courted in spite of their actual sex and coloration. At close range, pheromones seem to be more important than color patterns in sex recognition, but female coloration is also useful at long range to deter aggressive responses of males and to elicit courtship in conjunction with pheromones (Lo´pez & Martı´n, 2001a). True individual recognition has received little attention, although several studies suggest discrimination by lizards between their own scent and those of other conspecific individuals (Figure 6.4), as occurs in blue-tongued skinks (Tiliqua scincoides) (Graves & Halpern, 1991), desert iguanas (D. dorsalis) (Alberts, 1992a), male broad-headed
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FIGURE 6.4 Individual chemosensory recognition in lizards. Number (mean SE) of directed tongue flicks (TFs) emitted in one minute by male Iberian rock lizards (Iberolacerta cyreni) in response to the presentation of cotton swabs bearing deionized water (odorless control), or scent from their own femoral gland secretions, or from femoral gland secretions of familiar (Fam.) or unfamiliar males (Unfam.). (J. Martı´n & P. Lo´pez, unpublished data).
skinks (E. laticeps) (Cooper, 1996), male and female Cape girdled lizards (C. cordylus) (Cooper, Van Wyk, & Mouton, 1999), males of the lacertid Iberian rock lizard (I. cyreni) (Arago´n et al., 2001b), several species of the tropidurid lizard (Liolaemus spp.) (Labra & Niemeyer, 1999; Labra, Beltra´n, & Niemeyer, 2001; Labra, Cortez, & Niemeyer, 2003), and males of the anguid slow worm (Anguis fragilis) (Gonzalo, Cabido, Martı´n, & Lo´pez, 2004). Further, discrimination between the scent of familiar and unfamiliar individual males (Figure 6.4) has been suggested in male desert iguanas (D. dorsalis) (Glinski & Krekorian, 1985), green iguanas (I. iguana) (Alberts & Werner, 1993), male Iberian wall lizards (P. hispanica) (Lo´pez & Martı´n, 2002; Carazo, Font, & Desfilis, 2008), and male rock lizards (I. cyreni) (Arago´n et al., 2001a; 2001b; Arago´n, Lo´pez, & Martı´n, 2003), and discrimination between scents of familiar and unfamiliar individuals of the opposite sex has been suggested in broad-headed skinks (E. laticeps) (Cooper, 1996) and male geckos (E. macularius) (Steele & Cooper, 1997). The ability of territorial lizards to discriminate between neighbors (familiar) and non-neighbors (unfamiliar) might help to stabilize social systems by
reducing the frequency and intensity of aggressive encounters between males (Glinski & Krekorian, 1985) or by favoring mate location (Cooper, 1996). Most studies of familiar chemical discrimination, however, were accomplished by housing animals together to create familiar individuals or by maintaining animals individually to create non-familiar individuals. There is little direct empirical support for pheromonal discrimination among individuals whose actual spatial and social relationships in their natural environment are known. A study that combined field observations and laboratory experiments found that male Iberian rock lizards (I. cyreni) discriminate between the scent of familiar males (those whose home ranges actually overlap in the field) and unfamiliar males (those whose home ranges are far apart) (Arago´n et al., 2001a). Behavioral responses are dependent on relative body size differences between the responding and the unfamiliar male, suggesting that body size can be estimated by chemosensory cues alone and that relatively larger unfamiliar males are considered a higher threat (i.e., their scents elicit stronger chemosensory responses). In contrast, when the donor of the scent is a familiar male, the
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TF rate is not dependent on body size differences, suggesting individual discrimination of familiar neighbors with a previously established dominance hierarchy (Arago´n et al., 2001a). 5.2.2.2. Trailing Pheromone-mediated location of conspecifics has been documented extensively in snakes (e.g., Ford, 1986; LeMaster & Mason, 2001; see Section 5.3.2.1) but is poorly documented in other reptiles. In lizards, a few studies showed the use of scent trails to locate mating partners and to facilitate mate guarding in male broad-headed skinks (E. laticeps) (Cooper & Vitt, 1986), male snow skinks (Niveoscincus microlepidus) (Olsson & Shine, 1998), female sleepy lizards (Tiliqua rugosa) (Bull & Lindle, 2002), and males of the anguid slow worm (A. fragilis) (Gonzalo et al., 2004). However, pheromone trailing may be widespread among lizards, because many lizards can detect substrate scent marks of conspecifics (e.g., Cooper et al., 1996; Arago´n et al., 2001a). Chemosensory recognition of scent trails by males may facilitate location of new females, but also may allow prolonged mate guarding of a given female partner (Olsson & Shine, 1998). In the snow skink (N. microlepidus), males and females form ‘pairs’ for long periods and males preferentially follow the scent trail of their vitellogenic female ‘partner’ rather than that of another vitellogenic female, thus maintaining prolonged partnership (Olsson & Shine, 1998). Conversely, during the mating season, female sleepy lizards (T. rugosa) that are more frequently found with their male partner are more likely to follow the scent path of their male partner than are less strongly bonded females (Bull & Lindle, 2002). However, after mating has finished, females no longer scent trail. Males do not follow their female partner in any season. These results suggest that female sleepy lizards play an active role in maintaining the partnership and monogamy. 5.2.2.3. Kin recognition Kin recognition may allow lizards to recognize offspring and tolerate relatives, to maintain stable family associations of mother and offspring or of sibling juveniles, to reduce kin competition, or to avoid inbreeding. Mother–offspring recognition has been found in common lizards (L. vivipara) (Lena & de Fraipont, 1998) and the Australian skinks E. stokesii and T. rugosa (Bull et al., 1994; Main & Bull, 1996). Sibling recognition has been described in hatchling green iguanas (Werner et al., 1987) and juvenile skinks E. striolata (Bull et al., 2001). Recognition is presumably based on pheromonal cues, but in these studies the use of additional cues was not discounted. Kin discrimination suggests that individuals must have unique signals (e.g., Alberts et al., 1993), and that they can
Hormones and Reproduction of Vertebrates
discriminate between different signals. The mechanisms that allow kin recognition might be either familiarity with signals of individuals associated with kin in the early part of life, or, more interestingly, phenotype matching, in which close relatives have genetically similar signals and are recognized just because of that similarity (Waldman, 1987). The highly variable major histocompatibility complex (MHC) may lead to different pheromones in each individual and more similar pheromones in related individuals, which may be involved in kin and individual recognition and in mate choice (Penn & Potts, 1999).
5.2.3. Evolution of pheromonal mate choice Female mate choice has been thought to be rare in lizards (Olsson & Madsen, 1995; Tokarz, 1995; Olsson & Madsen, 1998). Most studies only examined mate choice based on visual traits without finding indications of mate choice, perhaps because in many cases these visual traits do not provide reliable information on the male characteristics useful to females (Olsson & Madsen, 1995; 1998). However, as it occurs in many other animals (Johansson & Jones, 2007), discriminations based on pheromones might be more reliable and provide more detailed information about males than might be obtained from visual traits alone. However, this possibility has been largely ignored in most studies of mate choice (Tokarz, 1995), despite pheromonal recognition being widespread among lizards. Recent studies have found evidence of female preferences for substrates scent-marked by particular individual males, which suggests active mate choice based on pheromones (e.g., Martı´n & Lo´pez, 2000; Lo´pez, Mun˜oz, & Martı´n, 2002; Lo´pez, Arago´n, & Martı´n, 2003; Olsson et al., 2003; Lo´pez & Martı´n, 2005a, Martı´n & Lo´pez, 2006). Femoral secretions might transmit chemical information about the characteristics of males, which females may use to choose mates. The characteristics of pheromones seem to be affected by the ‘quality’ and health state of the individual male that produces them, and this might be the basis of mate choice (Penn & Potts, 1998). By deciding where to establish their home ranges based on chemical characteristics of scent marks made by males, females may increase the probability of mating with certain individual males (Figure 6.5). The quality of male pheromones could communicate the heritable genetic quality of a male to females and thereby serve as the basis for adaptive female choice. Females may use this information because in many cases the only benefit of mate choice to female lizards may be the genetic quality of the male. In Iberian rock lizards (I. cyreni), when scent from two males of similar body size are offered, females preferentially associate with the scents of males with low levels of fluctuating asymmetry (FA) in their femoral pores, and also with the scents of relatively heavier males and males with
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FIGURE 6.5 Mating of male and female Iberian rock lizards (Iberolacerta cyreni). The male is biting the ventrolateral side of the female. The female is accepting the mating of this individual male, while advances by other individual males may be actively rejected. Photograph by J. Martin.
a higher number of femoral pores (Martı´n & Lo´pez, 2000; 2006b; Lo´pez et al., 2002b). This suggests that females are able to discriminate the FA and other characteristics of males by pheromones alone, or rather that females select males that secrete certain pheromones and those males are more symmetric or have better condition (Martı´n & Lo´pez, 2006b). Female lizards may obtain other information from male pheromones. Female sand lizards (Lacerta agilis) prefer to associate with the scent of males with a MHC genotype more different than their own MHC genotypes (Olsson et al., 2003). A similar way to avoid inbreeding might function in other lizard species (Bull & Cooper, 1999). Selective mating with non-kin or unrelated pairs may confer genetic benefits because the new combinations of immunocompetence in offspring will defend them more effectively against evolving parasites (Penn & Potts, 1999). When scents of two male Iberian rock lizards (I. cyreni) of different ages are offered, females associate preferentially with scents of older males (Lo´pez et al., 2003a). This suggests that females can assess the age of males by pheromones alone, and that females prefer to be in areas scent-marked by older territorial males. Thus, females may increase their opportunities to mate with older males or may avoid harassment by sneaking young males. This agrees with field observations of females mating with old males and rejecting advances by young males (Lo´pez et al., 2003a). However, there is little knowledge on the role of specific chemicals (pheromones) as sexual signals, and on how the
characteristics of pheromones can mediate mate choice. Female Iberian rock lizards (I. cyreni) show stronger chemosensory responses and prefer the scent of individual males of presumably high quality (i.e., those more symmetric and with a better immune response) (Martı´n & Lo´pez, 2006b). Chemical analyses showed that these preferred males allocate relatively more cholesta-5,7-dien-3-ol (provitamin D3) and ergosterol (provitamin D2) to femoral secretions, which suggests that females use these chemicals to choose between males’ scents. Further experiments confirmed that females can discriminate these steroids, and changes in their concentration, from similar chemicals (i.e., cholesterol) also found in males’ secretions. Moreover, females are more attracted to areas experimentally manipulated to increase the proportion of ergosterol in natural scent marks of males (Martı´n & Lo´pez, 2006b). These results suggest that femoral secretions with higher proportions of cholesta-5,7-dien-3-ol and ergosterol might be reliable advertisements of the quality of a male, that females could use to select mates. Cholesta-5,7-dien-3-ol is a precursor for vitamin D3 and is often found in the skin, where it will transform into the vitamin after exposition to sun UVB irradiation (Fraser, 1995; Carman, Ferguson, Gehrmann, Chen, & Holick, 2000). Vitamin D3 is essential in calcium metabolism (Laing & Fraser, 1999) and for regulation of the immune system (Hayes, Nashold, Spach, & Pedersen, 2003). However, very often, the synthesis of vitamin D3 in the skin is not sufficient to meet physiological requirements, and lizards require dietary intake of vitamin D (Ferguson et al.,
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2005). Under these conditions, vitamin D is an essential nutrient for lizards. Therefore, by allocating provitamin D to femoral secretions, male lizards might need to divert vitamin D from metabolism. Thus, diet quality may affect the quality of the pheromones. After experimental supplementation of dietary vitamin D, male Iberian rock lizards (I. cyreni) increase the proportion of provitamin D3 in femoral secretions and females prefer areas scent-marked by these males with more provitamin D3 (Martı´n & Lo´pez, 2006a). This suggests that allocation of this provitamin to secretions is costly and dependent on the ability of a male to obtain enough vitamin D in the diet, which may explain why females select these males. 5.2.3.1. Evolutionary mechanisms Empirical research on the mechanisms that confer reliability to sexual signals has focused almost exclusively on visual signals, but little is known about the mechanisms that may maintain pheromones as reliable sexual signals. Some theoretical models of mate choice without direct benefits have predicted that signals used in sexual selection can only be evolutionarily stable if they are honest and conditiondependent or costly to the signaler and if the cost is correlated with the signaler’s quality (Zahavi, 1975; Pomiankowski, 1988; Grafen, 1990). One possible cost is the energetic expenditure of producing and maintaining the immune system to fight against parasites, which may have a major effect on condition, thus creating a tradeoff because resources are used by both the condition-dependent sexual advertisement and the immune system (Wedekind &
Hormones and Reproduction of Vertebrates
Folstad, 1994; Sheldon & Verhulst, 1996). Therefore, only individuals in good condition could mount a strong immune defense and produce an extravagant sexual ornament (Westneat & Birkhead, 1998). Evidence for parasite-mediated sexual selection has been found in many animals that use visual ornaments to attract females. Studies with rodents and insects suggest that parasitic infections and immunocompetence may also affect the information content of pheromones (Penn & Potts, 1998; Rantala, Jokinen, Kortet, Vainikka, & Suhonen, 2002; Rantala, Kortet, Kotiaho, Vainikka, & Suhonen, 2003). Parasites and health state may affect pheromones of lizards too. For example, male common wall lizards (Podarcis muralis) with fewer parasites and a greater immune response have femoral secretions with higher proportions of two esters of octadecenoic acid (Martı´n, Amo, & Lo´pez, 2008), which suggests that pheromones might signal the parasite load and health state of a male. The signal would be reliable because only healthier males seem able to allocate presumably costly chemicals to secretions. Thus, pheromones might function in parasite-mediated sexual selection and females might use these relationships to select males. For example, female Iberian wall lizards (P. hispanica) select scents of males with higher proportions of cholesta-5,7-dien-3-ol in femoral secretions (Lo´pez & Martı´n, 2005a) (Figure 6.6). This can be explained because the proportions of cholesta5,7-dien-3-ol of males are related to their T-cell-mediated immune response such that only males with a good immune system may allocate higher amounts of this chemical to signaling.
FIGURE 6.6 Female mate choice in lizards. Relationships between the relative proportions of cholesta-5,7-dien-3-ol (pro vitamin D3) in femoral gland secretions of male Iberian wall lizards (Podarcis hispanica) and (a) the magnitude of their T-cell immune response (estimated with the phytohemagglutinin (PHA) delayed-type hypersensitivity test, or (b) the attractiveness of their pheromones to females (estimated from the number of females that preferred substrates scent-marked by a particular male) (J. Martı´n and P. Lo´pez, unpublished data).
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Similarly, female lizards Ps. algirus (Fam. Lacertidae) respond differently to the femoral gland secretions of males according to the parasite load and health of these males (Martı´n, Civantos, Amo, & Lo´pez, 2007b). Scents of healthier males elicit more TFs by females, suggesting that these scents are more attractive. Chemical analyses showed that parasite load and the T-cell-mediated immune response are related to the proportions of some lipids (alcohols and fatty acids) in the secretions of males. Further trials with chemical standards indicated that females actually discriminate the chemicals related to males’ health from other chemicals found in secretions (Martı´n et al., 2007b). These results suggest that pheromones may provide reliable information on the health and degree of parasitic infection of a male, and that females may prefer pheromones of males with a better health state. Pheromones might be honest signals if there were a tradeoff between sexual advertisement and the immune system, independent of the negative effects of parasites. In an experiment, the immune system of male Iberian wall lizards (P. hispanica) was challenged with a bacterial antigen (lipopolysaccharides (LPS) from the cell wall of the bacterium E. coli), without pathogenic effects, to explore whether immune activation per se affected chemical ornaments (Lo´pez, Gabirot, & Martı´n, 2009). The immune activation results in decreased proportions of provitamin D3 in femoral secretions (Lo´pez & Martı´n, 2005a). The active form of this steroid, vitamin D, has a variety of important effects on immune system function (Hayes et al., 2003). This suggests the existence of a tradeoff between physiological regulation of the immune system and the allocation of essential nutrients (e.g., vitamins or other ‘costly’ lipids) to sexual chemical ornaments in male lizards. Another possible cost of pheromone production may be related to the immunosuppressive effects of testosterone (Folstad & Karter, 1992; Wedekind & Folstad, 1994). In some lizards tested, the experimental elevation of T levels results in decreased immune responses (Belliure, Smith, & Sorci, 2004; Oppliger, Giorgi, Conelli, Nembrini, & John-Alder, 2004). Given that gland secretions are under direct androgenic control and vary seasonally with androgen production (Alberts et al., 1992a; 1992b), producing pheromones may require an increase in the levels of circulating T with subsequent immunosuppressive effects. Thus, only high-quality males could produce pheromones without detrimental effects on their immune system. This was tested in an experiment where T levels of male Iberian wall lizards (P. hispanica) were increased using silastic implants (Martı´n, Lo´pez, Gabirot, & Pilz, 2007). Different TF rates indicated that females discriminate and show greater responses to femoral secretions of T-implanted males than to control males, which suggests that T increases the concentration
of chemical signals. Chemical analyses indicated that T supplementation induces qualitative changes in lipid composition (i.e., the proportion of cholesta-5,7-dien-3-ol decreases) of femoral secretions (Martı´n et al., 2007a). These changes might be related to the immunosuppressive effect of T (Belliure et al., 2004; Oppliger et al., 2004). Variations in T levels have been implicated in lizards and other taxa in modulating immunity and lipid and steroid biochemistry (e.g., Sheridan, 1994; Lacy, Sheridan, & Moore, 2002). Further experiments on choice of males’ scent showed that females neither prefer nor avoid the scent marks of T-implanted males, but prefer males that maintain higher proportions of cholesta5,7-dien-3-ol in secretions, regardless of experimental manipulation (Martı´n et al., 2007a). Thus, because this steroid is negatively affected by T, there might be tradeoffs between increasing T levels to increase the production of chemical secretions, maintaining metabolism, and attracting females. Other theories of mate choice propose that males may evolve non-honest signals that exploit the sensory system of females (e.g., Fuller, Houle, & Travis, 2005). A preexisting sensory bias for food chemicals might explain the chemosensory preferences of females for male scents. When the hunger levels of female Iberian rock lizards (I. cyreni) are manipulated, food-deprived females exhibit increased chemosensory responses to chemical stimuli from both invertebrate prey and femoral secretions of males, but not to control water (Martı´n & Lo´pez, 2008a). Further tests suggested that provitamin D3, a lipid found in both prey and males’ scent, may be one of the chemicals eliciting these responses. Moreover, hungry females spend more time on male scent marks that have experimentally increased provitamin D3 than on scent marks of males alone, whereas for control females this effect is not so clear. These results suggest that preexisting sensory bias for essential nutrients (i.e., provitamin D3) may be the origin of similar female responses to male pheromones. However, sensory traps might evolve into honest signals if they are different costwise for males (Macı´as-Garcı´a & Ramirez, 2005). Previous studies have suggested that the allocation of these chemicals to ornaments is costly and only highquality males can afford it (Martı´n & Lo´pez, 2006a; 2006b; Lo´pez et al., 2009). Therefore, pre-existing sensory bias for essential nutrients may further allow the evolution and maintenance of honest pheromones used in mate choice.
5.2.4. Evolution of male social dominance based on pheromones Male competition over females often favors the evolution of male attributes that confer fighting ability and correlated
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conspicuous status-signaling traits (Andersson, 1994). When males of different dominance status exhibit different badges, individuals may use these badges to judge relative fighting ability and to modulate their own behavior accordingly, avoiding the costs of escalated aggressive interactions (Enquist & Leimar, 1983). In some lizards, pheromones may signal a male’s dominance status, or characteristics related to dominance, such as body size, through productivity rates and/or the quality of secretions (Alberts et al., 1992b; Lo´pez et al., 2003b; Moreira, Lo´pez, & Martı´n, 2006). Substrate scent marks can provide information on the individuality of familiar males, but also on body size and associated fighting ability of unfamiliar males (Arago´n et al., 2000; 2001a; Labra, 2006; Carazo, Font, & Desfilis, 2007). This information may influence behavior and space use by other males (Arago´n, Lo´pez, & Martı´n, 2001c; Arago´n et al., 2003; Labra, 2006). Pheromonal cues from dominant green iguana males (I. iguana) can elevate corticoid steroid levels in juveniles, which indicates stress, and this may suppress growth and assertion displays in juveniles (Alberts, Jackintell, & Phillips, 1994). Male Iberian wall lizards (P. hispanica) are attracted to areas scent-marked by males of higher competitive ability (i.e., larger size) but not to areas of smaller males (Carazo et al., 2007). This shows that males can assess rival competitive ability (i.e., rival size) from scent marks, and suggests that scent marks do not function as chemical barriers to deter intruders and that they may also inform on the quality of a territory. These studies suggest that males can process information from pheromones, and that decisions made during the ‘pre-confrontation’ stage are based on the assessment of the relative fighting abilities of potential opponents. During agonistic encounters between males, species and individual recognition and rival assessment based on pheromones may also be useful (Cooper & Vitt, 1987). The assessment of the probable outcome of future encounters with the same individual may be the best way of decreasing the costs of fighting (Lo´pez & Martı´n, 2001b). Pheromones are involved in rival recognition during agonistic interactions between male Iberian wall lizards (P. hispanica) (Lo´pez & Martı´n, 2002). Intruding familiar males experimentally impregnated with scents from unfamiliar males elicit aggressive responses by resident males similar to those observed for a new unfamiliar male with an unfamiliar scent. This suggests that males are unable to recognize familiar males when their own scents are removed. In contrast, responding males are less aggressive towards familiar males impregnated with their own scent or towards unfamiliar males impregnated with scents of familiar males, suggesting that, when two males have already interacted, their scents become familiar for both males and that the detection in successive encounters of the familiar scent reduces the aggressive responses (Lo´pez &
Hormones and Reproduction of Vertebrates
Martı´n, 2002). Therefore, pheromonal recognition mechanisms during agonistic encounters may reduce the intensity and costs of fighting and may play an important role in the organization of social systems. The chemical basis of rival assessment is little known, however, but this may be related to changes in concentrations of some chemicals in scents related to fighting ability (Alberts et al., 1992b). Proportions of cholesterol in the femoral secretions of male Iberian rock lizards (I. cyreni) increase with body size (Lo´pez et al., 2006). Males discriminate chemically and respond aggressively to cholesterol stimuli, and responses depend on their own body size (Martı´n & Lo´pez, 2007; 2008b). Moreover, in staged encounters in neutral cages between two unfamiliar and size-matched males, focal males lose more agonistic interactions against males manipulated to increase cholesterol in their scent than in control tests (Martı´n & Lo´pez, 2007). This may be explained because social dominance, lipid metabolism, femoral gland production, and percentage of lipids in secretions depend on androgen levels, and because testosterone upregulates cholesterol (Alberts et al., 1992b; Sheridan, 1994). Thus, it is likely that the proportions of cholesterol in secretions depends on sex steroid levels, which would make it a reliable signal of aggressiveness. Signaling social dominance may also be useful in other contexts. Male Iberian rock lizards (Iberolacerta monticola; formerly Lacerta monticola monticola) discriminate their own femoral secretions, and scent from their own copulatory plugs, from those of other males. Males also discriminate dominance of other males based on pheromones from femoral secretions and from copulatory plugs (Moreira et al., 2006). Femoral secretions signaling dominance may function to establish dominance hierarchies through the scent-marking of territories. Copulatory plugs and femoral secretions might allow males to scent-mark the female body during mating, and this behavior might influence male and female reproductive decisions under the selective pressures of sperm competition. Nevertheless, there is little knowledge regarding the mechanisms and evolutionary relationships between dominance status and pheromones. In lizards, both production of femoral gland secretion (Alberts et al., 1992b) and male dominance (Moore & Lindzey, 1992) are dependent on androgen levels. The immunosuppressive effect of testosterone (Folstad & Karter, 1992; Wedekind & Folstad, 1994; Belliure et al., 2004) may provide a link between dominance status and pheromones, such that only genuinely dominant males can maintain a good immune response and produce pheromones of dominance. Social costs may also be imposed by the targeted receivers. Thus, if the status signal is incongruent with behavior, deception may be detected and punished by genuinely dominant individuals (Olsson, 1994; Martı´n & Forsman, 1999).
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An experiment has examined whether femoral gland secretions of males may signal dominance status and explored the evolutionary mechanism on which this could be based. Staged encounters showed that larger male Iberian rock lizards (I. monticola) are dominant over smaller ones, but, after controlling for body size, dominant males are those that produce femoral secretions with higher proportions of two alcohols: hexadecanol and octadecanol (Martı´n et al., 2007c). In these experiments, males discriminate different concentrations of hexadecanol from other chemicals found in secretions and respond aggressively towards hexadecanol according to their own dominance status while responding neutrally towards other chemicals. These results suggest that hexadecanol may be a reliable status badge. Moreover, because hexadecanol elicits aggressive behavior, subordinate males signaling high status (‘cheaters’) may end up paying higher fighting costs, because honesty of the signal would be checked by social control from genuinely dominant males (Olsson, 1994; Martı´n & Forsman, 1999). Instead, subordinates might be better off avoiding the signaling of their inferior dominance status (not allocating hexadecanol to secretions). Similarly, dominant male green iguanas (I. iguana) produced larger amounts of femoral gland secretions than subordinates (Alberts et al., 1992b). In addition, male Iberian rock lizards with higher dominance status and that allocate higher proportions of hexadecanol to femoral secretions have greater T-cell immune responses (Martı´n et al., 2007c). This suggests a possible link between the quality of the immune system and the pheromones signaling dominance. Secretion of hexadecanol might be physiologically costly, so that only males in good condition can afford to signal high dominance status.
5.3. Snakes 5.3.1. Secretory glands and potential pheromones 5.3.1.1. Skin Chemical analyses of the skin or shed skin samples of many snake species have found a diversity of compounds such as fatty acids, steroids, alcohols, aldehydes, waxy esters, hydrocarbons, and methyl ketones (e.g., Roberts & Lillywhite, 1980; Schell & Weldon, 1985; Mason, Chinn, & Crews, 1987; Mason et al., 1989; Jacob, Ziemsen, & Hoppe, 1993; reviewed in Mason, 1992; Weldon et al., 2008). Lipids in the skin are mainly considered useful to avoid water loss (e.g., Burken, Wertz, & Downing, 1985), but some lipids may play an important role as pheromones, as suggested by observations of trailing, aggregation, and courtship (e.g., Mason, 1993). In amphibious sea snake
species (Laticauda spp.), skin lipid profiles differ between species and this allows interspecific chemosensory recognition and species isolation within sympatric congeners (Shine, Reed, Shetty, LeMaster, & Mason, 2002). The homologous series of relatively unvolatile longchain saturated and u-9-cis-unsaturated methyl ketones from the skin lipids of female garter snakes (Thamnophis sirtalis parietalis) have been characterized chemically and synthesized in the laboratory, and their role in attracting males and eliciting courtship is well-studied (Garstka & Crews, 1981; Mason et al., 1987; 1989; Mason, Jones, Fales, Pannell, & Crews, 1990; Mason, 1993; see Section 5.3.2.2). Similar series of ketones have been described in the skin of brown tree snakes (Boiga irregularis) (Murata et al., 1991). If male snakes do not detect these sexual attractiveness female pheromones, courtship will not be initiated and subsequent mating will not occur (Mason, 1993; Greene & Mason, 1998). Further, there is interpopulational variation in female garter snake pheromones (i.e., relative concentrations of individual unsaturated methyl ketones), associated with preferences of males of one population for courting and following scent trails of females of their own population (LeMaster & Mason, 2003). This suggests that pheromone-mediated sexual isolation exists to a degree among denning populations. 5.3.1.2. Cloacal scent glands Almost all snake species have a pair of elongated glands, located in the base of the tail, which secrete mainly lipids through two ducts located along the cloacal orifice. Secretions are mainly considered as defensive malodorous chemicals discharged to deter predators or even alarm pheromones for conspecifics (Graves & Duvall, 1988; Weldon, Sampson, Wong, & Lloyd, 1991; Mason, 1992; Weldon et al., 2008). However, cloacal secretions may also have other functions. Female brown tree snakes (B. irregularis) that are being courted by males sometimes release cloacal secretions that inhibit male courtship, serving to reject unpreferred mates (Greene & Mason, 2000; 2003). When the cloacal secretion of females is experimentally added into an arena where a pair of snakes have been placed, the time that males spend courting females and the intensity of courtship decrease compared with a control (Greene & Mason, 2003). Adding the cloacal secretion of males does not affect courtship time of males. These data suggest that a volatile pheromone in cloacal secretions of females acts in concert with the female skin sex pheromone to regulate the events leading to copulation. 5.3.1.3. Nasal glands These are a pair of glands found only in some colubrid snake species of the Subfam. Psammophiinae that are located
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lateral to the nasal cavity and that secrete close to the external nares (Dunson, Dunson, & Keith, 1978, de Haan, 2003). Secretions, consisting of lipids and proteins, are extended over the skin and serve to protect against water evaporation, but, also, it has been suggested that these snakes might perform self-rubbing to enable subsequent scent-marking of substrate and conspecific individuals (de Haan, 2003).
5.3.2. Chemosensory recognition 5.3.2.1. Sex discrimination and trailing Most snakes have the chemosensory ability to follow substrate-deposited scent trails for many functions, such as to locate sexual partners, overwintering hibernacula, or hidden prey, or to avoid conspecifics or predators (Ford & Low, 1984; Ford, 1986; Constanzo, 1989; Mason, 1992; Greene, Stark, & Mason, 2001; LeMaster & Mason, 2001). The ability of snakes to orient and locate mates based on pheromonal cues in the environment can have significant consequences for their reproductive success (Shine, O’Donnell, Langkilde, Wall, & Mason, 2005). Many female snake species, within at least five families, produce pheromone trails that guide males to their exact location during the mating season (e.g., Ford & Low, 1984; LeMaster & Mason, 2001; LeMaster, Moore, & Mason, 2001). Pheromone trails inform on the species identity, sex, sexual attractiveness, mating status, and travel direction of the individual signaler (Ford, 1982; Ford & Schofield, 1984; O’Donnell, Ford, Shine, & Mason, 2004). For example, male red-sided garter snakes (T. s. parietalis) tested on a Y-maze can detect and follow the skin lipid trails of females, but do not follow male trails. This suggests that the female pheromone responsible for mediating male trailing is the same sexual attractiveness skin pheromone responsible for releasing male courtship behavior (LeMaster & Mason, 2001). Most research on this topic was based on artificial situations, however, testing the ability of captive snakes to follow continuous trails over structurally simple homogeneous substrates. A field study tested the response of adult garter snakes (T. s. parietalis) to pheromone trails on the natural substrate under naturally occurring conditions. The results showed that during the spring breeding season males detect and follow female trails, whereas neither sex displayed trailing behavior during the autumn migration to the hibernacula (LeMaster et al., 2001). Another field experiment showed that male garter snakes follow a female’s trail less successfully if she has recently been followed by rival males, suggesting that trailing males obscured the cues left by the female (Shine, Webb, Lane, & Mason, 2005). Further, trail-following males switch from chemical to visual cues as soon as they are close to a female, enhancing speed but decreasing the accuracy of mate location (Shine
Hormones and Reproduction of Vertebrates
et al., 2005c). Because garter snakes mate in large aggregations, pheromones may be so widely distributed throughout the den area that it may be difficult for males to use pheromones to locate females. Experimental tests showed that males also use other visual (body size, muddiness) and thermal cues (very cold snakes are often females) to discriminate between males and females; but, after a mating ball forms around a female, pheromones may be the most important cue (Shine & Mason, 2001). The importance of pheromones for mate location and identification may also depend on the habitat of each species. Male turtle-headed sea snakes (Emydocephalus annulatus) use visual cues (size, movement, and coloration) to locate and assess whether any snake-shaped objects are potential sexual partners. Pheromonal skin lipids are important only after the male comes into physical contact with the stimulus (Shine, 2005). 5.3.2.2. Mate assessment Pheromones may play a major role in reproductive behavior in many snake species (Andren, 1982; Greene & Mason, 1998). Red-sided garter snakes (T. s. parietalis) in southern Manitoba (Canada) court and mate in large aggregations after emergence from overwintering dens and before dispersing to their summer ranges (Mason, 1993; Shine et al., 2001a). Chemoreception is the primary means by which male garter snakes assess another snake’s species, population, sex, body size, condition, and mated status (Devine, 1977a; Mason, 1993; Shine, Olsson, & Mason, 2000; Shine, O’Connor, & Mason, 2000c; LeMaster & Mason, 2003; Shine, Phillips, Waye, LeMaster, & Mason, 2003a; O’Donnell et al., 2004). Pheromonal cues not only enable males to discriminate between suitable and unsuitable mates, but pheromones may also provide many other types of information. For example, male garter snakes prefer to court larger females because they are likely to produce more or larger offspring. Males can discriminate among females of varying sizes by means of the skin sexual attractiveness pheromone of females. In laboratory tests, males prefer to display courtship behavior to skin lipid extracts from large females, showing that pheromone properties allow determination of female size (LeMaster & Mason, 2002). In the field, males not only direct more courtship to longer and heavier-bodied females, but also court most vigorously in response to lipids extracted from the skins of such females (Shine, Phillips, Waye, LeMaster, & Mason, 2003b). Chemical analyses showed that smaller females have skin pheromone profiles composed predominantly of saturated metyl ketones, while larger females express profiles dominated by unsaturated methyl ketones (LeMaster & Mason, 2002). Courtship and mating are size-assortative, however: larger male snakes court only large females, whereas small
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males court small and large females. Experiments in which body sizes and scents were manipulated showed that both vision and scent (sex pheromones) are important. Large males direct intense courtship only when the stimuli provide both visual and pheromonal evidence of large body size, whereas small males are much less discriminating in both respects (Shine et al., 2003a). Thus, size-assortative mating is generated not by larger males excluding their smaller rivals from the largest females (as occurs in other reptiles), but by a size-related shift in the visual and pheromonal cues that elicit courtship. Male garter snakes prefer to court newly emerged (weak) rather than dispersing (postrecovery) females. Experimental trials showed that papers impregnated with lipids from newly emerged females attract more intense male courtship than the scent of postrecovery females (Shine, Wall, Langkilde, & Mason, 2005a). This suggests that males use female skin pheromones to predict time since emergence, rather than other variables that reflect a recent emergence such as a low body temperature or high breathing rates. Chemoreception may also be used by male garter snakes to assess whether or not a female has recently mated (Devine, 1977b; Ross & Crews, 1977; Shine et al., 2000a; O’Donnell et al., 2004). Males deposit a thick gelatinous plug that occludes the female cloaca after copulation and physically prevents re-mating by the female (Shine et al., 2000a). Pheromonal cues from copulatory plugs were thought to discourage rival males from trying additional matings (Ross & Crews, 1977). However, experimental work supports the hypothesis that pheromonal information arises from fluids associated with copulation rather than with the plug per se (Shine et al., 2000a).
6. SUGGESTIONS FOR FUTURE RESEARCH
5.3.2.3. Pheromonal female mimicry In red-sided garter snakes (T. s. parietalis), there are some males, called ‘she-males,’ that mimic females (Mason & Crews, 1985; 1986). Males direct intense courtship to shemales as well as to females (Shine, Harlow, LeMaster, Moore, & Mason, 2000; Shine, O’Connor, & Mason, 2000d) (Figure 6.7). Experiments using hexane extracts showed that this effect is due to the skin lipids of she-males (Mason et al., 1989). Chemical analyses of female and shemale skin lipid extracts collected during the breeding season reveal a similar pheromone profile, one that is not observed in the skin lipids of males (Mason, 1993). The ‘she-maleness’ is an intrinsic property of a male rather than an artifact of lipid transfer from females (Shine et al., 2000b). ‘She-maleness’ is not, however, a permanent characteristic of a particular subset of males, but a transitory phase that most (perhaps all) male snakes pass through after they have emerged from the winter den (Shine et al., 2000b).
The results of these mainly recent experiments, which have suggested an important role of chemical cues in the reproductive behavior of reptiles, prompt a reexamination of how pheromones may affect many social and sexual behaviors such as mate choice, intrasexual interactions, etc. in other reptilian species. In most previous studies, there has been a probable ‘researcher’s sensory bias’ due to the prevalence of visual signals for human observers (even when pheromones might be more important for humans than we admit). However, the importance of the ‘hidden’ chemical signals should be reconsidered in many cases. The design of new studies and experiments that specifically look for pheromonal effects on reproductive behavior could provide new insights on this subject. We should increase and use knowledge on the glandular chemicals of reptiles to explore the functions of these chemicals and their potential role(s) as pheromones. This also suggests that future studies should be multidisciplinary and analyze simultaneously the chemistry of potential pheromones and their effects on
Recently emerged males have to restore their physiological performance (e.g., locomotion, fighting, and courtship ability) and spend the first day relatively inactive and without courting females. Interestingly, the experimental application of female skin lipids on to normal males decreases their courtship levels. These results suggest that recently emerged males may benefit from mimicking female skin lipids because it ‘switches off’ the male’s own (energetically expensive) courtship until they restore performance and courtship abilities. Female mimicking during this time might also disadvantage his rivals by distracting them from females and increasing their energy expenditure (Shine et al., 2000b). Nevertheless, she-males provide sufficient attraction and attract vigorous courtship of males only when females are not present, and she-males are abandoned immediately if a real female is found (Shine, Langkilde, & Mason, 2003c). This suggests that males search for the most female-like pheromonal stimulus. The costs of misdirected courtship to other males impose selection on the chemosensory abilities of male snakes to discriminate between sexes (Mason et al., 1987; 1989; 1990; Shine et al., 2003c). Thus, it has been suggested that she-males would attract other males, not to obtain a mating advantage within mating balls around females, but because courtship would provide warmth and protection to otherwise solitary she-males (Shine, Phillips, Waye, LeMaster, & Mason, 2001b).
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FIGURE 6.7 Pheromonal female mimicry in garter snakes. Relative attractiveness (mean SE) of females, normal males (he-males), and she-males to free-ranging male garter snakes (Thamnophis sirtalis parietalis). Attractiveness was assessed by measuring the proportion of males showing courtship behavior to the ‘target’ snake. Males were tested either when they were part of a ‘mating ball’ (group) or when they were solitary (lone). Reproduced from Shine, Harlow, LeMaster, Moore, and Mason, 2000.
behavior of reptiles. This has been traditionally done in studies of the chemical ecology of invertebrates, especially those with economic interests such as insect pests, but it is rarely done in studies of vertebrates and particularly reptiles. Most current studies are based on a few ‘model’ species (e.g., rock lizards and garter snakes), but we need to study more species representative of other groups, and more species within each group, if we want to know the extent of generalization of the known results or whether different alternatives exist. Knowing more about a diversity of species would allow comparative studies that would lead to a better understanding of the functional and evolutionary role of pheromones in reptiles. The interaction of chemical signals with other types of signals (visual, acoustic, etc.) should also be considered. One interesting promising field is the understanding of the influence of the environment on the use and relative importance of different types of communication system (e.g., visual vs. chemical). Multiple types of signal may be used by the same animal, and we need to understand whether multiple traits signal different characteristics or are redundant. Reptiles are especially good models for this question as they use both visual and chemical signals, and it
is likely that different types of signal deliver similar messages but in different contexts (e.g., long vs. short distance, or in different microhabitats). An interesting research field may be to understand the evolution of pheromones as signals. We need to know the evolutionary and physiological mechanisms that allow the receivers to rely on the message of the pheromone (why are pheromones reliable signals?) or, alternatively, that allow the senders to exploit the sensory physiology of the receivers with the pheromones (sensory bias). This is a current and rapidly growing topic in the case of visual signals based on carotenoids or other pigments, especially in birds, but it is almost unknown in the case of chemical signals. However, it is likely that the mechanisms involved are similar and, moreover, that the chemicals used to ‘construct’ visual signals are but a particular subset of chemical signals and that the only difference is the sensory system (either the visual or the vomeronasal system) used by the receivers to detect the presence and concentration of these chemicals. The studies of reproductive behavior and signaling systems based on pheromones may, thus, be not just a ‘curiosity’ of some ‘peculiar’ animals, but may help in understanding how intraspecific communication of most animals has evolved.
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Pheromones and Reproduction in Reptiles
ACKNOWLEDGEMENTS We thank W.E. Cooper and A. Salvador for ‘inducing’ us to study chemical communication in reptiles, and to all our friends that have worked with us to understand the pheromones of reptiles: L. Amo, P. Arago´n, C. Cabido, E. Civantos, M. Cuadrado, M. Gabirot, A. Gonzalo, R. Kopena, J.J. Luque-Larena, P.L. Moreira, K.M. Pilz, and N. Polo-Cavia. We also thank ‘El Ventorrillo’ MNCN Field Station for the long-term use of their facilities and Nino for taking care of and feeding lizards and researchers. Financial support during writing was provided by the projects MEC-CGL2005-00391/BOS and MCI-CGL2008-02119/BOS.
ABBREVIATIONS FA LPS MHC PHA T TF VNO
Fluctuating asymmetry Lipopolysaccharides Major histocompatibility complex Phytohemagglutinin Testosterone Tongue-flick Vomeronasal organ
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Shine, R., O’Connor, D., & Mason, R. T. (2000c). The problem with courting a cylindrical object: how does an amorous male snake determine which end is which? Behaviour, 137, 727–739. Shine, R., O’Connor, D., & Mason, R. T. (2000d). Female mimicry in garter snakes: behavioural tactics of "she-males" and the males that court them. Can. J. Zool., 78, 1391–1396. Shine, R., Langkilde, T., & Mason, R. T. (2003c). Confusion within ‘mating balls’ of garter snakes: does misdirected courtship impose selection on male tactics? Anim. Behav., 55, 1011–1017. Shine, R., Olsson, M., & Mason, R. T. (2000a). Chastity belts in garter snakes: the functional significance of mating plugs. Biol. J. Linn. Soc., 70, 377–390. Shine, R., Wall, M., Langkilde, T., & Mason, R. T. (2005a). Battle of the sexes: forcibly inseminating male garter snakes target courtship to more vulnerable females. Anim. Behav., 70, 1133–1140. Shine, R., Harlow, P. S., LeMaster, M. P., Moore, I., & Mason, R. T. (2000b). The transvestite serpent: why do male garter snakes court (some) other males? Anim. Behav., 59, 349–359. Shine, R., Elphick, M. J., Harlow, P. S., Moore, I. T., LeMaster, M. P., & Mason, R. T. (2001a). Movements, mating and dispersal of red-sided garter snakes from a communal den in Manitoba. Copeia, 2001, 82–91. Shine, R., Phillips, B., Waye, H., LeMaster, M., & Mason, R. T. (2001b). Advantage of female mimicry to snakes. Nature, 414, 267. Shine, R., Reed, R. N., Shetty, S., LeMaster, M., & Mason, R. T. (2002). Reproductive isolating mechanisms between two sympatric sibling species of sea-snakes. Evolution, 56, 1655–1662. Shine, R., Phillips, B., Waye, H., LeMaster, M., & Mason, R. T. (2003a). The lexicon of love: what cues cause size-assortative courtship by male garter snakes? Behav. Ecol. Sociobiol., 53, 234–237. Shine, R., Phillips, B., Waye, H., LeMaster, M., & Mason, R. T. (2003b). Chemosensory cues allow courting male garter snakes to assess body length and body condition of potential mates. Behav. Ecol. Sociobiol., 54, 162–166. Shine, R., O’Donnell, R. P., Langkilde, T., Wall, M. D., & Mason, R. T. (2005b). Snakes in search of sex: the relationship between matelocating ability and mating success in male garter snakes. Anim. Behav., 69, 1251–1258. Shine, R., Webb, J. K., Lane, A., & Mason, R. T. (2005c). Mate location tactics in garter snakes: effects of rival males, interrupted trails and non-pheromonal cues. Funct. Ecol., 19, 1017–1024. Solomon, S. E. (1984). The characterisation and distribution of cells lining the axillary gland of the adult green turtle (Chelonia mydas L.). J. Anat., 138, 267–279. Steele, L. J., & Cooper, W. E., Jr. (1997). Investigation of pheromonal discrimination between conspecific individuals by male and female leopard geckos (Eublepharis macularis). Herpetol., 53, 475–484. Tokarz, R. R. (1995). Mate choice in lizards: a review. Herp. Monogr., 9, 17–40. Trauth, S. E., Cooper, W. E., Jr., Vitt, L. J., & Perrill, S. A. (1987). Cloacal anatomy of the broad-headed skink, Eumeces laticeps, with a description of a female pheromone gland. Herpetol., 43, 458–466. Van Wyk, J. H. (1990). Seasonal testicular activity and morphometric variation in the femoral glands of the lizard Cordylus polyzonus polyzonus (Sauria: Cordylidae). J. Herp., 24, 405–409.
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Waldman, B. (1987). Mechanisms of kin recognition. J. Theor. Biol., 128, 159–185. Wedekind, C., & Folstad, I. (1994). Adaptive or nonadaptive immunosuppression by sex hormones? Am. Nat., 143, 936–938. Weldon, P. J., & Bangall, D. (1987). A survey of polar and nonpolar skin lipids from lizards by thin-layer chromatography. Comp. Biochem. Physiol. B, 87, 345–349. Weldon, P. J., & Ferguson, M. J. (1993). Chemoreception in crocodilians: Anatomy, natural history, and empirical results. Brain Behav. Evol., 41, 239–245. Weldon, P. J., & Sampson, H. W. (1988). The gular glands of Alligator mississippiensis histology and preliminary-analysis of lipoidal secretions. Copeia, 1988, 80–86. Weldon, P. J., & Tanner, M. J. (1990). Lipids in the Rathke’s gland secretions of hatchling loggerhead sea turtles (Caretta caretta). Copeia, 1990, 575–578. Weldon, P. J., & Tanner, M. J. (1991). Gular and paracloacal gland secretions of crocodilians: A comparative analysis by thin-layer chromatography. Biochem. Syst. Ecol., 19, 133–141. Weldon, P. J., & Wheeler, J. W. (2000). The chemistry of crocodilian skin glands. In G. C. Grigg, F. Seebacher, & C. E. Franklin (Eds.), ‘‘Crocodilian Biology and Evolution’’ (pp. 286–296). Chipping Norton, New South Wales, Australia: Surrey Beatty & Sons. Weldon, P. J., Flachsbarth, B., & Schulz, S. (2008). Natural products from the integument of nonavian reptiles. Nat. Prod. Rep., 25, 738–756. Weldon, P. J., Shafagati, A., & Wheeler, J. W. (1987). Lipids in the gular gland secretion of the American alligator (Alligator mississippiensis). Z. Naturforsch. C, 42, 1345–1346. Weldon, P. J., Shafagati, A., & Wheeler, J. W. (1988). Lipids from the paracloacal glands of the American alligator (Alligator mississippiensis). Lipids, 23, 727–729. Weldon, P. J., Scott, T. P., & Tanner, M. J. (1990b). Analysis of gular and paracloacal gland secretions of the American alligator (Alligator mississippiensis) by thin-layer chromatography: gland, sex and individual differences in lipid components. J. Chem. Ecol., 16, 3–12.
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Weldon, P. J., Dunn, B. S., McDaniel, C. A., & Werner, D. I. (1990a). Lipids in the femoral gland secretions of the green iguana (Iguana iguana). Comp. Biochem. Physiol. B, 95, 541–543. Weldon, P. J., Sampson, H. W., Wong, L., & Lloyd, H. A. (1991). Histology and biochemistry of the scent glands of the yellowbellied sea-snake (Pelamis platurus, Hydrophiidae). J. Herp., 25, 367–370. Weldon, P. J., Swenson, D. J., Olson, J. K., & Brinkmeier, W. G. (1990c). The American alligator detects food chemicals in aquatic and terrestrial environments. Ethology, 85, 191–198. Werner, D. I., Baker, E. M., Gonzales, E. C., & Sosa, I. R. (1987). Kinship recognition and grouping in hatchling green iguanas. Behav. Ecol. Sociobiol., 21, 83–89. Westneat, D. F., & Birkhead, T. R. (1998). Alternative hypothesis linking the immune system and mate choice for good genes. Proc. R. Soc. Lond. B, 265, 1065–1073. Wheeler, J. W., Ibrahim, S. A., & Weldon, P. J. (1999). 2,3Dihydrofarnesyl and citronellyl esters in the paracloacal gland secretions of the brown caiman (Caiman crocodilus fuscus) from Costa Rica. Biochem. Syst. Ecol., 27, 27–32. Whiting, A. M. (1967). Amphisbaenian cloacal glands. Am. Zool., 7, 776. Whyte, A., Yang, Z., Weldon, P. J., Eisner, T., & Meinwald, J. (1999). Reptilian chemistry: Characterization of dianeackerone, a secretory product from a crocodile. Proc. Natl. Acad. Sci. USA, 96, 12246– 12250. Winokur, R. M., & Legler, J. M. (1975). Chelonian mental glands. J. Morphol., 147, 275–292. Wyatt, T. D. (2003). Pheromones and Animal Behaviour. Cambridge: Cambridge University Press. Yang, Z., Whyte, A., Attygalle, A. B., Weldon, P. J., Eisner, T., & Meinwald, J. (1999). Reptilian chemistry: Characterization of a family of dianeackerone-related steroidal esters from a crocodile secretion. Proc. Natl. Acad. Sci. USA, 96, 12251–12256. Zahavi, A. (1975). Mate selectiondA selection for a handicap. J. Theor. Biol., 53, 205–214.
Chapter 7
Stress and Reproduction in Reptiles Richard R. Tokarz* and Cliff H. Summersy *
University of Miami, Coral Gables, FL, USA, y University of South Dakota, Vermillion, SD, USA
SUMMARY This review (1) describes behavioral, endocrine, and neural stress responses and their effects on reproduction in reptiles; (2) proposes a functional definition of stress for both acute and chronic conditions; (3) discusses the effects of stress on reproduction within the context of natural life histories; (4) provides a summary of the pertinent findings of the reviewed literature; and (5) offers ideas on future research directions to further our understanding of the effects of stress on reproduction. Studies of reptiles have shown that a variety of stressors will induce a stress response with an increase in plasma corticosterone levels and that this increase in corticosterone may affect gonadal function, reproductive behavior, pregnancy and egg-laying success, survival, and other aspects of fitness. There is also evidence that the effects of stress in reptiles may be modulated by environment, sex, reproductive state, season, mating strategy, and tradeoffs between survival probability and reproductive success.
1. INTRODUCTION 1.1. Objectives This chapter attempts to provide a comprehensive review in the sense that not only the findings of relatively recent studies but also those of the older literature (reviewed in Greenberg & Wingfield, 1987; Lance, 1990; Guillette, Cree, & Rooney, 1995; Greenberg, 2002; Romero, 2002; Moore & Jessup, 2003) are discussed. More space has been allocated to discussing the results of more recent studies because they have not been reviewed previously.
1.2. Definitions The word ‘stress’ has been used in a variety of ways and this has led to ambiguity in its meaning (Wingfield & Ramenofsky, 1999; Creel, 2001; Dallman, 2003; McEwen & Wingfield, 2003; Romero, 2004). Romero (2004, p. 250) in his discussion of what constitutes stress drew
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attention to this problem by noting that the term stress has been used at various times to refer to: ‘(i) the noxious stimuli that an individual is exposed to; (ii) the physiological and behavioral coping responses to those stimuli; and (iii) the overstimulation of the coping responses that results in disease.’ Clearly stress has been used to describe related but distinctly different phenomena. As the word stress has been and continues to be used in so many different ways, Creel (2001) even suggested that in general it would be clearer if the word were avoided in favor of referring explicitly to stressors and stress responses. Although there is some merit to this suggestion, we feel that such a solution is unnecessary if the word stress is defined carefully and used appropriately. Moreover, as the title of this and other reviews indicate, it is not possible to forego using the word stress. In this review, stress is defined (as in Pottinger, 1997) as the internal state that occurs when an animal is exposed to a stressor; stressors as external conditions that are capable of disrupting an animal’s homeostasis; and a stress response as the physiological and behavioral mechanisms that act to restore an animal’s homeostasis. As in Pottinger (1997), this chapter considers the effects of stress on reproduction in reptiles in those situations in which exposure to a stressor results in the activation of a physiological stress response and secretion of glucocorticoids, in this case corticosterone (CORT), the primary glucocorticoid of reptiles (Hanke & Kloas, 1995). Increased secretion of glucocorticoids has been used commonly as an indicator of a stress response (Greenberg & Wingfield, 1987; Wingfield & Ramenofsky, 1999; Creel, 2001; Moore & Jessop, 2003; Romero, 2004). As discussed in Creel (2001), glucocorticoid secretion is a useful indicator of a stress response because glucocorticoids have been measured in a relatively broad range of species and because the physiological and behavioral effects of glucocorticoids are fairly well known.
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Stress, or the internal state produced in response to a stressor, engages neural circuits that integrate specific physiological and behavioral responses (Summers & Winberg, 2006). This integrative response to stressors plays an important role in the concept that the stress response is a strategy by which animals cope with stressful social and physical environments (Koolhass et al., 1999; McEwen & Wingfield, 2003; described in Section 3.4). The response to stressful environments also has a temporal aspect, making it important to understand the differences between acute and chronic stresses. The definition of acute stress should be determined by the intersection of the timing of neural and physiological stress responses with the behavioral changes that accompany them. Thus, acute stress is functionally relevant at the specific time point when a neural or physiological component of the stress response has changed from baseline (e.g., increased concentrations of brain corticotropin-releasing factor (CRF)) and behavioral or motivational change is manifest (e.g., increased sensitivity to aggressive display and/or decline in courtship behavior) after an animal has been exposed to a stressor. This is difficult, because in many cases no temporal framework has been established relating neural and physiological responses to stressors with behavioral responses to stressors. However, both social and physical stressors have been linked temporally to neuroendocrine changes in some species of lizard (Matter, Ronan, & Summers, 1998; Summers, Larson, Summers, Renner, & Greenberg, 1998; Summers et al., 2003a; 2005a; 2005b), and the results of these studies may provide some framework with which to judge the differences between acute vs. chronic stress. The normal temporal pattern of neuroendocrine stress responsiveness in examined lizards appears to be biphasic (see Section 2.1 on brain mechanisms mediating stress response for specific details), which may indicate a natural demarcation between acute and chronic stress (Summers, 2002). Whereas the exact timing is likely to be different for each species, and also to be modified by allostatic load (see Section 1.2, the paragraph below), acute stress appears to be evident during motivational and/or behavioral stages that last up to approximately 60 minutes, whereas chronic stress may functionally be defined by stressors that last from 60 minutes to many weeks. Homeostasis is the maintenance of the status quo via feedback mechanisms. Adapting ideas originally developed from human clinical research (Sterling & Eyer, 1988), McEwen and Wingfield (2003, p. 2) have emphasized the importance in biology and medicine of the concept of ‘allostasis,’ defined as ‘maintaining stability through change,’ and have provided a new set of terms related to this concept. They defined ‘allostatic load’ as ‘the cumulative cost to the body of allostasis,’ and ‘allostatic
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overload’ as ‘being a state in which serious pathophysiology can occur.’ This new nomenclature reflects the idea that there is a difference between an animal’s response to a predictable and non-life-threatening stressor and to an unpredictable, potentially life-threatening stressor. McEwen and Wingfield (2003, p. 4) used the term ‘stress’ to ‘describe events that are threatening to an individual and which elicit physiological and behavioral responses as part of allostasis in addition to that imposed by the normal life cycle.’ Wingfield and Ramenofsky (1999) previously had termed these facultative behavioral and physiological responses triggered by unpredictable events in the environment an ‘emergency life-history stage’ and noted that these responses appear to be mediated by increases in glucocorticoids. In general, the concept of allostasis as developed by McEwen and Wingfield (2003) has been favorably received (Dallman, 2003; Schulkin, 2003; Walsberg, 2003; Romero, 2004). Schulkin (2003) noted that the allostatic concept brings together both the biomedical and ecological approaches to studying stress and promises to be especially helpful in understanding the biological consequences of stress in an ecological context. Some aspects of the allostatic concept have received criticism, however. For example, Dallman (2003) has questioned (1) whether the new terms (‘allostasis,’ ‘allostatic load,’ ‘allostatic overload’) are really needed; (2) whether the idea that the allostatic response systems are not essential for maintenance of life is valid; and (3) whether the proposed allostatic response system differs in kind from homeostatic response systems. Dallman (2003) also emphasized the importance of explicitly including central neural mechanisms in any consideration of stressors and the stress response. In the view of Walsberg (2003), the primary weakness of the allostatic concept is that it places too much emphasis upon the balance between an animal’s energy expenditure and energy intake. Specifically, Walsberg (2003) points out that animals in nature are rarely in energy balance and that an animal’s energy balance can be greatly modified without necessarily inducing stress. Thus, according to Walsberg (2003), energy balance may be of limited utility as an index of physiological stress. A final matter relating to definitions concerns the naming of the glands in reptiles that synthesize the stress hormones. In reptiles the steroidogenic tissue that synthesizes the glucocorticoid hormones and the chromaffin tissue that synthesizes the catecholamine hormones are intermingled to varying degrees within paired glands (Lofts & Bern, 1972; Lofts, 1978; Chester Jones & Phillips, 1986; Norris, 2007). Because these tissues in many reptiles (chelonians, crocodilians, and most snakes) are positioned, as in mammals, above the kidneys (Norris, 2007), we will refer to them as adrenal glands.
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1.3. Importance of Studying Reptiles The extant reptiles consist of the tortoises, turtles, and terrapins (order Testudines or Chelonia); the tuatara (order Rhynchocephalia or Sphenodontida); the snakes, lizards, and amphisbaenids (order Squamata); and the crocodiles, consisting of the gavials (or gharials), crocodiles, and alligators (order Crocodilia) (Tudge, 2000; Vitt & Cardwell, 2008). The reptiles are considered to be an important group in vertebrate evolution, based on their close relationship to birds and common ancestry with a group that gave rise to mammals (Benton, 2005). There are several reasons why it is important to study the effects of stress on reproduction in reptiles. Firstly, such studies are necessary for the conservation of these vertebrates. Like amphibians, many wild populations of reptiles are declining and some species are considered to be in danger of extinction (see reviews by Gibbons et al., 2000; Cockrem, 2005; Irwin & Irwin, 2006). The decline in population size and/or range has prompted studies of how stressors affect reproduction in the tuatara, Sphenodon punctatus (Tyrell & Cree, 1998); desert tortoise, Gopherus agassizi (Tracy et al., 2006); olive ridley turtle, Lepidochelys olivacea (Valverde, Owens, MacKenzie, & Amoss, 1999); Kemp’s ridley turtle, Lepidochelys kempii (Gregory & Schmid, 2001); loggerhead turtle, Caretta caretta (Whittier, Corrie, & Limpus, 1997); hawksbill turtle, Eretmochelys imbricata (Jessop, 2001); green turtle, Chelonia mydas (Jessop, Hamann, Read, & Limpus, 2000; Jessop, 2001; Jessop, Knapp, Whittier, & Limpus, 2002); and Texas horned lizard, Phyrnosoma cornutum (Wack, Fox, Hellgren, & Lovern, 2008). Studies of the effects of stress on reproduction in reptiles might also contribute to the control of harmful invasive species such as, for example, the brown tree snake, Boiga irregularis (Moore et al., 2005). Knowledge obtained from studies of the effects of stress on reproduction in reptiles also may lead to better management of captive reptiles in zoos as well as more humane treatment of reptiles in laboratory research (Langkilde & Shine, 2006). Squamate reptiles (lizards and snakes) exhibit unique reproductive patterns and have many characteristics useful for studying the effects of stress on reproduction. For example, in some species of lizards and snakes, males, and in some cases females, exhibit alternative reproductive tactics (Sinervo & Lively, 1996; Moore, Hews, & Knapp, 1998; Knapp, 2004; Calsbeek & Sinervo, 2008). Species with alternative reproductive tactics may be of special utility in demonstrating how exposure to stressors affects tradeoffs between mating success and individual survival (Knapp & Moore, 1996; Knapp & Moore, 1997a; Miles, Sinervo, Hazard, Svensson, & Costa, 2007b; Lancaster, Hazard, Clobert, & Sinervo, 2008). In addition, there are oviparous, viviparous (Blackburn, 2000; Thompson &
Blackburn, 2006), bisexual, and unisexual species of lizards and snakes (Crews, Gustafson, & Tokarz, 1983; Summers, 1984b), as well as several sex-determining mechanisms (Bull, 1980; Viets, Ewert, Talent, & Nelson, 1994; Sarre, Georges, & Quinn, 2004; Janzen & Phillips, 2006). Associated and dissociated reproductive patterns have been described (Crews, 1984; Crews et al., 1984). Finally, it is important to study the effects of stress on reproduction in reptiles because so much remains to be learned. Although it is quite clear that the adrenocortical response to acute stress in reptiles in terms of the release of stress hormones is similar to that observed in other vertebrates (see Romero, 2002; Moore & Jessup, 2003; Wingfield & Sapolsky, 2003), relatively little is known about the mechanisms that mediate the stress response in these vertebrates in terms of cellular receptors, plasma binding proteins, etc. (Moore & Jessup, 2003).
1.4. Relationship of Stress to Reproduction in Reptilian Life Histories In considering the effects of stress on reproduction in reptiles, it is important to consider these effects in terms of natural life histories (see Chapters 10–13, this volume). Although many studies of stress in reptiles and other vertebrates have tended to focus on those instances when animals suddenly experience highly aversive conditions in their environment, such as predation attempts or storms, stressful conditions may arise on a day-to-day basis, involve a variety of different stressors, and have an impact on a variety of life-history traits (Greenberg, Carr, & Summers, 2002). As knowledge of the physiological mechanisms that govern the stress response is still minimal, we believe that studies of the effects of stress on reproduction in reptiles (at least at this point in time) are likely to be of most value in testing hypotheses related to behavior, ecology, and evolution rather than in testing hypotheses related to the proximate mechanisms that govern the stress response. For this reason we believe that studies that examine the effects of stress on reproduction in reptiles within the context of the life history are of particular value. Studies of reptiles that have examined, for example, how exposure to stressors affects offspring fitness (Belliure, Meylan, & Clobert, 2004; Preest, Cree, & Tyrrell, 2005; Weiss, Johnston, & Moore, 2007) as well as the possible tradeoff between reproductive success and survival (e.g., Shine, 1980; French, DeNardo, & Moore, 2007a; French & Moore 2008; Lancaster et al., 2008; Mills et al., 2008) illustrate such a life-history approach. Whereas global climatic changes are likely to influence the reproductive success of whole populations or multiple species, relatively small changes in microhabitat such as differences in soil moisture or local humidity may
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adversely affect reproductive function (Jones, Summers, & Lopez, 1983; Summers, Austin, & Jones, 1985; Summers, 1988) in a subset of a reptilian population. Glucocorticoids affect and are in turn affected by an animal’s feeding and energetics (Cash & Holberton, 1999; Woodley, Painter, Moore, Wikelski, & Romero, 2003; Cote, Clobert, Meylan, & Fitze, 2006). These glucocorticoid-induced changes in feeding and energetics may also have an effect on gonadal function (Moore & Jessop, 2003; Cote et al., 2006) and even offspring fitness (Meylan & Clobert, 2005; Cote et al., 2006; Vercken, de Fraipont, Dufty, & Clobert, 2007; Lancaster et al., 2008). Stress responsiveness and coping strategies in reptiles are also affected by predation (Berger, Wikelski, Romero, Kalko, & Ro¨dl, 2007; Ro¨dl, Berger, Romero, & Wikelski, 2007), by territorial behavior, and by social rank (Korzan & Summers, 2007) as well as by an individual’s sex (Shine, 2005). For example, while both male and female reptiles exhibit aggressive behavior in an attempt to attain territories and elevated social rank, the purpose and advantages of aggression may be sex-specific (Summers & Greenberg, 1994; Summers, Suedkamp, & Grant, 1995; Andrews & Summers, 1996; Korzan, Øverli, & Summers, 2006). It is important to consider the ecological and social aspects of reptilian life history, because the effects of stress on reproduction may vary based on sex, size, stage of development, social status, opponent recognition, daily and annual cycles, and/or environmental conditions such as food or water availability (Jones et al., 1983; DauphinVillemant & Xavier, 1987; Summers, 1988; Summers & Norman, 1988; Summers, 1995; Summers et al., 1995; Summers, Hunter, & Summers, 1997; Forster, Watt, Korzan, Renner, & Summers, 2005; Korzan & Summers, 2007; French, Fokidis, & Moore, 2008).
2. MECHANISMS MEDIATING STRESS RESPONSE 2.1. Brain The release of adrenal glucocorticoid hormones into plasma is often the primary or only focus of studies on stress, and this is also true for studies that examine the effects of stress on reproduction. However, it is critical to keep in mind that (1) the release of glucocorticoid hormones is governed by the actions of the hypothalamic– pituitary–adrenal (HPA) axis; (2) the HPA axis is regulated by both the central and peripheral nervous systems (Capaldo et al., 2003); and (3) the hormones of the HPA axis in turn may affect the central nervous system (Summers et al., 2000; Summers et al., 2003b). No neurotransmitter systems, with the possible exception of those that involve CRF, are specifically
Hormones and Reproduction of Vertebrates
associated with stress, yet stress-related neurocircuitry overlaps and interacts with other behavioral and reproductive neurocircuits (Summers & Winberg, 2006). These neurocircuits in mammals are primarily glutamatergic, g-amino butyric acid (GABA)-ergic, and CRFergic, and involve limbic brain regions such as the hippocampus, amygdala, bed nucleus of the stria terminalis, hypothalamus, raphe, and locus ceruleus (Herman & Cullinan, 1997; Curtis, Pavcovich, & Valentino, 1999; Forster et al., 2008; Mo, Feng, Renner, & Forster, 2008). Similar neurotransmitter systems and brain regions are involved in reptilian stress responses (Bugnon et al., 1984; Greenberg, 2003; Summers et al., 2005a; Summers & Winberg, 2006; Øverli et al. 2007). For example, in the green anole lizard (Anolis carolinensis) stress resulting from social interactions in males elevates expression of glutamatergic N-methyl-D-aspartic acid (NMDA) receptor subunit (NR2) in the lizard medial cortex (Meyer, Keifer, Korzan, & Summers, 2004) and also influences the relative ratio of NR2A to NR2B subunit concentration in this hippocampal region (Summers et al., 2005a), thereby modifying receptor function and presumably postsynaptic current. The stress resulting from anoxia modifies cortical cell NMDA current in turtles (Chryemys picta belli (Shin & Buck, 2003)) and increases GABA concentrations (Chrysemys scripta elegans (Hitzig, Kneussl, Shih, Brandstetter, & Kazemi, 1985)). These glutamate and GABA circuits influence and are influenced by other transmitter systems, such as serotonin (5-HT), dopamine (DA), and norepinephrine (NE). For example, GABA inhibits 5-HT release in the hippocampus of A. carolinensis (Summers, 2001; Summers et al., 2003b). Stress in response to heat exposure stimulates increased brain 5-HT in the lizard, Agama stellio (Mohamed & Rahman, 1982). Stress resulting from restraint also stimulates serotonergic activity in numerous brain regions, including nucleus accumbens, hippocampus, amygdala, and locus ceruleus, but surprisingly not in the raphe, where 5-HT perikarya are located (Emerson, Kappenman, Ronan, Renner, & Summers, 2000; Ling et al., 2009). The stress resulting from social interactions in male lizards rapidly increases serotonergic activity in brain regions associated with stress neurocircuitry (e.g., hippocampus, amygdala, hypothalamus, raphe, and locus ceruleus), in a pattern differentiated by social rank (Summers & Greenberg, 1995; Matter et al., 1998; Summers et al., 1998; Korzan, Summers, & Summers, 2000; Korzan, Summers, Ronan, Renner, & Summers, 2001; Greenberg, 2003; Summers et al., 2003a; Korzan & Summers, 2004; Summers et al., 2005a; 2005b; Summers & Winberg, 2006; Watt, Forster, Korzan, Renner, & Summers, 2007). Social interactions and stress hormones follow a similar temporal schedule as rank-defined serotonergic activity (Summers, 2001; 2002; Summers et al., 2005c). Glucocorticoids both systemically
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Stress and Reproduction in Reptiles
and locally modify 5-HT release and activity in the hippocampus of A. carolinensis (Summers et al., 2000, Summers, 2001; Summers et al., 2003b). For social interactions, 5-HT appears to be an important stress-related neurotransmitter because the inhibition of the reuptake of 5-HT reversed social status in male A. carolinensis (Larson & Summers, 2001). Stress resulting from hyper-osmotic conditions and dehydration influences 5-HT, but also NE, epinephrine (E), and CORT in the softshelled turtle, Lissemys punctata punctata, (Mahapatra, Mahata, & Maiti, 1991). Similarly, stress resulting from physical restraint elevates central catecholamines in the stress circuitry of the lizard A. carolinensis (Waters et al., 2005; Ling et al., 2009). While DA and other central catecholamines are stress-responsive (Korzan et al., 2000; Korzan et al., 2001; Korzan, Forster, Watt, & Summers, 2006; Ling et al., 2009), the role for DA appears to be more closely related to memory and motivation (Korzan & Summers, 2007; Korzan et al., 2007). Motivation and memory are important during stress, and brain regions associated with motivation, such as the nucleus accumbens, are modified in reptiles (Summers et al., 2003a; Summers et al., 2005c) and mammals (Lukkes, Forster, Renner, & Summers, 2008) by activation of stress circuitry and neurotransmitters. As might be presumed, neuroendocrine stress responses due to social interactions can be very rapid. In the mountain spiny lizard, Sceloporus jarrovi, neurochemical changes can be measured within 0.5 minutes (Matter et al., 1998), or as rapidly as 0.42–1.50 minutes after exposure to physical or social stressors in A. carolinensis (Emerson et al., 2000; Waters et al., 2005; Watt et al., 2007; Ling et al., 2009). Following a very rapid neural response in A. carolinensis, a discernable glucocorticoid response to stressors is evident within 1.5 minutes (Summers et al., 2005c; Watt, Korzan, Summers, & Forster, 2005; Ling et al., 2009). The results from both neural and endocrine measurements of male A. carolinensis that were exposed to social stressors for an extended period suggest a biphasic stress response with the initial phase ending somewhere between 20 and 60 minutes (Summers et al., 2003a; Summers et al., 2005c). A second neural and endocrine stress response phase begins between 40 and 60 minutes, depending on social status, brain region examined, and neural or endocrine stress parameter measured (Summers, 2002; Summers et al., 2003a). The temporal range of the two putative stress phases suggested by these results have a similar time frame to that of the pulsatile secretion of the HPA axis hormones CRF, corticotropin (ACTH), and CORT in mammals (Lightman et al., 2002). For example, in rats, half of one full pulse phase from nadir to peak of one CORT pulse in plasma or brain lasts the order of 30 minutes (Lightman et al., 2002; Droste et al., 2008). This pulsatile ultradian pattern defines the normal circadian rhythm, and the frequency of
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glucocorticoid pulses is increased by chronic stress (Lightman, 2008). In lizards, chronic stress also alters the circadian phase relationship of HPA secretion by increasing the frequency of CORT pulses (Summers & Norman, 1988). The normal phase relationships and biphasic pattern of neuroendocrine stress responsiveness observed in many vertebrates including reptiles suggests a natural demarcation between acute and chronic stress (Summers, 2002). While the exact timing is likely to be different for each species, and also to be modified by allostatic load, acute stress appears to begin approximately 0.4 minutes after exposure of an animal to a stressor and to last up to 60 minutes, whereas chronic stress may last from 60 minutes to many weeks.
2.2. Hypothalamic–Pituitary–Adrenal (HPA) Axis The reptilian endocrine stress response appears to include similar elements to the HPA cascade in other vertebrates, including mammals (see Volume 5, Chapter 8). Corticotropin-releasing factor, one of the key initial elements of the HPA axis and an important central neurotransmitter, is present in the brain of A. carolinensis (Ten Eyck, Adams, Matter, Lowry, & Summers, 1996), the water snake Natrix maura (Mancera, Lo´pez Avalos, Pe´rezFı´gares, & Ferna´ndez-Llebrez, 1991), and the red-eared turtle, Trachemys scripta elegans (Bugnon et al., 1984; Fellmann et al., 1984). The distribution of CRF in the reptilian brain appears to be similar to that of mammals, and specifically includes paraventricular neurons with terminals in the median eminence, suggesting delivery of CRF to the adenohypophysis for the HPA cascade. In the water snake, N. maura, CRF is co-localized with arginine vasotocin (AVT) (Mancera et al., 1991), much as it is with the potent synergistic ACTH secretogogue arginine vasopressin (AVP) in mammals (Plotsky & Sawchenko, 1987; Mouri et al., 1993). Additional evidence of reptilian CRF in the HPA cascade comes from the Keeled Indian Mabuya, Mabuya carinata, in which a general CRF receptor antagonist (a-helical CRF) blocks stressrelated effects on ovarian recrudescence (Ganesh & Yajurvedi, 2002b). In the turtle T. s. elegans, adrenalectomy was used to demonstrate normal HPA negative feedback on hypothalamic hormonal CRF production (Fellmann et al., 1984). It is likely that the reptilian CRF stimulates the cells of the adenohypophysis that produce ACTH (i.e., corticotropes), because such cells have been described in the adenohypophysis of the teiid lizard, Cnemidophorus lemniscatus (Del Conte, 1980). Pituitary corticotropes synthesize and release ACTH, which has been
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demonstrated to stimulate adrenal corticosteroid synthesis and release in several lizards (Sceloporus undulatus (Carsia & John-Alder, 2003), Amblyrhynchus cristatus (Romero & Wikelski, 2006), Hoplodactylus maculatus (Preest et al., 2005), Egernia whitii (Cartledge & Jones, 2007)); the caiman, Caiman crocodilus (Gist & Kaplan, 1976); but not in the turtle, Pseudemys scripta (Sanford & Stephens, 1988). Unlike ACTH, which is a potent stimulator of glucocorticoid synthesis in reptiles, the natriuretic peptides appear to have an inhibitory effect on glucocorticoid synthesis in reptiles because these peptides inhibited in vitro steroidogenesis in dispersed adrenocortical cells that were obtained from the Eastern fence lizard, S. undulatus (Carsia & John-Alder, 2006). Reptiles appear to possess a glucocorticoid plasmabinding protein (Jennings, Moore, Knapp, Matthews, & Orchinik, 2000). Two plasma steroid-binding globulins have been identified in male tree lizards, Urosaurus ornatus, and one of these binds androgens and C-21 steroids such as CORT and has been termed androgen-glucocorticoid-binding globulin (AGBG); the other is a sex hormone-binding globulin (SHBG) that binds androgens and E2 with high affinity (Jennings et al., 2000). Moreover, the binding capacity of AGBG (but not of SHBG) has been shown to be significantly greater in territorial males than in non-territorial males in U. ornatus (Jennings et al., 2000).
2.3. Hypothalamic–Pituitary–Gonadal (HPG) Axis The hypothalamic–pituitary–gonadal (HPG) axis in reptiles is similar to that described in other vertebrates (Licht & Porter, 1987; Licht, 1995). It is important to note, however, that little is understood about the neural systems regulating the HPG axis in reptiles, and important clarifications with respect to neurohormones and gonadotropins (GTHs) are still to be made. As in mammals, NE appears to play an important role in ovarian function in female lizards (Jones, Desan, Lopez, & Austin, 1990). Noradrenergic activity in the contralateral hypothalamus predicts the pattern of ovarian growth and ovulation of A. carolinensis (Desan, Lopez, Austin, & Jones, 1992), which in female anoles entails ovulation of a single egg at a time and an alternating pattern in egg production between the left and right ovaries in successive ovulations (Smith, Sinelnik, Fawcett, & Jones, 1972). This hypothalamic regulatory control of ovarian function in A. carolinensis appears to be informed by afferent input from the ovaries to the brain (Jones et al., 1997). Ovariectomy influences not only noradrenergic activity but also brain hemisphere-specific serotonergic and dopaminergic activity.
Hormones and Reproduction of Vertebrates
Recent work in the leopard gecko, Eublepharis macularius, suggests that the most ubiquitous form of gonadotropin-releasing hormone (GnRH) in vertebrates, type II or chicken II (cGnRH-II or cGnRH2) shows gene, peptide, and receptor expression in reptiles (Ikemoto & Park, 2003; Ikemoto, Enomoto, & Park, 2004). However, like other vertebrate classes, there are multiple GnRH isoforms in reptiles. For example, Ikemoto and Park (2007) have identified two distinct forms of GnRH (cGnRH-I and cGNRH-II) and three forms of GnRH receptor in the leopard gecko, E. macularius. However, in A. carolinensis, there may be just one form, cGnRH II (Lescheid et al., 1997). According to Tsai and Licht (1993a), turtles such as T. scripta have at least two GnRH types (cGnRH-I and II), with cGnRH-I being the most abundant type in the median eminence. In addition to one of two GnRHs, another neurohormone, GTH-release inhibiting hormone (GnIH), appears to be present in reptiles as well as other vertebrates (Tsutui & Osugi, 2009; see also Volume 4, Chapter 2; Volume 5, Chapter 2). A variety of GnRH subtypes are capable of stimulating luteinizing hormone (LH) release from pituitary gonadotropes in the turtle T. scripta (Tsai & Licht, 1993b). The pituitary gonadotropes of other reptiles, such as the North African spiny-tailed lizard, Uromastyx acanthinura, have been characterized as having only follicle-stimulating hormone (FSH)-like secretory capacity, at least as far as immunoreactivity to human LH and FSH antibodies may demonstrate (Hammouche, Gernigon, & Exbrayat, 2007). In the Keeled Indian Mabuya, M. carinata, the physiological effects of FSH in males, including the full complement of gonadotropic and steroidogenic effects, are subject to inhibition by stress and glucocorticoids (Yajurvedi & Nijagal, 2000; Yajurvedi & Menon, 2005). In female M. carinata, FSH also stimulates ovarian function (recrudescence) but is inhibited (as in mammals) by b-endorphin (Ganesh & Yajurvedi, 2003). Although two GTHs, homologous to FSH and LH, are part of the generalized reptilian HPG axis, early studies had difficulty demonstrating separate reproductive functions or even that there were two GTH in some lizards (Licht et al., 1976; Licht, Wood, Owens, & Wood, 1979). In the green sea turtle, C. mydas, FSH and LH in females were demonstrated to be distinctly regulated and to have different functions in mating and oviposition (Licht et al., 1979). Similarly, in a more recent study in the lizard Calotes versicolor, a combined treatment of LH and FSH was more effective in stimulating testicular recrudescence than FSH alone, suggesting distinctive functions in males (Vijaykumar, Ramjaneyulu, Sharanabasappa, & Patil, 2002). In addition to the role of GTHs in stimulating gametogenesis and gonadal steroid hormone synthesis, there is some exciting new evidence from a study of the lizard Uta stansburiana that these pituitary hormones also
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Stress and Reproduction in Reptiles
may act directly to modulate a variety of physiological, morphological, and behavioral traits (Mills et al., 2008). There is some evidence that the seasonality of reproductive function in reptiles is mediated by changes at the level of the hypothalamus (in terms of the areas that regulate GnRH release and thus pituitary GTH release) rather than at the level of the gonads (in terms of gonadal sensitivity to the actions of GTH). For example, FSH treatment of A. carolinensis was effective in stimulating testicular growth in males during the so-called refractory period, which occurs immediately after testicular regression in the late summer and fall (Summers, 1984a). In addition, evidence exists that opioid peptides may play a role in regulating seasonal reproductive activity in the lizard Podarcis sicula sicula as treatment of males of this species with the opioid antagonist naltrexone increased androgen levels in both plasma and testis (Ciarcia et al., 1994).
3. EFFECTS OF STRESS ON REPRODUCTIVE FUNCTION 3.1. Neurotransmitters As brain NE, DA, and 5-HT systems have been demonstrated to influence gonadal function in reptiles (Jones et al., 1990; Jones et al., 1997; Desan et al., 1992), it is likely that stressor-induced changes in these neurotransmitter systems also affect reproductive processes. In female A. carolinensis, the stress associated with social interactions diminished telencephalic serotonergic and dopaminergic activity but elevated brainstem serotonergic activity, suggesting negative feedback in the 5-HT system, probably at the raphe nucleus (Summers et al., 1997). Courtship behavior and ovarian cycles of these socially stressed subordinate females also were reduced relative to controls (Summers et al. 1995). Further, combined treatment with CORT and with a 5-HT2A-receptor antagonist reduced male courtship behavior in the red-sided garter snake, Thamnophis sirtalis parietalis (Lutterschmidt, LeMaster, & Mason, 2004).
3.2. Reproductive Hormones One of the more common adverse effects of a prolonged stress response is the disruption of reproductive physiology and behavior (Greenberg & Wingfield, 1987; Wingfield & Sapolsky, 2003). Stress hormones released from the HPA axis can act at multiple levels to inhibit the secretion and/or action of hormones necessary for gonadal function and reproductive behavior. There is strong evidence in many species of reptile that plasma androgen levels in males decline after males are
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exposed to stressors (see Figure 7.1 and Table 7.1). However, some species of reptile show little or no change in androgen levels when exposed to relatively short-term or less intense stressors (e.g., Tiliqua [Trachydosaurus] rugosa (Bourne, Taylor, & Watson, 1986), S. punctatus (Cree, Guillette, Cockrem, & Joss, 1990), and bearded dragon, Pogona barbata (Cree, Amey, & Whittier, 2000)). The effect of stress on circulating testosterone levels in the tree lizard, U. ornatus, has been of special interest because these lizards have two alternative male reproductive morphs: non-territorial males with orange dewlaps (‘orange males’) and territorial males with orange dewlaps containing a central bluish-green spot (‘orange-blue males’) (Moore & Thompson, 1990). In a study of changes in T and CORT levels in response to acute and chronic stress in male U. ornatus of the orange-blue morph type, Moore, Thompson, and Marler (1991) found that T levels were negatively correlated with CORT levels within individuals in males exposed to chronic stress but not to acute stress. Knapp and Moore (1997a) treated males of both morph types with exogenous CORT or sesame oil (placebo) delivered via dermal patches (see Knapp & Moore, 1997b). The plasma levels of CORT in males of both morphs that received CORT patches did not differ significantly at the end of the experiment (Knapp & Moore, 1997a), and, importantly, the CORT levels were similar to those observed in male U. ornatus exposed to stressors (Moore et al., 1991). The T levels in males of both morph types that received CORT were significantly lower than in controls; however, T levels in males of the non-territorial orange morph were significantly lower than in males of the territorial orange-blue morph. Like in tree lizards, the relationship between plasma CORT and androgen levels in males differs with male mating tactics in the collard lizard, Crotaphytus collaris (Baird & Hews, 2007), although it is unclear to what extent this difference is mediated by stress. According to Baird and Hews (2007), plasma CORT levels in male C. collaris were negatively correlated with both T and dihydrotestosterone (DHT) levels in territorial males, but did not co-vary significantly with these androgens in non-territorial males. In some species of lizard and snake, plasma levels of corticosterone and/or testosterone have been measured in males following staged agonistic interactions (Greenberg & Crews, 1990; Knapp & Moore, 1995; Schuett al., 1996; Klukowski & Nelson, 1998; Smith & John-Alder, 1999). The importance of these studies is that they consider how changes in the levels of these hormones correlate with social dominance and thus potential breeding opportunities. The finding in most studies that exposure to stressors increases circulating CORT levels and decreases circulating androgen levels in male reptiles suggests a means by which stress can inhibit male reproductive function. It also
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Plasma corticosterone ng/ml
25 20 b
b
15 10 5 a
Plasma testosterone ng/ml
0
7.5 a
5
b 2.5
b
1
2
3
0
Treatment Group FIGURE 7.1 Mean plasma corticosterone (CORT) in ng/ml (upper panel) and mean plasma T in ng/ml (lower panel) in male alligators immediately after capture and at two hours post-capture. Group 1 (n¼18; left bar) represents samples immediately at capture, group 2 (n¼18; middle bar) represents samples collected at two hours following the immediate sample, and group 3 (n¼18; right bar) represents samples collected at two hours only. The standard errors of the means (SEM) are indicated by the lines at the top of the bars. Lower case letters when different indicate differences are highly significant (P < 0.001). Reproduced from Lance, Elsey, Butterstein, and Trosclair (2004).
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TABLE 7.1 Relative changes in plasma testosterone levels in males following exposure to stressors or stress hormones in different groups of reptile Species
Relative changes in plasma testosterone (T) levels
Reference
Painted turtle (Chrysemys picta)
T levels declined from initial values within 24 hours of capture
Licht et al. (1985)
Musk turtle (Sternotherus odoratus)
T levels declined 35–60% from initial values within 24 hours of capture, depending on season
Mendonc¸a and Licht (1986)
Snapping turtle (Chelydra serpentina)
T levels increased above initial values within 24 hours of capture but declined below initial values by 168 hours post capture
Mahmoud et al. (1989)
Snapping turtle (Chelydra serpentina)
T levels gradually declined from initial values within 24 hours of capture in males obtained in June and July, regardless of temperature or phase of spermatogenic cycle
Mahmoud and Licht (1997)
Hawksbill turtle (Eretmochelys imbricata)
T levels did not decline significantly from initial values in immature males at 1 hour and 5 hours post capture
Jessop et al. (2004)
American alligator (Alligator mississippiensis)
T levels were lower than initial values at all sample time periods after capture, with T levels being 50% of initial values by 4 hours and less than 10% by 24 hours post capture
Lance and Elsey (1986)
American alligator (Alligator mississippiensis)
T levels declined in sub adult males treated with ACTH or with saline within 6 hours of treatment
Mahmoud et al. (1996)
American alligator (Alligator mississippiensis)
T levels in juvenile males from a contaminated lake were lower than from a control lake but were no different than initial values after 2 hours of restraint following capture
Guillette et al. (1997a)
American alligator (Alligator mississippiensis)
T levels in sub-adult and adult males decreased from initial values by 2 hours post capture
Lance et al. (2004)
Tuatara (Sphenodon punctatus)
T levels did not differ significantly from controls within 3 hours of capture but were lower in males exposed to repeated blood sampling for 24 hours
Cree et al. (1990b)
Shingleback skink (Tiliqua [Trachydosaurus] rugosa)
T levels did not differ significantly from controls within 2–3 minutes or 24 hours of capture, although androgens were abnormally low in males held in captivity for several months
Bourne et al. (1986)
Brown anole (Anolis sagrei)
T levels were lower after 7 days of exposure to CORT delivered from subcutaneous pellets than in placebo-treated males
Tokarz (1987)
Tree lizard (Urosaurus ornatus)
T levels in males (orange-blue morph) that were held in collecting bags were not significantly different from initial values after 10 minutes but were lower by 240 min, and T levels in males exposed to captivity in cages for days declined slightly but nonsignificantly
Moore et al. (1991)
Side-blotched lizard (Uta stansburiana)
T levels in males that were exposed to CORT delivered from Silastic implants for at least 7 days were lower than in sham-treated males
DeNardo and Licht (1993)
Italian wall lizard (Podarcis sicula sicula)
T levels declined from initial values within 48 minutes of capture in males obtained in April, May, and June
Manzo et al. (1994)
Tree lizard (Urosaurus ornatus)
T levels in males that were captured and physically restrained for 10 or 30 minutes were negatively correlated with CORT levels in males of the orange morph, and T levels in males of both the orange and orange-blue morphs that had received exogenous CORT from dermal patches for a day prior to capture were lower than in controls with orange males having lower T levels than orange-blue males
Knapp and Moore (1997a)
(Continued)
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Hormones and Reproduction of Vertebrates
TABLE 7.1 Relative changes in plasma testosterone levels in males following exposure to stressors or stress hormones in different groups of reptiledcont’d Species
Relative changes in plasma testosterone (T) levels
Reference
Keeled Indian Mabuya (Mabuya carinata)
T levels were lower in males that received FSH and CORT injections over 30 days than in males that received only FSH injections
Yajurvedi and Nijagal (2000)
Common gecko (Hoplodactylus maculatus)
T levels in males did not differ significantly from initial values at 4 and 24 hours post capture
Cree et al. (2003)
White’s skink (Egernia whitii)
T levels were lower than initial values at 60 and 240 minutes post capture in males held in cloth bags following capture but T levels did not differ significantly from initial values in males held in captivity from 1 to 28 days
Jones and Bell (2004)
Keeled Indian Mabuya (Mabuya carinata)
T levels in males exposed for 30 days to a variety of stressors or to a variety of stressors and FSH injections were lower than in males that received only FSH injections
Yajurvedi and Menon (2005)
Red-sided garter snake (Thamnophis sirtalis parietalis)
T levels in males that were captured in the spring and held in cloth bags for up to 4 hours were lower at 4 hours post capture than in control males that were captured and bled within 90 seconds of capture
Moore et al. (2000a)
Red-sided garter snake (Thamnophis sirtalis parietalis)
T levels in males that were captured and subjected to serial blood sampling were lower than initial values at 1 hour post capture in males captured in the summer, and at 4 hours in males captured in the fall, but were not significantly different from initial values at any sample time in males that were captured in the early or late spring
Moore et al. (2001)
Red-spotted garter snake (Thamnophis sirtalis concinnus)
T levels in males that were captured and subjected to serial blood sampling were lower than initial values at 4 hours post capture in males captured in the spring of 1995 and of 1999 and at 1 hour post capture in males captured in the fall of 1999, but were not significantly different from initial values in males captured in the summer of 1999
Moore et al. (2001)
Brown tree snake (Boiga irregularis)
T levels in males captured in the field from a wild population were lower in monthly samples than in males in a captive population
Moore et al. (2005)
ACTH, corticotropin; CORT, corticosterone; FSH, follicle-stimulating hormone; T, testosterone.
suggests that the energetics–hormone–vocalization model proposed by Emerson (2001) to explain the relationship between corticosteroid and androgen levels and advertisement call characteristics and reproductive behavior of male frogs might be useful in understanding the effects of stress in male reptiles because many display behaviors in male reptiles also are androgen-dependent. Very little information is available on what effect exposure to stressors has on other reproductive hormones in male reptiles. Licht, Breitenbach, & Congdon (1985) reported that in the painted turtle, C. picta, plasma FSH levels declined significantly in males within a day of capture. Further, Cree et al. (1990b) in a study of the tuatara found that plasma progesterone (P4) levels in males were significantly elevated at 3, 12, and 24 hours post-capture and concluded that the increase in plasma P4 after capture
was most likely due to a stress-induced increase in adrenal synthesis of P4. Surprisingly, relatively few studies have investigated the effects of stress on the reproductive hormones of female reptiles. Results from available studies suggest that the effects of stress on plasma levels of estradiol (E2) and P4 vary by species and female reproductive condition. In the snapping turtle, Chelydra serpentina, females captured in June had significantly elevated plasma P4 levels six hours after capture and significantly elevated E2 levels 24 hours after capture (Mahmoud, Guillette, McAsey, & Cady, 1989). The levels of these two steroid hormones returned to baseline within seven days of capture. When analyzed by reproductive condition, gravid females had a significant increase in E2 whereas non-gravid females did not (Mahmoud et al., 1989). Although P4 levels increased
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significantly in both gravid and non-gravid females, the increase was unexpectedly greater in non-gravid than in gravid females. It was suggested that the adrenal gland as well as ovarian follicles were the sources of P4 (Mahmoud et al., 1989). In the tuatara, P4 levels were significantly higher in vitellogenic females captured in January and subjected to acute stress (3 hours of confinement) than in freely roaming females (Cree, Guillette, Cockrem, Brown, & Chambers, 1990a). However, plasma levels of E2 and T in these captive females did not differ significantly from those found in freely roaming females. In adult female alligators (A. mississippiensis) captured during the breeding season and subjected to restraint and blood sampling for 48 hours, plasma E2 levels decreased significantly below initial values by 22 hours after capture and then remained unchanged (Elsey, Lance, Joanen, & McNease, 1991). Corticosterone secretion had a biphasic pattern in that plasma CORT continued to increase for 16 hours, then declined, and then rose again significantly from 24 to 48 hours. Stress may inhibit ovarian E2 synthesis in the Keeled Indian Mabuya, M. carinata. Female M. carinata treated for 30 days with FSH (to induce ovarian recrudescence) and exposed daily to stressors (handling, chasing, and noise) had significantly lower serum E2 levels than did females that were treated with FSH alone (Ganesh & Yajurvedi, 2002a). In contrast to studies that have reported an effect of short-term stress on female reproductive hormones, no similar effect has been observed in female red-sided garter snakes, T. s. parietalis (Whittier, Mason, & Crews, 1987). Whittier et al. (1987) reported that the plasma levels of E2, P4, T, and CORT did not differ significantly among female T. s. parietalis captured in the fall and allowed to hibernate in the laboratory. Females placed under summer-like conditions in the laboratory or females captured in the field after emergence from hibernation, maintained under fluctuating conditions, and then transferred to the laboratory during the first five weeks after emergence also were not different. Because the hormone cycles observed in these two groups of females were so similar, it was suggested that female garter snakes may be relatively insensitive to the disruption of neuroendocrine events that are induced by handling and captivity over time periods of hours and days (Whittier et al., 1987). Moore et al. (2005) have suggested that an introduced population of brown tree snakes (B. irregularis) on Guam may be experiencing stress-induced reproductive suppression because of overcrowding and overexploitation of food resources. Three lines of evidence support this claim (Moore et al., 2005). Firstly, male and female B. irregularis captured on Guam had a significantly lower body condition relative to freely living
snakes captured from their native range in Australia, and females (but not males) also had a significantly lower body condition relative to a captive population of snakes maintained at Oregon State University. Secondly, studies of a wild population of B. irregularis on Guam found relatively few breeding individuals (Rodda, Fritts, McCoid, & Campbell, 1999; Mathies, Felix, & Lance, 2001). The third line of evidence is based on the monthly baseline levels of plasma CORT and sex steroids in freely living and captive males and females during the annual cycle (Moore et al., 2005). Plasma CORT levels in fieldcaptured males were higher and plasma T levels were lower than in captive males in most months. Similarly, plasma CORT levels were higher and plasma E2 and P4 levels lower in field-captured females than in captive females (Figure 7.2). Moore et al. (2005) suggested that these differences in baseline hormone levels are consistent with the hypothesis that the Guam populations of B. irregularis are living under stressful conditions and are thus reproductively suppressed. If true, this would be an example of stress directly inhibiting reproductive activity in a wild reptile. However, it is extremely difficult to draw strong inferences when comparing the plasma levels of hormones from freely living animals to those from captive animals. Thus, additional studies are needed before the hypothesis that B. irregularis in Guam are being reproductively suppressed because of stress can be confirmed.
3.3. Gonads The effects of stress on gonadal function in reptiles have been considered in several excellent reviews (Greenberg & Wingfield, 1987; Guillette et al., 1995; Pottinger, 1997). Since the publication of these reviews, new information has become available on how stress affects gametogenesis in the Keeled Indian Mabuya, M. carinata (Yajurvedi & Nijagal, 2000; Ganesh & Yajurvedi, 2002a; 2002b; 2003; Yajurvedi & Menon, 2005). Similar studies in other lizards as well as crocodilians, snakes, turtles, and the tuatara are needed.
3.3.1. Ovarian function There is convincing evidence that stress affects ovarian function in lizards (Crews, 1974; Summers & Norman, 1988; Summers, 1995; Summers et al., 1995; Moralez & Sa´nchez, 1996; Ganesh & Yajurvedi, 2002a; 2002b; 2003; see also Chapter 4, this volume). Reproductively inactive female A. carolinensis exposed to stimulatory environmental conditions and to dominant males exhibiting more inter-male aggressive behavior than courtship behavior had a reduced rate of ovarian recrudescence (percentage of females with yolky follicles and median ovarian condition)
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(a)
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Hormones and Reproduction of Vertebrates
0.4 0.3 0.2 0.1
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FIGURE 7.2 Annual cycles of (a) estradiol (E2), (b) progesterone (P4), and (c) corticosterone (CORT) for female captive and freely living populations of brown tree snakes (Boiga irregularis) from Guam. Numbers next to the points are the sample sizes for the free-living group. Sample size for the captive group was seven for all months. Reproduced from Moore et al. (2005).
in comparison to control females not exposed to male aggressive behavior (Crews, 1974). The inhibition of ovarian recrudescence in female A. carolinensis exposed to inter-male aggressive behavior was not due to the absence of male courtship behavior because females that were exposed to castrated males, which did not court females, exhibited normal ovarian recrudescence (Crews, 1974). Thus, exposure of females to male aggressive behavior during the period of ovarian recrudescence appears to be a social stressor in A. carolinensis. Female density, another social stressor, also affects the rate of ovarian recrudescence in female A. carolinensis.
Seasonally non-reproductive female A. carolinensis that were housed in a group of five females and one male and exposed to stimulatory environmental conditions for one month had smaller ovaries and less oviductal mass than did females housed singly with a male and exposed to stimulatory environmental conditions (Summers et al., 1995). Interestingly, this inhibitory effect of female density was size-dependent as females weighing more than 2.7 g exhibited normal ovarian recrudescence independently of whether or not they were housed with females. Additional evidence indicated that female body size is an important factor governing the effects of social stressors during
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Stress and Reproduction in Reptiles
ovarian recrudescence in this species. In female A. carolinensis treated with ACTH once a day for three weeks, ovarian and oviductal growth was inhibited in small females (< 2.6 g) but enhanced in large females (> 2.6 g) (Summers, 1995). Environmental stressors also influence the rate of environmentally induced ovarian recrudescence in female A. carolinensis. Seasonally reproductively inactive females housed under relatively low humidity conditions (< 35%) and stimulatory temperature and photoperiod regimes had reduced ovarian growth and oviductal growth in comparison with females that were housed under relatively high humidity conditions (> 80%) and stimulatory environmental conditions (Summers & Norman, 1988). Dehydration of females in the relatively low humidity treatment group might have acted to inhibit reproduction either directly, or indirectly via the release of stress hormones (Summers & Norman, 1988). Holding reptiles in captivity appears to be a potent stressor in many species (reviewed in Guraya, 1989; Morales & Sa´nchez, 1996; Romero 2002) and is associated with reduced vitellogenesis and ovarian development in A. carolinesis, Anolis pulchellus, and C. serpentina. In a series of studies, the effects of stress on seasonal ovarian development have been examined in the Keeled Indian Mabuya (Ganesh & Yajurvedi, 2002a; 2002b; 2003). Female M. carinata exposed to stimulatory environmental conditions and to physical stressors (handling, chasing, and noise applied randomly five times per day for one month) during the recrudescence phase of the ovarian cycle (November) had significantly reduced numbers of oocytes and primordial follicles compared to untreated control females and completely lacked vitellogenic follicles (Ganesh & Yajurvedi, 2002a). Female M. carinata exposed to the same stressors but also treated with FSH on alternate days for 30 days during the non-breeding regression phase of the ovarian cycle (August) had significantly fewer oogonia, primary oocytes, and primordial follicles than did females that were treated only with FSH. Females exposed to stressors as well as FSH had no vitellogenic follicles and had significantly lower serum E2 levels and liver weights than did females treated only with FSH. Based on these findings, Ganesh and Yajurvedi (2002a) suggested that the growth of vitellogenic follicles was inhibited by a stressinduced decrease in E2 synthesis that was not prevented by FSH treatment. Two lines of evidence suggest that the inhibitory effects of stress on ovarian development in FSH-treated female M. carinata are mediated by the activation of the adrenal gland (Ganesh & Yajurvedi, 2002a). Firstly, females exposed to stressors had a significant increase in the nuclear diameter of the steroidogenic cells of the adrenal cortex, and, secondly, there was a significant decrease in the number of islands of white pulp in the spleen, the main site
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of lymphocyte production. Ganesh and Yajurvedi (2002a) suggested that stressor-induced activation of the adrenal gland in female M. carinata stimulated CORT synthesis, and the resulting increase in CORT may have inhibited ovarian development and suppressed the immune system by decreasing lymphocyte production. Stress-induced inhibition of ovarian recrudescence in female M. carinata requires the activation of the HPA axis (Ganesh & Yajurvedi, 2002b). Treatment of female M. carinata with an antagonist (a-helical CRF9-41) to mammalian CRF largely prevented the inhibition of ovarian and oviductal development in females exposed to stressors during the recrudescence phase of the ovarian cycle (Ganesh & Yajurvedi, 2002b). Specifically, females exposed both to stressors and the CRF antagonist had significantly heavier ovaries and oviducts (relative to body size), and significantly more oocytes, primordial follicles, and stage 2 follicles (previtellogenic follicles) than did females exposed only to stressors. Unlike the females exposed only to stressors and which lacked vitellogenic follicles (stage 4 and stage 5), females that were exposed to stressors but that also received the CRF antagonist had vitellogenic follicles. Treatment with the CRF antagonist also inhibited the stressor-induced increase in the nuclear diameter of the steroidogenic adrenal cortical cells and the decrease in the number of islands of white pulp in the spleen (Ganesh & Yajurvedi, 2002b). A further indication of the involvement of the HPA axis in the stress-induced inhibition of seasonal recrudescence in M. carinata is the finding that treatment of females with b-endorphin inhibits both seasonal and FSH-induced ovarian recrudescence (Ganesh & Yajurvedi, 2003). Endogenous opioids including b-endorphin are released as part of the stress response in mammals (reviewed in Drolet et al., 2001; see also Volume 5, Chapter 8) and may be operational in reptiles as well. Treatment of female M. carinata with b-endorphin daily for 30 days during the period of seasonal ovarian recrudescence inhibited vitellogenesis compared to control females treated with distilled water (Ganesh & Yajurvedi, 2003). Germinal bed activity was also inhibited in the ovaries of females that received either the 0.5 or 1.0 mg (but not 0.1 mg) dose of b-endorphin in that they had significantly fewer primordial follicles than controls. When administered concurrently with FSH, b-endorphin also inhibited FSH-induced ovarian recrudescence and vitellogenesis. This finding led Ganesh and Yajurvedi (2003) to suggest that b-endorphin in female M. carinata may have an inhibitory effect at the level of the ovary, independent of an effect on the release of pituitary FSH. Specifically, these investigators suggest that b-endorphin might prevent vitellogenic follicular development by inhibiting E2 synthesis by ovarian follicles. Interestingly, b-endorphin treatment stimulated rather than inhibited E2
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synthesis in-vitro by ovaries obtained from the Italian wall lizard, P. s. sicula (Polzonetti-Magni et al., 1994). This difference in the effects of b-endorphin treatment may be due to the fact that the Ganesh and Yajurvedi (2003) study was an in-vivo study that used relatively high doses (i.e., 0.1, 0.5, or 1.0 mg) of b-endorphin, whereas the Polzonetti-Magni et al. (1994) study was an in-vitro study that used a relatively low dose (4 pmol) of b-endorphin.
3.3.2. Testicular function Exposure to stressors or to the stress hormone CORT affects testicular function in several species of lizard (Tokarz, 1987; Manzo, Zerani, Gobbetti, Di Fiore, & Angelini, 1994; Dunlap & Schall, 1995; Yajurvedi & Nijagal, 2000; Yajurvedi & Menon, 2005) and in a turtle (Mahmoud & Licht, 1997). (Details of testicular function in reptiles are found in Chapter 3, this volume.) For example, in the brown anole, Anolis sagrei, reproductively active males that were implanted for three weeks with CORT pellets had significantly reduced testis weight, a less active spermatogenic stage (spermatozoa abundant but spermatids and spermatocytes greatly reduced; see Licht (1967), stage 7), and significantly lower plasma T levels than did placebo-treated controls (Tokarz, 1987). Similarly, in the fence lizard, Sceloporus occidentalis, males that were implanted with CORT had significantly lower T levels and testis mass compared to males that received empty implants (Dunlap & Schall, 1995). Yajurvedi and Nijagal (2000) also documented a dose-dependent inhibitory effect of CORT treatment on normal and FSH-induced testicular recrudescence in M. carinata. Male M. carinata in the recrudescence phase of the testicular cycle that were treated on alternate days for 30 days with the highest dose of CORT (40 mg) had significantly fewer primary spermatocytes, secondary spermatocytes, and spermatids in comparison to treatment controls (oil-treated), although testes weight did not differ from that found in the controls (Yajurvedi & Nijagal, 2000). In a second experiment, male M. carinata in the postbreeding regression phase of the testicular cycle that were treated with both CORT (40 mg) and FSH on alternate days for 30 days had significantly fewer primary spermatocytes, secondary spermatocytes, and spermatids than males that were treated only with FSH (Yajurvedi & Nijagal, 2000). Moreover, males that received both CORT and FSH had significantly lighter testes and lower serum T levels than did males treated only with FSH; they also had fewer spermatozoa in the seminiferous tubule lumina in comparison to FSH-treated males. Because CORT treatment of FSH-treated males lowered serum T levels and only affected the relatively advanced stages of spermatogenesis and spermiogenesis,
Hormones and Reproduction of Vertebrates
Yajurvedi and Nijagal (2000) suggested that in male M. carinata CORT treatment had inhibited FSH-induced T secretion by the testes. This is a reasonable suggestion given that the transition of spermatocytes into spermatozoa is known to be highly androgen-dependent in reptiles (Lance, 1984). Environmental stressors also inhibit testicular activity in male M. carinata. Yajurvedi and Menon (2005) exposed male M. carinata, which were in the beginning of the recrudescence phase of the annual testicular cycle, to either mild acute stressors (handling, chasing, noise applied randomly for five minutes each, five times a day), to FSH (on alternative days), to stressors plus FSH, or to saline (treatment control) for 30 days. Males exposed to the stressors had significantly fewer secondary spermatocytes and comparatively fewer spermatozoa in the seminiferous tubules than did treatment control males. The effects of exposure to stressors were much more evident in males that also had been treated with FSH in that they had significantly fewer primary and secondary spermatocytes as well as fewer spermatids than did males that were treated with just FSH. However, they did not differ significantly in the number of spermatogonia, a result suggesting that the proliferation of spermatogonia may be relatively insensitive to stress. Males that were exposed to both stressors and FSH also had significantly lower serum T levels than did FSH-treated males. Based on these findings, Yajurvedi and Menon (2005) argued that stress in male M. carinata has less of an effect on spermatogonial proliferation and entrance into meiosis (forming primary spermatocytes) than it does on the later stages of spermatogenesis (formation of secondary spermatocytes) and spermiogenesis. This is a reasonable argument in light of the evidence that spermatogonial proliferation and early stages of meiosis in reptiles are FSH-dependent processes, whereas the later stages of spermatogenesis and spermiogenesis are largely androgen-sensitive processes (Lance, 1984). In the snapping turtle C. serpentina, however, there is some indirect evidence that the stress resulting from capture and captivity might interfere with the initiation of spermatogenesis (Mahmoud & Licht, 1997). Male C. serpentina that were captured in the spring and early summer maintained their testes in a regressed state during captivity, whereas males that were captured later in the year, after testicular growth had begun, showed normal testicular enlargement and spermiation in captivity (Mahmoud & Licht, 1997). In contrast to studies that have indicated that stress inhibits testicular function, a study of the lizard A. carolinensis failed to detect such an inhibitory effect of stress (Greenberg, Chen, & Crews, 1984). In this study, no significant relationship was found to exist between testicular function (as determined by spermatogenic stage) and
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Stress and Reproduction in Reptiles
either social status (dominant vs. subordinate males) or relative CORT levels (high vs. low). Although male A. carolinensis that were judged in male–male pairings to be subordinate individuals (based on specific acts such as wins, losses, and draws) had significantly higher CORT levels (mean ¼ 18.8 ng/ml) than did males that were judged to be dominant (mean ¼ 4.9 ng/ml), they did not differ from dominant males in spermatogenic stage as assessed by the relative proportions of spermatogonia, primary spermatocytes, spermatids, and spermatozoa in the seminiferous tubules. It is unclear why there was no significant difference in spermatogenic stage between subordinate and dominant males given that they differed significantly in CORT levels. One possible explanation is that CORT levels in subordinate males may not have been elevated for a sufficient period of time to affect testicular function. Another possibility put forth by Greenberg et al. (1984) is that it might be highly adaptive for subordinate males not to differ from dominant males in spermatogenic stage because it would permit them to breed relatively quickly if they were able to become the dominant male in an area either because they successfully supplanted other dominant males after aggressive interactions or because the former dominant males were removed by predators. A similar phenomenon has been noted in birds in which the gonads and accessory organs of animals that are exposed to shortterm stressors due to storms are maintained in a near functional state so that birds are able to re-nest quickly after a storm is over (Wingfield & Romero, 2001).
3.4. Behavior Exposure of animals to unpredictable environmental stressors elicits what has been traditionally termed an acute stress response but more recently has been termed an ‘emergency life-history stage’ (Wingfield et al., 1998; Wingfield & Ramenofsky, 1999; Wingfield & Romero, 2001). An emergency life-history stage consists of a suite of facultative behavioral and physiological responses that are largely mediated by the activation of the HPA axis and release of glucocorticoids (Wingfield, Breuner, & Jacobs, 1997; Wingfield et al., 1998; Wingfield & Ramenofsky, 1999; Landys, Ramenofsky, & Wingfield, 2006). During an emergency life-history stage (Wingfield et al., 1998), an animal might abandon its current life-history state, especially if it is a relatively energetically expensive state such as reproduction, and adopt an alternative strategy such as moving away, seeking refuge, or seeking refuge and then moving away if conditions do not improve (Wingfield & Ramenofsky, 1999). This concept that animals in an emergency life-history stage change their behavior in response to the short-term effects of glucocorticoids (i.e., over minutes to hours) has been demonstrated especially well in field studies of birds in which individuals that were
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exhibiting reproductive behavior changed their behavior in response to rapid and unexpected environmental perturbations such as severe storms (for examples see Wingfield, 1988; Wingfield & Romero, 2001). To our knowledge there are no studies of reptiles comparable to those of birds that have documented a change in reproductive behavior in freely living animals due to an emergency life-history stage caused by unpredictable, short-term environmental stressors. There are, however, studies that have sought to ascertain whether male or female reptiles alter their reproductive behavior when treated experimentally with CORT so as to elevate plasma levels of CORT to those that would likely occur in an emergency life-history stage. Using this experimental approach, CORT treatment has been shown to inhibit male aggressive and territorial behavior in lizards (Tokarz, 1987; DeNardo & Licht, 1993; DeNardo & Sinervo, 1994a; 1994b) and male mating behavior in snakes (Moore & Mason, 2001; Lutterschmidt et al., 2004). In many species of lizard, male territorial behavior is an important factor affecting male mating success (reviewed in Stamps, 1983; Olsson & Madsen, 1998), and several studies have shown that CORT treatment inhibits aspects of male territorial behavior. In the brown anole, A. sagrei, males implanted with CORT pellets exhibited significantly fewer displays, approaches, and bites in response to stimulus males than did placebo-treated controls; they also erected a dorsal crest less frequently and had significantly lower plasma T levels than did placebo-treated males (Tokarz, 1987). Similarly, DeNardo and Licht (1993) in a laboratory study of the side-blotched lizard, U. stansburiana, found that CORT-implanted males in comparison to salineimplanted males had significantly reduced display and attack behavior when challenged by an intruder male as well as significantly lower circulating T levels. The inhibitory effects of CORT on aggressive behavior in male U. stansburiana appear to be mediated directly by CORT rather than by a CORT-induced lowering of T levels because males treated with both CORT and T implants still showed reduced male aggressive behavior (DeNardo & Licht, 1993). Although CORT treatment inhibited male aggressive behavior, it did not inhibit male courtship or copulatory behavior when CORT-treated males were tested with estrogen-treated and thus presumably sexually receptive females (DeNardo & Licht, 1993). Corticosterone treatment also inhibited territorial behavior in freely living male U. stansburiana (DeNardo & Sinervo, 1994a). In this study, male U. stansburiana at a field site that were implanted with CORT were significantly less active and had significantly smaller home ranges than saline-implanted males, but only when their neighbors were saline-treated and thus normally aggressive males. Because CORT-implanted males lost space to saline-implanted males, DeNardo and Sinervo (1994a) suggested that elevated CORT levels put males at
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a competitive disadvantage. Consistent with the suggestion that CORT directly inhibits male aggressive behavior independent of its effects on plasma T levels (DeNardo & Licht, 1993), CORT treatment of male U. stansburiana inhibited territorial behavior in males that were also treated with T. Males that received both T and CORT implants had a reduction in activity and in home-range size similar to that previously reported for males that were implanted only with CORT, whereas males that were implanted with T had a significant increase in activity and home range size compared to saline-implanted males (DeNardo & Sinervo, 1994b). Treatment with CORT also affects male locomotor performance and endurance in U. stansburiana (Miles, Calsbeek, & Sinervo, 2007a). Male U. stansburiana (blue morph) that were given long-term implants of CORT had increased locomotor performance (stamina) and a lower resting metabolic rate in comparison to sham-treated control males. It was suggested that this observed CORTinduced increase in locomotor performance and decrease in resting metabolic rate might be adaptive by increasing the ability of subordinate males to move to new areas and to conserve energy necessary for survival (Miles et al., 2007a). In the tree lizard, U. ornatus, stressor-induced changes in CORT and T levels may be a hormonal means of switching male reproductive tactics and thus behaviors (Knapp & Moore, 1997a; Knapp, Hews, Thompson, Ray, & Moore, 2003; Oliveira, Cana´rio, & Ros, 2008). In male U. ornatus, plasma T levels declined in response to CORT treatment, but T levels declined to a greater extent in the non-territorial orange males than in the territorial orangeblue males (Knapp & Moore, 1996; 1997a). In addition, the hormonal responses to stressful environmental conditions such as dry years appear to differ in the two types of male morphs in U. ornatus. During drought years, plasma CORT levels were elevated in both morph types but T levels were only suppressed in the non-territorial orange morph (Figure 7.3), which switched from a sedentary satellite tactic to a nomadic tactic during the dry years (Knapp et al., 2003; Oliveira et al., 2008). However, in a recent study of anti-predator tactics in U. ornatus, the two male morph types did not differ in mean CORT levels (or T levels) at 6–10 minutes after a staged encounter with a predator (collard lizard, Crotaphytus nebrius); however, flight initiation distances were associated with postencounter CORT levels (but not T levels) with males with higher CORT levels generally having greater flight initiation distances (Thaker, Lima, & Hews, 2009). Thus, individual variation in escape responses might be directly correlated with corticosterone levels in male U. ornatus (Thaker et al., 2009). Stressor-induced increases in plasma CORT levels may also affect how an animal responds in future social interactions. The blocking of CORT synthesis with injections of
Hormones and Reproduction of Vertebrates
the drug metyrapone inhibited male habituation to repeated aggressive stimuli in A. carolinensis (Yang & Wilczynski, 2003). This finding, if confirmed, would fit with the idea that elevated levels of CORT play an important role in modifying an animal’s subsequent social behavior. In the copperhead, Agkistrodon contortrix, a nonterritorial species of snake, males compete with other males for priority access to females (Schuett, Harlow, Rose, Van Kirk, & Murdoch, 1996; Schuett & Grober, 2000). Several studies of male A. contortrix have examined whether the losers and winners of staged fights differ in hormone levels. One hour after staged fights between males, losers of fights had significantly higher plasma CORT levels than did winners of fights, or in males of a control group in which males were paired with a female, although losers and winners as well as control males did not differ significantly in plasma T levels (Schuett et al., 1996). Moreover, plasma levels of CORT and of lactate in male A. contortrix 60 minutes after fights were significantly higher in losers than in winners of fights or in control males that were not used in fights (Schuett & Grober, 2000) (Figure 7.4). Schuett and Grober (2000) suggested that losers of fights had higher levels of lactate and CORT than winners because of differences in psychoneuroendocrine factors rather than because of differences in exercise during fights. These investigators also proposed that the elevated levels of CORT in losers might retard metabolic recovery and perhaps inhibit subsequent aggressive behavior. Treatment with CORT or a stressor-induced increase in plasma CORT levels inhibits mating behavior in several species of snake (Schuett, 1996; Moore & Mason, 2001; Lutterschmidt et al., 2004). For example, CORT treatment has been found to inhibit male courtship behavior in the red-sided garter snake, T. s. parietalis (Moore & Mason, 2001; Lutterschmidt et al., 2004). This species of snake is unusual because male courtship behavior is not dependent upon the activational effects of sex-steroid hormones (Crews, 1984; Crews et al. 1984; Crews, 1991). Moore and Mason (2001) found that CORT treatment of male T. s. parietalis suppressed mating behavior in a threshold manner. Specifically, Moore and Mason (2001) found that the proportion of males that courted unmated, sexually attractive females in an arena was significantly lower in males that were captured one month after spring emergence and treated with intra-peritoneal injections of either 25 mg, 50 mg, or 100 mg (but not 10 mg) of CORT than in males treated with vehicle or in untreated males (Figure 7.5). Because CORT treatment of male T. s. parietalis had no effect on plasma T levels, Moore and Mason (2001) concluded that CORT is most likely not directly inhibiting the HPG axis. Lutterschmidt et al. (2004) also found that CORT injections significantly reduced courtship behavior in a dose-dependent manner in freely living male T. s.
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Stress and Reproduction in Reptiles
Monthly Rainfall (cm)
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FIGURE 7.3 (a) Total monthly rainfall in central Arizona for January through March for wetter 1993 (gray filled circles or bars) and drier 1995 (white circles or bars), two years for which hormone data are available for male tree lizards. These months represent the late winter/early spring period that establishes yearly food resources for tree lizards. Both orange and orange-blue males exhibited elevated plasma corticosterone levels in the drier 1993 (b), but only orange males exhibited associated yearly variation in testosterone levels (c). The data are back-transformed adjusted means and standard errors from the ANCOVAs (or simply back-transformed means from the ANOVA for orange-blue testosterone levels). Sample sizes are shown within each bar. *, P < 0.05; **, P < 0.01. Reproduced from Knapp, Hews, Thompson, Ray, and Moore (2003).
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Hormones and Reproduction of Vertebrates
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Treatment FIGURE 7.4 Effects of fight outcome on (a) mean plasma lactate and (b) mean plasma CORT in male copperhead snakes (Agkistrodon contortix). Histogram bars denote the mean standard error of the mean (SEM). Sample sizes for both histograms are provided in (b). Letters above error bars represent significant differences, P < 0.05. Reproduced from Schuett and Grober (2000).
parietalis that were captured during the month following their emergence from hibernacula, a time when mating and plasma T levels are declining (Krohmer, Grassman, & Crews, 1987). In addition to examining the effects of CORT treatment, Lutterschmidt et al. (2004) investigated what effects melatonin and the serotonergic type 2A (5-HT2A) receptor antagonist, ketanserin, have on male courtship behavior in T. s. parietalis. The experiments were designed to determine whether melatonin modulates the behavioral and hormonal responses of males to exogenous CORT. Males in four pretreatment groups received a single intraperitoneal injection of vehicle, 0.03 mg melatonin (low dose), 0.3 mg melatonin (high dose), or ketanserin (0.045 mg). Thirty minutes after pretreatment, males within in each group received no CORT (vehicle), 15 mg of CORT, or 60 mg of CORT, and then were exposed in an arena to
unmated, sexually attractive females. Within each pretreatment group CORT treatment significantly decreased average courtship scores. Pre-treatment with melatonin prior to CORT treatment further suppressed male courtship behavior. Melatonin pretreatment when followed by vehicle also significantly inhibited male courtship behavior. These findings led Lutterschmidt et al. (2004) to suggest that melatonin and CORT have an additive inhibitory effect on male courtship behavior in T. s. parietalis. Pretreatment of male T. s. parietalis with the 5-HT2A receptor antagonist ketanserin followed by CORT treatment reduced male courtship behavior. However, pretreatment with ketanserin when followed by vehicle treatment did not reduce male courtship behavior. The inhibitory effects of melatonin and CORT on male courtship behavior do not appear to be mediated by androgens because
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Percentage of males exhibiting sexual behavior
80 70 60 50 40 30 20 10 0 Control (N=53)
Saline (N=53)
B 10µg (N=27)
B 25µg (N=24)
B 50µg (N=24)
B 100µg (N=27)
Treatments FIGURE 7.5 Percentage of male red-sided garter snakes, Thamnophis sirtalis parietalis, exhibiting sexual behavior in groups of males that were treated with different doses of CORT, saline, or were untreated. Groups underneath the solid lines are not significantly different from one another. Reproduced from Moore and Mason (2001).
treatment with either melatonin or CORT did not significantly affect the circulating concentrations of T or of DHT. Based on the observed effects of melatonin, CORT, and ketanserin, Lutterschmidt et al. (2004) argued that CORT and melatonin modulate male courtship behavior in T. s. parietalis via their actions on a serotonergic neural system. However, it should be noted that Lutterschmidt and Mason (2005) in a subsequent study of male T. s. parietalis found no evidence that melatonin plays a role in the modulation of the stress response. A stressor-induced increase in plasma CORT also appears to inhibit male courtship behavior in the copperhead, A. contortix (Schuett, 1996). Male copperheads fight for priority access to females during the two mating periods in the spring and late fall (Schuett, 1996; Schuett et al., 1996). In staged fights between male A. contortix, males that lost fights in comparison to males that won fights exhibited significantly less courtship behavior in trials with females, which occurred 30 minutes after fights (Schuett, 1996). This inhibition of male courtship behavior in losers was relatively long lasting because it was still present when losers were retested at 24 hours post-fights, although courtship scores did recover by 7 days post-fights (Schuett, 1996). To our knowledge there are no examples in reptiles of females terminating or modifying their reproductive behavior in response to short-term stressors. However, there are examples of females continuing to maintain
reproductive behaviors even when exposed to extreme stressors (Moore & Jessop, 2003). In the green sea turtle C. mydas, for example, females that had been seriously injured by sharks maintained nesting behaviors (Jessop, 2000). Similarly, in T. s. parietalis, females have been observed to mate despite sustaining injuries or being exposed to adverse environmental conditions (Whittier et al., 1987; Moore 1999). Elsey, Joanen, McNease, and Lance (1990) also noted that, in A. mississippiensis, plasma CORT levels in females that were actively defending their nests (in breeding pens) did not differ significantly from females that were not attending their nests, a result suggesting that females may be relatively resistant to stress associated with nest defense. There are several possible reasons why females might be less likely than males to modify their reproductive behavior in response to short-term stress. Firstly, female reptiles have a greater energy investment in their eggs than do males. Thus, it may benefit females more than males to maximize their current reproductive event, even though under certain situations it could entail costs to female survival (Moore & Jessup, 2003). Secondly, females, in comparison with males, may require much higher levels of plasma CORT before there is a change in behavior in response to a stressor (Moore & Jessop, 2003). Finally, sex-related differences in response to stressors may develop because the stress response frequently varies more with female reproductive stage than with male reproductive stage (Cartledge & Jones, 2007).
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4. SEASONAL CHANGES IN CIRCULATING CORTICOSTERONE (CORT) LEVELS 4.1. Baseline and Stress Levels Baseline and stressor-induced glucocorticoid levels vary seasonally in some species of amphibian, reptile, bird, and mammal (reviewed in Romero, 2002; see also Volume 2, Chapter 6; Volume 4, Chapter 5; Volume 5, Chapter 8). There is also some evidence for seasonal differences in baseline and stressor-induced glucocorticoid levels in some teleosts (Wendelaar Bonga, 1997) and elasmobranchs (Manire, Rasmussen, Maruska, & Tricas, 2007). In his review of seasonal changes in plasma glucocorticoid concentrations in freely living vertebrates, Romero (2002) considered glucocorticoid levels to be baseline if they were obtained from animals sampled within several minutes of capture and to be stressor-induced if they were obtained from animals beyond this time period. In a subsequent review, Romero and Reed (2005, p. 78) addressed the validity of this assumption in birds and a reptile. These investigators examined CORT levels in samples collected at various times from freely living individuals of five species of bird and one species of lizard and found that the available evidence strongly supports the idea ‘that samples collected in less than 2 min can be assumed to reflect unstressed concentrations with a high degree of confidence, and samples collected within 3 min most likely reflect unstressed concentrations.’ It was also noted by Romero (2002), however, that in some species of reptiles a stressor-induced increase in CORT might not occur until after 10 minutes of exposure to a stressor. However, a number of very recent studies in the lizard A. carolinensis have demonstrated that a stressor may induce a significant elevation in plasma CORT levels within 1.5 minutes, and that the speed of this response may be dependent on territorial or social status (Summers et al., 2005c; Watt et al., 2005; Ling et al., 2009). In addition, it may be important to consider stressor-induced circadian variation in this definition, since the desert iguana, Dipsosaurus dorsalis; the marine iguana, A. cristatus; the European common lizard, Lacerta vivipara; and the green anole, A. carolinensis show diurnal variation in CORT secretion, which can be modified by stress (Chan & Callard, 1972; Dauphin-Villemant & Xavier, 1987; Summers & Norman, 1988; Romero & Wikelski, 2006). Romero (2002) has suggested that ‘baseline’ is a more appropriate term than ‘basal’ for describing glucocorticoid levels in animals that were sampled within several minutes of capture because basal levels would be those in unstressed animals prior to capture. Of course, in some cases, baseline levels may not differ significantly from basal levels, especially if blood samples are taken very
Hormones and Reproduction of Vertebrates
quickly after animal capture and/or the subject species exhibits a delayed stress response. It has been suggested that seasonal basal glucocorticoid levels meet the demands normally associated with a predictable life-history stage, whereas stress-related levels of glucocorticoids respond to life-threatening and unpredictable events associated with an emergency lifehistory stage (Wingfield et al., 1997; 1998; Wingfield & Ramenofsky, 1999; Wingfield & Romero, 2001; Landys et al., 2006). Most studies have found that diel and seasonal elevations in baseline glucocorticoid levels are much lower than stress-related levels, and it is thought that these lower levels of glucocorticoids play a key role in the regulation of feeding, locomotor activity, and energy metabolism associated with predictable life-history cycles (Landys et al., 2006). Basal glucocorticoid levels are also believed to mediate important ontogenetic transitions such as dispersal behavior in mammals, birds, and reptiles (Wada, 2008).
4.2. Pre-breeding, Breeding, and Post-breeding periods Baseline levels of CORT during the breeding season have been found to be higher, lower, or no different than in the non-breeding season in a variety of reptiles (see review by Romero, 2002) (Table 7.2). To make it easier to discern possible patterns in a dataset with high variability in assay methods, sampling times after animal capture, and type of glucocorticoid measured, Romero (2002) ranked measured glucocorticoid levels within a season as being either high, medium, or low relative to the levels at other seasons for the same sex and species. The seasons were pre-breeding, breeding, post-breeding, and other. Seasons were assigned different relative ranks for glucocorticoid levels only when a significant difference was present between seasons in measured glucocorticoid levels. On the other hand, if glucocorticoid concentrations did not vary significantly with season, a ranking of medium was assigned to each season measured for glucocorticoid levels. Romero (2002) found that baseline CORT levels in reptiles varied seasonally for the majority of examined species with seasonal reproductive cycles and that plasma CORT levels were most often highest during the breeding season relative to other seasons. According to Romero (2002), the assertion that baseline CORT levels in many reptiles tend to be highest during the breeding season is strengthened by the results of earlier studies that showed that the size (mass and/or volume) of the adrenal glands was greatest in animals sacrificed during the breeding season (see references in Guillette et al., 1995). Romero (2002) cites the results of the Manzo et al. (1994) study of the Italian wall lizard, P. s. sicula, as evidence that the
Sex
Relative plasma corticosterone (CORT) levels
Reference
Loggerhead sea turtle (Caretta caretta)
Male and female
Baseline and stressor-induced CORT in small turtles caught by trawl were higher in the summer breeding season than in the winter non-breeding season, and in small turtles baseline CORT was not significantly different between seasons but stressorinduced CORT was higher in the breeding season
Gregory et al. (1996)
Gala´pagos tortoise (Geochelone nigra)
Male
Baseline CORT was higher in the breeding season than in the non-breeding season in animals living under semi-natural conditions
Schramm et al. (1999)
Gopher tortoise (Gopherus polyphemus)
Male and female
Baseline CORT did not differ significantly during the active period from May through October, a period that includes most of the mating season
Ott et al. (2000)
Desert tortoise (Gopherus agassizii)
Male and female
Baseline CORT was higher in the breeding season than in the non-breeding season in captive animals
Lance et al. (2001)
Green sea turtle (Chelonia mydas)
Male
Stressor-induced CORT was lower in migrating breeding males than in non-breeding males
Jessop et al. (2002)
American alligator (Alligator mississippiensis)
Female
Baseline CORT did not vary significantly among sampled months or with reproductive stage
Guillette et al. (1997b)
Tuatara (Sphenodon punctatus)
Male and female
Baseline CORT varied among the four seasons of the year and in females varied between reproductive states
Tyrrell and Cree (1998)
European common lizard (Lacerta vivipara)
Male and female
Baseline CORT in males did not vary significantly during the active period and baseline CORT in females was higher in the breeding season and was influenced by female reproductive stage
Dauphin-Villemant et al. (1990)
Whiptail lizard (Cnemidophorus uniparens)
Female
Baseline CORT was lower in the breeding season than in the non-breeding season
Grassman and Crews (1990)
Six-lined race runner (Cnemidophorus sexlineatus)
Male and female
Baseline CORT in males was higher in the breeding season than in the non-breeding season and baseline CORT in females did not differ significantly between seasons
Grassman and Hess (1992a)
Side-blotched lizard (Uta stansburiana)
Female
Baseline CORT was higher in the breeding season than in the non-breeding season and female reproductive mass was positively correlated with CORT levels
Wilson and Wingfield (1992)
Italian wall lizard (Podarcis sicula sicula)
Male
Baseline and stressor-induced CORT were higher in the breeding season than in the non-breeding season
Manzo et al. (1994)
Side-blotched lizard (Uta stansburiana)
Male and female
Baseline CORT was higher in the breeding season than in the non-breeding season
Wilson and Wingfield (1994)
Western fence lizard (Sceloporus occidentalis)
Male and female
Baseline CORT did not differ significantly between seasons and stressor-induced CORT was higher during the breeding season in April, with greater responses in peripheral than central populations of lizards
Dunlap and Wingfield (1995)
Stress and Reproduction in Reptiles
Species
Chapter | 7
TABLE 7.2 Relative baseline and stressor-induced corticosterone levels during the breeding and non-breeding seasons in different groups of reptile
(Continued)
189
190
TABLE 7.2 Relative baseline and stressor-induced corticosterone levels during the breeding and non-breeding seasons in different groups of reptiledcont’d Sex
Relative plasma corticosterone (CORT) levels
Reference
Common gecko (Hoplodactylus maculatus)
Female
Baseline CORT varied significantly with season but was independent of reproductive state
Girling and Cree (1995)
Brown anole (Anolis sagrei)
Male
Baseline CORT was lower in the breeding season than in the non-breeding season
Tokarz et al. (1998)
Eastern fence lizard (Sceloporus undulatus)
Male
Stressor-induce CORT was higher in the breeding season than in the non-breeding season in males exposed to male and female conspecifics
Smith and John-Alder (1999)
Bearded dragon lizard (Pogona barbata)
Male and female
Baseline CORT did not vary significantly with time of year or reproductive condition
Amey and Whittier (2000)
Gala´pagos marine iguana (Amblyrhynchus cristatus)
Female
Baseline CORT was higher in the breeding season than in the non-breeding season
Rubenstein and Wikelski (2005)
Green anole lizard (Anolis carolinensis)
Male
Baseline CORT was higher in the post-breeding season than in the breeding season in both lightweight and heavyweight males
Husak et al. (2007)
Texas horned lizard (Phrynosoma cornutum)
Male and female
Baseline CORT was higher in both sexes in the breeding season than in the nonbreeding season, and stressor-induced CORT in females was higher in the breeding season than in the non-breeding season
Wack et al. (2008)
Eastern fence lizard (Sceloporus undulatus)
Female
Baseline CORT was higher in the breeding season than in the non-breeding season, whereas stressor-induced CORT did not differ significantly between the seasons
Phillips and Klukowski (2008)
Red-sided garter snake (Thamnophis sirtalis parietalis)
Male
Baseline CORT was higher in the breeding season upon emergence from the den than in the non-breeding season
Krohmer et al. (1987)
Red-sided garter snake (Thamnophis sirtalis parietalis)
Male
Baseline CORT was higher in the spring breeding season than in the non-breeding season, and stressor-induced CORT was higher in the summer at 1 hour post-capture
Moore et al. (2001)
Red-spotted garter snake (Thamnophis sirtalis concinnus)
Male
Baseline CORT was lower in the breeding season than in the non-breeding season
Moore et al. (2000b)
Red-spotted garter snake (Thamnophis sirtalis concinnus)
Male
Baseline CORT did not differ significantly between the spring breeding season and the non-breeding season in 1999 and stressor-induced CORT was higher in the spring of 1995 and 1999 at 4 hours post-capture and in the summer and fall of 1999 at 1 hour post-capture
Moore et al. (2001)
Western diamondback rattlesnake (Crotalus atrox)
Female
Baseline CORT was higher in the breeding season than in the non-breeding season and varied with stage of the reproductive cycle
Taylor et al. (2004)
Hormones and Reproduction of Vertebrates
Species
Chapter | 7
Stress and Reproduction in Reptiles
condition of the adrenal glands can in some cases be used as a reliable indicator of CORT secretion. Manzo et al. (1994) found that in male P. s. sicula the in-vitro pattern of adrenal gland CORT secretion paralleled the in-vivo pattern of CORT secretion in that the highest secretion rates occurred in May during the mating phase. Since Romero’s review in 2002, seasonal changes in baseline levels of CORT in relation to reproduction have been measured in the western diamondback rattlesnake, Crotalus atrox (Taylor, DeNardo, & Jennings, 2004); the green anole lizard, A. carolinensis (Husak, Irschick, Meyers, Lailvaux, & Moore, 2007); and in the Texas horned lizard, Phrynosoma cornutum (Wack et al., 2008). In the study of C. atrox, Taylor et al. (2004) directed freely living snakes into a plastic tube and bled them from the caudal vein within five minutes of capture, a short enough interval of time to prevent a stressor-induced rise in CORT (G. Schuett & E. Taylor, unpublished data). The results of this study showed that plasma CORT levels in male C. atrox do not vary significantly with season (Taylor et al., 2004). In female C. atrox, non-reproductive females had relatively constant CORT levels, whereas reproductive females showed a slight rise in CORT in the spring when females are undergoing vitellogenesis and mating, and a significant increase in CORT levels at the end of gestation (Figure 7.6). Taylor et al. (2004) suggested that the rise in CORT levels in the spring in the viviparous C. atrox might have a role in mobilizing energy for both vitellogenesis and parturition. It was also proposed that CORT might be the hormonal stimulus that initiates parturition in this snake because CORT levels in females that were captured and maintained in the laboratory increased prior to parturition, peaked at parturition, and then precipitously declined following parturition (Taylor et al., 2004). In light of the finding that CORT levels in males were unchanged across the nonbreeding and breeding periods and did not have a negative effect on T levels during the breeding period, Taylor et al. (2004) suggested that either the energy requirements of males during the breeding season are not substantial or that males have special features (e.g., low basal metabolic rates and energy requirements) that allow them to meet their energy requirements without a CORT-induced increase in energy mobilization. In A. carolinensis, baseline CORT levels were higher during the post-breeding season (October) than during the breeding season (May) (Husak et al., 2007). This betweenseason difference in baseline CORT levels with higher levels in the post-breeding season than in the breeding season was present in males that were defined as heavyweights (snout-to-vent length (SVL) > 64 mm) and in males that were defined as lightweights (SVL < 64 mm), although within a season the two morphs did not differ significantly from each other in CORT levels. Interestingly, T levels were negatively correlated with CORT levels for
191
lightweight males but there was no significant relationship between the two steroid hormones for heavyweight males. Wack et al. (2008) in a study of P. cornutum measured baseline levels of CORT and other sex steroid hormones in freely ranging adult females captured in the breeding season (April–May), egg-laying season (June), and nonbreeding season (July–September), and in adult males captured in the breeding season (April–May) and non-breeding season (June–September). Plasma CORT levels in female P. cornutum varied significantly with season with the peak in CORT concentrations occurring during the egg-laying period. When gravid vs. non-gravid female P. cornutum were compared across all time periods and within the egg-laying period, CORT levels were significantly higher in gravid females than in non-gravid females. In male P. cornutum, CORT levels were marginally (P < 0.055) higher in breeding males than in nonbreeding males. Wack et al. (2008) indicated that their findings are similar to those of other studies of reptiles (see Table 7.2) in that they found a seasonal peak in CORT during the breeding season in males and during the breeding and egg-laying periods in females. These investigators also suggested that the observed increase in CORT levels in male and female P. cornutum during the breeding season might help meet the energetic needs of individuals during breeding. In addition, they discussed the possibility that the relative peak in CORT during the egg-laying period of females might be due to stressors associated with digging multiple nests to find an appropriate place to oviposit eggs, an idea that was originally suggested in Tyrrell and Cree (1998) to explain the high levels of CORT during egg-laying in the tuatara S. punctatus. Stressor-induced levels of CORT in reptiles also tend to vary seasonally and to be elevated during the breeding season, although this pattern is less well supported than for baseline CORT levels because it is based on the results of relatively few studies (see Romero, 2002) (Table 7.2). Another problem in establishing a seasonal pattern in stressor-induced glucocorticoid levels is that an animal’s stress response can be modulated by many factors including sex, age, health, body mass, reproduction condition, and time of year (reviewed in Moore & Jessop, 2003). Three major hypotheses have been advanced to explain seasonal differences in glucocorticoid levels (Romero, 2002). These are the energy mobilization hypothesis (Wingfield & Ramenofsky, 1999), the behavior hypothesis (Wingfield & Romero, 2001), and the more recently proposed preparative hypothesis (Romero, 2002). The energy mobilization hypothesis suggests that glucocorticoid concentrations will be highest during energetically costly periods. Thus, glucocorticoid levels would be expected to be relatively high during reproduction to meet the increased energetic demands associated with this
192
Hormones and Reproduction of Vertebrates
(b)
(a) vitell.
gestation
40 30 20 10 0 Mar Apr May Jun Jul
mating
vitell.
gestation
30 20 10 0
Aug Sep
Mar Apr May Jun Jul
Aug Sep
(d)
(c) 160
mating
vitell.
gestation
25
Testosterone (ng/ml)
Corticosterone (ng/ml)
40
Progesterone (ng/ml)
17β β-Estradiol (ng/ml)
50
mating
140 120 100 80 60 40 20
mating
vitell.
gestation
20 15 10 5 0
0 Mar Apr May Jun Jul
Aug Sep
Mar Apr May Jun Jul Aug Sep
FIGURE 7.6 Monthly plasma levels of (a) 17b-estradiol (E2), (b) progesterone (P4), (c) corticosterone (CORT), and (d) testosterone (T) in reproductive (black circles, solid lines) and non-reproductive (white triangles, dashed lines) female western diamondback rattlesnakes (Crotalus atrox). Hormone levels in nonreproductive snakes were relatively low throughout the year, but were high in reproductive snakes at certain times in the reproductive cycle (significant differences are marked with *). Values are presented as means 1 standard errors of the means (SEM). Approximate timing of spring mating, vitellogenesis (vitell.), and gestation are indicated in the bars above the graphs. Reproduced from Taylor, DeNardo, and Jennings (2004).
life-history stage (reviewed in Harshman & Zera, 2007). The behavior hypothesis posits that seasonal changes in glucocorticoid levels are due to an animal’s need to modify its behavior across seasons (Romero, 2002). For example, during the breeding season a short-term increase in glucocorticoid levels due to adverse weather conditions might inhibit an animal’s reproductive behavior and induce dispersal behavior, a phenomenon that has been especially well documented in birds (Wingfield et al., 1998; Wingfield & Ramenofsky, 1999; Wingfield & Romero, 2001). The preparative hypothesis suggests that annual changes in glucocorticoid levels act to modulate the priming of stress pathways in relation to differences in the likelihood of being exposed to stressors (Romero, 2002). Thus, based on the preparative hypothesis, baseline glucocorticoid levels should be relatively high in the breeding season if the risk of being exposed to stressors during this period is greater than at other times of the year. An increase in baseline glucocorticoid levels at the beginning of the breeding season could have permissive effects that would allow an animal to cope more efficiently when exposed to subsequent stressors. Similar permissive effects of glucocorticoids have been demonstrated for aggressive territorial behavior in
A. carolinensis (Summers et al., 2005c), which may suggest a specific purpose for elevated plasma CORT concentrations during the breeding season, at least in males. The permissive effects of glucocorticoids are believed to be mediated by baseline glucocorticoid levels acting via the high-affinity mineralocorticoid receptor (type I, MR), whereas the stimulatory, suppressive, and preparatory effects of glucocorticoids are for the most part thought to be mediated by stress levels of glucocorticoids acting via the low-affinity glucocorticoid receptor (type II, GR) (Sapolsky, Romero, & Munck, 2000; Romero, 2004). However, the necessary corticosteroid contribution to territorial aggressive behavior in A. carolinensis appears to be mediated by GR, since the GR antagonist mifepristone inhibits early stages of aggression (Summers et al., 2005c).
5. MODULATION OF STRESS RESPONSE DURING REPRODUCTION 5.1. Evidence One of the more important advances in our understanding of how animals respond to stressors was the finding that the
Chapter | 7
Stress and Reproduction in Reptiles
stress response in some situations can be modulated (Wingfield et al., 1998; Wingfield & Ramenofsky, 1999; Wingfield & Romero, 2001). The process by which animals modulate changes in the rate, duration, and magnitude of glucocorticoid release has been termed adrenocortical modulation (Wingfield & Ramenofsky, 1999; Wingfield & Romero, 2001). The ability of animals to modulate the activity of their HPA axis in response to environmental and social stressors may be particularly important during energetically demanding activities such as reproduction (Wingfield et al., 1998). In reptiles, as in other vertebrates, a number of factors such as sex, age, body condition, health, season, population, and reproductive state affect the likelihood that an animal will modulate its stress response (reviewed in Moore & Jessop, 2003). Here we will consider the evidence for adrenocortical modulation in reptiles in terms of its impact on reproduction and discuss the results of studies that have been published since the Moore and Jessop (2003) review. A difference in the stress response of breeding vs. nonbreeding animals has been particularly well documented in turtles (Jessop et al., 2000; Jessop, 2001) and viviparous snakes (Moore, Lerner, Lerner, & Mason, 2000; Moore, Greene, & Mason, 2001; Cease, Lutterschmidt, & Mason, 2007). The response of females to acute stress as measured by circulating CORT levels was suppressed significantly during the breeding season in the green sea turtle, C. mydas (Jessop et al., 2000; Jessop, 2001) and in the hawksbill turtle, Eretmochelys imbricata (Jessop, 2001). Males of these species show a similar reduced stress response during the breeding season, but to a much lesser extent than females (Jessop, 2001). The adrenocortical response of males to capture also appears to be modulated in the redsided garter snake (Moore et al., 2001; Lutterschmidt & Mason, 2005; Cease et al., 2007). Moore, LeMaster, and Mason (2000a) reported that male T. s. parietalis captured in 1997 during the spring mating season (May) in the Interlake region of Manitoba, Canada had elevated plasma CORT levels and decreased plasma T levels after four hours of capture relative to baseline levels of these hormones measured at the time of capture. However, in a subsequent study, Moore et al. (2001) found that male T. s. parietalis that were also captured during the spring mating season in the same region of Manitoba, Canada did not have a significant increase in CORT levels or a significant decrease in T relative to baseline levels after one or four hours of capture stress. The investigators suggested that these apparently contradictory results might be explained by the fact that the initial baseline CORT levels differed between the two studies. According to Moore et al. (2001), the initial CORT levels observed in the Moore et al. (2000a) study (62 ng/ml) were significantly lower than the initial CORT levels they observed in their study (129 ng/ml), although initial T levels were similar. Moore et al. (2001)
193
hypothesized that there may be some critical threshold level of CORT at which the stress response in terms of a suppression of T levels is suppressed. Romero (2004) has discussed the difficulty of trying to compare stress responses in a situation in which the compared populations differ in baseline levels of glucocorticoids, and posed the question of whether it is more meaningful to compare the absolute stressor-induced glucocorticoid levels or the percent change from baseline levels. In another study of male T. s. parietalis, obtained from the Interlake region of Manitoba in Canada, CORT and T levels did not change significantly relative to baseline levels four hours after capture in males that were captured during the spring or during the fall when males have a brief period of mating prior to entering their dens (Lutterschmidt & Mason, 2005). Cease et al. (2007) examined the adrenocortical response of male T. s. parietalis in relation to the distance males had traveled from their winter dens in Manitoba, Canada. These investigators found that actively courting males, that had emerged from the winter den in the spring, had a significantly reduced stress response compared to males that had ceased courting and were dispersing from the den to feeding grounds. Specifically, courting males captured at the den or within the den perimeter did not exhibit an increase in plasma CORT levels when measured at either one or four hours after capture, whereas dispersing, non-courting males captured approximately 0.6 m from the den did exhibit a significant increase in CORT (Figure 7.7). This significant CORT increase in dispersing males, and lower baseline CORT levels in these males compared with courting males (which did not show an increase in stressor-induced CORT), suggests that animals with relatively lower basal CORT levels may show a relatively greater stressor-induced increase in CORT. Several studies have examined the adrenocortical stress response of the red-spotted garter snake, Thamnophis sirtalis concinnus (Moore et al., 2001; Lutterschmidt & Mason, 2005). This garter snake lives at mid-latitudes and has a longer breeding season than does T. s. parietalis, which lives in more northern latitudes (Moore et al., 2001; Lutterschmidt & Mason, 2005). Male T. s. concinnus that were captured in western Oregon during the spring, summer, and fall had a similar elevation in plasma CORT levels after one or four hours of capture as compared to baseline levels, whereas plasma T levels only declined in males captured during the spring (Moore et al., 2001). Thus the stressor-induced decline in T levels in animals that were captured in the spring appears to be suppressed during the summer and fall, when males are undergoing spermatogenesis (Moore et al., 2001). In a study of the Texas horned lizard, P. cornutum, Wack et al. (2008) examined the relationship between plasma CORT concentrations and sampling time (time from capture) in adult females captured in the breeding
194
Corticosterone Concentration (ng/ml)
Hormones and Reproduction of Vertebrates
80
60
Pre-stress
Post-stress
a
a,b
b 40
20
0
Den
Den Perimeter
0.6 km from Den
FIGURE 7.7 Hormonal responses of male red-sided garter snakes (Thamnophis sirtalis parietalis) to capture stress during the spring mating season. This study was conducted during the last quarter of the mating season (May 20–25, 2004), a time when snakes can be found transitioning from active courtship behavior to dispersal to the feeding grounds. Courting male garter snakes were collected from the den and den perimeter; snakes collected approximately 0.6 km from the den were dispersing to the feeding grounds. For ease of visual interpretation, and because the duration of capture stress did not significantly influence hormonal stress responses, the one hour and four hour capture stress treatment groups have been collapsed as shown (n ¼ 20 for each location). Mean plasma corticosterone (CORT) concentrations. Standard errors (þ1) are shown by the vertical lines. Letters above each group indicate statistically significant differences in hormonal responses to capture stress among males collected from different sites. Modified from Cease, Lutterschmidt, and Mason (2007).
season (April–May), egg-laying season (June), and nonbreeding season (July–September), and in adult males captured in the breeding season and non-breeding season (June–September). No overall significant relationship was found between sampling times and CORT levels across season and sex. Although a significant positive correlation between CORT levels and sampling times was detected within non-breeding males, this relationship became nonsignificant with the removal of one outlier value from the dataset. Corticosterone levels and sampling times were, however, significantly and positively related within females during the egg-laying season. Because basal CORT levels were already elevated in egg-laying females, it was suggested that the HPA axis during the egg-laying period might be more sensitive to an acute stressor (Wack et al., 2008). The adrenocortical response to stressors can vary with specific reproductive events in some reptiles (see Table 7.3). For example, in the olive ridley sea turtle, L. olivacea, breeding females undergoing mass nesting (arribada) as well as females that were solitary nesters responded more slowly to the stressor of turning than did basking, nonnesting females (Valverde et al., 1999). On the other hand, in the green turtle, C. mydas, the stress response of females that abandoned their nests because of competition for nest sites
did not differ from that observed in females that persisted in their nesting activities (Jessop & Hamann, 2004). In the tree lizard, U. ornatus, vitellogenic females that were subjected to stress resulting from handling had significantly higher levels of CORT than gravid females handled in the same manner (Woodley & Moore, 2002). In addition, Cartledge and Jones (2007) observed that in the viviparous White’s skink, E. whitii, postovulatory females had the most conservative response to stress, and, although gestating females showed a large initial response, it was not prolonged. In marked contrast, in the viviparous gecko, H. maculatus, the CORT response of pregnant females to capture and confinement stressors was not suppressed in comparison to that observed in non-pregnant, vitellogenic females (Cree, Tyrrell, Preest, Thorburn, & Guillette, 2003). The adrenocortical response to stressors in reptiles has also been found to vary in some species with sex (see Table 7.4), body condition, and health (reviewed in Moore & Jessop, 2003). In breeding male C mydas, a reciprocal relationship appears to exist between stressor-induced levels of CORT and relative body condition (Jessop et al., 2002). In this sea turtle, migrant breeding males that were in the lowest quartile of body condition had significantly higher plasma CORT levels than did males in the highest
Chapter | 7
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Stress and Reproduction in Reptiles
TABLE 7.3 Adrenocortical modulation of the stress response in relation to reproductive condition in different groups of reptiles Corticosterone (CORT) levels and reproductive condition
Species
Modulation
Loggerhead sea turtles (Caretta caretta)
Possible
CORT levels did not increase in several large turtles captured during the summer breeding season
Gregory et al. (1996)
Olive ridley sea turtle (Lepidochelys olivacea)
Yes
In nesting female turtles (both mass ‘arribada’ and solitary nesters), CORT levels increased more slowly than in non-breeding basking turtles within 2 hours of the turtles being turned onto their backs
Valverde et al. (1999)
Green turtle (Chelonia mydas)
Yes
In breeding females exposed either to capture or to capture and an ecological heat stressor, CORT levels were lower than in non-breeding females exposed only to capture
Jessop et al. (2000)
Green turtle (Chelonia mydas)
Yes
CORT levels were lower in breeding females than in non-breeding females after capture
Jessop (2001)
Hawksbill turtle (Eretmochelys imbricata)
Yes
CORT levels were lower in breeding females than in non-breeding females after capture
Jessop (2001)
Bearded dragon lizard (Pogona barbata)
No
CORT levels were relatively low after capture and were unrelated to female reproductive stage
Cree et al. (2000)
Tree lizard (Urosaurus ornatus)
Yes
After capture and 10 minutes of handling, CORT levels in vitellogenic but not gravid females were higher than in control females bled immediately after capture
Woodley and Moore (2002)
Common gecko (Hoplodactylus maculatus)
No
CORT levels in response to capture and confinement did not differ significantly between vitellogenic females and pregnant females
Cree et al. (2003)
White’s skink (Egernia whitii)
Yes
CORT levels varied with female reproductive stage with post-ovulatory females showing the smallest increase in CORT within 4 hours of capture
Cartledge and Jones (2007)
Texas horned lizard (Phrynosoma cornutum)
Yes
CORT levels in breeding males, unlike nonbreeding males, did not increase in response to capture and handling
Wack et al. (2008)
Red-sided garter snake (Thamnophis sirtalis parietalis)
Yes
Pre- and post-stress CORT levels did not differ significantly in reproductively active males captured at or near the den but did differ in dispersing males captured approximately 0.6 km from the den
Cease et al. (2007)
quartile of body condition after eight hours of capture and restraint. The dynamics of the association between body condition and stressor-induced increases in CORT levels vary, with some species of reptile exhibiting an inverse linear relationship and others a nonlinear, threshold relationship (Moore & Jessop, 2003). In addition, the adrenocortical response to capture may be independent of body condition in some reptiles (Moore & Jessop, 2003).
Reference
In addition to body condition, an animal’s health can affect its response to acute stress. For example, in the western fence lizard, S. occidentalis, CORT levels in reproductively active males one hour after capture and confinement in a cloth bag were higher in males that were infected with a malarial parasite (Plasmodium mexicanum) than in uninfected males (Dunlap & Schall, 1995).
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Hormones and Reproduction of Vertebrates
TABLE 7.4 Sex differences in adrenocortical stress response following exposure to stressors in different groups of reptiles Species
Relative plasma corticosterone (CORT) levels
Reference
Red-eared slider turtle (Trachemys scripta elegans)
Males and females did not differ significantly in CORT levels at any sampling time after capture and handling
Cash et al. (1997)
Olive ridley sea turtle (Lepidochelys olivacea)
Breeding females had lower CORT levels than mating males 20 minutes after turtles had been turned onto their backs
Valverde et al. (1999)
Green sea turtle (Chelonia mydas)
Females had lower CORT levels than males in breeding turtles after capture, restraint, and blood sampling
Jessop (2001)
Hawksbill turtle (Eretmochelys imbricata)
Females had lower CORT levels than males in sampling breeding turtles after capture, restraint, and blood sampling
Jessop (2001)
Hawksbill turtle (Eretmochelys imbricata)
In immature turtles, males and females did not differ significantly in CORT levels after exposure to a capture protocol causing stress
Jessop et al. (2004)
American alligator (Alligator mississippiensis)
Females had lower CORT levels than males in adult alligators from both wild and captive populations within 15 minutes of capture, restraint, and blood sampling
Elsey et al. (1990)
Australian freshwater crocodile (Crocodylus johnstoni)
Males and females of several age classes did not differ significantly in CORT levels after capture, restraint and blood sampling
Jessop et al. (2003)
Tuatara (Sphenodon punctatus)
Males and vitellogenic females did not differ significantly in CORT levels after capture and confinement for 3 hours
Tyrrell and Cree (1998)
Six-lined racerunner (Cnemidophorus sexlineatus)
Females had higher CORT levels than males in intact, reproductively mature lizards after being handled for 30 minutes
Grassman and Hess (1992b)
Marine iguana (Amblyrhynchus cristatus)
Males and females did not differ significantly in CORT levels after capture and restraint for 15 or 30 minutes
Romero and Wikelski (2001)
Tree lizard (Urosaurus ornatus)
Vitellogenic females had a greater increase in CORT levels than males after capture and handling for 10 minutes
Woodley and Moore (2002)
Common gecko (Hoplodactylus maculatus)
Vitellogenic females had higher CORT levels than males following capture and confinement for 24 hours
Cree et al. (2003)
Yellow-bellied water-skink (Eulamprus heatwolei)
Females consistently had at least threefold higher CORT levels than males in reproductively active lizards 1 hour following exposure to a variety of potential stressors
Langkilde and Shine (2006)
White’s skink (Egernia whitii)
Gestating and postovulatory females had lower CORT levels than reproductively quiescent males after capture and being held in cloth bags for up to 240 min
Cartledge and Jones (2007)
Moore and Jessop (2003) posed the question of why it might be advantageous for amphibians and reptiles to suppress their adrenocortical response to stressors during reproduction. They suggested that, like in some species of desert and arctic birds (see Wingfield & Romero, 2001), species of reptile with relatively short breeding seasons may maximize their reproductive success by suppressing their adrenocortical response to stressors in order to continue to breed even in the face of severe stressors.
There is some support in reptiles for this suggestion. Male garter snakes captured from a northern population with a relatively short breeding season have been found to exhibit a less intense adrenocortical response to capture than males captured from a southern population with a longer breeding season (Moore et al., 2001). However, as pointed out in Moore and Jessop (2003), the duration of the reproductive season is unlikely to explain why breeding females in at least three species of sea turtle
Chapter | 7
Stress and Reproduction in Reptiles
have a reduced or slower adrenocortical response to capture than do non-breeding females, because these reptiles typically have extended breeding seasons and in some cases breed throughout the year. However, these sea turtles do vary in reproductive life-history traits, such as age to sexual maturity, timing of seasonal nesting episodes, and relative investment in particular reproductive events (Moore & Jessop, 2003), which might explain why breeding females exhibit a reduced or slower adrenocortical response to stressors than do non-breeding females.
5.2. Mechanisms One important but largely unanswered question is how animals, including reptiles, modulate their stress responses (Moore & Jessup, 2003; Romero, 2004). In other words, what are the physiological mechanisms that cause a stress response to be suppressed under some conditions but not others? It has been suggested that the type and numbers of glucocorticoid receptors, glucocorticoid plasma binding proteins, and/or activity of enzymes such as 11b-hydroxysteroid dehydrogenase that convert glucocorticoids to inactive metabolites might play a key role in the modulation of the adrenocortical stress response (Wingfield & Romero, 2001; Breuner & Orchinik, 2002; Moore & Jessup, 2003; Romero, 2004; Landys et al., 2006). This is so because they affect the plasma levels of glucocorticoids, their action on target tissues, and the physiology of the glucocorticoid negative feedback systems in the brain and pituitary (Moore & Jessop, 2003; Romero, 2004; Fietta & Fietta, 2007). Is there any direct evidence that any of these mechanisms play a role in adrenocortical modulation in reptiles? Unfortunately, to our knowledge, gene or protein sequences for intracellular glucocorticoid receptors have not been identified in reptiles, although these receptors do appear to have a similar brain distribution to that in mammals and other vertebrates, based on immunochemical staining using antibodies to mammalian receptors (Summers, Adams, Anderson, & Ten Eyck, 1994). Thus, we can assume that reptiles are similar to other vertebrates in having a high-affinity mineralocorticoid (type I) receptor and a low-affinity glucocorticoid (type II) receptor that bind glucocorticoids (Romero, 2004; Landys et al., 2006), and perhaps a non-genomic membrane glucocorticoid receptor as well (Moore & Evans, 1999; Borski, 2000). Differences in the binding capacity of the plasma glucocorticoid binding proteins may also play a role in the modulation of the stress response in reptiles. Two plasma steroid-binding globulins have been identified in male tree lizards, U. ornatus: one of these binds androgens and C-21
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steroids such as CORT and has been termed androgen– glucocorticoid-binding globulin (AGBG) and the other is a sex hormone-binding globulin (SHBG) that binds androgens and E2 with high affinity (Jennings et al., 2000). It has been suggested that the difference in the binding capacity of AGBG in male morph types in U. ornatus may explain why ‘free’ CORT levels in non-territorial males are higher than in territorial males during a stress response and why T levels are depressed to a greater extent in nonterritorial males than in territorial males (Jennings et al., 2000). Indirect evidence indicates that either the pituitary and/ or brain may play a role in the modulation of the adrenocortical stress response in reptiles. In the olive ridley sea turtle, L. olivacea, the mechanism dampening the stress response of nesting females to capture appears to be at the level of the pituitary or brain rather than the adrenal gland because nesting females that show little CORT response to capture exhibit a marked increase in CORT levels when treated with ACTH (Valverde et al., 1999). There is also evidence that estrogen levels may modulate the response of females to ACTH in the viviparous skink, E. whitii (Cartledge & Jones, 2007). Female E. Whitii treated with E2 responded to ACTH treatment with a marked increase in plasma CORT levels in comparison to females that were treated with saline rather than this estrogen. Because E2 levels in most squamates peak during the late vitellogenic stage of the reproductive cycle and then decline markedly during early gestation (Edwards & Jones, 2001), Cartledge and Jones (2007) suggested that these changes in circulating E2 levels could perhaps be the mechanism for the modulation of the stress response across reproductive phases in viviparous reptiles. Lutterschmidt and Mason (2005) examined the effects of ketanserin (a serotonergic type 2A receptor antagonist), 5-hydroxytryptophan (a precursor in 5-HT and melatonin synthesis), and melatonin (either a low or high dose) on the hormonal responses (changes in CORT and androgen levels) of males exposed to the stressors associated with four hours of capture (snakes held in cloth bags for four hours) in two populations of garter snake (T. s. parietalis and T. s. concinnus). When ketanserin, 5-hydroxytryptophan, and melatonin were administered to males prior to their being held in cloth bags for four hours, these agents had no significant effect on CORT or androgen levels in male T. s. parietalis after four hours of capture. However, in male T. s. concinnus, treatment with ketanserin prior to four hours of capture prevented the significant decline in androgen levels that normally occurs in males of this subspecies that are captured during the spring and subjected to the stressors associated with four hours of capture. Based on these results, Lutterschmidt and Mason (2005) suggested that a serotonin-regulated system (but not melatonin) might modulate the activity of the HPG axis
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during the physiological stress response of T. s. concinnus but not of T. s. parietalis.
6. FITNESS EFFECTS OF STRESS DURING REPRODUCTION 6.1. Adults As pointed out by Greenberg and Wingfield (1987), the physiological stress response cannot be assumed to be invariably harmful, and may effectively be beneficial, and thus stress depending on a variety of factors may have negative or positive fitness consequences. Congruent with this idea is the finding that stress in reptiles can have either negative or positive effects on survival and other components of fitness, depending on species (Romero & Wikelski, 2001; Cote al., 2006), sex (Cote al., 2006), and even alternative mating strategies (Comendant, Sinervo, Svensson, & Wingfield, 2003; Lancaster et al., 2008). Moreover, in as much as stress hormones such as glucocorticoids can influence condition-dependent sexually selected traits that serve as indicators of mate quality, stress hormones may also play a role in mate choice (Husak & Moore, 2008). The effects of stress, either positive or negative, are likely to be especially important during periods of reproduction because of the high energetic costs associated with successful reproduction (see review by Harshman & Zera, 2007). These energetic costs in female reptiles include the energetic costs of gamete production as well as the indirect costs of reproduction, including the increased metabolic costs of maintenance or activity (Angilletta & Sears, 2000; Olsson, Shine, & Bak-Olsson, 2000; Shine, 2003a; 2003b; French et al., 2007a; French, McLemore, Vernon, Johnston, & Moore, 2007b; French & Moore, 2008; Zani, Neuhaus, Jones, & Milgrom, 2008). Although gamete production in male reptiles is less costly than in females (French & Moore, 2008), there are also relatively high costs associated with male reproductive behavior, which, depending on species, may include the costs of producing displays, acquiring and guarding territories, and aggressive interactions with other males (Marler & Moore, 1988; 1989; Sinervo, Miles, Frankino, Klukowski, & DeNardo, 2000; French & Moore, 2008). Because of the relatively high costs of reproduction, tradeoffs may occur between reproductive activities on the one hand and activities such as somatic growth, self-maintenance, and immune function on the other (Berger et al., 2005; French et al., 2007a; 2007b; French & Moore, 2008; Lancaster et al., 2008; Mills et al., 2008). Although the production of offspring during a given reproductive event is obviously a key component to an individual’s fitness, it is also true
Hormones and Reproduction of Vertebrates
that an individual’s long-term survival will affect its fitness (French & Moore, 2008). Tradeoffs exist between reproductive activity and the functioning of the immune system in a number of species of lizard. In a study of the highly polygynous Gala´pagos marine iguana, A. cristatus, Berger et al. (2005) measured the immune response of males of three distinct behavioral and morphological types (territorial, satellite, bachelor) during the peak of the breeding season. Berger et al. (2005) captured adult male A. cristatus and injected them into the toe-web of the back foot with either the antigen phytohemagglutinin (PHA) or with saline (control). After initial treatment and measurements, males were released and recaptured just prior to the next measurement period (at 6, 12, 18, 24, and 48 hours after injection) so that the responses to PHA could be ascertained in freely living animals under natural conditions. The large, highly ornamented territorial males that secure the majority of matings (95%) were found to have a lower immune response than the smaller satellite males that attempt to force copulations with females or the non-reproductive bachelor males (Berger et al., 2005). Territorial males also had a significantly lower body condition and higher plasma CORT levels than did satellites or bachelors (Berger et al., 2005). Moreover, males of all three types that had their CORT levels elevated experimentally by being exposed to restraint or to CORT injections had reduced immune responses as compared to control animals (Berger et al., 2005). These findings are consistent with the idea that elevated CORT levels can suppress immune function in a reptile during reproduction, thus allowing an animal to continue its reproductive activities even though there may be a cost to its long-term fitness. Stress inhibits wound healing in the tree lizard, U. ornatus (French, Matt, & Moore, 2006; French & Moore, 2008). French and Moore (2008) have argued that wound healing can be used as a measure of innate immune function and that wound healing is a highly biologically relevant process in U. ornatus. These investigators found that wound healing in male and female U. ornatus varies significantly with reproductive stage as well as with context (field or laboratory). However, French and Moore (2008) detected no significant relationship between plasma levels of E2, T, and CORT and healing rate for individuals in any reproductive stage or context. Previous studies of females U. ornatus have demonstrated that food availability is a key factor affecting the ability of female U. ornatus to sustain reproduction and immune function in stressed females (French et al., 2007a; 2007b). In the side-blotched lizard, males as well as females exhibit genetically based reproductive strategy polymorphisms (Sinervo & Lively, 1996; Lancaster et al., 2008). Female U. stansburiana within a population exhibit
Chapter | 7
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Stress and Reproduction in Reptiles
either of two alternative mating strategies. Orangethroated females (hereafter termed orange females) are ‘r-strategists’ because they produce relative large clutches of small-sized offspring, whereas yellow-throated females (hereafter termed yellow females) are ‘K-strategists’ because they lay relatively small clutches of large-sized offspring (Lancaster et al., 2008). Lancaster et al. (2008) experimentally manipulated CORT levels (high-end basal, stress-related, or unchanged) in adult female U. stansburiana as well as the timing of CORT exposure to determine the effects of this stress hormone on female survival and reproductive success in relationship to female mating strategy. As predicted, the effects of CORT treatment differed with female mating strategy. Corticosterone linearly affected survival into the reproductive period but differentially by female mating strategy. The chances of survival decreased in orange females with increasing CORT levels, whereas the chances of survival increased in yellow females with increasing CORT levels (Lancaster et al., 2008). However, in those females that survived, the chances of reproducing increased in orange females with increasing CORT levels, whereas the chances of reproducing decreased in yellow females with increasing CORT levels (Lancaster et al., 2008). Thus, CORT appears to mediate the tradeoff between reproduction and survival in the two female mating strategies in U. stansburiana (Lancaster et al., 2008). The differential nature of the tradeoffs observed in female U. stansburiana after manipulation of CORT levels may at least in part enhance the competitive ability of females in each mating strategy because CORT increased in both orange and yellow females in nature in response to crowding by conspecifics (Comendant et al., 2003). Thus, according to Lancaster et al. (2008), r-strategist orange females, which face increased survival costs with increasing CORT levels, may benefit from laying eggs earlier in the breeding season because they produce relatively large clutches with small hatchlings, which may do better by emerging earlier in the breeding season when there is likely to be a lower density of competitors than later in the breeding season. The same is not true for K-strategist yellow females because they do not suffer increased survival costs with increasing CORT levels and because they produce relatively small clutches with large hatchlings, which may be able to compete successfully for resources under the high-density conditions that are likely to occur later in the breeding season (Lancaster et al., 2008). Another interesting finding from the Lancaster et al. (2008) study of the effect of CORT manipulation on reproductive tradeoffs relates to how CORT affected energy mobilization to meet the energy demands of reproduction. For females of both mating strategies in U. stansburiana, CORT acted to increase the feeding rate of females during
oogenesis rather than to mobilize fat reserves. Thus, Lancaster et al. (2008) suggested that this is an example of income breeding vs. capital breeding, as described in Jo¨nsson (1997). The best direct evidence of the negative effects of longterm stress on adult survival and thus reproductive success in reptiles comes from a study of the Gala´pagos marine iguana, A. cristatus, by Romero and Wikelski (2001) and from a study of the common lizard, L. vivipara, by Cote et al. (2006). Although Romero and Wikelski (2001) did not compare animals based on reproductive condition but rather selected animals randomly, they found that during the El Nin˜o of 1998 when A. cristatus on most islands of the Gala´pagos faced famine because of reduced algae forage, baseline and stressor-induced CORT levels were significantly higher in iguanas on five of the six examined islands than they were in the year following the El Nin˜o event. Moreover, the CORT levels measured after 15 minutes of handling predicted survival of iguanas on islands that were differentially affected by El Nin˜o conditions (Romero & Wikelski, 2001). Cote et al. (2006) assessed the effects of experimentally elevated levels of CORT on the survival in L. vivipara by monitoring survival and behavior of CORT-treated and of placebo-treated lizards that were housed in outdoor enclosures from September to April. Corticosterone-treated males had a significantly higher survival rate than placebotreated males, although the survival of CORT-treated females did not differ from that of placebo-treated females. Guillette et al. (1995) noted that stress could delay or even prevent oviposition or parturition in reptiles. Difficulty in successfully ovipositing (laying eggs) in oviparous species or parturition (giving birth) in viviparous species can have negative fitness effects, especially in chelonians and squamates in which problems during egg laying most frequently occur (Frye, 1991). The effects of stress on the timing of oviposition or parturition in reptiles can be mediated not only by changes in the plasma levels of hormones such as CORT but also by the activation of the sympathetic nervous system (see references in Guillette et al., 1995).
6.2. Mothers and Offspring In a study of the viviparous New Zealand common gecko, H. maculatus, Cree et al. (2003) exposed pregnant females to CORT and found that this treatment harmed the embryos and prevented successful births. Specifically, four out of nine examined embryos from two of five female H. maculatus treated with CORT pellets during pregnancy showed developmental abnormalities at three weeks and none of the CORT-treated females successfully gave birth at the beginning of the fourth week (Cree et al., 2003). The significance of this finding, however, is unclear because
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gravid females were exposed to potentially nonphysiological levels of CORT (360 ng/ml at three weeks). In another study of H. maculatus in which CORT levels in ACTH-treated pregnant females were within the physiological range seen during acute stress, females had normal pregnancies (Preest et al., 2005). Specifically, the CORT levels in ACTH-treated female H. maculatus (mean ¼ 62 ng/ml) were seven-fold greater than basal levels and were similar to levels observed in pregnant females following capture and confinement (Preest et al., 2005). However, these elevated levels of CORT did not significantly affect the duration and success of pregnancy (Preest et al., 2005). Meylan and Clobert (2005) also found no effect of CORT treatment of pregnant females on the number of live births in the viviparous common lizard, L. vivipara. In this study the mean plasma level of CORT was 280.9 ng/ml in the CORT-treated pregnant females (Meylan & Clobert, 2005). In the oviparous eastern three-lined skink, Bassiana duperreyi, the transdermal application of CORT to gravid females caused females to oviposit their eggs sooner (on average 7.5 days earlier) and at an earlier embryonic stage than did control females that were treated with oil vehicle (Radder, Elphichk, Warner, Pike, & Shine, 2008). At oviposition, CORT-treated females had significantly higher CORT levels (mean ¼ 246.2 ng/ml) than did control females (79.0 ng/ml), although CORT levels in the egg yolk of offspring were unaffected by maternal treatment. Radder et al. (2008) posited that the observed premature oviposition by CORT-treated females might be adaptive in terms of protecting the eggs from the possible harmful effects of CORT. On the other hand, in the side-blotched lizard, U. stansburiana, treatment of gravid females with a low dose (imitated high-end baseline physiological levels) or a high dose (imitated stress-related levels; i.e., equal to or greater than double baseline levels) of CORT delivered from implants did not accelerate or delay the timing of egg laying (Lancaster et al., 2008). There is also evidence that super-physiological levels of CORT present in egg yolk at the time of oviposition reduce hatching success in U. stansburiana (Johnston & Moore, unpublished data; Lucas & Weiss, unpublished data; both cited in Weiss et al., 2007), a finding that was attributed to the gross detrimental effects of CORT on the embryo rather than on the hatching event itself (Weiss et al., 2007). The implication of this finding is that tree lizards are unlikely to develop to fullterm in extremely high-CORT environments (Weiss et al., 2007). However, it is unclear whether such high CORT levels occur in nature. The effects of treating oviposited eggs with CORT rather than treating gravid females have been studied in the tree lizard, U. ornatus (Weiss et al., 2007). The rationale behind this study was the unexpected finding that RU-486, a P4 and glucocorticoid antagonist, when given to nearly
Hormones and Reproduction of Vertebrates
full-term embryos adversely affected hatching success in a dose-dependent manner. This finding led Weiss et al. (2007) to hypothesize that CORT plays an important role in the successful hatching of viable U. ornatus as it does in mammalian parturition (see Volume 5, Chapter 6). These investigators found that oviposited eggs that were treated with CORT on day 30 of incubation hatched significantly sooner than vehicle-treated eggs and untreated eggs. Based on this discovery, Weiss et al. (2007) suggested that CORT produced in the embryo in response to environmental stressors might mobilize the energy stores needed to fuel hatching and/or to stimulate lung maturation and thus accelerate the timing of hatching in U. ornatus. More broadly, they suggested that embryos within oviposited eggs might be able to detect environmental stressors and accelerate their time of hatching to escape these stressors. The results of Weiss et al. (2007) are noteworthy because they raise the issue of the relative importance of CORT derived from the mother vs. CORT that is synthesized by the developing embryo itself. The extent to which glucocorticoids pass from mothers to their eggs and the functional significance of such a transfer is currently an active area of research in oviparous species (Romero, 2004; Radder, 2007). Well-designed studies of more species of lizard as well as other reptiles are required before it will be possible to make meaningful generalizations about the effects of CORT on the timing and success of oviposition and parturition in reptiles.
6.3. Offspring One of the most exciting findings related to the effects of stress is that maternal stress in a variety of vertebrates has been shown to affect offspring quality (reviewed in Francis et al., 1996; Weinstock, 2001; Breuner, 2008). Several studies have explored the effects of maternal stress on offspring fitness in lizards. In a study of the viviparous lizard, L. vivipara, de Fraipont, Clobert, John-Alder, and Meylan (2000) found that treating pregnant females with CORT had a significant inhibitory effect on the tendency of their offspring to disperse from their natal area. This is an important finding because the dispersal or lack of dispersal of offspring from their natal site is known to be a significant factor affecting an individual’s fitness in juvenile lizards (Warner & Shine, 2008). Offspring of CORT-treated female L. vivipara were found to move in lower numbers to an enclosure that did not house their mother compared to the offspring of control females (de Fraipont et al., 2000). Based largely on this finding, de Fraipont et al. (2000) proposed that increased prenatal maternal CORT inhibits dispersal (and thus promotes philopatry) and
Chapter | 7
Stress and Reproduction in Reptiles
suggested that CORT is one of the proximate mechanisms in the prenatal control of juvenile dispersal in L. vivipara. According to these investigators, if relatively high plasma CORT levels decrease the likelihood of maternal survival in L. vivipara, offspring fitness could be increased by not dispersing because their mothers would not be present at the site of their birth and thus there would be an absence of mother–offspring competition (de Fraipont et al., 2000). Belliure et al. (2004) also reported that the offspring of CORT-treated female L. vivipara spent significantly less time moving, a finding consistent with the earlier report of de Fraipont et al. (2000) that the offspring of CORT-treated females tended to exhibit philopatry. In the Belliure et al. (2004) study, CORT was delivered to pregnant female L. vivipara transdermally (see Knapp & Moore, 1997b), whereas, in the earlier study of de Fraipont et al. (2000), pregnant females received Silastic implants containing crystalline CORT. Meylan, Belliure, Clobert, and de Fraipont (2002) also found evidence in L. vivipara that prenatal CORT treatment (via transdermal delivery (see Meylan, Dufty, & Clobert, 2003)) decreased the tendency of offspring to disperse; however, in this study there was an interaction between maternal condition (corpulence) and prenatal CORT treatment, with dispersal only decreasing in offspring of corpulent, CORT-treated females. Similarly, Meylan, de Fraimont, and Clobert (2004) found that the dispersal of offspring from small-sized (young) mothers that had been treated with CORT was significantly greater than for the offspring from large-sized (old) mothers that had been treated with CORT. Taken together, these findings suggest that the tendency of offspring to disperse in L. vivipara is mediated by an interaction between CORT levels and the prenatal body condition of the mother. Interestingly, Vercken et al. (2007) found that natal dispersal in L. vivipara was not affected by CORT exposure or by treatment timing, but it was significantly affected by the interaction between the duration of CORT treatment and clutch sex ratio. Specifically, the proportion of offspring that dispersed was higher in female-biased clutches (male/female ratio < 0.5) and lower in malebiased clutches in offspring whose mothers had been briefly exposed to CORT during gestation. However, opposite trends were observed in offspring of mothers that had been chronically exposed to CORT. Exposure of pregnant females to CORT influences other aspects of offspring fitness. Meylan and Clobert (2004), for example, found that offspring from CORT-treated female L. vivipara had decreased sprint speed but normal endurance in comparison to the offspring of placebo-treated control females. These investigators proposed that the lower sprint speed of offspring of CORT-treated females could be a behavioral adjustment rather than a shift in
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physiological performance because the offspring required more stimuli to run. Belliure et al. (2004) also noted in L. vivipara that prenatal CORT treatment increased the time spent basking by juveniles from large mothers but not small mothers, a finding that again illustrates the importance that the body condition of pregnant females plays in modulating the effects of CORT. However, unlike in L. vivipara, offspring quality does not appear to be affected by elevated maternal CORT levels in the gecko H. maculatus (Preest et al., 2005). In a study in which CORT levels were increased in pregnant females to those levels observed during acute stress by treating pregnant females with ACTH, sprint speed and body growth in offspring of ACTH-treated pregnant females did not differ significantly from those in offspring of placebo-treated pregnant females (Preest et al., 2005). Although it is difficult to compare the findings of different studies because they vary in so many ways, it seems likely based on the findings to date that there are species differences in the effects of maternal CORT on offspring fitness in reptiles. In a study of L. vivipara, Meylan and Clobert (2005) not only recorded the morphological characteristics of offspring from CORT- and from placebo-treated females at birth but also, importantly, followed the growth and survival of offspring in the field until they hibernated. These investigators found that the offspring of CORTtreated females compared to the offspring of placebotreated females had shorter SVLs, a leaner body condition (residual from a regression of mass against SVL), and a lower growth rate prior to hibernation. Significantly, Meylan and Clobert (2005) also found that CORT treatment of pregnant female L. vivipara increased the probability of offspring survival in a sex-specific manner. Juvenile males born from CORT-treated females had a higher survival rate than did males that were born from placebo-treated females. On the other hand, juvenile females born from CORT- or from placebo-treated mothers did not differ significantly in survival rate. These findings led Meylan and Clobert (2005) to conclude that CORT treatment of pregnant females does not have detrimental effects on offspring survival and that CORT may have an adaptive function in L. vivipara. Uller, Meylan, de Fraipont, and Clobert (2005) also found that CORT treatment of pregnant female L. vivipara did not significantly affect clutch size, offspring sex ratio, or offspring sexual dimorphism (as indicated by the number of ventral scales, which differs between the sexes in some reptiles). However, in a study in which CORT was directly administered to the developing eggs of pregnant female L. vivipara, the anti-predator behavior of hatchlings was altered in that CORT-treated animals stayed in a shelter for a significantly longer time after a simulated predator attack than did controls (Uller & Olsson, 2006).
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Treatment of recently oviposited eggs (within 24 hours) with CORT affected offspring growth and sex ratios in two species of oviparous lizard with environmental sex determination (Warner, Radder, & Shine, 2009). Specifically, CORT application to eggs reduced hatchling growth rates in the jacky lizard, Amphibolurus muricatus, but enhanced hatchling growth rates in a scincid lizard, B. duperreyi. This type of CORT treatment also resulted in a significant female-biased sex ratio in A. muricatus hatchlings and a non-significant male-biased sex ratio in B. duperreyi hatchlings. In a recent study of the effects of maternal CORT levels on offspring in the garter snake T. elegans, Robert, Vleck, and Bronikowski (2009) found that pregnant females treated with CORT had fewer successful births and a higher offspring mortality rate within the first month of offspring life than did vehicle-treated control mothers that were not treated with CORT. In addition, the body length (SVL) of female offspring from corticosteronetreated females was significantly less than that of offspring from all other groups. Offspring behavior was also affected, as there was a significant effect of maternal treatment (CORT vs. vehicle treatment) on offspring predator escape behavior (reversal of locomotion direction and tail lashing) that was dependent upon maternal ecotype (slow growth or fast growth).
7. CONCLUSIONS AND RESEARCH DIRECTIONS 7.1. Conclusions Exposure of reptiles to a variety of types of stressor causes an elevation in the circulating levels of CORT, the most important glucocorticoid in reptiles. This stressor-induced increase in CORT is often associated with changes in the circulating levels of gonadal steroids. In male reptiles, an increase in plasma CORT above baseline levels is usually followed by a decrease in baseline T levels (e.g., Lance, Elsey, Butterstein, & Trosclair, 2004). The relationship between plasma CORT levels and the levels of E2 and of P4 following exposure of female reptiles to a stressor vary greatly with species and female reproductive state. However, exposure of females to stressors may lower E2 levels in some species (e.g., Elsey et al., 1991; Ganesh & Yajurvedi, 2002a). In the Keeled Indian Mabuya, CORT treatment or exposure to stressors inhibits gonadal recrudescence in females (Ganesh & Yajurvedi, 2002a) as well as males (Yajurvedi & Nijagal, 2000; Yajurvedi & Menon, 2005). Importantly, other HPA axis factors such as CRF and b-endorphin play key roles in the stressor-induced inhibition of gonadal recrudescence (Ganesh & Yajurvedi, 2002b; 2003).
Hormones and Reproduction of Vertebrates
Stress in reptiles can inhibit male behaviors that are necessary for successful reproduction. Corticosterone treatment, for example, inhibits male territorial behavior in many species of lizard. Whether or not there are cases in reptiles similar to those observed in some other vertebrates (reviewed in Creel, 2001), in which more dominant individuals have higher basal and stressor-induced levels in CORT, remains to be determined. In reptiles, CORT appears to inhibit male behavior by mechanisms that are largely independent of changes in circulating androgens. Evidence from studies of the green anole lizard suggests that stressor-induced increases in plasma CORT might affect how males respond in future social interactions (Yang & Wilczynski, 2003; Korzan et al., 2006b). Interestingly, in species of reptile with alternative mating strategies, such as the tree lizard, stressor-induced changes in CORT and T levels may be a hormonal means of switching male reproductive tactics and thus behaviors (Knapp & Moore, 1996; 1997a; Moore et al., 1998; Knapp, 2004). Stress may also inhibit male courtship behavior in some reptiles. Corticosterone treatment or the stress associated with losing fights has been found to inhibit male courtship behavior in several species of snake (Schuett, 1996; Moore & Mason, 2001; Lutterschmidt et al., 2004). In these studies, CORT apparently inhibited male behavior without affecting plasma androgen levels (Schuett et al., 1996; Schuett & Grober, 2000). Baseline CORT levels in the majority of examined species of reptiles which have seasonal reproductive cycles, are highest during the breeding season relative to other seasons. The noted increase in baseline glucocorticoid levels in most reptiles during the reproductive season suggests that elevated levels of stress hormones are necessary, and perhaps beneficial, for reproduction, although exceptionally high CORT concentrations may inhibit reproductive events. For example, the increase in the baseline levels of CORT in females at the end of gestation that has been observed in the western diamondback rattlesnake might serve to mobilize energy for both vitellogenesis and parturition (Taylor et al., 2004). This would be consistent with the energy mobilization hypothesis of Wingfield and Ramenofsky (1999). In addition, a recent study of the Texas horned lizard found that baseline CORT levels in gravid females were highest during the egg-laying period, and this elevation in CORT levels might be due to stressors associated with digging multiple nests to find an appropriate place to oviposit eggs (Wack et al., 2008). Stressor-induced levels of CORT may also be highest during the breeding season, although this possibility is based on the results of relatively few studies (Moore & Jessop, 2003). The magnitude of the stress response during reproduction may be reduced in some reptiles. In the red-sided garter snake, for example, males suppress their stress response during reproduction (e.g., Moore et al., 2001; Cease et al.,
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2007). In females, the magnitude of the acute stress response also varies with specific stages of female reproduction in some species of lizard (e.g., Dauphin-Villemant & Xavier, 1987; Woodley & Moore, 2002; Cartledge & Jones, 2007) and turtle (e.g., Valverde et al., 1999). Much remains to be learned about the mechanisms that act to modulate the extent of the stress response in reproducing reptiles. The mechanism dampening the stress response of nesting females to capture in olive ridley sea turtles does not appear to be at the level of the adrenal glands because females that showed little response to the stress associated with capture exhibited a marked increase in CORT levels when treated with ACTH (Valverde et al., 1999). There is also evidence in the viviparous skink E. whitii that an elevation in plasma E2 levels increased the responsiveness of females to ACTH in terms of increasing plasma CORT levels, and thus the relative concentrations of E2 throughout the reproductive cycle may play a key role in the modulation of the stress response in this and perhaps other viviparous species (Cartledge & Jones, 2007). A serotonin-regulated system within the brain may also be of importance in modulating the response of the HPG axis to the stress response because treatment of male red-spotted garter snakes with a serotonergic type 2A receptor antagonist prior to capture prevented a stressor-induced decline in androgen levels following capture (Lutterschmidt & Mason, 2005). The relatively high costs of reproduction may be increased in reptiles that are exposed to stress (e.g., Berger et al., 2005; French et al., 2007a; French & Moore, 2008; Lancaster et al., 2008; Mills et al., 2008). In the sideblotched lizard, CORT levels may mediate the tradeoff between fitness and reproduction in a mating strategydependent manner (Lancaster et al., 2008). Corticosterone also affects pregnancy success and/or timing of egg laying in some lizards (e.g., Cree et al., 2003; Weiss et al., 2007; Radder et al., 2008) but not in others (Meylan & Clobert, 2005; Preest et al., 2005; Lancaster et al., 2008). Further, in the tree lizard, an oviparous species, CORT treatment of eggs with late-term embryos caused eggs to hatch significantly sooner than control eggs, and this may illustrate a mechanism by which the stress resulting from environmental stressors could allow embryos to hatch sooner and thereby possibly escape environmental stressors (Weiss et al., 2007). Treatment of recently oviposited eggs with CORT also affected offspring growth and sex ratios in two species of lizard with environmental sex determination (Warner et al., 2009). Studies of the viviparous common European lizard have shown that CORT treatment of pregnant females or their developing eggs can affect offspring dispersal, sprint speeds, anti-predator behavior, and the probability of survival (e.g., de Fraipont et al., 2000; Belliure et al., 2004; Meylan & Clobert, 2004; Meylan et al., 2004; Meylan & Clobert, 2005; Uller et al.,
2005; Uller & Olsson, 2006; Vercken et al., 2007). However, exposing mothers to stress during the prenatal period does not appear to affect clutch size, sex ratio, or sexual dimorphism in these lizards (Uller et al., 2005). In the common gecko, another viviparous lizard, elevated CORT levels in pregnant females did not affect offspring fitness (Preest et al., 2005). Thus, the effects that stress during pregnancy may have on offspring fitness in viviparous lizards appear to vary with species.
7.2. Future Research Directions Although it is generally assumed that exposure to stressors has an inhibitory effect on reproduction, relatively little empirical evidence exists to support this assumption in freely living reptiles (see Guillette et al., 1995). As most work has been based largely on the results of studies of captured animals, additional studies are needed to assess the effects of environmental and social stressors on the reproductive activity of freely living reptiles. An example of this type of approach is seen in a study that examined reproductive activity and hormone levels in a population of brown tree snakes believed to be living in stressful environmental conditions (Moore et al., 2005). It is also important in future studies to investigate whether a tradeoff between reproduction and survival occurs in reptiles that have been exposed to natural stressors rather than to CORT treatment (see Lancaster et al., 2008), and the extent to which individuals differ in the modulation of their stress response during reproduction (see Lendvai, Giraudeau, & Chastel, 2007). A series of studies of a viviparous lizard documented that CORT treatment of pregnant females influences offspring behavior, physiological performance, and offspring survival (e.g., Vercken et al., 2007). However, studies are needed to verify that the fitness and survival of offspring are affected when pregnant females are exposed to naturally occurring stressors in the environment. In addition, studies are needed that assess the extent to which glucocorticoids pass from mothers to their eggs and the functional significance of such a transfer (Radder, 2007). Even though many potential pitfalls exist in using CORT levels to evaluate the health of a population (see Romero, 2004), the plasma levels of this stress hormone have been shown to be an important factor in successfully assessing the relative health of populations of wild birds (see Kitaysky, Piatt, & Wingfield, 2007) and of wild reptiles (Romero & Wikelski, 2001). Additional studies of this nature are needed to firmly establish the utility of using circulating CORT levels to assess the relative reproductive health of wild populations of reptiles. The data from such studies may also serve as useful benchmarks to assess whether captive populations of reptiles are stressed. For
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studies that do focus on the glucocorticoid aspect of the stress response, Breuner, Patterson, and Hahn (2008, p. 288) have argued that the studies should include measures of performance and fitness, and incorporate and test ‘ideas of context dependency, coping strategies, and possible fluctuating selection pressures when considering the fitness benefits of the acute [glucocorticoid] response.’ These factors should also be considered when animals are exposed to chronic stress. There is a clear need for studies of the proximate mechanisms governing the stress response in reptiles. A plasma protein that binds glucocorticoids has been identified in a lizard (Jennings et al., 2000); however, studies are needed to establish the presence, location, and number of these receptors in other reptiles. Studies of the enzymes that mediate the synthesis and inactivation of CORT are also needed. In addition, studies of the sympathoadrenal component of the stress response are required as there is very little information on how this component of the stress response affects reproduction in reptiles. The majority of studies that have investigated the effects of stress on reproduction in reptiles have focused on the actions of CORT. Moreover, there has been a tendency, without substantiating evidence, to interpret differing sensitivities to CORT as being indicative of species differences in stress responsiveness. This is especially a problem because the doses and/or mode of administration of CORT often vary between studies. Further, upstream stress molecules of either central or endocrine origin (e.g., 5-HT, NE, CRF, ACTH) may have significantly different effects, and the entire endocrine stress axis may behave differently than does CORT alone. Neural and peripheral specificity, resulting in distinctive local actions of transmitters and hormones, indicate the problems of relying on plasma CORT levels alone to explain the stress status of an animal. Additionally, hormonal and/or genetic pleiotropy should be considered, in which related systems manifest multiple effects that modify the specific adaptive context, potentially incorporated into other adaptive traits (Greenberg, 2003). Indeed, it is striking how versatile and flexible the system of stress-sensitive genetic, epigenetic, neural, and endocrine physiological responses are (Greenberg et al., 2002). Dallman (2003) and others have argued that central neural mechanisms must be considered in addition to other physiological components of the stress response, but rarely in studies of reptiles have these mechanisms been considered within the framework of stress and reproduction. While in some squamates 5-HT may modulate HPG activity during the physiological stress response, differential telencephalic and brainstem serotonergic activity induced by social stress predicts gonadal function (Summers et al., 1997; Lutterschmidt & Mason, 2005). We suggest that studies of these central neural mechanisms in reptiles are required if we are to better
Hormones and Reproduction of Vertebrates
understand how the stress response can be modulated in relation to reproductive stage and function.
ABBREVIATIONS 5-HT ACTH AGBG AVP AVT CORT CRF CRH DA DHT E E2 FSH GABA GnIH GnRH GR GTH HPA HPG LH MR NE NMDA NR2 P4 PHA RU-486 SHBG SVL
Serotonin (5-hydroxytryptamine) Corticotropin Androgen–glucocorticoid-binding globulin Arginine vasopressin Arginine vasotocin Corticosterone Corticotropin-releasing factor (see CRH) Corticotropin-releasing hormone (same as CRF) Dopamine Dihydrotestosterone Epinephrine Estradiol Follicle-stimulating hormone g-Amino butyric acid Gonadotropin release-inhibiting hormone Gonadotropin-releasing hormone Glucocorticoid receptor Gonadotropin Hypothalamus–pituitary–adrenal Hypothalamus–pituitary–gonadal Luteinizing hormone Mineralocorticoid receptor Norepinephrine N-Methyl-D-aspartic acid Glutamatergic NMDA receptor subunit Progesterone Phytohemagglutinin Mifepristone Sex hormone-binding globulin Snout-to-vent length
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Chapter | 7
Stress and Reproduction in Reptiles
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Tokarz, R. R. (1987). Effects of corticosterone treatment on male aggressive behavior in a lizard (Anolis sagrei). Horm. Behav., 21, 358–370. Tokarz, R. R., McMann, S., Seitz, L., & John-Alder, H. (1998). Plasma corticosterone and testosterone levels during the annual reproductive cycle of male brown anoles (Anolis sagrei). Physiol. Zool., 71, 139–146. Tracy, C. R., Nussear, K. E., Esque, T. C., Dean-Bradley, K., Tracy, C. R., DeFalco, L. A., Castle, K. T., Zimmerman, L. C., Espinoza, R. E., & Barber, A. M. (2006). The importance of physiological ecology in conservation biology. Integr. Comp. Biol., 46, 1191–1205. Tsai, P.-S., & Licht, P. (1993a). Differential distribution of chicken-I and chicken-II GnRH in the turtle brain. Peptides, 14, 221–226. Tsai, P.-S., & Licht, P. (1993b). In vivo GnRH responsiveness of LH secretion in the female turtle, Trachemys scripta, in relation to the reproductive stage. Gen. Comp. Endocrinol., 90, 328–337. Tsutsui, K., & Osugi, T. (2009). Evolutionary origin and divergence of GnIH and its homologous peptides. Gen. Comp. Endocrinol, 161, 30–33. Tudge, C. (2000). ‘‘The Variety of Life’’. New York: Oxford University Press, Inc. Tyrrell, C. L., & Cree, A. (1998). Relationships between corticosterone concentration and season, time of day and confinement in a wild reptile (Tuatara, Sphenodon punctatus). Gen. Comp. Endocrinol., 110, 97–108. Uller, T., & Olsson, M. (2006). Direct exposure to corticosterone during embryonic development influences behaviour in an ovoviviparous lizard. Ethology, 112, 390–397. Uller, T., Meylan, S., de Fraipont, M., & Clobert, J. (2005). Is sexual dimorphism affected by the combined action of prenatal stress and sex ratio? J. Exp. Zool. Part A Comp. Exp. Biol., 303A, 1110–1114. Valverde, R. A., Owens, D. W., MacKenzie, D. S., & Amoss, M. S. (1999). Basal and stress-induced corticosterone levels in olive ridley sea turtles (Lepidochelys olivacea) in relation to their mass nesting behavior. J. Exp. Zool., 284, 652–662. Vercken, E., de Fraipont, M., Dufty, A. M., Jr., & Clobert, J. (2007). Mother’s timing and duration of corticosterone exposure modulate offspring size and natal dispersal in the common lizard (Lacerta vivipara). Horm. Behav., 51, 379–386. Viets, B. E., Ewert, M. A., Talent, L. G., & Nelson, C. E. (1994). Sex-determining mechanisms in squamate reptiles. J. Exp. Zool., 270, 45–56. Vijaykumar, B., Ramjaneyulu, T., Sharanabasappa, A., & Patil, S. B. (2002). Effect of FSH and LH on testis during nonbreeding season in Calotes versicolor (Daud.). J. Environ. Biol., 23, 43–46. Vitt, L. J., & Cardwell, J. P. (2008). ‘‘Herpetology, An Introductory Biology of Amphibians and Reptiles’’. New York: Academic Press. Wack, C. L., Fox, S. F., Hellgren, E. C., & Lovern, M. B. (2008). Effects of sex, age, and season on plasma steroids in free-ranging Texas horned lizards (Phrynosoma cornutum). Gen. Comp. Endocrinol., 155, 589–596. Wada, H. (2008). Glucocorticoids: Mediators of vertebrate ontogenetic transitions. Gen. Comp. Endocrinol., 156, 441–453. Walsberg, G. E. (2003). How useful is energy balance as a overall index of stress in animals? Horm. Behav., 43, 16–17. Warner, D. A., & Shine, R. (2008). Determinants of dispersal distance in free-ranging juvenile lizards. Ethology, 114, 361–368. Warner, D. A., Radder, R. S., & Shine, R. (2009). Corticosterone exposure during embryonic development affects offspring growth and sex ratios in opposing directions in two lizard species with environmental sex determination. Physiol. Biochem. Zool., 82, 363–371.
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Stress and Reproduction in Reptiles
Waters, R. P., Emerson, A. J., Watt, M. J., Forster, G. L., Swallow, J. G., & Summers, C. H. (2005). Stress induces rapid changes in central catecholaminergic activity in Anolis carolinensis: Restraint and forced physical activity. Brain Res. Bull., 67, 210–218. Watt, M. J., Korzan, W. J., Summers, C. H., & Forster, G. L. (2005). Rapid systemic response to restraint stress differ between winners and losers of social interactions. Soc. Neurosci. Abstr., 47, 133. Watt, M. J., Forster, G. L., Korzan, W. J., Renner, K. J., & Summers, C. H. (2007). Rapid neuroendocrine responses evoked at the onset of social challenge. Physiol. Behav., 90, 567–575. Weinstock, M. (2001). Alterations induced by gestational stress in brain morphology and behavior of the offspring. Prog. Neurobiol., 65, 427–451. Weiss, S. L., Johnston, G., & Moore, M. C. (2007). Corticosterone stimulates hatching of late-term tree lizard embryos. Comp. Biochem. Physiol., Part A Mol. Integr. Physiol., 146, 360–365. Wendelaar Bonga, S. E. (1997). The stress response in fish. Phsyiol. Rev., 77, 591–625. Whittier, J. M., Mason, R. T., & Crews, D. (1987). Plasma steroid hormone levels of female red-sided garter snakes, Thamnophis sirtalis parietalis: Relationships to mating and gestation. Gen. Comp. Endocrinol., 67, 33–43. Whittier, J. M., Corrie, F., & Limpus, C. (1997). Plasma steroid profiles in nesting loggerhead turtles (Caretta caretta) in Queensland, Australia: relationship to nesting episode and season. Gen. Comp. Endocrinol., 106, 39–47. Wilson, B. S., & Wingfield, J. C. (1992). Correlation between female reproductive condition and plasma corticosterone in the lizard Uta stansburiana. Copeia, 1992, 691–697. Wilson, B. S., & Wingfield, J. C. (1994). Seasonal and interpopulational variation in plasma levels of corticosterone in the side-blotched lizard (Uta stansburiana). Physiol. Zool., 67, 1025–1049. Wingfield, J. C. (1988). Changes in reproductive function of free-living birds in direct response to environmental perturbations. In M. H. Stetson (Ed.), ‘‘Processing of Environmental Information in Vertebrates’’ (pp. 121–148). Berlin: Springer-Verlag.
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Wingfield, J. C., & Ramenofsky, M. (1999). Hormones and the behavioral ecology of stress. In P. H. M. Balm (Ed.), ‘‘Stress Physiology in Animals’’ (pp. 1–51). Sheffield: Sheffield Academic Press. Wingfield, J. C., & Romero, L. M. (2001). Adrenocortical responses to stress and their modulation in free-living vertebrates. In B. S. McEwen, & H. M. Goodman (Eds.), ‘‘Handbook of Physiology; Section 7: The Endocrine System; Volume IV: Coping with the Environment: Neural and Endocrine Mechanisms’’ (pp. 211–234). New York: Oxford University Press. Wingfield, J. C., & Sapolsky, R. M. (2003). Reproduction and resistance to stress: When and how. J. Neuroendocrinol., 15, 711–724. Wingfield, J. C., Breuner, C., & Jacobs, J. (1997). Corticosterone and behavioral responses to unpredictable events. In S. Harvey, & R. J. Etches (Eds.), ‘‘Perspectives in Avian Endocrinology’’ (pp. 267–278). Bristol: Society of Endocrinology. Wingfield, J. C., Maney, D. L., Breuner, C. W., Jacobs, J. D., Lynn, S., Ramenofsky, M., & Richardson, R. D. (1998). Ecological bases of hormone-behavior interactions: the "emergency life history stage". Am. Zool., 38, 191–206. Woodley, S. K., & Moore, M. C. (2002). Plasma corticosterone response to an acute stressor varies according to reproductive condition in female tree lizards (Urosaurus ornatus). Gen. Comp. Endocrinol., 128, 143–148. Woodley, S. K., Painter, D. L., Moore, M. C., Wikelski, M., & Romero, L. M. (2003). Effect of tidal cycle and food intake on the baseline plasma corticosterone rhythm in intertidally foraging marine iguanas. Gen. Comp. Endocrinol., 132, 216–222. Yajurvedi, H. N., & Nijagal, B. S. (2000). Corticosterone inhibits normal and FSH-induced testicular recrudescence in the lizard, Mabuya carinata. Gen. Comp. Endocrinol., 120, 283–288. Yajurvedi, H. N., & Menon, S. (2005). Influence of stress on gonadotrophin induced testicular recrudescence in the lizard Mabuya carinata. J. Exp. Zool., 303A, 534–540. Yang, E.-J., & Wilczynski, W. (2003). Interaction effects of corticosterone and experience on aggressive behavior in the green anole lizard. Horm. Behav., 44, 281–292. Zani, P. A., Neuhaus, R. A., Jones, T. D., & Milgrom, J. E. (2008). Effects of reproductive burden on endurance performance in side-blotched lizards (Uta stansburiana). J. Herpetol., 42, 76–81.
Chapter 8
Hormones and Behavior of Reptiles Barry Sinervo* and Donald B. Milesy *
University of California, Santa Cruz, CA, USA, y Ohio University, Athens, OH, USA
SUMMARY The role of the endocrine system in regulating the expression of behavior in vertebrates has become the paradigm for studies at both proximate and ultimate levels of analysis. This chapter reviews variation in reptilian reproductive behavior that is induced by different endocrine networks. We focus on the roles of natural and sexual selection to provide the structure for understanding the functional role of the various components of the endocrine system at the proximate level. We specifically review models for the evolution of male–male interactions, ideas underlying the evolution of density regulation (r-K selection) and viviparity, parental care, and recent models for the evolution of sociality. We highlight empirical results that indicate that reptiles, and in particular lizards, display considerable variation in social systems. Notably, we also describe patterns of sociality that include both nuclear family structure in large groups and cooperation (e.g., greenbeard selection), which until recently were thought to be restricted to mammals, birds, and social insects. While details of the endocrine control of endocrine systems related to male behaviors have been quite well studied in reptiles (testosterone (T), arginine vasotocin (AVT), neuroendocrine), we point out other systems that have received little attention (e.g., prolactin (PRL)) and suggest likely control points for the endocrine regulation of alternative reproductive strategies.
1. INTRODUCTION The role of the endocrine system in regulating the expression of behavior in vertebrates has become the paradigm for studies at both the proximate level of analysis, which includes a molecular analysis of behavior, and the ultimate level of analysis, which includes an understanding of selection. This chapter reviews the modulation of reproductive behavior of reptiles by endocrine systems as shaped by the ultimate level of natural and sexual selection. Studies of natural and sexual selection provide the structure for understanding the functional role of the various components of the endocrine system at the proximate level. We specifically review models for the evolution of male–male interactions, ideas underlying the evolution of density
Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
regulation (r-K selection) and viviparity, new emerging models for the evolution of sociality, and network models of such social interactions. New empirical results indicate that reptiles, and in particular lizards, exhibit a diversity of social systems; in particular, we describe advanced forms of sociality including both nuclear family structure in large groups (Chapple, 2003; O’Connor & Shine, 2003) and greenbeard selection (Sinervo & Clobert, 2003; Sinervo et al., 2006b; 2007), which until recently were thought to be restricted to mammals, birds, and social insects. Throughout this review, we highlight the generic control networks of the neuroendocrine system of vertebrates and then reference what is known from reptilian studies of behavior and hormones. We finish our review of hormones and the behavior of reptiles by synthesizing the results in terms of regulatory networks applied to social systems and their endocrine control. Many of these social systems involve alternative social strategies for reproduction and throughout our review we highlight findings from species with alternative reproductive strategies. We also highlight key research opportunities that have not been tapped in studies of the hormonal control of behavior in reptiles. In particular, we specifically posit that the hormones arginine vasotocin (AVT) and prolactin (PRL) and their receptors are the most likely control mechanisms for the evolution of diverse behavioral phenomena including alternative reproductive strategies, territoriality, viviparity, dispersal, migration, sociality, and coloniality. The order in which we highlight items in our overview of reptilian hormones and behavior roughly follows the role of hormones during development, beginning with early organizational events, proceeding to activational events, and ending with the effects of social neighbors on the expression of hormones and behavior. An understanding of the roles of hormones in effectuating behavioral responses requires a consideration of two very different timescales. Over evolutionary time, behavior is shaped by natural and sexual selection to generate ever more optimal responses in endocrine function. Over the lifespan of an organism, behavior changes in response to the environment and social
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context, and the endocrine system modulates many of these changes through interactions with the neuroendocrine system. Complicating this interaction of timescales are feedback loops that act between behavioral responses and the endocrine system to shape the organism in a plastic fashion, via social cues. Such plasticity can by favored by selection and generates new coping strategies under a variety of social circumstances, as the social system evolves. At the end of this review we integrate social system interactions with the endocrine control of individual behaviors. In this chapter, we highlight the utility of network systems analysis (Milo et al., 2002) in providing a comprehensive synthesis of these various timescales that shape the endocrine control of behavior. We compare and link social networks and endocrine networks to unravel the dimensions involved in the hormonal control of behavior. Network theory provides a framework in which we can unify ultimate forces of selection with complex proximate sources generated by endocrine systems and temporal regulation of gene activation (gene cascades) on the expression of behaviors, their selection, and associated tradeoffs.
1.1. Organization and Other Developmental Effects of Reptilian Hormones on Behavior The degree to which an organism is responsive to change is also genetically programmed and dictated in a large measure by ontogenetic stages from the juvenile to adult phases. The organizational effects of hormones (Phoenix, Goy, Gerall, & Young, 1959), which act during embryogenesis to pattern the basic behavioral repertoire of male vs. female types, and the activational effects of hormones, which trigger the expression of male vs. female behavior at maturity, accomplish these ontogenetic changes (Caro & Bateson, 1986). Juvenile behavior also arises in a physiological background where sex steroids are not yet circulating at significant levels. In this context, many aspects of the juvenile behavior of reptiles are regulated by neuroendocrine systems such as the interplay between the control of behavioral thermoregulation (Seebacher, 2005) and thyroid hormones (e.g., metabolic regulation by thyroxine (Sinervo & Dunlap, 1995), which we only mention briefly but which may play a very important role). Many organizational events arise during the earliest stages of development in both live-bearing (viviparous) and egg-laying (oviparous) forms of reptile. In lizards, model systems with alternative reproductive strategies (ARSs) have been investigated to specifically understand the organizational role of hormones in generating basic behaviors within a given sex. In Urosaurus ornatus, orange males have an orange dewlap (throat), are non-territorial, and are larger than blue-orange males that are territorial and have a blue patch on their orange dewlap
Hormones and Reproduction of Vertebrates
(Thompson & Moore, 1991). Castration early in prematurational development results in a high frequency of the orange, non-territorial morph, and supplemental testosterone (T) results in a high frequency of the blue-orange morph (Hews & Moore, 1995), suggesting a possible organizational role for T. However, subsequent experiments revealed that a perinatal surge in progesterone (P4) of adrenal origin (higher in blue-orange males) governs the organization of the two male strategies, and exogenous P4 mimics the effects of T on generation of the blue-orange type (Weiss & Moore, 2004) (Figure 8.1(a)). Despite these salient details on hormones in organizing the phenotype of male tree lizards, female tree lizards (and many other lizard species described below) exhibit conspicuous color polymorphism associated with alternative reproductive strategies (Zucker & Boeklen, 1989) and the interaction between the ARS locus of tree lizards with sex chromosomes is poorly understood (Figure 8.1(a)). Besides endogenous sources of hormones that govern organization, maternally provided yolk steroids may provide a developmental mechanism for altering the behavior of progeny. The organizational role of such early acting hormones plays a clear role, for example, in birds where maternally provided yolk steroids alter differential expression of aggression in chicks (Schwabl, 1993). Noteworthy in this regard is the maternal modulation in yolk steroids observed in female side-blotched lizards, Uta stansburiana, which expresses three alternative reproductive strategies that, as in U. ornatus, are coded by throat color. Genetic crosses and preliminary gene mapping studies using a field pedigree suggest that the polymorphism is due to a single gene (Sinervo, Bleay, Adamopoulou, 2001; Sinervo et al., 2006b). Both male and female side-blotched lizards can be yellow-, orange- or blue-throated. In males, color reflects alternative mating strategies. Orange males are territorial and aggressive and their territories overlap with multiple females. Blue males tend to have a single female overlapping their territory and repel rival males who might attempt to copulate with the resident female (mate guarders). This mate-guarding behavior entails cooperative behavior with other blue males (Sinervo et al., 2006b). Finally, yellow males are sneakers that lack a territory, mimic the appearance and behavior of females, and copulate with females by stealth (Sinervo & Lively, 1996; Zamudio & Sinervo, 2000; Sinervo et al., 2006b). In females, alternative throat colors correlate with alternative egg-laying strategies. Orange females lay larger clutches of small eggs. Yellow and blue females lay small clutches of large eggs (Sinervo et al., 2000b). Throat color and life-history traits are heritable (Sinervo, Miles, Frankino, Klukowski, & DeNardo, 2000; Sinervo & Zamudio, 2001). In U. stansburiana, females deposit greater quantities of yolk 17b-estradiol (E2) when the female mates with
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Hormones and Behavior of Reptiles
(c)
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FIGURE 8.1 Organizational effects of steroid hormones on behavior for some model reptilian systems (see Section 1 for details and references). (a) Progesterone (P4) secretion by the adrenals organizes alternative reproductive strategies (ARSs) of the ornate tree lizard, Urosaurus ornatus. (b) Sex differences in behavior are organized by temperature-dependent sex determination (TSD) in the leopard gecko, Eublepharis macularius. (c) Yolk estradiol organizes dorsal patterns, an anti-predator signal, in the North American common side-blotched lizard, Uta stansburiana, and yolk volume independently organizes morphology and escape behavior. These two organizational effects, in tandem, produce highly adaptive and functionally integrated ARSs in both sexes. (d) Exogenous corticosterone application to female European common lizards, Lacerta vivipara, organizes juvenile dispersal syndromes, but the relationship to ARS of females is unknown. Moreover, intrauterine steroids secreted by embryos (i.e., sibs) affects development of sexual dimorphism. Arrows describe positive effects of each mechanism (or inferred pathways of action) on endocrine function, and fitness-related traits in the case of (c) and (d). Octagonal symbols indicate the action of key genes: ARS loci, dorsal pattern loci, sex chromosomes (XX/XY or WW/ZW systems of heterogamy), or, in the case of TSD, tdf, a hypothetical testis-determining factor that is temperature-sensitive. The ? symbol identifies processes that are poorly understood. P4, progesterone; CRF, corticosterone-releasing factor; CNS, central nervous system; ACTH, adrenocorticotropin hormone.
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a yellow sire and, thus, progeny carry yellow alleles (Lancaster, McAdam, Wingfield, & Sinervo, 2007). Females also deposited increased yolk E2 in response to crowding by orange neighbors. Thus, there are two distinct inducing cues that alter levels of yolk E2dyellow alleles from the sire and crowding by orange female neighborsdand these two yolk E2 pathways alter dorsal patterns of progeny in interaction with the color locus (Figure 8.1(b)). Increased yolk E2 in combination with yellow sire alleles enhanced the barred dorsal pattern (transverse dorsal bars) phenotype of progeny, which is quite cryptic in grass and, thus, highly adaptive for yellow sneaker genotypes. Increased yolk E2 induced by orange crowding increased the prominence of striped dorsal patterns (longitudinal dorsal stripes), but in a sex-specific manner that interacted with throat color genes. In response to yolk E2 induced by social crowding, stripes were increased in sons destined to be orange and daughters destined to be yellow or blue. These changes were highly adaptive and enhanced progeny survival to maturity of the induced phenotypes. The effects were also causally verified with exogenous E2 ectopically applied to eggs (Lancaster et al., 2007). Yolk E2 could also have organizational effects on the central nervous system (CNS) akin to those described below (see Section 1.3) for temperature-dependent sex determination (TSD) in geckos, and perhaps even play a major role in promoting the conspicuous and stereotypical female mimicking behaviors observed in yellow male morphs. Squamates with ARSs provide a number of potential research avenues for investigating the classic organizational effect of hormones on basic neural structures between the sexes and between the alternative male types. Consider the neural basis of spatial abilities, which has been identified as being influenced by sex differences in the volume of the hippocampus in other vertebrates such as birds and mammals (Clayton, 2001; LaDage, Riggs, Sinervo, & Pravosudov, 2009). The medial and dorsal cortices are the putative reptilian homologs of the mammalian hippocampus (LaDage et al., 2009), yet few studies have examined the relationship between these brain areas and different spatial use strategies in reptiles. Recent studies indicate that spatial use strategies of male side-blotched lizards (U. stansburiana) are linked to the volume of the medial and dorsal cortical regions of the brain, the analog of the mammalian hippocampus in reptiles. Orange males, which defend large territories (Sinervo et al., 2000a), had larger dorsal cortical volumes compared with nonterritorial yellow males, and the blue males with small territories were intermediate. Future studies on alternative polymorphisms should ascertain whether this effect is due to the organizational effect of endogenous hormone produced by the embryo (sensu Phoenix et al.,
Hormones and Reproduction of Vertebrates
1959), the yolk-steroid effect and maternal programming (sensu Lancaster et al., 2007), activational differences between morphs that are manifest at maturity (Sinervo et al., 2000a), or simply a consequence of the feedback role of territory size, which differs among the morphs, thereby inducing increases in hippocampal volumes during ontogeny and adulthood. The basic sex differences in the expression of male vs. female behavior have been a classic question explored in model systems of reptiles that express TSD (Figure 8.1(c)). For example, incubation experiments on the leopard gecko, E. macularius, demonstrate induction of female clutches at low temperatures (26 C), female-biased sex ratio at 30 C, male-biased clutches at intermediate temperatures (32.5 C), and nearly all females at the very high temperatures (34–35 C) (Crews & Moore, 2005). Crews and colleagues (Crews, 1998; Sakata & Crews, 2003) have discovered that TSD has a classic organizing effect on male vs. female behaviors, akin to those first elucidated in mammals (e.g., Phoenix et al., 1959; see also methods in Vom Saal, 1983). For example, males from female-biased temperatures are less likely to adopt an offensive attack posture, a stereotypical male behavior, than those raised at male-biased temperatures. Conversely, females from malebiased temperatures are more aggressive than females from female-biased temperatures, although females are less aggressive than males overall. Additional experiments with gonadectomy followed by restorative hormone treatment indicate that expression of male vs. female stereotypical behaviors is influenced by egg-rearing temperature independently from sex-steroid levels (Crews & Moore, 2005). In the turtle Chrysemys picta (Bowden, Ewert, & Nelson, 2000), another species with TSD, the ratio in the levels of maternally provided E2 to T varies seasonally and this variation contributes to the production of female-biased clutches late in the season. In this experiment all eggs were incubated at 28 C, and the ratio of females shifted seasonally from 72% male to 76% female. The adaptive significance of environmental sex determination has been a subject of much debate (reviewed in Shine, 1999; see also Chapter 1, this volume), but it is generally thought that females can maximize their fitness by giving progeny an opportunity to develop into the gender that will perform best given the environmental conditions. In the case of a mechanism coupled to yolk estrogens, it is possible for females to fine-tune both the sex ratio produced and the phenotypic expression of male vs. female behaviors. Bowden et al. (2000) suggest that other aspects of offspring phenotype, such as the association between egg size and TSD (Ewert, Jackson, & Nelson, 1994; Roosenburg & Kelley, 1996) might be fine tuned with the addition of a mechanism linked to egg size. The endocrine control of egg size and the ensuing effects on progeny behavior and fitness have been studied
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Hormones and Behavior of Reptiles
most thoroughly in U. stansburiana, where a battery of endocrine manipulations and normative studies have determined both the proximate mechanisms governing egg size regulation and adaptive consequences for the progeny of each gender (Figure 8.1(c)). In yellow and blue females, but not orange females, increased egg size (which leads to increased progeny size) leads to enhanced progeny survival, reflecting an egg size by genotype interaction (Lancaster, Hazard, Clobert, & Sinervo, 2008). One reason for this is that large vs. small progeny exploit different escape behaviors (Lancaster, McAdam, & Sinervo, in review), and escape behaviors exhibited by smaller progeny benefit orange progeny (Lancaster et al., 2010). The effects of maternally provided yolk steroids noted above are likely to play a synergistic role with egg size in altering progeny escape behavior, but experimental manipulations in which yolk is withdrawn from eggs with a syringe indicate that egg size alone alters progeny morphology and escape behavior through modification of body size and limb allometry. The local frequency of throat colors in the social environment plastically affects maternal egg size allocation strategy via the impacts of social cues on the hypothalamus– pituitary–adrenal (HPA) axis (discussed in Section 1.7). Specifically, orange females exhibit decreased corticosterone (CORT) in response to orange-throated neighbors in the wild (Comendant et al., 2003). Conversely, yellow and blue females exhibit increased CORT when crowded by yellow- and blue-throated individuals (Comendant, Sinervo, Svensson, & Wingfield, 2003). Chronically elevated CORT leads to an increase in females’ average egg mass without a concomitant decrease in clutch size (Lancaster et al., 2008). This likely occurs because of CORT’s effect of increasing appetite (Jo¨nsson, 1997), so females experiencing chronically elevated CORT are likely to feed more during oogenesis. This mechanism allows each maternal morph to appropriately adjust offspring size in response to social cues, which can inform her of her offspring’s likely genotype. Small size benefits orange hatchlings, and orange females lay small eggs. Orange females further decrease egg size via reducing plasma CORT when the local frequency of orange throat color is high (indicating that offspring will likely inherit that orange color through the sire as well as the dam). Large size benefits yellow and blue hatchlings. Yellow and blue females further increase egg size when the social environment predicts that offspring are also likely to inherit yellow and blue throat color from the sire. Lancaster et al. (in review) performed a second experiment assigning females to controlled mating in the laboratory. Females mated to yellow-throated sires, but not sires of other throat colors, responded by increasing average egg size (Lancaster et al., in review). Adrenal-mediated allocation plasticity is an adaptive mechanism by which females respond to the tradeoff between different aspects of offspring quality. Females use
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social cues to predict offspring inherited traits (Alonzo & Sinervo, 2001; Weiss & Moore, 2004; Alonzo & Sinervo, 2007) and then use that information, via endocrine modulation, to alter progeny phenotypes and behavior. One common tradeoff that is rarely considered from a lifehistory perspective is the tradeoff between the diverse aspects of offspring quality. Traditional life-history theory considers offspring quality to be synonymous with offspring size (as in the classic offspring size–number tradeoff) (Smith & Fretwell, 1974). This is because offspring size is under maternal endocrine control and a direct function of her resource allocation to reproduction. However, the size–number model does not take into account genetic variation among offspring, which could render maternal resource investment more or less beneficial, depending on progeny genotype. The Trivers–Willard (1973) model expanded on this early theory and explicitly treated resource allocation to each progeny sex owing to differential forces of sexual selection in sons vs. natural selection in daughters (discussed in Section 1.3). This is a form of parent–offspring conflict and interaction among egg size, incubation temperature, yolk steroids, and TSD are likely evolved mechanisms to ameliorate any mismatch between progeny genotypes (e.g., gender or other genotype effects such as alternative strategies) and resource allocation (Janzen & Phillips, 2006). When considered from the offspring’s perspective, parent–offspring conflict is known as ontogenetic conflict (Rice & Chippindale, 2001), and the most intuitive example of this is intersexual ontogenetic conflict, in which traits that are beneficial when inherited by one sex are detrimental when inherited by the other sex (Rice and Chippindale, 2001). For an example from reptiles, in the side-blotched lizard, genes that enhance clutch size are related to levels of gonadotropin hormones (GTH). A high level of expression for the genes for clutch size not only increases fecundity but also female progeny survival prior to maturity (Sinervo & McAdam, 2008). These same genes, when expressed at high levels in males, increase sexually selected male traits at maturity (Mills et al., 2008) but also decrease male survival from birth to maturity (Sinervo & McAdam, 2008). Male progeny inherit these genes for clutch size (GTH) from the female and male parents, but male progeny do not express clutch size; rather, the ‘clutch size genes’ serve to enhance male sexual phenotypes such as display behaviors, plasma T, and gular color (Mills et al., 2008). Thus, the positive selection on genes for clutch size (controlled by GTH) in females is counteracted by the negative effects of such genes on the survival of male progeny. In the context of organizational effects of gender differences, unless early-acting steroids due to maternal causes are precisely targeted towards the expression of traits in one sex but not the other (e.g., in the case of yolk steroids or intrauterine steroidal effects in viviparous
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lizards; see Section 1.2), ontogenetic conflict due to organizational events can generate adaptive effects in one sex but maladaptive phenotypes in the opposite sex. Selection due to such ontogenetic conflict continually refines the organizational effect of hormones on male vs. female phenotypes. Moreover, ontogenetic conflict can occur with respect to any genotype that varies among offspring, and is not limited to genotypes associated with sex, such as those found within sexes and due to the action of ARSs. Natural selection studies on the side-blotched lizard indicate that the endocrine-modulated progeny phenotypes are highly adaptive (e.g., anti-predator behaviors due to the synergism of escape behavior and egg size (Lancaster et al., in review) or dorsal patterns and yolk E2 (Lancaster et al., 2008)). When we discuss the structure of the male and female endocrine systems, influenced by activational events, we will return to the kinds of natural vs. sexual selection that act on the two sexes.
1.2. Endocrine Effects on Behavior in Viviparous Species Eutherian mammals are all viviparous, and intrauterine steroids from neighboring fetuses in mammals govern organization of salient aggressive behaviors between the sexes (Vom Saal et al., 1983). In contrast to the single origin of viviparity in mammals, viviparity has evolved over 100 times in squamate reptiles (Guillette, 1993; see also Chapter 11, this volume). Recent studies on the role of hormones in modulating progeny behavior in the viviparous European common lizard, L. vivipara, suggest this may be a potent avenue by which a female can program progeny behavior. These studies suggest roles for both fetal steroid hormones and maternal steroid hormones. For example, the maternal environment affects dispersal behavior in L. vivipara, and the glucocorticoid hormone CORT is a key factor influencing dispersal vs. philopatry (Figure 8.1(d)). In an experimental study, de Fraipont, Clobert, John-Alder, and Meylan (2000) manipulated circulating levels of CORT in females during the gestation period by applying this oil-soluble hormone to the surface of the dorsal skin. High levels of ectopic CORT supplied to the mother during gestation resulted in neonates that were largely philopatric and also were more attracted to the mother’s odor than were offspring from mothers with low levels of CORT during gestation. As in the synergisms between egg size and yolk steroids or maternal steroids in oviparous species noted above, the effects of CORT were also modulated by juvenile nutrition, as manipulated by differences in feeding to the mothers. Juveniles in good condition, or born from mothers in good condition, also have been found to disperse more (Clobert, Massot, Lecomte, Sorci, de Fraipont, & Barbault, 1994). In the
Hormones and Reproduction of Vertebrates
European common lizard, L. vivipara, there are three dispersal strategies (Cote, Clobert, Meylan, & Fitze, 2006): one type of juvenile disperses to empty habitats, another type to low-density habitats, and a third type to densely populated habitats. All types exhibit marked differences in social behavior varying from asociality to cooperation and from neophobia to neophilia. Most of the behavioral characteristics appear to be under maternal influence, although a direct genetic control cannot be excluded in some cases. These basic differences are, thus, under the control of maternal endocrine and nutritional environment. These basic dispersal strategies interact with the mechanisms governing the evolution of sociality (see Section 3). In the case of viviparity, the consequences of maternal uterine environment need not be strictly adaptive and may arise from constraints. Ullner and Olsson (2003) studied the impact of male-biased, female-biased, and equal sex ratio in litters from the viviparous L. vivipara. They found an effect of female-biased vs. male-biased litters on a sexually dimorphic trait, head size to snout-to-vent length, and this effect persisted after the growth period. In particular, males from female-biased litters had smaller heads in relation to snout-to-vent length, and Uller and Olsson (2003) suggest such effects would be maladaptive in male L. vivipara, which are sexually selected for dimorphic expression of body size and other traits such as head size. Thus, the evolution of viviparity imposes an adaptive constraint on the organization of male and female phenotypes in the shared fetal endocrine environment of the uterus. One mechanism by which such potential negative effects could be ameliorated is with the evolution of a yolk-steroid deposition mechanism that counteracts the negative impacts of fetally derived steroid hormones. For example, in L. vivipara, the female is the heterogametic sex (Figure 8.1(d)) and, thus, when eggs are ovulated, the female determines sex ratio by the ovulation of Z vs. W follicles. If females evolved a mechanism to alter the quantity of yolk steroids (e.g., E2 to T ratio) available to the growing follicles (which already have sex determined), the adaptive constraints of littermate sex could be counteracted. For example, the addition of yolk T to eggs fated to be sons (ZZ) or yolk E2 to eggs fated to be daughters (ZW) would allow for egg steroids to counteract the feminizing or masculinizing effect of littermates on progeny as they complete gender development in the uterus. As noted above, the 100þ origins of viviparity provide an amazing opportunity to explore such hypotheses. The phylogeny for L. vivipara indicates that the three to four origins of viviparity in this species range from two to half a million years ago. Two oviparous clades of L. vivipara are found in only two regions, the south in the Pyrenees of France and in the Slovenian and Italian Alps. The four viviparous clades occur throughout the rest of the species range. It is possible to study the evolution of yolk
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Hormones and Behavior of Reptiles
steroid and fetal steroid interactions in geographically isolated populations that exhibit different modes of reproduction (e.g., viviparity vs. oviparity).
1.3. Activational Events Our understanding of the role of hormones has benefited from three very different but complementary perspectives: laboratory and field endocrinology (Bradshaw, 2007), selection analyses and studies of function (Arnold, 1983), and behavioral ecology and game theory (Maynard Smith, 1982). In brief, endocrine systems are shaped by simple forms of selection and more complex forms involving competition among behavioral strategies (Figure 8.2). The basic strategies of females involve density-dependent and
Field and Laboratory Endocrinology
life-history allocation strategies, and the basic strategies of males include territoriality and alternatives to territoriality such as sneaker tactics or scramble competition. In any given population, males might express continuous variation between two strategy types or be fixed for a simple strategy. In populations with ARS, males or females might express discrete types. Whereas the organizational effects of steroids are critical for the neurodevelopment of male vs. female behaviors and the organization of alternative strategies within a given sex (Crews & Moore, 2005), steroids also have activational effects that are manifest at maturity when the sexes initiate reproduction. Later-acting activational effects trigger changes in behavior and physiology in adults. Dramatic changes in behavior can be acted on by natural and sexual
Selection Analyses
Behavioral Ecology
supraoptic amygdala nucleus AVT AVT complex AVT CORT
PHASES OF SEXUAL SELECTION
Gonadal T Hormone Response Elements (HRE)
Survival
Signals Coloration/Size
MALE–MALE COMPETITION
Cooperation Territoriality
Behaviors: a. Male–male territoriality b. Male displays to females
BEHAVIORAL STRATEGIES
Aggression
Deception
Mate Opportunities
Physiology: a. Hematocrit b. Bite Force c. Stamina d. Sprint Speed
MATE CHOICE Survival Mate Siring Acquisition Success Paternity Success
Active Choice
Cryptic Choice
Allocation Strategies
r K
Care
FIGURE 8.2 The understanding of hormonal effects on behaviors has benefited from the tripartite approaches of (1) laboratory and field endocrinology of proximate mechanisms (reviewed in Crews & Moore, 2005; Bradshaw, 2007); (2) selection analyses of adaptive function (Arnold, 1983); and (3) behavior ecology of fitness in social networks (e.g., game theory (Maynard Smith, 1982)). Hormones have pleiotropic behavioral effects. A useful starting point in linking hormones to behavior is an analysis of hormones such as testosterone (T), which have pleiotropic effects on three classes of trait: (1) signaling traits under metabolic control (e.g., coloration controlled by carotenoids, pteridines, or melatonin systems, or size controlled by growth hormones); (2) behavioral traits under neuroendocrine control (e.g., male–male territoriality, male displays to females); and (3) physiological traits that affect performance (e.g., hematocrit, stamina, speed, bite force). The gonadal steroids T and E alter gene transcription via hormone response elements (HREs/EREs) on DNA, thereby altering gene expression in target tissues and thus diverse traits. Testosterone generates effects in interaction with other endocrine systems such as the adrenal steroid corticosterone (CORT), the major glucocorticoid of reptiles involved in stress responses, and arginine vasotocin (AVT), a neuropeptide hormone secreted by many brain regions such as the supraoptic nucleus of the hypothalamus and the amygdalic complex, which innervates the hypothalamus.
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selection. Selection on activational events is under ‘direct selection’ in that the survival or reproduction of males is temporally associated with behavioral changes during maturation. In contrast, direct selection on earlier-acting organizational effects is less likely as differentiation is not yet complete. However, direct selection on activational events can lead to indirect selection on organizational effects if activational and organizational effects are governed by the same sets of genes. Thus, direct selection on activational events and indirect selection on organizational events may primarily take place during maturation when activational events are manifest. The early organizing effect of steroids is likely to occur via selection generated by ontogenetic conflict on determination of male vs. female phenotypes. Likewise, selection on the sex-limited expression of activational events will likely act via ontogenetic conflict generated by the expression of the same genes in the sexes at maturity (Sinervo & McAdam, 2008). Before proceeding to our discussion of activation, we will summarize the effects of adrenal hormones and their interactions with GTHs in terms of network diagrams. Selection on most behaviors will involve selection in social networks
(a)
so the formalism of network diagrams will allow us to understand the proximate control of behavior due to endocrine networks and the ultimate forms of selection arising from social networks; this will be outlined at the end of our review. Here we consider the examples discussed thus far and summarize the hypothalamic–pituitary–gonadal (HPG) axis as an introduction to activational events, and how endocrine systems can be plastically modified by the HPA axis.
1.4. Endocrine Network Theory and the Regulation of Behaviors Endocrine networks generate homeostasis and alter energy flow and metabolism among competing life-history functions (Sinervo & Lancaster, 2009). At maturity, once an appropriate size is reached, activational events are triggered, typically in response to photoperiod cues that are monitored by the pineal, precipitating the release of gonadotropin-releasing hormone (GnRH) by the hypothalamus. Gonadotropin-releasing hormone induces the release of GTHs, which are secreted by the anterior pituitary (Figure 8.3) (Phillips et al., 1987). The GTHs
(b)
Abiotic Trigger of Reproduction (Photoperiod)
STRESSORS OFF
Abiotic Trigger of Reproduction (Photoperiod)
GATED SWITCH via Direct versus Indirect Pathways
HP GnRH
CRF
ne tive
ve
siti
po
E follicles Gonad
Energy Regulation
Optimal Investment Reproduction
Survival TRADEOFF
HP GnRH
ACTH 1. CORT reduces GnRH & FSH
1. and 2. ARE DIRECT NEGATIVE REGULATION
ga
FSH REGULATORY FEEDBACK LOOP:
STRESSOR ON
CNS Reproductive Behaviors 2. CORT binds to nuclear or membrane receptors to extinguish E reproductive behavior
FSH
CORT Adrenal 3. CORT affects other systems to redirect energy flow to survival systems
follicles Gonad Energy Regulation
Survival
Reproduction
3. INDIRECT NEGATIVE REGULATION TRADEOFF
FIGURE 8.3 The structure of endocrine networks and impacts on energy allocation. (a) A positive–negative feedback loop regulating optimal allocation to reproduction as described in Section 1.4. Solid lines denote positive effects (upregulation). Dashed lines denote negative effects (downregulation). (b) The same network modified to include the effects of the adrenal glucocorticoid corticosterone (labeled CORT), which is triggered in response to a stressor that stimulates corticotropin-releasing factor (CRF) secretion, which in turn stimulates corticotropin (ACTH) secretion. The stressor can be either social, biotic, or abiotic. Glucocorticoids act as a gated switch at three regulatory points to (1) shut off gonadotropin hormone (GTH) production in the hypothalamus (HP), (2) directly bind to receptors in the central nervous system (CNS) to alter behaviors, or (3) indirectly redirect energy from reproduction to other critical metabolic functions that enhance survival. The effects of the hypothalamus–pituitary–gonadal (HPG) and hypothalamus–pituitary– adrenal (HPA) axes interact to alter behavior and also fitness through effects on the tradeoffs of survival and reproduction.
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Hormones and Behavior of Reptiles
luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are essential for reproduction through their regulation of gonadal function. In mammals, LH stimulates T secretion in the testes (Saez, 1994; Habert, Lejeune, & Saez, 2001) and FSH stimulates sperm production (Simoni, Gromoll, & Nieschlag, 1997). The action of LH and FSH is modulated by the hormone prolactin (PRL), which increases LH receptor number in Leydig cells and FSH receptor number in Sertoli cells (Bole-Feysot, Goffin, Edery, Binart, & Kelly, 1998). Less information is available regarding the roles of LH and FSH in reptiles and what is known is based on heterologous GTHs from mammals. Mammalian LH increases androgen production in immature water snakes, Nerodia sipedon (Krohmer, 1986), yet FSH stimulates androgen secretion in the turtle Chelonia mydas (Licht, Breitenbach, & Congdon, 1985). In lizards, the synergism of mammalian FSH and LH increase plasma T in the side-blotched lizard U. stansburiana (Mills et al., 2008), but mammalian FSH and LH have distinct effects on subsets of male traits. The activational effects of gonadal steroids include changes in levels of aggression (Moore & Crews, 1986; Marler & Moore, 1988) and changes of morphology and physiology (Sinervo et al., 2000a; Miles, Sinervo, Hazard, Svensson, & Costa, 2007). In females, mammalian LH positively regulates the maintenance of corpora lutea and ovulated follicles secrete P4, which is yet another feedback loop controlling the length of the reproductive cycle and likely played a role in the evolution of viviparity from ancestral oviparity (Guillette, 1993). The positive (e.g., stimulatory effect of LH and FSH on gonads) and negative endocrine regulation (e.g., negative effect of rising T and E produced by the gonad on FSH and LH production by the HPG axis) are also subject to the action of other axes associated with the HPA that generate a ‘gated switch.’ ‘Gated switch’ is a term used in networks (Milo et al., 2002) where one network component (in this case the HPA axis) modulates whether or not the HPG has effects on salient behaviors. Thus, the HPA axis can turn off the behavioral effects of the HPG axis or allow for behaviors to be induced by the HPG axis in a switch-like fashion. The HPG axis interacts with the HPA axis via the stress hormone corticosterone (CORT), the primary glucocorticoid of reptiles (Sapolsky, 1992; Summers et al., 2005). This stress system modulates the expression of behaviors in a variety of social contexts and as a result of the genetic background of an individual. For example, hormones of the HPA and HPG axes also interact in combination with the ARS locus of female lizards (Comendant et al., 2003) (Figures 8.1(c) and 8.3) and male lizards (discussed in Section 2.2). The HPA and HPG axes interact to determine male behaviors, as evinced by the interactive effects of CORT and T on male territorial
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behavior (DeNardo & Licht, 1993; DeNardo & Sinervo, 1994a; DeNardo & Sinervo, 1994b) or in male–male contests (Knapp, 2003; Knapp, Hews, Thompson, Ray, & Moore, 2003). Adrenal glucocorticoids have a dual function in both metabolic regulation and the regulation of stress, including social stress (Korte, Koolhaas, Wingfield, & McEwen, 2005). In response to various abiotic and/or biotic stressors, the CNS triggers the hypothalamus to secrete corticotropinreleasing factor (CRF), which in turn stimulates the release of corticotropin (ACTH) from the pituitary. Corticotropin travels via the bloodstream to stimulate release of CORT by the adrenals. Corticosterone travels to various tissues and can be bound by corticosterone-binding globulins (CBGs) (Jennings, Moore, Knapp, Matthews, & Orchinick, 2000). Corticosterone has diverse tissue targets. For example, it binds to both mineralocorticoid and glucocorticoid receptors in the hypothalamus to alter levels of FSH, and binds to components of the CNS to cause alterations in behavior in vertebrates (Korte et al., 2005). Corticosterone is associated with color in lizards, and suppresses the effect of T on the expression of male behavior (DeNardo & Licht, 1993; DeNardo & Sinervo, 1994a; 1994b). Thus, CORT can directly switch off either the endocrine circuits that control the gonads or the behaviors that control receptivity of sexual behaviors (Figure 8.3). We will use the formalism of network diagrams to summarize all of the relevant endocrine systems in reptiles and draw parallels between reptiles and other vertebrates. Each figure will include a triangle that is comprised of a regulatory feedback loop along one side of the triangle and a gated switch at the opposing vertex. The complete networks and selection on network structure are likely to be quite complex and involve the HPG/HPA axes, selection on reproductive function, and other systems such as immune function. A brief summary of the role of lifehistory tradeoffs and plasticity in structuring endocrine systems is given in Figure 8.4.
2. THE FORM OF NATURAL, SEXUAL, AND SOCIAL SELECTION The patterns of selection on the behaviors of sexes and on juvenile vs. adult phases are very different. The organizational events in reptiles, discussed in Section 1.1, provide a mechanism to fine tune progeny phenotypes and ameliorate ontogenetic conflict (Sinervo et al., 2008). In addition, TSD has been hypothesized to ameliorate such conflict in situations where differences in growth or maturation are linked to developmental temperature regime (Janzen & Phillips, 2006). Though not universally true in vertebrates (e.g., not in the case of sex role reversal or in the case of hermaphrodites), reptilian males are under sexual and natural selection while females tend to be under natural
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Hormones and Reproduction of Vertebrates
LEGEND Estrogen Response Elements ERE
2. Abiotic Inputs (e.g. photoperiod triggers reproduction)
1. Biotic Inputs Social crowding by female morphs: r-K regulatory Network
Gonadotropin-Releasing Hormone GnRH Corticotropin-Releasing Factor CRF Gonadotropin Hormones GTH Follicle-Stimulating Hormone FSH Luteinizing Hormone LH
Hypothalamus– Pituitary GnRH CRF GTH
ACTH
+ve -ve CORT Stress
Life history plasticity
Adrenocorticotropic hormone ACTH Corticosterone CORT Progesterone P Estrogen E
1. Endocrine Regulatory System/Organ(s) 2. Endocrine-Gene Cascade 3. Positive–Negative Feedback Loops
-ve
+ve
Life History Tradeoff
+ve FSH -ve LH E T LH
CORT Energy +ve
Adrenal
Gonadal
Corpora Liver cells Lutea
E
Vitellogenin ERE Reproductive
E P
CORT
3. Extended Phenotype of the Male Territory
Follicles
FSH
Effort FSH Clutch Size
Yolk Egg Size
P Oviducal Egg Rention
Timing a. Incubation Time b. Lay Date Progeny Survival
Cell-mediated
Humoral
Dispersal Philopatry Cooperation
Female Survival
MHC Immune function
4. Social Neighborhood Settlement Choice
OFFSPRING QUANTITY VS. QUALITY TRADEOFF
SOCIAL TRADE-OFFS
COSTS OF REPRODUCTION TRADEOFF
Life History Traits Behavioral Traits
FIGURE 8.4 The hypothalamus–pituitary–gonadal (HPG) axis, which regulates clutch size and progeny size, is associated with selection and lifehistory tradeoffs in reptiles (Sinervo, 1999) and other vertebrates (Oksanen, Koskela et al., 2002), based on endocrine manipulations. An example of an endocrine cascade for egg size and egg number in lizards is depicted (Gonadotropin-releasing hormone (GnRH) / gonadotropic hormone (GTH) / follicle proliferation / theca of follicles produce E2 / E2 travels to liver / initiates gene transcription via an estrogen response element (ERE) / to effectuate vitellogenin transcription and translation / vitellogenin is packaged in many small ova or a few large ova by ovaries). A single cascade regulates tradeoffs in egg and clutch size. Additional endocrine cascades in this system affect levels of progesterone (P4) via stimulation of the corpora lutea by GTH. However, clutch size and thus GTH determine the number of corpora lutea. Progesterone affects the degree of egg retention and incubation time in reptiles. These endocrine system interactions effect a tradeoff in females: progeny survival and costs of reproduction for the female parent (Sinervo, 1999). Reproductive hormones affecting life-history traits are modulated by the adrenal system, the same system that plastically regulates territorial behavior of males. More complex theories of tradeoffs implicate immune function effects on reproductive costs through effects on resource allocation and metabolic pathways (Lochmiller & Deerenberg, 2000; Costa & Sinervo, 2004). For example, in the side-blotched lizard, we have observed a strong tradeoff between immune function vs. survival in the r-selected vs. K-selected color morphs of females. A single gene of major effect called the oby locus, which is named for the colors orange, blue, and yellow, governs female throat color and reproductive strategy (r- vs. K-selected). The strong negative genetic correlation (Sinervo, Svensson, 2001a; 2001b), indicative of a genetic tradeoff between immune function and survival costs (Svensson et al., 2002; 2009), arises from two physiological pathways: (1) adrenal / immune system / survival, which interacts with (2) adrenal / gonadal / clutch size vs. egg mass (Lancaster, McAdam, Wingfield, & Sinervo, 2007; Lancaster, Hazard, Clobert, & Sinervo, 2008; Lancaster et al., 2010). In summary, while the offspring size–number tradeoff appears to arise from multiple effects of a single endocrine cascade, the costs of reproduction tradeoffs arise from several endocrine cascades. One of these cascades is the hypothalamus–pituitary–adrenal (HPA) axis of reproductive females. In this case the HPA axis modulates life-history plasticity in the context of social stressors of female density and female morph frequency (Comendant, Sinervo, Svensson, & Wingfield, 2003). However, there are also far more complex life-history tradeoffs that involve egg size, oviposition date, incubation time and density regulation (Svensson & Sinervo, 2000; Calsbeek & Sinervo, 2007), and the choice of territory by the female (Calsbeek & Sinervo, 2004). In addition, male progeny experience very complex social tradeoffs of altruism, mutualism, and competition that involve offspring size and offspring settlement strategies (reviewed in Sinervo et al., 2008). All of these complex behaviors are regulated by endocrine systems.
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Hormones and Behavior of Reptiles
selection. There are few overt differences in the form of sexual selection found among reptilian groups, with most exhibiting polygynous mating systems, and multiple mating is extremely common in squamate reptiles (Olsson & Madsen, 1998). Squamate reptiles provide examples of resource defense polygyny (e.g., Uta palmeri (Hews, 1993)); lekking behaviors (e.g., Amblyrhynchus cristatus (Wikelski, Steiger, Gall, & Nelson, 2005)); scramble competition and mating balls (Thamnophis sirtalis (Shine, Langkilde, Wall, & Mason, 2005)); mate guarding (Vipera berus (Luiselli, 1995)); and territorial cooperation (U. stansburiana (Sinervo et al., 2006b)). Multiple alternative mating strategies within a single species are common in lizards and are associated with color and pattern polymorphism expressed on either the ventral (e.g., L. vivipara (Sinervo et al., 2007; Vercken, Massot, Sinervo, & Clobert, 2007)); gular (U. ornatus (Carpenter, 1995), U. stansburiana (Sinervo & Lively, 1996), Podarcis muralis, Podarcis millisenensis (Huyghe, Vanhooydonck, Herrel, Tadic, & Van Damme, 2007)); or dorsal surfaces (Egernia spp. (Chapple, 2003), U. stansburiana (Lancaster et al., 2007); Anolis sagrei (Calsbeek, Bonvini, & Cox, 2009)). These colors have a genetic basis in two species examined (female L. vivipara (Vercken et al., 2007), Anolis sagrei (and male and female U. stansburiana (Sinervo, 2001)). As is common in the case of color polymorphism in other vertebrates (Hoffman & Blouin, 2000), the color of U. stansburiana has a Mendelian pattern of inheritance (linkage mapping and controlled crosses suggests a single factor controls color). The biochemical basis of color arises from a combination of yellow and orange carotenoids acquired from food and endogenously produced pteridines (in the colors orange, blue, and yellow), and structural colors for melanin and blue pigments (Morrison, Rand, & Frost-Mason, 1995; Morrison, Sherbrooke, & Frost-Mason, 1996). Color polymorphism is not restricted to males; it is also prominently expressed in females in all of the species listed above. There is one example, Sceloporus pyrocephalus, that is unusual in that females of this species express greater color polymorphism than males (Calisi & Hews, 2007). The expression of sexually dichromatic color, and alternative colors within a sex, during maturation is one of the most obvious factors influencing behaviors controlled by hormones, since color is used as a signal in animals. Expression of color in females has been hypothesized to be a signal to males advertising lack of receptivity when the female is gravid (Cooper & Greenberg, 1992) or to advertise receptivity and stimulate male courtship (Chan, Stuart-Fox, & Jessop, 2009). However, the clear role of color in social selection among females (Sinervo, Svensson, & Comendant, 2000; Comendant et al., 2003; Vercken et al., 2007; Vercken & Clobert, 2008b) indicates that it also is likely to have the same role as that of color in
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males, which is used as a badge of status in lizards (Thompson & Moore, 1991). The expression of sexually dimorphic color in vertebrates is under endocrine control, and a number of studies on reptiles indicate a positive correlation between the expression of color and steroid hormones (Cooper & Greenberg, 1992; Calisi & Hews, 2007). Only a few studies have experimentally manipulated the endocrine system to alter color (Cooper, Mendonc¸a, & Vitt, 1987) (Table 8.1). Mills et al. (2008) manipulated ovine FSH and LH in a factorial design and found that blue throat color in the homozygous yellow color genotype (yy) of the side-blotched lizard intensified in response to an activational effect of LH, the by color genotype intensified in response to FSH stimulation, whereas the other color genotypes (oo, bb, bo) did not respond to either hormone treatment. This result is consistent with plasticity in reproductive strategy of the yellow color genotypes (yy, by). In particular, the blue color in the by color genotype can intensify during the breeding season as this morph transforms from a sneaker strategy and begins defending a larger territory, with increases in plasma T. Similarly, new findings on the yy color morph suggest that the light grey melanic stripes that provide a base to the yellow throat color can darken dramatically (Mills et al., 2008), particularly in the absence of male neighbors of the territorial color genotypes (oo, bo, bb, by). Given that plasma T is modulated by mammalian FSH and LH (Mills et al., 2008), the association of color and plasma steroids in other reptiles is likely under the control of both the GTH and steroids. However, separate LH-like and FSH-like GTHs have not been demonstrated conclusively in squamates. Throat color has also been manipulated in experiments investigating the organization of the alternative strategies discussed in Section 1.1. Early studies on the morphs of U. ornatus indicated that the male types did not differ in circulating levels of plasma T during the reproductive season (Moore, Hews, & Knapp, 1998) despite obvious differences in aggression (Hover, 1985), which led Moore on a search for organizational effects (Crews & Moore, 2005). The polymorphism in U. ornatus is fixed in adults and color does not change in response to T implants (Thompson & Moore, 1992); however, manipulations of T in hatchling U. ornatus have successfully altered throat color development (Hews & Moore, 1995) indicating that organizational events contribute to color in U. ornatus (Figure 8.1(a)). We suspect similar effects are in operation in U. stansburiana because while we have modulated color expression of blue in by genotypes and the darker melanic colors of yy, we have been unable to phenocopy the basic difference between blue (bb) and orange lizards (oo, yo, bo) with activational experiments (e.g., FSH, LH, and T have not altered the basic colors). It is likely that orange-yellow vs. blue differences are organized early in development because, even though throat color is not expressed until
226
TABLE 8.1 Reptilian species having intraspecific polymorphisms conveying different social statuses Family
Species
Type of Polymorphism
Testudines
Emydidae
Trachemys scripta
Size and color
Squamata
Agamidae
Agama agama
Color morphs
Madsen and Loman (1987)
Amphibolurus maculosus
Color morphs
Mitchell (1973)
Ctenophorus pictus
Color morphs
Olsson et al. (2007)
Polychrotidae
Anolis carolinensis
Size morphs
Lailvaux et al. (2004), Husak et al. (2009)
Phrynosomatidae
Uta stansburiana
Color morph
Sinervo and Lively (1996)
Urosaurus ornatus
Color morph
Thompson and Moore (1991; 1992)
Sceloporus undulatus erythrocheilus
Color morphs
Rand (1990); Morrison et al. (1995)
Microlophus albemarlensis
Size morphs
Stone et al. (2003)
Microlophus delanonis
Size morphs
Werner (1978)
Iguanidae
Amblyrhynchus cristatus
Behavioral morphs: territorial, satellite, sneaker
Wikelski et al. (2005)
Lacertidae
Podarcis muralis
Color morphs
Sacchi et al. (2007), J. Clobert (Pers. Comm.)
Podarcis melisellensis
Color morphs
Huyghe et al. (2007)
Lacerta vivipara
Color morphs
Sinervo et al. (2007)
Psammodromus algirus
Color morphs
Salvador et al. (1997)
Cordylidae
Platysaurus broadleyi
Color morphs
Whiting et al. (2003)
Colubridae
Thamnophis sirtalis parietalis
Behavioral morphs
Shine et al. (2005)
Viperidae
Vipera berus
Color morphs
Forsman (1995)
Tropiduridae
Reference
Hormones and Reproduction of Vertebrates
Order
Chapter | 8
Hormones and Behavior of Reptiles
maturity, behaviors of juveniles and selection on juveniles are markedly different among progeny genotypes (Sinervo & Zamudio, 2001). In addition to a role for GTH and T in controlling some color expression at maturity, and P4 and T, which act during organizational events, the hormone melatonin governs expression of dark melanic color. For example, in U. ornatus, a-melanocyte-stimulating hormone (a-MSH) is the melanotropin that governs color change (Castrucci, Sherbrooke, & Zucker, 1997). The dark dorsal melanin is used as a badge of status in territorial U. ornatus (Zucker, 1989).
2.1. Hormone Modulation of Male Reproductive Behaviors An organism’s fitness is ultimately defined as the number of progeny produced over its lifespan. Fitness therefore depends on the ability to grow and reach adult (reproductive) body size, survival, number of reproductive episodes (clutch or litter), progeny produced per episode, and survival of young. In many reptilian species, a suite of behaviors governs reproductive success. These behaviors in turn are likely to vary with the type of mating system. Regardless of mating systems, the two major limitations to breeding success in many reptiles are the ability to defend a territory or repel rivals and the ability to court females. Territorial status is determined by agonistic interactions among competing males. Dominant males, as conveyed by aggressive behaviors, are more likely to have territories than less aggressive, subordinate individuals. In addition, territory size often is tied to the number of potential mates, at least in polygynous species (e.g., U. stansburiana). A larger territory has the potential to overlap with more females, which increases the number of progeny a male may sire. However, extra-pair fertilizations may reduce the number of offspring sired by a male with multiple mates or even in monogamous males. Hence, mate-guarding behaviors are likely to evolve in monogamous species as a strategy to reduce the likelihood of males sneaking copulations from a territorial male. The discovery of cooperative behavior in certain morphs within lizards (Sinervo & Clobert, 2003) suggests the importance of hormones that facilitate filiative interactions. Subordinate males without a territory may adopt other behaviors, e.g., sneaker strategy, in order to furtively copulate with females. Finally, other reptilian groups, for example snakes, rarely defend territories (Shine et al., 2005). Therefore, the reproductive success of these males may entail outcompeting other rival males for access to females. A second limitation is the ability to court and attract a female. Males may require specific signals (i.e., color pattern) or behaviors that convey dominance status to repel
227
rivals as well as signal their attractiveness to females. Males enhance their attractiveness to females by simultaneously using phenotypic signals, such as badges, and courtship behaviors, such as pushup displays. Territorial and courtship behaviors may be distinct. Thus, agonistic and threat displays (biting, chasing, and other aggressive behaviors) may be used to chase rivals out of a territory, whereas courtship displays may entail other behaviors. The behaviors tied to dominance and mate acquisition entail two phases of sexual selection, namely intra-sexual competition (male–male interactions) and mate choice. Testosterone and other hormones modulate behaviors of males. Territorial behaviors and dominant interactions comprise one phase of sexual selection (antecedent phase), which determines reproductive opportunities for males. Copulation and sperm transfer make up the second phase of sexual selection: mate choice. Selection operating on the HPG or HPA axis will vary according to the magnitude of male–male competition or female mate choice in affecting reproductive success. As described above, each life-history stage is regulated by specific endocrine cascades that influence the success of the individual in various behavioral challenges, such as territory acquisition, courtship, and parental care. Hormones are key for modulating the timing and expression of behaviors tied to fitness. Coordination via external cues synchronizes male courtship and copulatory behavior with female receptivity, timing of maturation of gametes, and territoriality. Because reptiles display a remarkable diversity of reproductive tactics, they are model organisms for investigating how hormones are involved in reproductive behavior. In this section we review the various reproductive behaviors influenced by T, which is the most thoroughly studied set of hormone effects. We also describe emerging details regarding the interaction between T and AVT in governing reproductive and sexual behaviors. We briefly summarize the differences between the sexes and selection on their respective endocrine systems.
2.2. Sexual Selection, Life History, and Correlated SelectiondHormones, Behavior, and Fitness The endocrine cascades depicted in Figure 8.2 reveal the complex linkages between T, CORT, and other hormones with numerous phenotypic traits and life-history attributes. In the pre-maturational phase, T may either enhance or inhibit growth (Cox & John-Alder, 2005). The action of T therefore depends on genetically determined patterns of energy allocation that may impact fitness. If body size covaries with fitness, then it may be advantageous for males to optimize growth so as to increase the chance of acquiring
228
territories (through dominance interactions) and enhance mating opportunities. In contrast, T may favor reallocating energy to other competing functions, for example musculature, which enhance physiological performance, or other traits that signal dominance status. By jointly influencing two trait groups, the action of T enhances the resourceholding potential of a male and increases the probability of mating. The nature of the effects of T entails an interplay between traits that confer advantages for survival (avoidance of predators, foraging success) and maximizing reproductive success. Thus, T affects multiple trait complexes (Figure 8.2) in interaction with other hormones, e.g., CORT and AVT (Figure 8.5). Species with alternative mating strategies offer the opportunity to dissect the role of hormones in affecting fitness. Morphs may be conditional or fixed (Rhen & Crews, 2002) and occur in a diversity of species in reptiles, including lizards, snakes, and turtles (Table 8.1). Conditional traits tend to be related to body size (e.g., marine iguanas, A. cristatus (Wikelski et al., 2005)), age or ontogenetic polymorphisms (Lailvaux, Herrel, Vanhooydonck, Meyers, & Irschick, 2004), or genetic polymorphisms (e.g., U. stansburiana (Sinervo, 2001)). Most species have two alternative mating strategies, one practiced by dominant males and the other by sneaker or satellite males. In other species there may be three morphs (e.g., U. stansburiana) occurring in a population. In particular, discrete morphs within a population adventitiously introduce variation that facilitates determination of how hormones, physiology, behavior, and fitness are integrated (Miles et al., 2007). Comparing variation in traits among morphs generates insights into the multidimensional effects of sex steroids on sexual ornaments and behavior, the corresponding influences on other traits tied to dominance, such as physiological performance, and their role in generating life-history tradeoffs. Further, fitness differences between morphs create the ideal system for identifying the targets of selection on hormones (Figure 8.2).
2.3. Testosterone (T) and SignalsdColors and Badges Substantial evidence is available supporting the importance of T in the expression of color patterns in male reptiles (Cooper & Greenberg, 1992; Cox, Skelly, Leo, & JohnAlder, 2005; Cox, Zilberman, & John-Alder, 2008; see also related references Section 2.1–2.2). Variation in color patterns among morphs also depends on androgens (Moore et al., 1998, Knapp et al., 2003; Knapp, 2004), although other hormones, e.g., P4, may modify the expression of some morphs. As noted above, differences in color likely provide a signal (badge) to rival males and females regarding dominance status and male quality.
Hormones and Reproduction of Vertebrates
2.4. Aggression and Territoriality The connection between T and aggressive behavior, dominance status, and territoriality has received considerable attention. Rising levels of T at the beginning of the breeding season correspond with increased frequency of agonistic displays and other forms of territorial behavior (Moore & Lindzey, 1992; Tokarz, McMann, Smith, & John-Alder, 2002; Watt, Forster, & Joss, 2003; Wade, 2005). Male Anolis sagrei treated with exogenous T during the breeding season exhibited significantly greater dewlap extensions than castrated individuals in response to the presence of rival males (Tokarz et al., 2002). To what extent, however, is individual variation in levels of T related to the intensity of aggression? A recent study compared aggressive displays and T levels in U. ornatus based on unmanipulated males and castrated males given exogenous T (Kabelik, Weiss, & Moore, 2008). Size-matched males were paired in an arena and the number of fullshow displays (aggressive display in Urosaurus) and display intensity were quantified. Fullshow displays were positively correlated with plasma T levels (Kabelik et al., 2008). To our knowledge there is scant experimental evidence that T increases physiological characteristics associated with dominance. Identifying the link between T and physiological traits tied to dominance is key, since several studies have implicated T as a mediating factor between dominance and it has been shown to affect display capacities in male U. stansburiana. Mills et al. (2008) demonstrated a significant positive correlation between stamina and levels of T. Little information is available on the effects of T and aggression in snakes. Testosterone levels increased in copperheads (Agkistrodon contortrix) during the onset of the breeding season (Schuett, Harlow, Rose, Van Kirk, & Murdoch, 1996; 1997). However, T levels did not differ between winner and loser snakes at the end of staged agonistic encounters (Schuett et al. 1996). Territorial status is critical for lizards to obtain copulations, and the literature is replete with examples showing that territorial males have elevated levels of T (DeNardo & Sinervo, 1994b; Watt et al., 2003; Wikelski et al., 2005). Male A. sagrei treated with cyproterone exhibited fewer dewlap extensions than sham controls and spent more time in a low quality habitat patch (Tokarz, 1995). However, in some species, territorial status is unrelated to T. For example, T levels in older territorial male collared lizards (Crotaphytus collaris) did not differ from younger, nonterritorial males (Baird & Hews, 2007). Similarly, blueorange and orange morphs in the tree lizard do not differ in levels of T (Knapp et al., 2003), but other hormones, e.g., P4, determine whether a male adopts a nomadic behavior or remains sedentary.
Chapter | 8
Pituitary Hypothalmus CRF
ACTH
CORT
Adrenal
229
Hormones and Behavior of Reptiles
Supraoptic nucleus AVT
STRESSOR (ABIOTIC, SOCIAL)
GnRH
CNS Amygdala complex AVT POA Mineralocorticoid AVT sensitive neurons glucocorticoid Dominant/Subordinate receptors
GTH LH FSH
T
CORT
AVT
Gonadal
T Hormone Response Elements (HRE) Signal Coloration Behavior: a. Male–male territoriality b. Male displays to females Physiology: a. Hematocrit b. Stamina c. Sprint Speed FIGURE 8.5 The details of endocrine system regulation (e.g., negative feedback loops and gated switches; see Figure 8.3) for plasma testosterone (T). Levels of plasma T secreted by the gonads are positively regulated by gonadotropin (GTH) (while the putative analogs of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have not been definitively isolated in reptiles, injections of orthogolous mammalian FSH and LH have distinct biological effects in lizards (Mills et al., 2008)), which is in turn negatively regulated by high plasma T. The adrenal steroid corticosterone (CORT) is under positive regulation by corticotropin (ACTH), which is triggered by corticotropin-releasing factor (CRF) in the hypothalamus, in response to diverse stressors of abiotic (e.g., thermal, hydric) and biotic (e.g., social) origin. Corticosterone can also affect neuronal activity in the central nervous system (CNS) via mineralocorticoid receptors and glucocorticoid receptors to effectuate changes in the secretion of many neuroendocrine hormones such as arginine vasotocin (AVT) and other neurotransmitters (serotonergic systems and catecholaminergic systems, not shown; see Section 2.8 for references). Arginine vasotocin can affect dominant vs. subordinate behaviors of males via differences in receptor density in the anterior vs. posterior preoptic area (POA), thereby adjusting social strategy.
230
2.5. Testosterone (T) and Courtship/ Copulations Considerable evidence is available showing that male courtship behaviors are induced by T (Moore & Lindzey, 1992). The onset of reproductive behaviors in the majority of reptiles coincides with a seasonal rise in androgens (Woolley, Sakata, & Crews, 2004). The association between a seasonal rise of T (and 5a-dihydrotestosterone (DHT)) and the initiation of courtship behavior in reptiles has been documented in many groups (see Table 8.2). The role for T controlling sexual behavior has been substantiated by experimental manipulations involving castration followed with exogenous T or androgen receptor blockers (e.g., flutamide) and inhibitors of androgen synthesis (e.g., cyproterone acetate), and provides additional evidence of the role of androgens in courtship and copulation behaviors (Haider & Rai, 1986). The cessation of courtship behaviors after castration has been established in A. sagrei (Tokarz, 1995); Anolis carolinensis (Mason & Adkins, 1976); Sceloporus jarrovi (Moore, 1987); E. macularius (Rhen & Crews, 1999); and Cnemidophorus inornatus (Lindzey & Crews, 1986). For example, the musculature necessary for the dewlap display used by male anoles to attract females is responsive to levels of T and differs between the sexes (Wade, 2005). Moreover, the hemipenes increase in size in response to rising levels of T (Wade, 2005). The frequency of courting behavior is also sensitive to T.
2.6. IntegrationdHormones, Performance, Behavior, and Fitness Linking the role of natural and sexual selection on structuring hormone networks requires the quantification of individual hormone levels and the behavioral consequences of variation in hormones, and the ascertainment of the covariation between the behavioral trait and various components of fitness (for natural selection) or mating opportunities (for sexual selection). Thus, not only is it necessary to corroborate that T influences traits that signal dominance (badges) and aggressive behaviors establishing dominance, but also that dominant males either acquire more mating opportunities or sire more offspring (Figure 8.2). Disparate evidence is available supporting individual paths in the endocrine cascade, but relatively few studies have linked hormones and fitness. Male marine iguanas may adopt one of three mating strategies: dominant male holding a small display territory, satellite male, and sneaker (Wikelski et al., 2005). Territorial males have access to a higher number of females, but the overlap with females drops when the action of T is temporarily blocked by flutamide.
Hormones and Reproduction of Vertebrates
The link between dominance, territory size, and fitness has been demonstrated in only a few species. The fitness consequences of dominance have been clearly established in U. stansburiana (Sinervo et al., 2000a; Zamudio & Sinervo, 2000). In addition, the long-term work of JohnAlder and associates has linked hormones with growth, aggression, territory size, overlap with females, and number of progeny sired (Haenel, Smith, & John-Adler, 2003). Males with larger territories overlap with more females (Haenel et al., 2003). Elevated T enhances territory size and activity time (Cox, Skelly, & John-Alder, 2005). Interestingly, the number of clutches sired and number of progeny sired are both associated with territory size. However, T is unrelated to fitness; rather, the paternity data show a significant, positive association with levels of CORT.
2.7. Modulation of Aggressive and Courtship Behavior: Arginine Vasotocin (AVT) Understanding how T can mediate sexual signals, aggressive behavior, territoriality, and divergent mating strategies is a fundamental goal in evolutionary endocrinology. Recent evidence suggests that steroid-sensitive neuropeptides are critical for regulating social behaviors (Kabelik et al., 2008). Arginine vasotocin is a neuropeptide that modulates a variety of physiological and behavioral traits (Goodson & Bass, 2001; Goodson, 2008). Most information about the sexual behavioral consequence of AVT (or the mammalian analog arginine vasopressin (AVP)) in males is based on experiments with fish, amphibians, birds, and mammals. The dearth of studies conducted on reptiles makes generalizations about the influence of AVT on reptilian sexual behavior difficult. Details regarding the roles of AVT in social and reproductive behaviors are based on neuroendocrine studies showing areas of the brain that have high populations of AVT-ir neurons and manipulative experiments involving exogenous AVT. Production of AVT in vertebrates occurs in neurons found in the preoptic area (POA) and the anterior hypothalamus (AH) (AdkinsRegan, 2005). In reptiles, AVT-producing neurons also occur in the supraoptic nucleus of the hypothalamus (Goodson, 2008). Despite the apparent conserved distribution of AVT-associated neurons (Moore & Lowry, 1998), there is considerable diversity among vertebrates in the locations of other neurons that produce AVT, which may provide substantial diversity in patterns of aggression and sociality (Goodson, 2008). Notably, there are populations of AVT neurons associated with the medial bed nucleus of the stria terminalis (BSTm) and amygdala, two areas of the brain that mediate a suite of social behaviors (De Vries & Panzica, 2006). The sexes differ in the density of cell populations in these regions, with males having higher
Chapter | 8
Order
Family
Species
Reference
Rhynchocephalia
Sphenodontidae
Sphenodon punctatus
Bradshaw et al. (1988)
Testudines
Squamata
Crocodilia
Licht (1982) Emydidae
Chrysemys picta
Callard et al. (1976)
Testudinidae
Gopherus polyphemus
Ott et al. (2000)
Cheloniidae
Caretta caretta
Wibbels et al. (1990)
Agamidae
Amphibolurus nuchali
Bradshaw et al. (1991)
Amphibolurus caudicinctus
Bradshaw et al. (1991)
Polychrotidae
Anolis sagrei
Tokarz et al. (1998)
Scincidae
Tiliqua rugosa
Bourne et al. (1986)
Tiliqua nigrolutea
Edwards and Jones (2001)
Varanidae
Varanus albigularis
Phillips and Millar (1998)
Teiidae
Cnemidophorus inornatus
Lindzey and Crews (1986)
Colubridae
Thamnophis sirtalis
Clesson et al. (2002)
Crotalidae
Agkistrodon contortrix
Schuett et al. (1997)
Viperidae
Vipera aspis
Saint Girons et al. (1993)
Crocodylidae
Alligator mississippiensis
Bradshaw et al. (1988)
Hormones and Behavior of Reptiles
TABLE 8.2 Reptilian species in which testosterone (T) has been identified in the initiation of courtship behavior
231
232
numbers than females in pythons (Python regius), pit vipers (Bothrops jaracara), gekkos (Gecko gecko), anoles (A. carolinensis), chameleons (Chameleon chameleon), and turtles (Trachemys scripta) (De Vries & Panzica, 2006). Recent evidence suggests that these areas are also sensitive to sex steroids, suggesting a role for T or E2 in mediating aggressive or social behaviors. Manipulation of AVT via exogenous administration affects a variety of behaviors ranging from aggression and courtship display behavior to parental care (Goodson & Bass, 2001). Comparison of the action of AVT in territorial vs. colonial birds revealed contradictory responses (summarized in Goodson, 2008). Arginine vasotocin reduced aggression in territorial species but enhanced aggressive behaviors in colonial species. In reptiles, the investigation of AVT initially focused on its physiological role as an anti-diuretic hormone (Bradshaw, 2007). Individuals that are water-stressed will reset their preferred body temperature to avoid water loss (Bradshaw, 2007). Arginine vasotocin is critical in adjusting thermoregulatory behavior in the context of water balance. Recent evidence in reptiles suggests that the expression of AVT in the brain is affected by T (Goodson & Bass, 2001). Testosterone regulates mating behaviors, but AVT either influences initiation or continuation of sexual behavior in males (e.g., Kime, Whitney, Davis, & Marler (2007) for multiple references based on amphibian models; see also Hillsman, Sanderson, & Crews (2007)). An early manipulative study on reptiles found that injection of AVT into adult male Podarcis sicula inhibited completion of spermatogenesis and reduced the endocrine activity of the gonads (Ciarcia, Angelini, Picariello, & Botte, 1983). Recent studies provide evidence for a role of AVT in modulating social status and courtship behavior in species with alternative reproductive tactics (Goodson, 2008; Greenwood, Wark, Fernald, & Hofmann, 2008). Current data suggest that AVT mediates aggressive and territorial behavior as well as courtship behavior (Goodson & Bass, 2001; Santangelo & Bass, 2006). Curiously, AVT promotes or inhibits aggression depending on the social context. Hence, its action appears to vary in the context of the social/ mating system. Arginine vasotocin promotes calling behavior of satellite males and has a role in divergent social strategies of satellite males vs. calling males in the cricket frog, Acris crepitans (Semsar, Klomberg, & Marler, 1998; Marier, Boyd, & Wilczynski, 1999). Comparison of mRNA expression in the brains of the cichlid fish Astatotilapia burtoni revealed differences between dominant and subordinate males. Arginine vasotocin expression in the posterior POA was higher in dominant males. In contrast, subordinate males expressed more AVT in the anterior POA (Greenwood et al., 2008). Non-territorial, subordinate males have small testes with low amounts of sperm. Because the expression of AVT
Hormones and Reproduction of Vertebrates
varies with male phenotype, the findings of Greenwood et al. (2008) suggest that AVT may regulate alternative strategies in males via maturation of testes and hence production of T. Although the role of AVT in modulating dominant and territorial behavior has been demonstrated in fishes (Santangelo & Bass, 2006), information on the role of AVT in regulating aggression and dominance behavior in reptiles is limited. Hillsman et al. (2007) compared AVT fiber densities in the brains of C. inornatus (sexual species) and Cnemidophorus uniparens (asexual species). Males have higher densities of AVT in the POA than females. Further, T-treated males had higher AVT-immunoreceptivity than sham-treated males. Hillsman et al. (2007) concluded that androgens affected expression of AVT in regions that are critical for male reproductive behavior; specifically, courtship and copulatory behaviors. Dominant and subordinate A. carolinensis lizards differ in the number of AVT immunoreactive cells in the POA, but not in other parts of the brain (Hattori & Wilczynski, 2009). Dominant males have higher counts than subordinates. In contrast, AVT-ir cell counts for subordinate individuals were lower than dominant males, males housed singly, or males paired with a female. The findings of Hattori and Wilcynski (2009) provide support for the proposition that the social context influences AVT. Unlike the work of Greenwood et al. (2008), the higher AVT-ir cell counts were unrelated to T levels. The lower number of AVT-ir cells in subordinate males deserves additional attention. It is unclear whether the reduced cell count is a consequence of the agonistic interactions. Unfortunately, Hattori & Wilcynski did not measure CORT levels. It is possible that the low number of AVT-ir cells is related to elevated CORT. Previous data have shown that the effects of AVT on sexual behavior may be modified by glucocorticoids. Data from mammals suggest that vasopressin mRNA receptor binding in the hypothalamus is affected by glucocorticoids (Viau, Chu, Soriano, & Dallman, 1999). Therefore, input and feedback from the HPA axis may be critical in regulating reproductive behavior as a consequence of extrinsic or intrinsic stressors (Goodson & Bass, 2001). Data regarding differences in AVT activity in reptiles with alternative mating strategies are limited. Recently, Kabelik et al. (2008) compared the association between T, aggression, and AVT in male morphs and female tree lizards (U. ornatus). Males had greater densities of AVT cells in the POA, AMY, bed nucleus of the stria terminalis, and nucleus accumbens than females, which is consistent with other studies (Goodson & Bass, 2001; Goodson, 2008). Further, AVT-ir activity was lower in castrated males receiving a sham implant compared to males receiving exogenous T, supporting a role for T. However, the dominant blue-orange morph and subordinate orange morph did not differ in AVT-ir. Testosterone affected both dominance
Chapter | 8
233
Hormones and Behavior of Reptiles
behavior and AVT-ir in tree lizards, but AVT-ir was unrelated to aggressive behavior.
2.8. Interactions with CNS Neuroendocrine Hormones Two other neuroendocrine systems have recently been linked to behavior in reptiles. The adrenergic and serotonergic systems are related to the regulation of aggressive behavior in vertebrates. The limited information on reptiles (Matter, Ronan, & Summers, 1998) indicates that male S. jarrovi engaged in aggressive territorial behaviors express higher levels of norepinephrine and dopamine (DA) and their metabolites, while satellite or subordinate males without territories express higher levels of serotonin (5-HT) and higher serotonergic turnover (e.g., ratio of 5-hydroxyindoleacetic acid to 5-HT). Territorial males at rest exhibit low levels of L-3,4-dihydroxyphenylalanine (L-DOPA), but diencephalic 3,4-dihydroxyphenylacetic acid, a metabolite of DA, rapidly increases after an agonistic interaction. In response to agonistic encounters, 5-HT levels of dominant males rapidly rise, but at rest the values are low compared to the elevated levels observed in satellite males. This finding is salient for this species because previous studies on S. jarrovi did not find elevated levels of plasma T and plasma CORT after staged encounters, suggesting that the increased territorial locomotor behavior (patrolling) and agonistic displays (pushup behaviors) are driven by the monoaminergic system. Other behavioral signals including a darkening of the eyespot in A. carolinenesis males that win encounters have been linked to the monoaminergic system, including elevated epinephrine and norepinephrine (Summers & Greenberg, 1994). Monoaminergic systems also regulate endocrine responsiveness (e.g., 5-HT stimulates CRF release).
2.9. Parental CaredCrocodilians as Model Organisms Parental care in the form of egg guarding is rare in reptiles (Shine, 1988). However, there are species that tend eggs or guard nests. Roughly 4% of squamate reptiles (lizards and snakes) display some form of egg guarding by females (Shine, 1988). In contrast, 38% of crocodilians have biparental care and 62% have female-only care (Shine, 1988; Platt & Thorbjarnarson, 2000; Reynolds, Goodwin, & Freckleton, 2002). A key question is: do hormones permit a switch in male behavior to tend young rather than pursue additional mating opportunities to increase fitness? There is evidence that T suppresses parental behavior in birds (Ketterson & Nolan, 1999; Hau, 2007), but the evidence is equivocal for other taxa. Oliveira, Hirschenhauser, Carneiro, and Canario (2002) found little support that T
reduces parental care behavior in reptiles and other ectothermic vertebrates (Oliveira, 2004). Moreover, Oliveira (2004) reviewed evidence suggesting that PRL is involved in paternal care behaviors in a diverse array of vertebrates including fishes, birds, and mammals (see also Schradin & Anzenberger, 1999). However, the possible role of PRL in biparental care in reptiles, and in particular crocodilians, remains to be elucidated.
3. SOCIAL NETWORKS AND ENDOCRINE NETWORKS Social system networks can be coordinated with endocrine networks because social interactions commonly affect endocrine interactions within individuals and vice versa (i.e., hormones affect social behavior and social cues affect hormones). As in endocrine networks (Figures 8.3–8.6), social networks (Figure 8.6) can be depicted as positive (solid arrows) and negative (dashed arrows) interactions that generate regulation in some aspect of system performance. Consider a population composed of an r-strategist, which produces large clutches of small eggs, and a Kstrategist, which produces small clutches of large eggs (Sinervo et al., 2000b). Throughout this discussion we highlight strategy types that have a characteristic genetic/ endocrine profile and gain a fitness advantage as a function of frequency or density of the alternative strategy types. Systems of r- and K-strategists can generate oscillatory density cycles, stabilize on a population density with both types present, fix on the r-strategist, or fix on the K-strategist. Under the oscillatory scenario, strong negative regulation of r-strategists on self types, when the population exceeds carrying capacity, allows the K-strategist to invade during the population crash. After the population has crashed, the K-strategist replaces the r-strategist. As the population recovers from low density, the r-strategist, by virtue of a release of self-regulation, can outcompete the Kstrategist. The life history of each strategy type becomes adapted to a density cycle. The corresponding social regulatory network is depicted in Figure 8.6(a). The stress hormone CORT responds to social stress and crowding differentially in r- vs. K-strategists. In sideblotched lizards, the r-strategist with an orange throat is much more responsive to crowding, and levels of CORT (Figure 8.4) are elevated in response to orange neighbors compared to the effects of either neighbor type on the yellow-throated K-strategy type (Comendant et al., 2003). As noted above, CORT has cascading effects on progeny behavior in L. vivipara and U. stansburiana. Thus, the full regulatory network for social and endocrine effects would couple Figure 8.6(a) to Figure 8.4 at the input labeled: ‘1. Biotic inputs: Social crowding by female morphs.’ Neighborhood frequency experienced by a female provides
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Hormones and Reproduction of Vertebrates
(a)
POPULATION REGULATION NETWORK
(b) HAWK–DOVE GAME NETWORK Dove is Neutral on Self
Strong negative self-regulation
DOVE=O
r-strategy REGULATORY FEEDBACK LOOP: positive versus negative population regulation
r-type
REGULATORY FEEDBACK LOOP: positive versus negative frequency regulation
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Hawk type
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(C) HAWK–DOVE–BULLY NETWORK
(d) GENERIC SOCIAL RPS NETWORK Positive self-regulation
Dove is nearly neutral on self
MULTIPLE REGULATORY FEEDBACK LOOPS: positive versus negative frequency and population regulation generate equilibrium or oscillations
Cooperation
DOVE = oo REGULATORY FEEDBACK LOOP: positive versus negative frequency regulation
Dove type HAWK
Hawk type
BULLY=yo
Strong negative self-regulation
C-type
Weak negative self-regulation D type
Strong negative A type self regulation
DOVE HAWK=yy BULLY PLASTICITY
Aggression Deception Deception has neutral effect on regulation of A type relative to self regulation of D type
Hawk type
(e) RPS of European Common Lizard
(f) RPS in side-blotched lizards
white allele
blue allele by PLASTICITY
w-genotype by play B
bb genotype
o genotype y genotype yellow allele
oo, bo, yo genotypes by, yy genotypes
orange allele
yellow allele
orange allele by play Y by PLASTICITY
FIGURE 8.6 Social networks of organisms can also be depicted in network diagrams with positive (solid line) and negative (dashed lines) regulation between non-self types or between self types (see Section 3). (a) Network diagram for a system of density genotypes, r- and K-strategists, such as the side-blotched lizard or European common lizard. (b) Network diagram for a system of Hawks and Doves in which Hawk is strongly self-regulating but benefits from a high frequency of Dove. The Hawk–Dove system is likely to govern the alternative reproductive strategy (ARS) of the ornate tree lizard, Urosaurus ornatus. (c) Network diagram for a system of three genotypes (oo, oy, yy) that generate Hawk (yy), Dove (oo), and Bully (yo) phenotypes for a social system of female morphs of the European common lizard, which involve density strategies. The Bully strategy is heterozygous for both alleles and can plastically change phenotype depending on social situation (Vercken, Massot, Sinervo, & Clobert, 2007; Vercken et al., 2008; in press). (d) Network diagram for a system of three density types: a cooperative type that exhibits positive self regulation, an aggressive type that exhibits negative self regulation, and a deceptive type that exhibits weaker self-regulation. This system is observed in nature in (e) European common lizards, which express o, w and y color alleles, and (f) side-blotched lizards, which express a parallel system of o, b, and y alleles. The heterozygote genotype in side-blotched lizards (by) reflect a Bully whose phenotype is plastically altered by social situation; e.g., when orange is common, by adopts a sneaker profile with low T; when orange is rare and yellow or blue is common, by adopts a territorial blue strategy and can even engage in cooperative interactions with blue males.
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Hormones and Behavior of Reptiles
the information necessary to predict future density-dependent selection on progeny (see Section 1.4). The conserved HPG and HPA endocrine network of vertebrates (Licht et al., 1977), exemplified by Uta, is likely to be similar across all vertebrates. With minor modifications, the network structure of r–K strategists can be applied to a system of aggressive and nonaggressive personality types involved in a Hawk–Dove dynamic (Smith, 1982) (Figure 6(b)). Korte et al. (2005) reviewed the endocrine, behavioral, and metabolic networks governed by the HPA axis that regulate the expression of Hawk-like and Dove-like behavioral strategies. Besides this regulatory control over Hawk–Dove behavior by the endocrine system, Hawks and Doves also regulate one another in a social network. Hawk exhibits strong negative self-regulation, but the frequency of Hawk responds positively to Doves. Dove on the other hand is neutral to self, but contracts in frequency when subjected to more Hawks. This system will equilibrate with Hawk and Dove if the negative effects between Hawks are great enough to allow invasion of Dove; otherwise, it fixes on Hawk. The lizard species listed above with dimorphisms are likely to exhibit Hawk–Dove games (Figure 8.6(b)), such as the two color morphs of U. ornatus (Table 8.1). Vercken et al. (2007) describe interactions among r-vs. K-strategists of female L. vivipara that resembles a variant of the Hawk–Dove game referred to as Hawk–Dove–Bully (Figure 8.6(c)), in which homozygote female L. vivipara oo and yy are Hawk and Dove, respectively (Vercken & Clobert, 2008a) and Bully is the heterozygous by genotype that can play either Hawk or Dove depending on the local frequency of each. When Hawks are common, Bully plays Dove, but, when Dove is common, Bully plays Hawk. This allows the heterozygous strategy to gain higher fitness and a heterozygote advantage. Although the endocrine induction of Bully in female L. vivipara is currently unclear, it is likely related to density and frequency effects modulated by CORT based on exogenous manipulations. These observations on morphs of female L. vivipara are salient for other plasticities observed in male ARSs in U. ornatus (Knapp et al., 2003; Knapp, 2004) and U. stansburiana (Sinervo et al., 2000a; Sinervo, 2001). In all these systems, social neighbors induce gated switch responses in behavior in specific genotypes; in males of both lizard species, these are the sneaker genotypes (Moore et al., 1998; Sinervo et al., 2000b). Hawk–Dove interactions of two players generalize to systems with three strategies (Sinervo & Lancaster, 2009) with the addition of a cooperative type, which often evolves in social systems. The cooperative type generates positive self-regulationdmore cooperators (in a group) enhance recruitment of self. Cooperation is vulnerable to invasion by aggression (i.e., Hawk-like type), which is self-limiting and generates negative self-regulation. Aggression is vulnerable to invasion by a deceptive (e.g., Dove-like
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strategy or sneaker strategy) type that is immune to or avoids aggression. Thus, non-aggressive deceptive types (e.g., sneakers), by virtue of lower levels of negative selfregulation, can increase in frequency when aggressive types are common. Cooperative types, which thwart deception via cooperation, benefit from a high frequency of deceptive types. A rock–paper–scissors (RPS) social system ensues from Hawk–Dove–Cooperator (e.g., aggressive– deceptive–cooperative) social interactions. The RPS systems can either stabilize with all three types preserved or it can cycle in frequency of all three types (Sinervo et al., 2007). Rock–paper–scissors networks are common in organisms (Figure 8.6(d–f)). For example, European common lizards (Figure 8.6(e)) and side-blotched lizards (Figure 8.6(f)) exhibit similar RPS frequency regulation. In general, any social organism demonstrating cooperation will generate similar frequency regulation and complex social tradeoffs (Figure 8.6(d)). Selection in such systems will operate on endocrine responses within and among individuals of each type. For example, cooperation is strongly stabilized by an endocrine response that attenuates aggression to self types, while aggressive types benefit from heightened agonistic behaviors induced by endocrine responses to all types (e.g., T-modulated), but particularly other territorial types, and this involves the adrenal response (Knapp & Moore, 1996). Deceptive strategies are likely to be under facultative or plastic control, being submissive when dominants are around but aggressive when confronting self types (Sinervo, 2001). Cooperative behavior is advantageous in interactions with deceptive types, provided aggressive self types are not present in local neighborhoods. Deception is advantageous in situations with a high frequency of aggressive types. The concepts of endocrine plasticity in Bully genotypes discussed above for female L. vivipara also generalize to the RPS of Uta and observed endocrine plasticity in the by genotype in particular (Figure 8.6(f)). When orange genotypes are common, by genotypes adopt a sneaker strategy, but, when orange genotypes are absent and thus blue and yellow are common, by genotypes adopt a blue throat color (Sinervo et al., 2000a) via GTH stimulation of T (Mills et al., 2008). Analogous behavioral plasticity occurs in the orangethroated genotype of U. ornatus, which switches between sedentary satellite and nomadic behavioral tactics (Moore et al., 1998). The switch between nomadic vs. satellite tactics is linked to the gated switch action of P4 and CORT (Knapp et al., 2003), which more strongly suppresses plasma T in the orange compared to blue-orange morph (Knapp & Moore, 1997). In this case social interactions do not directly induce the switch; instead, it may be triggered by drought conditions. However, it is still possible that the drought per se generates elevated agonistic social interactions within and between morphs that result in elevated
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CORT in both types and, by virtue of the more resistant T levels of the blue-orange genotype, induce the orange morph to adopt an alternative tactic. The actual control mechanisms may involve steroid hormone-binding globulin (SHBG) activity or levels. Jennings et al. (2000) found that U. ornatus possesses two SHBGs: an androgenglucocorticoid-binding globulin (AGBG) and a typical SHBG. The capacity of AGBG is higher in the territorial blue-orange morph than in the non-territorial orange morph and accounts for significant binding of both CORT and T. The behavior of the orange morph, due to the lower AGBG capacity, switches from satellite to nomadic type as CORT levels rise.
3.1. Social Groups, Monogamy, Filiative Behaviors, and the Suppression of ‘Aggression’ Whereas endocrine responses promoting heightened aggression are strongly favored by sexual selection in aggressive types, cooperative types are selected for the opposite strategy and a suppression of endocrine responses for heightened aggression (Sinervo et al., 2006b). Very little is known about the endocrine suppression of aggression in the context of cooperative behavior in vertebrates in general or reptiles in particular. Great attention has been focused on the role of adrenal glucocorticoids in the suppression of aggression in dominant–subordinate relations (Summers et al., 2005), which is distinctly different from the suppression of aggression required for advanced cooperation. Complex social systems have only recently been discovered in lizards, including both advanced nuclear family structure in large groups (Gardner, Bull, Cooper, & Duffield, 2001; Chapple, 2003; O’Connor & Shine, 2003) and greenbeard selection, defined in the following paragraph (Sinervo & Clobert, 2003; Sinervo et al., 2006b; Sinervo & Clobert, 2007). The blue male genotype of the side-blotched lizard exhibits remarkable cooperative behavior in which one male will sacrifice fitness for a partner when the cooperative dyad is attacked by an orange neighbor (Sinervo & Clobert, 2003; Sinervo et al., 2006b). The deep field pedigree (20 generations) for U. stansburiana reveals that neighboring blue cooperators are not related by genealogy, yet they resemble brothers in levels of gene sharing (Sinervo & Clobert, 2003; Sinervo et al., 2006b). Genes for attraction to the blue color will be shared between blue males that are mutually attracted to blue. Other genes for self-attraction enhance the stability and fitness of pairs of cooperative blue males. Thus, the blue genotype finds other genotypes with which they share a large number of genes, via such self-attraction loci. These genes were uncovered by gene mapping behavioral traits like
Hormones and Reproduction of Vertebrates
settlement preference for blue neighbors or other genes that stabilize and enhance blue cooperation (Sinervo et al. 2006b). Genes that control such social interactions are referred to as ‘greenbeards,’ and Hamilton (1964) theorized that true altruism, the expression of care between unrelated individuals, might evolve if a supergene simultaneously affected a signal and recognition of the signal, and if signal recognition elicited social acts costly to donors but beneficial to recipients. Dawkins (1976) coined the term ‘greenbeard’ for Hamilton’s social supergene in a hypothetical example of human altruists that sported a green beard distinct in color from other beards sported by non-altruists. Sinervo and Clobert (2003) demonstrated that greenbeard genes and the self-attraction loci can be distributed across the genome, thereby enhancing and stabilizing cooperation (Sinervo et al., 2006b). Hamiltonian greenbeards also pertain to concepts of kin relatedness, but they apply directly to single genes and shared alleles, not merely genealogical sources of gene sharing. Thus, the concepts of kin cooperation, coloniality, and observed nuclear family structure noted above are likely stabilized by suppression of aggression, as in the case of the greenbeard of U. stansburiana. The greenbeard cooperation of U. stansburiana is not unique. The lizard L. vivipara exhibits evidence of similar evolutionary cooperation and both species have a similar RPS social system despite 175 million years of independent evolution (Sinervo et al., 2007). A recent review of lizards in the family Phrynosomatidae indicates that the RPS colors have evolved 13 times independently and are present in 57 species out of 274 taxa/populations sampled and included in a composite phylogeny, based on ancestor reconstructions. Only dimorphic systems are more common, occurring in 65 extant taxa in our sample. Both Uta and Urosaurus exhibit trimorphic RPS social systems, but the RPS social system is a very common mating system in the genus of Sceloporus lizards, particularly in the viviparous species of Sceloporus lizards. The bb genotype of U. stansburiana can form large clusters of adjacent territorial males of up to 12 bb or by males. These clusters resemble a primordial colony structure; even though each individual blue male retains a territory, their territories overlap extensively. In some instances we have observed up to four bb males within a two-meter radius of one another, suggesting remarkable tolerance, particularly compared to the orange male genotype, which is completely intolerant of territorial incursions by either orange or blue genotypes (Sinervo & Clobert, 2003). Incipient coloniality in RPS species may provide a unique, but as yet completely unexploited, opportunity to study the suppression of the endocrine-based aggressive response. The endocrine system is likely to be controlled by the key set of genes by which suppression of aggression is achieved between cooperating and tolerant neighbors. By sharing
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Hormones and Behavior of Reptiles
endocrine-related alleles for suppressed aggression, for example, bb genotypes of U. stansburiana would form a much more stable territorial association. A likely place to search for such shared genes is among the factors that regulate gonadal steroid hormones like T, such as GTHs, AGBG, SHBG, or perhaps the steroidogenic enzymes that modulate metabolic conversion to T and metabolism of T (Knapp, 2003). It is unlikely that the suppression of aggression occurs via the adrenal response (Summers et al., 2005), which tends to shut down reproductive function in the context of dominance and subordinate relations. In reptiles with more advanced social systems, both sexes are under strong social selection. Social selection includes selection on behaviors that mediate settlement and spacing behavior, and behaviors for interactions among members of social groups. One of the key factors mediating the evolution of sociality in lizards may be the evolution of monogamy (Bull, 2000). For example, of the reptilian species that have evolved coloniality, many live in monogamous family groups (Bull, 2000; Chapple, 2003), and, intriguingly, most of these species are viviparous. The blue genotype of U. stansburiana also forms a genetic selfattraction mechanism (e.g., for neighboring blue genotypes) among males and females, owing to high levels of shared genes for self-attraction (Sinervo et al., 2006b) and this promotes higher levels of monogamy in the blue genotype (Zamudio & Sinervo, 2000) compared to orange or yellow types, which tend to be quite promiscuous. Interestingly, color polymorphism is common in colonial species of Egernia (Chapple, 2003), and positive assortative mating within morphotype has been reported in some populations of Egernia whitii (Milton, 1987; 1990) of the form observed for the three color morphs of side-blotched lizards (Sinervo et al., 2006b; Bleay & Sinervo, 2007). Here we suggest the novel hypothesis that viviparity per se predisposes lizards to an endocrine profile of suppressed aggression and the evolution of coloniality. An alternative route to proto-coloniality is the suppressed aggression exhibited by cooperative RPS genotypes. We have inferential evidence that PRL is responsible for both behavioral syndromes and here we present the arguments for the first time.
3.2. The Potential Role of Prolactin as the Master Regulator of Reptilian Behaviors Inferences from other vertebrate groups (birds, fishes, mammals) suggest that PRL may be the key hormone related to maternal and paternal care behaviors (Schradin & Anzenberger, 1999) (Figure 8.7). In viviparous reptile species, the association of mother and progeny would allow for natural filiative behaviors to be established by direct contact after birth, which is not as likely in oviparous
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species in which eggs are deposited in soil or cavities and progeny hatch at some time point after the female has gone through the hormonal stages in preparation for egg-laying. The evolution of viviparity has a number of salient endocrine changes that by inference link it to PRL regulation. For example, viviparity has been linked to changes in the regulation of prostaglandins in the uterus (Guillette, 1993). In other vertebrates, PRL upregulates prostaglandins via phospholipase A2 prostaglandin G/H synthase 2 (BoleFeysot et al., 1998). Prolactin also increases P4 receptor number in the uterus and decreases rates of P4 metabolism in mammals. Particularly noteworthy is PRL’s role in regulating FSH receptors and LH receptors in the gonads of other vertebrates (Schradin & Anzenberger, 1999). The broad spectrum of PRL’s effects make it a likely candidate not only in the evolution of alternative strategies such as viviparity but also in the evolution of alternative strategies seen in RPS social systems. The other key behavior necessary for regulation in the context of sociality is dispersal vs. philopatry (e.g., sociality is enhanced when philopatry generates family groups). Dispersal and migration behavior is also linked to PRL and thyroxine regulation in diverse vertebrates (Rankin, 1991). Whereas colonial lizard species exhibit marked philopatry (Chapple, 2003), variation in dispersal syndromes of RPS species such as L. vivipara and U. stansburiana are genetically correlated with morphs (Cote et al., 2006; Sinervo et al., 2006a). The sheer number of traits genetically correlated with endocrine systems regulated by PRL strongly suggests that PRL/PRL receptor (PRL-R) is the endocrine system responsible for both the RPS morphs and many instances of viviparity. Unfortunately, there is a dearth of reptilian studies on PRL and the PRL-R. A recent molecular analysis of the tissues in which PRL/PRL-R is expressed in the gecko indicates mRNA for PRL in the whole brain, pituitary, and oviduct, ovary, and testis, but not other tissue, and widespread presence of PRL-R mRNA in all tissues tested (Kato, Ikemoto, & Park, 2005). This indicates that PRL secretion by the brain has a classic endocrine role (Kato et al., 2005), in which it circulates to other tissues and effectuates changes in physiology and behavior, and potential paracrine and autocrine roles, in which it can also respond in the same or adjacent tissues. This new finding should allow reptilian behavioral endocrinologists to develop their own molecular probes to investigate the role of PRL in the evolution of viviparity, sociality, and ARS (e.g., RPS systems). Until now, mammalian analogs of many reptilian protein hormones have proven ineffective (e.g., receptor and hormone assays (see Knapp, 2003)), including the mammalian PRL (Mills & Sinervo, unpublished data). At the same time, great progress has been made in using heterologous GTHs in eliciting behavioral and endocrine effects in reptiles (results cited in Section 1.4).
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We suggest that the regulatory role of PRL should be a new focus of behavioral endocrinology in reptiles (Figure 8.7). We hypothesize that mutations in PRL, and in particular PRL-R, would be an extremely likely candidate for a gene controlling the evolution of the suite of traits related to alternative strategies, viviparity, and advanced coloniality observed in reptiles. Our hypothesis is advanced because of the suite of traits known to be genetically correlated (Sinervo & Svensson, 2002) with the color morph locus of U. stansburiana, including clutch size (regulated by GTH), male aggression and territorial behavior (regulated by T and GTH), dispersal behavior, immune function (regulated in part by PRL), and the expression of color with the reproductive shedding of skin (regulated by PRL in other vertebrates). (Prolactin effects in reptiles and other vertebrates are reviewed in Bole-Feysot et al. (1998).) In particular, the PRL-R is a classic membrane-bound signal transduction receptor. PRL-R also has been shown to be a member of the same family as the growth hormone (GH) receptor and also part of the larger class of receptors known as the class 1 cytokine receptor superfamily (Bole-Feysot et al., 1998). Although approaches to understanding the organizational effect of hormones on behavior in reptiles have identified a number of potential steroidal control points, all of these are potentially regulated by PRL/PRL-R, including the production of diverse steroid hormones by
Hormones and Reproduction of Vertebrates
the adrenal (Bole-Feysot et al., 1998), which, as noted above, have a role in organizing behaviors in embryos and juvenile mammals. The significance of PRL in regulating behavior of reptiles is greatly under-studied. The genes directly controlling alternative reproductive strategies in vertebrates have not yet been identified, despite an exhaustive search by many research laboratories, but, intriguingly, to our knowledge PRL/PRL-R has not yet been posited as a control system for such behavioral syndromes. The links between the evolution of viviparity and control over salient hormones (P4, prostaglandins) would provide a parsimonious pleiotropic endocrine mechanism, PRL/PRL-R, that links diverse phenomena, including the evolution of sociality, morphs, and viviparity. Our understanding of the endocrine control of reptilian behavior has largely been focused on the agonistic behaviors that evolve under sexual selection on male display behavior. As noted above, it is clear that the HPG axis regulates the expression of such male behavior. Less is known about the endocrine control that shapes adult female behavior, despite the interesting and nuanced behaviors that evolve under social selection to generate care. Network visualization generalizes concepts of regulation to social forces that govern selection and life-history tradeoffs that govern the evolution of endocrine system control of behavior. Social tradeoffs arise directly from social competition (density regulation) and cooperation that
< FIGURE 8.7 The endocrine control of behavior and life history, depicted for a generic vertebrate in which resource allocation to competing functions of growth regulation, metabolic regulation (homeostasis), immunocompetence and immune system regulation, reproduction, and life-history behaviors such as parental care and dispersal/migration. The topology of endocrine system network interactions that generate behaviors is multi-dimensional, which we depict folded as a tetrahedron (lower left). Endocrine systems at each vertex interact in positive and negative regulatory feedback loops, and the triangular ‘panes’ forming the sides of the tetrahedron meet at adjacent vertices where regulatory feedback loops can act. Such loops can also act along the edges separating the vertex on a given triangular pane (e.g., gonadotropin hormone (GTH)–gonadal). The opposing vertex (e.g., adrenal) forms a gated switch. Real networks are more complex than the tetrahedra we use to depict network interactions, but the examples we use are holistic in that many essential functions for homeostasis are included (e.g., thermoregulatory, metabolic, immunological, behavioral). Detailed discussion of the hierarchical structure of the endocrine system can be found in Figures 8.3, 8.4, and 8.5 and in Sinervo and Calsbeek (2004) and Sinervo and Clobert (2008). We have clustered those hormones that largely affect juvenile life history on the left, those that largely affect adult life history on the right, and those related to the immune system, which is generic to both stages, at the bottom. Major regulatory feedback loops. The endocrine regulators of behaviors and life-history function (progeny investment, care behavior, dispersal vs. philopatry, migration, territoriality, coloniality) are found in the hypothalamus and pituitary, which produce the releasing hormones (RH) such as prolactin-releasing hormone (PRL-RH), thyrotropin-releasing hormone (TRH), corticotropin-releasing factor (CRF), and gonadotropin-releasing hormone (GnRH). In vertebrates, growth regulation (and metamorphosis, if present) is achieved by the thyroid hormones, the metabolically active T3, and its precursor T4, in interaction with brain prolactin (PRL) pathways and/or growth hormones. The hormone PRL is an extremely important but as yet under-studied hormone from the perspective of reptilian behavior. Prolactin interacts with diverse endocrine systems including those controlling metamorphosis; the molt cycles of reptiles, amphibians, birds, and mammals (Boyle-Feysot et al., 1999); the regulation of dispersal and/or migration behaviors (Rankin, 1991); and the expression of parental care in both sexes of several vertebrate groups (Shraden & Anzenburger, 1999). Neuroendocrine interactions. While PRL plays a major role in the regulation of parental care in both sexes in mammals, neuroendocrine hormones such as arginine vasopressin (AVP) play a role in generating care behaviors in males whereas the octapeptide oxytocin plays a role in female mammals. Pair bonding in mammals arises in brain regions such as the nucleus accumbens and involves dopamine (DA) (Goodson & Bass, 2001). In reptiles, AVT, the reptilian homolog of AVP, has a more ancient role in regulating territorial behavior (see Section 2.7). Arginine vasotocin, in interaction with testosterone (T) and brain regions with AVT-sensitive neurons such as in the POA, generates behaviors related to territoriality and sexual behavior. Dominant and subordinate lizards differ in both AVT-sensitive neurons in the anterior vs. posterior preoptic area (POA), respectively (see Section 2.7). Besides AVT, DA turnover (catecholaminergic) and 5-HT turnover (serotonergic) in the diencephalon and telencephalon (metabolism of each neurotransmitter is shown) also differ between dominant and subordinate lizards.
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abound in natural systems (Sinervo et al., 2007; 2008). Social networks can also provide exogenous cues regarding the events that govern endogenous endocrine networks of organisms. In many cases, individuals in social networks induce gated switch effects in the internal endocrine networks of conspecifics through social stresses (e.g., aggressive types exert dominance and higher levels of CORT in neighbors). This review highlights the growing body of evidence demonstrating the impact of hormones on sexual behavior in reptiles. Clearly, sex steroids are critical in generating
Temperature Regulation
Juvenile PRL and TSH involved in molt of reptiles, amphibians, mammalian pelage, and avian plumage
Growth/Molt Regulation Growth
many patterns of sexual behaviors. However, other hormones, notably PRL, AVT, and glucocorticoids, modulate the action of sex steroids. The effects of hormones have major ramifications for individual fitness through multiple pathways, which govern life-history patterns and tradeoffs between life-history traits (Hau, 2007; Sinervo & Lancaster, 2009; Sinervo et al., 2008). Because sex steroids simultaneously modulate multiple trait complexes that ultimately influence lifetime reproductive success, natural and sexual selection will operate on the complex interactions among hormone systems
Pineal
Pituitary Hypothalmus PRL-RH PRH TRH GnRH CRH
PRL TSH
IGFI, IGF2 Insulin
Dispersal–Migration regulation
Adult
PRL has a role care in birds, fish, mammals, and perhaps amphibians (?)
Supraoptic nucleus AVT
Amygdala complex
AVT
Diverse NeuroEndocrine systems
CNS
PRLR cell Neurotransmitters membrane in Dom/Sub males: 5-HTP Receptor mineralocorticoid 5-HT - dopamine 5-HT - serotonin glucocorticoid receptors 3,4-DOPAC 5-HIAA
PRL
ACTH GTH
POA: AVT sensitive neurons Dom/Sub
Regulation of Territoriality
Thyroxine metabolism T3
Monoidonase T4
Tissue specificity
alpha & beta nuclear T 3 receptors (retinoid X)
T4
Thyroid
gene transcription
Metabolic Regulation
AVT Care
E CORT
FSH/LH Receptors
CORT
Adrenal
Oxytocin
P
Gondal
CORT
E
T (-ve Birds) (+ve Fish)
Stress CORT Regulation
Regulation of Care and Pair Bond in Mammals
AVP mammals
Estrogen Response Elements (ERE)
Many Genes
Hormone Response Elements (HRE)
HP tropins
Territoriality PRLR cell membrane Receptor macrophages superoxides
Adrenal
Gonadal
Thyroidal Growth
Reproductive System
Care
Immunological
Neuroendocrine
Humoral Cell-mediated
Immune System
Endocrine Hierarchy Master Endocrine Organ System of negative (dashed lines) and positive regulation (solid lines) Endocrine Organ Endocrine Series of Gene Receptors Organ Cascades Alternative Alternative Series Strategies of Gene Cascades
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through the process of correlation selection by which multi-trait combinations are favored (reviewed in Miles et al., 2007; Sinervo et al., 2008). The next phase in evolutionary behavioral endocrinology is to focus on how individual variation in sex steroids affects variation in behaviors and the physiological traits that are linked with behaviors. This research program (as highlighted in Figure 8.2) argues against sex steroids as constraints and rather highlights the potentiating (or gated response) roles of other hormone systems (adrenal, PRL, etc.) in promoting life-history variation and social and mating system diversity.
ABBREVIATIONS a-MSH 5-HT ACTH AGBG AH ARS AVP AVT BSTm CBG CNS CORT CRF CRH DA DHT E2 ERE FSH GnRH GTH HP HPA HPG HRE L-DOPA LH P4 POA PRL PRL-R RPS SHBG T TSD
a-melanocyte-stimulating hormone Serotonin Corticotropin Androgen-glucocorticoid-binding globulin Anterior hypothalamus Alternative reproductive strategy Arginine vasopressin Arginine vasotocin Bed nucleus of the stria terminalis Corticosterone-binding globulin Central nervous system Corticosterone Corticotropin-releasing factor Corticotropin-releasing hormone Dopamine Dihydrotestosterone 17b-estradiol see HRE Follicle-stimulating hormone Gonadotropin-releasing hormone Gonadotropin Hypothalamus Hypothalamus–pituitary–adrenal Hypothalamus–pituitary–gonadal Hormone response element L-3,4-dihydroxyphenylalanine Luteinizing hormone Progesterone Preoptic area Prolactin Prolactin receptor Rock–paper–scissors Steroid hormone-binding globulin Testosterone Temperature-dependent sex determination
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Chapter 9
Viviparity in Reptiles: Evolution and Reproductive Endocrinology Lori C. Albergotti and Louis J. Guillette Jr. University of Florida, Gainesville, FL, USA
SUMMARY Among vertebrates, squamate reptiles exhibit a significant number of transitions from oviparity to viviparity. This high frequency of occurrence coupled with a complete absence of viviparity in other reptiles has made the study of squamate viviparity intriguing for the past century. In order for viviparity to fully evolve, viviparous species must overcome three major morphological and/or physiological modifications. First, egg retention time must be increased so that the embryo completes development in utero. Second, eggshell thickness must be decreased to reduce the physical barrier between embryonic and maternal environments. Third, a placenta must develop to facilitate maternal and embryonic exchange. This chapter provides a brief overview of some of the work that has shaped our understanding of these modifications associated with the evolution of viviparity. Further, based on recent work in our laboratory, we present a new hypothesis for the evolution of endocrine extraembryonic membranes, including the placenta, of amniotes.
1. INTRODUCTION The evolution of viviparity has been of considerable interest for over 100 years. In part, this has been driven by an interest in understanding the reproductive adaptations in mammals that were seen as unique in vertebrates. These adaptations included retention of the embryo until development is largely completed; the development of a maternal–embryonic exchange organ, the placenta; the evolution of maternal chemical communication, including maternal recognition of pregnancy and placental–maternal endocrine regulation of maternal and embryonic physiology; and the development of mammary glands. Over the last three decades, research efforts have focused on each of these aspects and today we recognize that all of these ‘mammalian adaptations’ except mammary gland evolution, are, in fact, not uniquely mammalian, but present in other vertebrates. Work with various reptilian species, largely squamates, has helped to clarify our understanding Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
of the evolution of viviparity, and this chapter provides a relatively brief overview of some of the work that has changed our understanding. Further, based on recent work in our laboratory, we present a new hypothesis for the evolution of endocrine extraembryonic membranes, including the placenta, of amniotes.
2. DEFINING REPRODUCTIVE MODE The terminology associated with reproductive mode in squamates has varied over the last four to five decades, with three terms commonly used in the literature: ‘oviparity,’ ‘viviparity,’ and ‘ovoviviparity.’ For the sake of clarity, we will discuss each of these terms briefly and define our usage of them in this chapter. Here, oviparity is defined as the oviposition of an embryo within a shelled egg requiring some period of embryonic development outside of the female’s reproductive tract (Guillette, 1993). The state of embryonic development at which point the egg is oviposited varies dramatically within reptiles. Crocodilians (Ferguson, 1987), turtles (Crews, 2003), and tuatara (Cree, Thompson, & Daugherty, 1995) are at one end of the spectrum and lay eggs with embryos at relatively early stages of development, whereas the majority of oviparous squamates generally exhibit some degree of egg retention, with many retaining embryos in utero for approximately one third to one half of the total embryonic developmental period (Shine, 1983; Andrews & Mathies, 2000). Regardless of the exact developmental stage at the time of oviposition, oviparous embryos continue embryonic development in an external environment, generally in a nest of some kind. Viviparity is defined as the retention of an embryo in utero for the duration of embryonic development, resulting in the live birth of a fully developed neonate (Guillette, 1993). This requires some modifications in the maternal uterine environment to (1) maintain pregnancy (e.g., uterine quiescence to retain the developing embryo) 247
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and (2) facilitate gas and water exchange for the developing embryo. Thus, with viviparity we see the emergence of placentae and complete embryonic development within a maternal environment. These two phenomena are present in species demonstrating incipient viviparity, even though embryos are laid within a shell membrane and generally hatch within hours of oviposition (Guillette, 1993). Ovoviviparity has been historically described as the retention of a shelled embryo within the reproductive tract until the embryo hatches, resulting in live birth (Guillette, 1993). Guillette (1993) argued against the use of this term in describing squamate reproductive biology on the grounds that the totally independent development of a shelled embryo within the uterus cannot occur without some specialization: a placenta, in order to meet the demands of embryonic water and gas exchange. Ovoviviparity, as historically used in most herpetological studies over the last five decades, describes complete embryonic development in utero and the development of a placenta, thus fulfilling the definition of viviparity. Thus, we will not use the term ovoviviparity to describe a reproductive mode in reptiles.
3. EVOLUTION OF VIVIPARITY WITHIN SQUAMATES The order Squamata encompasses lizards, snakes, and amphisbaenians (‘worm lizards’) (Evans, 2003; Townsend, Larson, Louis, & Macey, 2004; Vidal & Hedges, 2005). Squamate phylogenies reject monophyly within the lizards, but have yet to resolve the precise position of snakes within this order (Townsend et al., 2004; Vidal & Hedges, 2005). Within squamates, it has been estimated that there have been five (Shine, 1985) to six (Blackburn, 1999) familylevel and 58 (Blackburn, 1999) to 65 (Shine, 1985) subgeneric origins of viviparity. If we look at the number of genera containing subgeneric origins, we find that viviparity has evolved an estimated 13 times within snakes (Shine, 1985; Blackburn, 1999) and 27 (Blackburn, 1999) or 28 (Shine, 1985) times within lizards. It is currently estimated that viviparity has evolved independently over 100 times within squamate reptiles (Shine, 1985). This transition in reproductive mode from oviparity to viviparity has occurred more frequently in squamates (Blackburn, 1999) than in any other vertebrate lineage including fishes (Dulvy & Reynolds, 1997; Goodwin, Dulvy, & Reynolds, 2002), amphibians (Reynolds, Goodwin, & Freckleton, 2002; Gower, Giri, Dharne, & Shouche, 2008), and mammals (Zeller, 1999). This high frequency of occurrence in squamates coupled with a complete absence of viviparity in other reptiles (Shine, 1985) has made the study of squamate viviparity intriguing for a century.
Hormones and Reproduction of Vertebrates
This rate of independent evolutionary change in reproductive mode sets up two fundamental questions: why and how has viviparity evolved in this order of vertebrates more than in any other? Further, if the estimates on the number of independent origins of viviparity are correct, this implies that the genetic, morphological, and physiological modifications required for the evolution of viviparity in squamate reptiles are readily ‘obtained’dalready presentdwith the correct selective pressures. The why question of viviparity has been approached primarily from an ecological perspective, whereas the question of how viviparity evolves has been tackled by examining attributes of physiology, anatomy, and endocrinology. The focus of this chapter concerns how viviparity has evolved, but we first will review briefly the proposed selective forces and ecological correlations associated with the evolution of viviparity in squamates.
4. SELECTIVE FORCES FOR THE EVOLUTION OF VIVIPARITY A number of factors have been proposed as selective forces for the origin of viviparity in squamates. Indeed, some of the earliest researchers of reptilian viviparity (e.g., Weekes, Gadow, and Sergev) observed that this reproductive mode appeared to evolve more often in cold climates, at higher elevations, and in more northern latitudes respectively (as cited in Tinkle & Gibbons, 1977). Many studies have shown temperature, elevation, and latitude to be highly correlated with the evolution of viviparity in squamates. However, correlation does not infer causation, and identifying a direct cause of viviparity in squamates has proved challenging. As a selective pressure for viviparity, the cold climate hypothesis, which proposes that viviparity evolved in cold climates, has received the bulk of scientific investigation. In 1929, Mell (as cited in Tinkle & Gibbons, 1977) hypothesized that, in cold environments, viviparity would confer a selective advantage by retaining the embryo within the reproductive tract. Because maternal behavioral and metabolic thermoregulation can maintain maternal body temperature above ambient, maternal incubation would provide the embryo with a warmer internal environment than that found in an external nest. Therefore, since embryos incubated at cooler temperatures develop more slowly, embryos in an external nest would require increased developmental time and might experience increased likelihood of mortality due to environmental factors such as predation, drought, or flooding. When evaluating the proposed selective forces of viviparity, we should consider whether an advantage for evolutionary intermediates is plausible. In the transition from oviparity to viviparity, the embryo is retained within
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the reproductive tract for increasing periods of time until ‘hatching’ occurs internally. Thus, a selective force should confer an advantage for the retention of an embryo in utero for increasing periods of time. In this respect, the cold climate hypothesis either alone or in combination with high altitude or northern latitude is attractive. It is logical to expect that embryos retained in utero for any extended amount of time in these conditions could benefit from an increased developmental rate due to a warmer incubation regime. In contrast, other factors such as egg predation and arboreal or aquatic life history, which also have been suggested as possible selective forces for viviparity in squamates, have received criticism due to the lack of generality (Tinkle & Gibbons, 1977) or an obvious advantage for evolutionary intermediates (Guillette, 1993). However, there are problems with inferring causation of cold environments (cold climate, high altitude, northern latitude) as selective forces of viviparity. Certainly, correlations have been found for each of these factors individually or in combination when looking at the proportion of viviparous species. However, since reptilian species richness generally decreases with each of these factors, percentages should be regarded with caution. For example, in a comprehensive review, Tinkle and Gibbons (1977) compared the geographic distribution of viviparity in squamates. In one analysis, they compared the numbers of oviparous and viviparous species in North America with latitude. They showed that oviparous and viviparous species can be found in the same latitude ranges except for at the most northern latitude examined (55–60 N), which consisted of a single viviparous snake species. In examining the worldwide distribution of viviparity, Tinkle and Gibbons (1977) concluded that viviparous species were most abundant at mid-latitudes and least abundant in cold and tropical environments. In fact, oviparous and viviparous species are predominately distributed in similar temperature, latitudinal, and elevational environments (Tinkle & Gibbons, 1977; Guillette, Jones, Fitzgerald, & Smith, 1980), suggesting that these factors are not the exclusive selective force in driving a shift in reproductive mode. It has been suggested, and is certainly plausible, that viviparity did not evolve in response to thermal conditions, but allowed viviparous species to expand their range and exploit a less hospitable environment than oviparity would have allowed (i.e., exaptation) (Neill, 1964; Tinkle & Gibbons, 1977). Tinkle and Gibbons (1977) suggested that, in temperate zones with more variable environmental conditions, selection might favor females that retain eggs in utero for some period until optimal environmental conditions for oviposition are met. This hypothesis, which we call the ‘optimal climate hypothesis,’ offers a parsimonious explanation for the frequency of viviparous species in temperate regions and allows for the expansion of viviparous species
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into colder climates, higher elevations, and more northern latitudes. This hypothesis also offers an explanation for the relative rarity of viviparous species inhabiting the tropics (Tinkle & Gibbons, 1977), but does not explain why some of the most advanced examples of viviparity are found in tropical (e.g., Mabuya) or Mediterranean (e.g., Chalcides) environments. Indeed, the presence of viviparous squamates in the tropics has been hard to explain under the cold climate hypothesis. Exaptation has been offered as one possible explanation for the existence of viviparous tropical squamates (Webb, Shine, & Christian, 2006), but it is not directly testable. A second explanation involves a hypothesis put forth by Shine: the maternal manipulation hypothesis (Shine, 1995; 2004). This hypothesis suggests that viviparity could evolve in any environment in which differences between maternal body temperature and nest temperature exist (Shine, 1995) and in which embryos would benefit from increased thermal precision provided by maternal incubation (Shine, 1995; 2004). Shine suggests that this hypothesis does not exclude the cold climate hypothesis; rather, the cold climate hypothesis would be a specific example of the broader maternal manipulation hypothesis. Webb et al. (2006) examined the maternal manipulation hypothesis in a tropical species of death adder (Acanthophis praelongus) and determined that more precise thermal regulation resulted in larger offspring than incubation at more variable maternal temperatures (Webb et al., 2006). While more investigation is needed, this hypothesis does offer a more unified explanation for the current distribution of viviparous squamates. In conclusion, determining the selective pressures resulting in the origin of viviparity in squamates is complex and could involve unidentified processes. For instance, embryonic responses to incubation conditions such as gas and water exchange recently have been proposed as a possible selective force for the evolution of viviparity in squamates (Shine & Thompson, 2006). In addition, we suggest that epigenetic modifications in response to environmental factors, in part dependent on modulation of signaling molecules stored in yolk (see Section 11), likely play a role in a transition of parity mode. In the discussion above we have considered only the advantages to the developing embryo in the transition from oviparity to viviparity. However, viviparity confers certain maternal fitness advantages and costs that must be considered. For example, increasing the length of maternal incubation of offspring could increase maternal fitness by decreasing the possibility of offspring predation at the nest. However, increasing the length of maternal incubation could also decrease maternal fitness if gestation results in (1) increased predation risk of the mother herself due to decreased mobility or increased basking requirements, (2) decreased foraging ability, (3) increased energetic costs due
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to an increase in the precision of thermoregulation, or (4) decreased reproductive opportunities in a breeding season (Tinkle & Gibbons, 1977; Shine, 1980; Seigel, Huggins, & Ford, 1987; Charland, 1995). Just as there are tradeoffs between the mother and the developing offspring in the evolution of viviparity, numerous advantages and disadvantages are evident for both oviparous and viviparous reproductive modes (Neill, 1964; Tinkle & Gibbons, 1977; Guillette et al., 1980; Shine, 1980), which leads us to consider the directionality of this transition. Viviparity is considered to be the derived condition and as such it is generally assumed that this is a unidirectional transition with viviparity evolving from an oviparous ancestor (Neill, 1964; Tinkle & Gibbons, 1977; Guillette et al., 1980). The reverse transition can be considered improbable under the theoretical framework that once an organ or structure has been lost through evolution, it will not re-evolve in precisely the same form in that lineage (Dollo’s law) (Lee & Shine, 1998). For example, Tinkle and Gibbons (1977) suggested that the evolution of viviparity from oviparity is more probable because this transition requires primarily structural or functional losses, such as uterine shell glands. In contrast, the evolution of oviparity from viviparity requires primarily structural or functional gains; i.e., uterine shell glands would have to be re-evolved for the secretion of an eggshell (Tinkle & Gibbons, 1977). Lee and Shine (1998) tested this general assumption by examining the direction of evolutionary transitions between oviparity and viviparity in reptiles. They concluded that evolution of viviparity from oviparity has occurred more frequently and is more strongly supported than the reverse (Lee & Shine, 1998). While a reversal to oviparity from viviparity does not appear to be impossible, it does appear to be a rare event (Lee & Shine, 1998). We, like others before us, suggest that the origin of viviparity is unlikely due to a single, environmental factor, and commonality in this evolutionary process may be improbable because of the multiple origins of this trait and the resulting diversity of viviparous squamates. As importantly, we hypothesize that many of the morphological and physiological traits we consider unique to viviparous squamates likely are already present in oviparous ancestors, making the process less onerous than once thought.
5. REPRODUCTIVE MORPHOLOGY AND PHYSIOLOGY: REQUIREMENTS OR REFINEMENTS OF VIVIPARITY? The evolution of viviparity is accompanied by certain key characteristics including the lengthening of gestation until embryonic development is complete, the reduction of the eggshell, and the development of a placenta. There has been much discussion surrounding whether these
Hormones and Reproduction of Vertebrates
characteristics are requirements for viviparity or refinements of viviparity. In terms of requirements vs. refinements, it remains a challenge to distinguish between characteristics that occurred before, after, or concurrently with the transition to viviparity in squamates. We can, however, establish some potential baselines by studying the appropriate model systems/species complexes. In order for viviparity to fully evolve, viviparous species must make three major morphological and/or physiological modifications. Egg retention time must be increased so that the embryo completes development in utero. Second, eggshell thickness must be decreased to reduce the physical barrier between embryonic and maternal environments so that, at the least, gas and water can effectively be exchanged. Third, a placenta must develop to facilitate maternal and embryonic exchange. Due to the diversity of viviparous squamates and the number of proposed independent origins, these processes could have been achieved through different mechanisms, but then again limited by physiological and morphological constraints. We will discuss each of these modifications for viviparity in the subsequent sections as we explore potential pathways for the evolution of viviparity in squamates.
6. EGG RETENTION AND THE LENGTHENING OF GESTATION By definition, viviparity requires that the time of egg retention is increased so that the embryo completes development in utero: how is this lengthening of gestation accomplished? In order to understand the lengthening of gestation in squamates, we must understand the physiology of pregnancy and, thus, first turn our attention to the corpus luteum (CL) and its possible role in egg retention. In all vertebrates, the CL is a transitory endocrine organ formed from the post-ovulatory follicle (see also Chapter 4, this volume). Following ovulation, the remnants of the ovarian follicle’s granulosa and thecal cell layers undergo luteinization, which involves changes in vascularization, hypertrophy, and the accumulation of lipids (Saidapur, 1982). The CL functions in the secretion of hormones for some predetermined time period before degenerating (luteolysis). The natural lifespan of the CL varies in vertebrates, but is short-lived in most mammalian species unless implantation occurs. If implantation is successful, the life of the CL is ‘rescued;’ i.e., extended by embryonic factors associated with the maternal recognition of pregnancy (Saidapur, 1982; Rothchild, 2003). The endocrine properties of the CL are most often associated with the secretion of progesterone (P4) in nonmammalian vertebrates (Callard et al., 1992; Koob & Callard, 1999; Girling & Jones, 2003; Powell, Kavanaugh, & Sower, 2006), but the CL also has been shown to secrete
Chapter | 9
Viviparity in Reptiles: Evolution and Reproductive Endocrinology
other steroid and peptide hormones in eutherian mammals (see Volume 5) and viviparous squamates (Arslan, Zaidi, Lobo, Zaidi, & Qazi, 1978; Gobbetti, Zerani, DiFiore, & Botte, 1993; Niswender, Juengel, McGuire, Belfiore, & Wiltbank, 1994). In eutherians, P4 is associated with uterine quiescence and, as such, P4 secretion by the CL plays an important role in the maintenance of pregnancy until synthesis of this steroid hormone is assumed by the placenta in many species (Simpson & MacDonald, 1981; Rothchild, 2003; Spencer & Bazer, 2004; Spencer, Johnson, Burghardt, & Bazer, 2004). The CL is a major source of P4 in reptiles; therefore, it has been suggested that the CL in squamates could provide some hormonal control of gravidity via the secretion of P4 (Callard et al., 1992; Guillette, 1993; Custodia-Lora & Callard, 2002). Likewise, changes in the secretory activity of the CL have been hypothesized to play a role in the timing of oviposition or parturition by affecting oviductal response to arginine vasotocin (AVT), which, like its mammalian homolog oxytocin, induces smooth muscle contractions in the oviduct (Norris, 2007). In the reptiles that reproduce strictly by oviparitydthe tuatara, crocodilians, and turtlesdthe life of the CL is approximately the length of time it takes to shell the eggs (a few days to a few weeks) and it deteriorates shortly before or after oviposition (Guillette et al., 1995; Guillette & Cree, 1997), whereas the CL of squamates is maintained for varying periods of gravidity that can last for weeks or months (Uribe, Omana, Quintero, & Guillette, 1995; Guarino et al., 1998; Cruz & Mendez-de la Cruz, 1999; Martinez-Torres, Hernandez-Caballero, AlvarezRodriguez, Luis-Diaz, & Ortiz-Lopez, 2003). In oviparous squamates, the presence of an active CL is strongly correlated with egg retention and the timing of oviposition coincides with the loss of luteal activity (Jones & Guillette, 1982; Guillette, 1993). However, viviparous squamates show a great deal of variation in the life of corpora lutea with luteolysis occurring well before parturition in some species, which precludes any gross generalizations concerning the role of the CL alone in the lengthening of egg retention in viviparous species. This observation has been further confounded by studies examining the effects of surgical or chemical removal of the CL on gravidity (Table 9.1). We would hypothesize that, if the CL is critical in determining the length of egg retention or gestation in squamates, then removal of the CL should result in premature oviposition or parturition. This is the pattern observed in the oviparous lizards Sceloporus undulatus (Roth, Jones, & Gerrard, 1973) and Cnemidophorus uniparens (Cuellar, 1979), where surgical deluteinization results in early oviposition. However, experimental manipulations of the CL in viviparous species have been far from clear-cut. For example, Badir (1968) reported no effect of deluteinization on the timing of parturition in
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Chalcides ocellatus, whereas Callard et al. (1972a) reported that ovariectomy resulted in stillbirths and delayed parturition in Sceloporus cyanogenys. These differences could reflect species differences, but also have been suggested to be the result of differences in the stage of pregnancy when deluteinization was performed (Guillette, 1993). In oviparous squamates, circulating P4 concentrations generally decrease following luteolysis and subsequent oviposition (Fox & Guillette, 1987), whereas circulating P4 concentrations often remain elevated in viviparous squamates for some period of gestation, but that time period varies in species comparisons (Figure 9.1 and Table 9.2). At one end of the spectrum, we find examples in which circulating concentrations of P4 increase significantly and remain elevated for the duration of gestation, declining just prior to parturition as in the blue-tongued skink, Tiliqua nigrolutea (Edwards & Jones, 2001). Another common pattern is for plasma P4 to increase during early gestation, peak at mid-pregnancy, but then decline thereafter, either steadily as in the garter snake (Thamnophis elegans) (Highfill & Mead, 1975b) or well before parturition as in the skink Niveoscincus metallicus (Jones & Swain, 1996). In the southern snow skink (Niveoscincus microlepidotus) (Girling, Jones, & Swain, 2002a), which has an extended gestation lasting for approximately one year, P4 concentrations peak in early pregnancy and decrease to basal levels prior to hibernation. Progesterone also peaks at ovulation and declines steadily through gestation until the time of parturition, as described in the Western diamondback rattlesnake, Crotalus atrox (Taylor, DeNardo, & Jennings, 2004). Maintenance of the CL as well as its correlation with circulating P4 concentrations is also variable in viviparous squamates. Many studies have determined the CL to be the primary source of P4 in viviparous squamates and have reported a strong positive correlation between the persistence of the CL during gestation and circulating P4 concentrations in viviparous lizards (Fergusson & Bradshaw, 1991; Jones & Swain, 1996; Martinez-Torres et al., 2003) and snakes (Chan, Ziegel, & Callard, 1973; Highfill & Mead, 1975b). However, in Sceloporus jarrovi (Guillette, Spielvogel, & Moore, 1981) and Chalcides chalcides (Guarino et al., 1998), plasma P4 concentrations remain elevated late in gestation following luteolysis. In S. jarrovi, Guillette et al. (1981) found luteolysis to occur in conjunction with the development of the chorioallantoic placenta. This observation led to the hypothesis that another source of P4dthe placentadcould be important in the maintenance of gestation (Guillette et al., 1981). Such a hypothesis mirrors the pattern found in many eutherian mammals in which P4 synthesis by the placenta replaces that of the CL during gestation (Rothchild, 2003). As hypothesized, the chorioallantoic placenta of S. jarrovi synthesizes P4 (Painter & Moore, 2005) (see further
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Hormones and Reproduction of Vertebrates
TABLE 9.1 Effects of the removal of the corpora lutea (CL) or the entire ovary on pregnancy or parturition of viviparous squamates Structure removed
Stage of pregnancy
Results
References
Thamnophis spp. and Nerodia spp.
Ovaries
All stages
Two females aborted Normal gestation for 25 days post-operation
Rahn, 1938
Nerodia spp.
Ovaries
Early Mid
Clausen, 1940
Late
Resorption of all embryos Resorption or abortion of all embryos Normal parturition
Species SNAKES
Thamnophis sirtalis
Ovaries
Late
Normal parturition
Clausen, 1940
Storeria dekayi
Ovaries
Mid
Resorption or abortion of all embryos
Clausen, 1940
Bothrops spp.
CL
Unknown
All embryos died; females died 10 days after surgery
Fraenkel et al., 1940
Nerodia sipedon
Ovaries
Mid
Development and parturition normal in one female of three. Two females died
Bragdon, 1951
Thamnophis elegans
CL
Early
Development normal; parturition delayed
Highfill and Mead, 1975b
Ovaries and CL (Unilateral) Ovaries
All stages All stages All stages
Panigel, 1956
CL
All stages
Normal development and parturition Parturition delayed in most females Parturition delayed in two of three females
Sham-operated
All stages
Badir, 1968
CL
All stages
Normal development and parturition Normal parturition
Ovaries
Unknown
Lien and Callard, 1968; Callard et al., 1972a
Ovaries
Early
Normal development; parturition delayed Majority showed normal course of development. Parturition delayed or prevented, resulting in some fetal death
Xantusia vigilis
Sham-operated Ovaries CL
Early and mid Early and mid Early and mid
Majority of embryos aborted Majority of embryos aborted Majority of embryos aborted
Yaron, 1972; 1977
Trachydosaurus rugosus
Ovaries
Early
Embryonic resorption; stillbirth parturition delayed
Bourne, 1972
Bradypodion (Chameleo) pumilis
CL
Mid-late
Veith, 1974
Ovaries
Early and mid
Late embryos normal; early embryos resorbed Majority of embryos aborted or resorbed
CL
Early
Normal development and parturition
Sekharappa and Devaraj Sarkar, 1978
LIZARDS Lacerta vivipara
Chalcides ocellatus
Sceloporus cyanogenys
Mabuya carinata
(Continued)
Chapter | 9
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Viviparity in Reptiles: Evolution and Reproductive Endocrinology
TABLE 9.1 Effects of the removal of the corpora lutea (CL) or the entire ovary on pregnancy or parturition of viviparous squamatesdCont’d Species
Structure removed
Stage of pregnancy
Results
References
Sceloporus jarrovi
CL
Early
Embryos aborted or resorbed
Guillette, unpublished
Mabuya capensis
CL
Early-mid
Normal development for four weeks post-surgery
Yaron and Tai, unpublished
discussion in Section 8). Regression of the CL and the resulting drop in hormone secretory activity also has been implicated in the timing of oviposition or parturition by affecting the oviductal response to interactions between endocrine and neural adrenergic systems. The neurohypophysial peptide hormone AVT is elevated in plasma at the time of oviposition or parturition in oviparous (Figler, MacKenzie, Owens, Licht, & Amoss, 1989; Guillette, Propper, Cree, & Dores, 1991) and viviparous (Fergusson & Bradshaw, 1991) reptiles. Arginine
vasotocin stimulates smooth muscle contractions involved in the mechanical process of uterine expulsion of the egg or embryo (Cree & Guillette, 1991; Guillette, Dubois, & Cree, 1991; Atkins, Jones, & Guillette, 2006). In squamates, exogenous treatment with AVT induces oviposition in oviparous species (Guillette & Jones, 1982) and parturition in viviparous species (Guillette, 1979; Guillette & Jones, 1982; Cree & Guillette, 1991; Girling, Jones, & Swain, 2002b). Arginine vasotocin also stimulates uterine contractions in vitro (Mahmoud, Cyrus, Wright, 1987;
FIGURE 9.1 Plasma progesterone concentrations at several reproductive stages, including follicular growth, early or late gravidity or pregnancy, and post partum, for two oviparous lizards (Crotophytus collaris, Sceloporous undulatus) and four viviparous species (Sceloporouscyanogenys, Sceloporous jarrovi, Niveoscincus metallicus, Niveoscincus microlepidotus). See Table 9.2 for a more complete presentation of these data.
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TABLE 9.2 Plasma progesterone (P4) concentration (ng/ml)a during the reproductive cycle of squamates Species
Follicular growth
Preovulatory
Early pregnancy
Mid pregnancy
Late pregnancy
Postpartum
References
Uromastix Hardwicki
–
1.66 0.30
13.41 1.43
–
–
–
Arslan et al., 1978
Crotophytus collaris
0.183 0.77d
–
4.4 0.87c
10.97 0.46d
2.1 0.30c
0.30 0.12d
c
OVIPAROUS
d
Eumeces obsoletus
0.137 0.05d
–
1.4 0.08c
11.41 6.24d
0.42 0.07c
0.513 0.08d
c d
Scelopores undulatas
0.268 0.18d
25.81 1.22e
12.51 2.73e
3.78 1.19e
0.321 0.14d
NDd
d e
Agama atra
2.14 0.50
2.97 0.99
7.45 1.72 b
Fox and Guillette, 1987 Masson, 1985 Fox and Guillette, 1987 Masson, 1985 Masson, 1985 Guillette and Herman, 1985
–
–
–
van Wyk, 1984
–
–
~2.5
Moore et al., 1985
–
Bonagallo et al., 1980
Cnemidophorus uniparens
< 1.0
~2.50
1.24–70.0
Naja naja
w1.4
w3.00
10.5 1.12
–
–
3.07 1.04
1.31 0.32
0.74 0.24
VIVIPAROUS Barisia imbricata imbricata
Martinez-Torres et al., 2003
–
57.90 4.81
132.65 16.79
216.22 17.92
347.20 17.38
3.71 0.56
Xavier, 1982
Sceloporus cyanogenys
0.70 0.15
0.90 0.38
3.302 0.48
–
3.5 0.34
1.6 0.22
Callard et al., 1972b; Lance and Callard, 1978
Sceloporus jarrovi
–
–
0.75 0.11
0.95 0.05
4.1 0.94
0.43 0.06
Guillette et al., 1981
Bradypodion (Chameleo) pumilis
0.86
0.95 0.71
4.95 3.9
2.30 0.34
–
–
Veith, 1974
Nerodia taxispilota
0.44 0.04
0.91 0.08
1.93 0.24
–
–
–
Chan et al., 1973
Nerodia sipedon
1.27 0.19
3.93 0.83
4.95 1.41
6.94 0.78
2.81 0.44
–
Chan et al., 1973
Niveoscincus metallicus
w1.8
9.1 1.33
11.5 1.93
Niveoscincus microlepidotus Thamnophis elegans
–
Tiligua nigrolutea Vipera aspis ND, not detectable; –, not measured. a Mean standard error (SE) b Values for entire period of gravidity c–e See references column
38.5 7.9
15.4 5.9
–
1.70 0.30
1–2 3.7
Jones and Swain, 1996 1.1 0.2
6.20 1.0
1.0 0.2
12.7 1.27
4.4 0.88
18.7
2.7 0.9
Girling et al., 2002a Highfill and Mead, 1975a
1–2
Edwards and Jones, 2001 Bonnet et al., 2001
Hormones and Reproduction of Vertebrates
Lacerta vivipara
Chapter | 9
Viviparity in Reptiles: Evolution and Reproductive Endocrinology
Cree & Guillette, 1991; Atkins et al., 2006). An increase in plasma concentrations of AVT at the time of oviposition or birth is correlated with an increase in plasma prostaglandin concentrations as well (Guillette, Cree, & Gross, 1990; Guillette et al., 1991a). Prostaglandins (PG) are eicosanoids, small lipid compounds synthesized from the essential fatty acid arachidonic acid (Norris, 2007). Prostaglandins stimulate smooth muscle contractions in the uterus of eutherian mammals and are associated with the onset of labor (see Volume 5, Chapter 7). The correlation between increasing concentrations of AVT and PGs in reptiles (Guillette et al., 1991b) led to the hypothesis that AVT stimulates the local production of PGs in the reptilian oviduct and as such, AVT and PGs are important modulators of oviductal contractions (Guillette, Gross, Matter, & Palmer, 1990). However, studies examining the role of AVT and PGs, specifically PGF2a, in the timing of oviposition or parturition have yielded disparate results. In oviparous lizards, exogenous AVT generally induces premature oviposition (Guillette & Jones, 1982), whereas exogenous PGF2a does not (Guillette, Masson, & Demarco, 1991). In contrast, treatment with either AVT or PGF2a induces early parturition in the viviparous spotted snow skink (Niveoscincus ocellatus) (Atkins et al., 2006) and S. jarrovi (Guillette, 1979; Guillette, Demarco, Palmer, & Masson, 1992). These differences in PGF2a response are not simply attributable to parity mode since AVT, but not PGF2a, stimulates parturition in the viviparous gecko, Hoplodactylus maculatus (Cree & Guillette, 1991). However, this study reported that if H. maculatus were pretreated with a b-adrenoreceptor antagonist, then exogenous PGF2a treatment resulted in rapid parturition. Likewise, studies have shown an unusual lack of sensitivity to AVT in Anolis carolinensis without prior treatment with a b-adrenergic blocker (Summers, Austin, & Jones, 1985; Jones, Lopez, Austin, Orlicky, Summers, 2006). Atkins et al. (2006) provided further evidence for the role of both AVT and PGF2a in parturition of the viviparous southern snow skink, N. microlepidotus, and proposed b-adrenergic modulation of the endocrine cascade regulating this process (Atkins et al., 2006). Such work suggests that the autonomic nervous system is important in the regulation of oviposition and parturition (Cree & Guillette, 1991; Rooney, Donald, & Guillette, 1997) and that AVT and PGF2a could be regulated differently in squamates by the b-adrenergic system. The mechanisms controlling the lengthening of egg retention in squamates are still poorly understood. In some species, correlational evidence presents a strong case for the role of the CL in the maintenance of gestation; however, in other species we can find no such correlation. Likewise, circulating concentrations of P4 during gestation are quite variable across viviparous species. Initiation of oviposition or birth appears to be modulated by a complex interplay of
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endocrine and autonomic factors. The complexity of squamate responses is not surprising, especially when we find similar disparity in eutherian mammals, which have a presumed single origin of viviparity (Vogel, 2005; Wildman et al., 2006). For example, in most eutherian mammals studied to date, P4 prevents parturition by maintaining uterine quiescence, and parturition requires removal of the so-called P4 block to allow uterine contractibility. However, in contrast with other eutherians, circulating P4 concentrations remain elevated at birth in humans and higher primates (Mesiano et al., 2002). This observation led to recent work proposing a functional P4 withdrawal at birth mediated through changing P4 receptor (PR) expression in the human myometrium (Mesiano et al., 2002). Very little is currently known about oviductal steroid and peptide hormone regulation via receptor regulation or tissue biotransformation of hormones in squamates. Modulation of signals by altered expression of hormone receptors and receptor co-factors is clearly another potential point of control in the lengthening of egg retention and timing of parturition. To date, a few studies have demonstrated the presence of progesterone receptor (PR), estrogen receptor (ER), and androgen receptor (AR) in the reptilian oviduct, but no studies have examined in detail the regulation of their expression or function (Kleis-San Francisco & Callard, 1986; Whittier, West, & Brenner, 1991; Giannoukos & Callard, 1996; Vonier, Guillette, McLachlan, & Arnold, 1997; Paolucci & Di Cristo, 2002; Selcer, Smith, Clemens, & Palmer, 2005).
7. THE REDUCTION OF THE EGGSHELL The reduction of the thickness and structure of the eggshell is another requisite change for the evolution of viviparity in squamates (Guillette, 1993; Heulin et al., 2005). Oviparous squamates generally lay flexible eggs composed of two layers: a thick inner shell membrane composed of proteinaceous fibers and an extremely thin external crust of calcium carbonate (Packard, Packard, & Boardman, 1982; Palmer & Guillette, 1991). Some viviparous species also have a shell membrane present for the duration of embryonic development, but an external calcified layer is either absent or greatly reduced (Guillette & Jones, 1985; Qualls, 1996; Heulin et al., 2005). All viviparous species examined demonstrate either a significant reduction in eggshell thickness or loss of the eggshell altogether (Guillette, 1987). However, the debate continues over the timing of eggshell reduction in terms of the evolutionary transition to viviparity. One hypothesis proposes that eggshell reduction was a refinement of viviparity occurring after complete egg retention had evolved, whereas the other and more generally accepted hypothesis proposes that a reduction in the eggshell evolved concurrently with increasing periods of egg retention and thus is a requirement of viviparity
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(Guillette, 1982; Guillette, 1993; Qualls, 1996). The latter hypothesis has been supported by experiments utilizing the lizard Lacerta vivipara, which demonstrates both parity modes in reproductively isolated populations. Heulin and colleagues successfully crossed individuals in the laboratory from oviparous and viviparous populations of L. vivipara, generating hybrid F1 females that laid eggs with incompletely calcified eggshells and an eggshell thickness (21 mm) intermediate between the completely calcified eggshells of oviparous parental population (40 mm) and the eggshell membrane (9 mm) of the viviparous parental population (Heulin, Arrayago, Bea, & Brana, 1992; Arrayago, Bea, & Heulin, 1996). Further hybridization experiments of L. vivipara performed by Arrayago et al. (1996) resulted in oviposition by hybrid F1 females at an embryonic stage of development also intermediate between the oviparous and viviparous populations. This artificial experiment directly connects increased time of egg retention with decreased thickness of the eggshell, although it does not show a causal relationship. Heulin et al. (2002) expanded on this study by comparing eggshell thickness of two oviparous populations of L. vivipara with different durations of egg retention and found again that lengthening of egg retention was associated with decreased eggshell thickness (Heulin, Ghielmi, Vogrin, SurgetGroba, & Guillaume, 2002). Guillette (1987) proposed the hypothesis that increased chemical communication between the developing embryo and the maternal uterus is one consequence of a reduced eggshell, thus precipitating physiological modifications that would drive uterine quiescence and extend gestation times. In other words, this might drive the evolution of maternal recognition of pregnancy. The experimental data obtained by Heulin and colleagues provide additional support for this hypothesis. Further evidence that reduction of the eggshell appears to evolve concurrently with increased durations of egg retention came from work examining the interrelationships between thickness of eggshell membrane, degree of calcification, and duration of egg retention in the Australian lizard, Lerista bougainvillii (Qualls, 1996). L. bougainvillii exhibits three distinct reproductive modes: oviparous, viviparous, and an oviparous intermediate with extended egg retention. An increase in the duration of egg retention was associated with decreased calcification of the eggshell and decreased thickness of the eggshell membrane in this species. The oviparous population laid calcified eggs at embryonic stage 33 with a mean eggshell membrane thickness of 23 mm; the intermediate oviparous population laid thinner calcified eggs (19 mm) at embryonic stage 36. Embryos of the viviparous population were born at stage 40 and were enveloped in an uncalcified eggshell membrane of only 6 mm (Qualls, 1996). Two nonexclusive hypotheses have been put forth to explain the adaptive significance of evolving reduced
Hormones and Reproduction of Vertebrates
eggshell thickness concurrently with prolonged periods of in-utero egg retention. The first proposes that decreasing the thickness of the eggshell would facilitate gas exchange between maternal and embryonic environments. This would be particularly important in later stages of development as oxygen requirements of the embryos increase drastically in the exponential growth phase of development (Guillette, 1982). However, this hypothesis was not supported when tested in oviparous species from the genus Sceloporus (Andrews & Mathies, 2000). Oviparous Sceloporus species exhibit differences in the amount of embryonic development that can be supported in utero past the ‘normal’ time of oviposition. Some species, such as Sceloporus scalaris, support advanced embryonic development during extended egg retention experiments, while other oviparous Sceloporus species do not. However, in contrast to the above proposed hypothesis, these differences in the possible amount of extended in utero development were not related to thickness of the eggshell (Andrews & Mathies, 2000). A second hypothesis suggests that reducing eggshell thickness would reduce diffusion distance between embryonic and maternal tissues, thereby facilitating the movement of chemical signals from the embryo to the mother that could play an important role in the maternal recognition of pregnancy (Guillette, 1987; Guillette, 1993). In addition, reducing the eggshell thickness would also reduce the diffusion distance for nutrient transfer from the mother to the developing offspring. These observations led researchers to investigate the mechanisms involved in the reduction of the eggshell. Early hypotheses posited that a reduction in the eggshell was accomplished by delayed shelling of the egg within the oviduct (Tinkle & Gibbons, 1977). Tinkle and Gibbons (1977) suggested that reduction of eggshell thickness was not necessarily an intermediate stage between oviparity and viviparity and only required that shelling of the egg be delayed until just prior to oviposition. However, this hypothesis has not been supported by studies examining the timing of eggshell deposition in squamates. In both oviparous and viviparous species, eggshell formation occurs early in embryonic development, immediately following ovulation (Palmer, Demarco, & Guillette, 1993; Corso, Delitala, & Carcupino, 2000; Adams, Biazik, Stewart, Murphy, & Thompson, 2007). A particularly striking example of this is shown by the lizard Sphenomorphus fragilis, which exhibits incipient viviparity. Embryos of S. fragilis are encased within a thin shell that is completed by early neurulation and remains intact throughout gestation (Guillette, 1992). A reduction in the number of uterine shell glands, which secrete the eggshell, also has been proposed as a mechanism for reducing eggshell thickness (Guillette, 1992; 1993; Heulin et al., 2005). Weekes (1935) was one of the first to observe that, in viviparous squamates, uterine glands
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Viviparity in Reptiles: Evolution and Reproductive Endocrinology
were reduced in number when compared to oviparous species. This pattern has since been observed in a number of studies (Guillette, 1992; Girling, Cree, & Guillette, 1998; Adams et al., 2007) including those examining reproductively bimodal sibling species and reproductively distinct populations of a single species. Guillette and Jones (1985) compared uterine shell glands in Sceloporus aeneus, which consists of two sibling species, and reported a lack of uterine shell glands in the viviparous S. aeneus bicanthalis when compared with the oviparous S. aeneus aeneus (Guillette & Jones, 1985). Likewise, in L. bougainvillii, uterine shell glands were observed in oviparous females during early gravidity; however, none were found in viviparous females at any stage of gestation (Adams et al., 2007). These reports suggest that a reduction in the eggshell could be due to a reduction in uterine shell gland recruitment that occurs during follicular growth prior to ovulation (Guillette, 1993). A recent study has suggested, however, that reduction of the eggshell in L. vivipara could be accomplished by a reduction in the size of uterine shell glands. This study did not directly compare the number of shell glands between oviparous and viviparous populations, but instead focused on the development of the uterine shell glands. Heulin et al. (2005) reported no differences in the size of the glandular layer containing the uterine shell glands prior to vitellogenesis or following ovulation, but found that during vitellogenesis the glandular layer from oviparous females (100.7 mm 16.7) was significantly larger than from those of viviparous females (63 mm 7.6). In addition, they reported that during early gestation the shell membrane was thicker in oviparous females (52–73 mm) than viviparous females (4–8 mm). They concluded that differences in the thickness of the eggshell membrane in L. vivipara were due to differences in the preovulatory growth of the uterine shell glands (Heulin et al., 2005). We suggest that the difference in size of uterine shell glands might well reflect differences in the amount of secretory material present in these glands. Palmer and Guillette (1991) demonstrated that proteinaceous fibers are secreted by these glands and it is likely that the size difference represents differences in stored material. Further, this difference is potentially related to post-estrogen stimulation events involving various growth factors important in gland development, such as insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) (Cox, 1994). Although a comparison of the number of shell glands was not included in this analysis, the authors did note that shell glands of viviparous L. vivipara were not densely packed in the lamina propria, as was observed in the oviparous form (Heulin et al., 2005). Whether or not eggshell thickness is decreased by a reduction in the number or size of uterine shell glands, it appears that these glands are important in this process. Uterine shell glandsdalso referred to as tubulo-aveolar
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glands or mucosal glandsdare generally prominent features of the uterus in oviparous squamates during vitellogenesis (Uribe, Velasco, Guillette, & Estrada, 1988; Perkins & Palmer, 1996; Heulin et al., 2005), but less so in viviparous squamates (Guillette, 1992; Girling, Cree, & Guillette, 1997). The shell glands undergo seasonal changes associated with the reproductive cycle (see also Chapter 12, this volume). In squamates, shell glands are formed at the beginning of each reproductive season, peak during vitellogenesis, and regress following ovulation (Heulin et al., 2005). The recruitment of new uterine shell glands occurs by an infolding of the epithelium, the innermost layer of the uterus (Ortiz & Morales, 1974; Palmer & Guillette, 1988; Guillette et al., 1989). This process appears to be regulated by estrogens secreted by the ovary during the period of vitellogenesis, as oviductal and shell gland development coincides with increased circulating concentrations of estrogens during the preovulatory period in reptiles (Palmer & Guillette, 1990; Motz & Callard, 1991; Edwards & Jones, 2001; Heulin et al., 2005). Likewise, exogenous treatment with 17b-estradiol (E2) typically results in oviductal hypertrophy and an increase in the number and size of uterine shell glands in reptiles (Mead, Eroschenko, & Highfill, 1981; Girling, Guillette, & Cree, 2000; Girling, 2002). Guillette (1993) hypothesized that a reduction in the number of uterine shell glands recruited during the preovulatory period could involve changes in estrogen regulation via one of the following mechanisms: reduction in circulating estrogens, reduction in number or disruption of ERs, post-transcriptional modifications, or reduction in shell gland stem cell numbers. Currently very little is known about any of these possibilities in squamates. However, a recent study tested the hypothesis that a reduction in the number of uterine shell glands in viviparous species was the result of lower concentrations of circulating E2 during the preovulatory period. In L. vivipara, oviparous populations have more pronounced shell glands than their viviparous counterparts (Heulin et al., 2005); yet, Heulin et al. (2005) did not find significant differences in plasma E2 concentrations between viviparous and oviparous females. They suggested that recruitment of uterine shell glands in L. vivipara could be primarily regulated at the level of the receptor but provided no evidence to support this conjecture. In light of what is known about the role of estrogens in ovarian and oviductal development and growth, it is not surprising that an overall reduction in circulating estrogens during vitellogenesis does not occur in viviparous species associated with a reduction in shell glands (see discussion by Guillette, 1993). However, because shell gland number or size is stimulated by estrogen treatment in reptiles, local regulation of the estrogenic response could be modified through altered expression of receptors or their cofactors as
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well as changes in the expression of genes responsive to estrogens and important in uterine shell gland recruitment. For example, a number of studies suggest that various estrogen-induced growth factors, such as IGF-1 and EGF, stimulate proliferation and differentiation of the female reproductive tract. Estrogens are essential for normal growth and differentiation of the vertebrate oviduct (Murphy & Murphy, 1994). However, observations that E2 initiated little or no response in isolated mammalian uterine epithelial cells (Cooke, Uchima, Fujii, Bern, & Cunha, 1986; Cunha & Young, 1992) have led to the hypothesis that paracrine growth factors mediate estrogen-induced proliferation and differentiation of the mammalian uterus (Zhu & Pollard, 2007; Maekawa, Takeuchi, Kanayama, & Takahashi, 2009). The demonstration that stromal tissue is necessary for estrogen responsiveness of uterine epithelia supports this hypothesis. A number of polypeptide growth factors have been suggested as paracrine mediators of estrogen-induced oviductal growth, including plateletderived growth factor (PDGF), transforming growth factor a (TGF-a), IGF-1, and EGF (Murphy & Murphy, 1994; Hom et al., 1998). Much of the mammalian research performed in vivo on growth factor mediation of uterine proliferation and differentiation has focused on EGF and IGF-1. It was first reported that EGF-mediated estrogeninduced uterine growth in ovariectomized mice (Nelson, Takahashi, Bossert, Walmer, & McLachlan, 1991) and EGF receptor null mice exhibited altered uterine and vaginal growth (Hom et al., 1998). Our laboratory has reported that the reptilian oviduct expresses IGF-1 during the vitellogenic phase of
Hormones and Reproduction of Vertebrates
reproduction when plasma estrogen concentrations are elevated (Cox & Guillette, 1993). We also have performed a study in which adult female Mediterranean geckos, Hemidactylus turcicus, were ovariectomized and implanted with control E2, IGF-1, or EGF pellets to test the hypothesis that IGF-1 and EGF mediate the effects of E2 in the reptilian oviduct. Oviductal morphological data (epithelial cell height, endometrial thickness, gland diameter, and myometrial thickness) were analyzed to determine whether significant variation existed among the four treatment groups. The heights of the epithelial cells in the oviducts of the animals treated with control pellets were significantly smaller than for those treated with IGF-1, EGF, or E2 (Cox, 1994). Epithelial cell heights of IGF-1 and EGF-treated oviducts were greater than the control but still significantly smaller (25%) than those of females treated with E2. There was no statistical difference in epithelial cell height between IGF-1- and EGF-treated oviducts. Endometrial thickness and gland diameter were significantly smaller in oviducts of ovariectomized control females vs. oviducts from females treated with E2, IGF-1, or EGF. Again, these measurements were significantly smaller in the oviducts from IGF-1- and EGF-treated animals when compared to E2-treated animals. No statistical differences were observed in myometrial thickness among the four treatment groups. These data suggest an important role for growth factors (IGF-1 and EGF), oviductal proliferation (see Figure 9.2), and differentiation in reptiles, and support the hypothesis that this pathway is evolutionarily conserved in amniote vertebrates. Further, changes in uterine growth factor expression are estrogen-mediated in mammals and, thus,
FIGURE 9.2 Model for the recruitment of shell glands in the reptilian uterus. Estrogens are essential for uterine hyperplasia and hypertrophy and the recruitment of uterine glands. Estrogens bind to estrogen receptors (ERs) that act as transcription factors, inducing gene transcription and translation. We hypothesize that major downstream factors associated with uterine gland induction are insulin-like growth factor (IGF-1) and epidermal growth factor (EGF) acting in a paracrine fashion (see Section 7). Changes in ER action would alter expression of these growth factors and thus the recruitment of glands as well as the production of shell fiber protein.
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Viviparity in Reptiles: Evolution and Reproductive Endocrinology
altered growth factor expression could provide a mechanism to explain either fewer shell glands or less secretory product in those glands. Thus, future studies need to examine the relationship between estrogen signaling and growth factor function in the reptilian oviduct, and its relationship to shell gland recruitment, development, and function. In birds, exogenous treatment with estrogens stimulates uterine shell gland development and growth (Oka & Schimke, 1969). In the chicken oviduct, Isola (1990) reported that ERs were localized exclusively in the glandular epithelial cells of estrogen-treated animals (Isola, 1990) and specifically that ERa was observed in the glandular epithelium of the uterine shell glands (Hansen et al., 2003). Further, ER localization changes with estrogen treatment and during normal oviductal development in the chicken. In the undifferentiated oviduct, ERs are present in both stromal and epithelial cell types; however, following differentiation either induced by exogenous estrogens or during puberty, ERs are found exclusively in the uterine shell glands (Joensuu & Tuohimaa, 1989). Like birds and mammals, reptiles have two forms of ER: ERa (ESR1) and ERb (ESR2), with both isoforms reported in crocodilians, turtles, and squamates (Sumida, Ooe, Saito, & Kaneko, 2001; Thornton, 2001; Katsu et al., 2004; Katsu et al., 2008). In mammals and birds, the relative expression of these isoforms can alter the response of a tissue. Thus, future studies need to examine the distribution and functioning of both isoforms of ER in reptilian reproductive tissues. The distribution of both ERs, as well as other factors such as EGF, IGF-1, and their receptors, in the shell glands of squamates needs to be determined, as the observation of a redistribution of ERs during avian oviductal development presents another plausible mechanism for regulating differences in shell gland recruitment between oviparous and viviparous species. Estrogens have been suggested to regulate the PR via ER in the glandular epithelial cells of the chick oviduct (Isola, 1990) as well as in the oviduct of the ostrich (Madekurozwa, 2004); therefore, the PR is potentially another point of regulation that should be investigated in the development of squamate shell glands. Recent work by Herbert, Lindsay, Murphy, & Thompson (2006) suggested that the loss of Ca2þATPase pumps in the uterine shell glands as another mechanism for the reduction and eventual loss of the calcified eggshell in viviparous squamates. This study observed differences in the distribution of uterine Ca2þATPase channels between oviparous and viviparous species, and suggested that these differences were associated with increasing placental complexity. The authors suggest that the redistribution of Ca2þATPase pumps from the uterine shell glands of oviparous species to the luminal epithelium of viviparous species functions in the transport of calcium across the uterine wall where Ca2þ can then be
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stored in the yolk in species with simple placentae or be continually transported throughout gestation in those with complex placentae (Herbert et al., 2006). In light of this finding, it would be interesting to explore the potential estrogen regulation of Ca2þATPase pumps in the squamate uterine shell glands. Since the uterine epithelium is the source of the cells induced for eggshell gland formation, the Ca2þATPase pumps present may always be present in these cells but ‘remain’ on the luminal epithelium in the viviparous uterus because invagination of the glands does not occur. Estrogens play a role in the regulation of calcium in a variety of organ systems (van Abel et al., 2002; Szemraj, Kawecka, Lachowicz, & Zylin˜ska, 2003), including the uterus of the rat (Kim, Lee, Ji, Choi, & Jeung, 2006) and the eggshell glands of the domestic chicken (Corradino, Smith, Krook, & Fullmer, 1993). Therefore, it is possible that changes in ER activity of squamate uterine shell glands regulate the expression of these Ca2þATPase pumps. A common finding when oviparous and viviparous species are compared is that the uterine shell glands are reduced in some capacity and that reduction is correlated with a reduction in eggshell thickness. The mechanism by which this occurs is still poorly understood but likely involves altered estrogen signaling. Uterine shell glands are prominent in all oviparous squamates studied to date, but are absent or greatly reduced in the majority of viviparous species. In the viviparous species with obvious uterine shell glands, a general reduction in size of the shell gland has been reported, as in L. vivipara (Heulin et al., 2005), as well as a reduction in the thickness of the endometrial stroma, as in Pseudemoia spenceri (Herbert et al., 2006). Shell glands undergo seasonal changes with reproductive cyclicity that match changes in the prominent sex steroid hormones, E2 and P4; therefore, future studies are needed to understand the mechanisms associated with shell gland formation and recruitment. Estrogens appear to be central to shell gland recruitment, proliferation, and synthesis of secretory material, and ERs serve as key modulators in this process. Further, the regulation of the expression of other important factors, such as membrane calcium pumps, needs to be examined. While the mechanisms by which uterine shell glands and the eggshell are reduced remains unresolved, there is evidence that reduction of the eggshell evolved concurrently with the evolution of extended egg retention and, as such, should be considered a requirement for the evolution of viviparity in squamates.
8. EMERGENCE OF THE PLACENTA Placentation is another key characteristic of viviparity in squamates. In all viviparous squamates examined, placentae are present and thus appear to be required for embryonic survival in utero (Guillette, 1993). Here, we
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define a placenta as a composite structure of maternal and embryonic origin. The embryonic contribution to the placenta consists of one or more of the four extraembryonic membranes (yolk sac, chorion, allantois, and amnion). These membranes function in the transfer of maternally derived nutrients, regulation of gas and water exchange, removal of metabolic wastes, and protection of the developing embryo (Kluge, 1977; Mossman, 1987). These basic requirements are crucial for embryonic survival and development regardless of whether the embryo is encased within a calcified egg in an external nest (oviparous) or maintained within the maternal uterus (viviparous) (Figure 9.3). Gas and water exchange with the environment is achieved by two vascularized extraembryonic membranes: the chorioallantois and the yolk sac membrane. The chorioallantoic membrane (CAM) is formed by the apposition of the non-vascularized chorion and highly vascularized allantois and serves as the major respiratory organ of the developing embryo. The yolk sac membrane transfers nutrients from the yolk, functions in water exchange, and has been proposed to act as a secondary gas exchange site, supplementing the chorioallantois (Guillette, 1993; Mess, Blackburn, & Zeller, 2003; Stewart & Thompson, 2003). To facilitate exchange with the environment, these
Hormones and Reproduction of Vertebrates
membranes line the inner surface of the eggshell with the surface area of the CAM, increasing dramatically during development to meet the gas demands of the embryo. In viviparous species, the loss of the calcified crust and reduction in the protein layer of the eggshell results in apposition of the eggshell or extraembryonic membranes to the vascularized uterus, allowing exchange between the embryo and the maternal environment. Viviparous squamates have two primary types of placentation present during gestation: a chorioallantoic placenta and some form of yolk sac placenta (Stewart & Blackburn, 1988). Both types of placenta are epitheliochorial in squamates reptiles, meaning that the extraembryonic membranes are in contact with the uterus through apposition but the embryonic tissue does not erode and invade the uterine lining (Thompson, Adams, Herbert, Biazik, & Murphy, 2004). The yolk sac placenta is typically formed at the abembryonic pole during the early stages of embryonic development and broadly describes the apposition of omphalopleure (yolk sac) to the uterine epithelium. The chorioallantoic placenta (allantoplacenta) is present later in development at the embryonic pole and is formed by apposition of the CAM to the uterus. Both the yolk sac and the chorioallantoic placenta demonstrate a wide range of morphological diversity and exhibit varying
FIGURE 9.3 The extraembryonic membranes of oviparous and viviparous squamates. Oviparous and viviparous amniotes share the same extraembryonic membranes: the amnion, chorion, allantois, and yolk sac. These structures perform similar functions regardless of reproductive mode, indicating that very little specialization of extraembryonic membranes is required in an initial transition to viviparity. Redrawn from Guillette (1993). Artwork by Patpilai Kasinpila.
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degrees of complexity across squamates (reviewed in Stewart & Thompson, 2000). Four major types of chorioallantoic placenta have been described in squamates and are differentiated by levels of morphological complexity. Weekes (1935) described the first three of these types and a fourth has been proposed by Blackburn et al. (Blackburn et al., 1984; Blackburn, 1993). The simplest and most common type of chorioallantoic placenta, Weekes type I, involves indirect apposition of the CAM to the uterus without any anatomical specializations in either structure. A reduced shell membrane encasing the embryo is present and it is this structure that lies in contact with the uterine epithelium. In types II and III, we see the emergence of the placentome, a specialized region that forms at the embryonic pole and brings the chorionic epithelium and uterine epithelium in direct apposition. Here, the chorioallantoic epithelium interdigitates with the uterine mucosa, and this region has been proposed as a site of histotrophic transfer due to the appearance of absorptive chorionic cells and a secretory uterine epithelium (Blackburn & Callard, 1997). The type IV chorioallantoic placenta is the most complex and has been described in the South American lizard genus Mabuya (Blackburn, 1993). This type of chorioallantoic placenta undergoes extensive transformation and demonstrates invagination of the CAM by uterine endometrium forming interlocking projections. The placentome and specialized chorionic cells called areolae have been suggested to function in nutrient transfer (Blackburn, 1993). Oviparous species and viviparous species with a simple placenta (type I) are predominately lecithotrophic, meaning that nourishment for embryonic development is primarily supplied by the yolk (Thompson & Speake, 2002). Therefore, in the transition to viviparity as the period of egg retention was increased and eggshell thickness decreased, the major requirement of a placental structure was to aid in gas exchange, which appears to be the primary role of the type I placenta. Species forming a more complex chorioallantoic placenta exhibit differentiation into specialized regions: the placentome and paraplacentome. The paraplacentome has been described as possibly functioning as a specialized gas exchange organ in Pseudemoia entrecasteauxii (Adams, Biazik, Thompson, & Murphy, 2005) and as another site of histotrophic transfer in Mabuya mabouya (Jerez & Ramirez-Pinilla, 2001). While the vast majority of viviparous squamates are lecithotrophic, it is common for these species to exhibit some degree of placental transport of organic and inorganic nutrients as well (Swain & Jones, 1997; Thompson, Stewart, & Speake, 2000; Thompson, Speake, Russell, & McCartney, 2001; Speake, Herbert, & Thompson, 2004). In contrast, the condition of matrotrophy is quite rare in squamates. Yet, a few species exhibit a high degree of matrotrophy, in which the majority of the nutrients required
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for embryonic development are provided by the mother via a placenta. These species have microlecithal eggs with little or no yolk present in the oocyte. The New World species of the genus Mabuya demonstrate the most extreme form of matrotrophy, which surpasses that found in any other squamate reptile and is similar to that of eutherian mammals (Jerez & Ramirez-Pinilla, 2001). The specialized type IV chorioallantoic placenta of Mabuya heathi provides approximately 99% of nutrients for embryonic development (Blackburn, Vitt, & Beuchat, 1984). In eutherian mammals, the placenta is an endocrine organ that synthesizes, metabolizes, and transports a suite of steroid and peptide hormones crucial for embryonic survival and development (Petraglia, Florio, Nappi, & Genazzani, 1996). Progesterone is a key placental hormone that plays a role in the maintenance of pregnancy (Henson, 1998) and timing of birth (Mesiano et al., 2002), and that promotes growth of the embryo (Mark, Smith, & Waddell, 2006) and of the placenta itself (Ogle, Mills, & Costoff, 1990; Jojovic, Wolf, & Mangold, 1998; Mark et al., 2006). The first indirect evidence of placental P4 synthesis in squamates came from observations in S. jarrovi. In this lizard species, maternal plasma P4 concentrations remain elevated after luteolysis. However, the chorioallantoic placenta is present during this time, suggesting that the placenta is a source of P4 (Guillette et al., 1981). The three-toed skink, C. chalcides, also demonstrates elevated plasma P4 following deterioration of the CL and formation of the placenta. The chorioallantoic placenta of this species has been shown to be capable of steroidogenesis (Guarino et al., 1998). Guarino et al. (1998) investigated the expression of the steroidogenic enzyme 3b-hydroxysteriod dehydrogenase (3b-HSD), which converts pregnenolone to P4, in the CL and chorioallantoic placenta. They found high 3b-HSD activity in the CL during early gestation but not at late pregnancy, whereas the placenta exhibited intense 3b-HSD reactivity only at late pregnancy. In-vitro synthesis of P4 by the CL was high during early, but not late, pregnancy, but P4 synthesis was highest at late pregnancy in the placenta. This work supports the hypothesis of an endocrine chorioallantoic placenta in C. chalcides and models a similar pattern found in many eutherian mammals where the placenta assumes the primary role of P4 synthesis following deterioration of the CL (Guarino et al., 1998). In-vitro production of P4 by the chorioallantoic placenta also has been demonstrated in the southern snow skink (N. microlepidotus). Girling and Jones (2003) reported that the chorioallantoic placenta of this species was capable of synthesizing P4 when pregnenolone precursor was added to the culture media; however, unlike for C. chalcides, this study did not find significant levels of P4 produced by the placenta in the absence of added pregnenolone.
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The relatively simple chorioallantoic placenta of S. jarrovi can perform in-vitro metabolism of steroid hormones and synthesis of P4 in the presence of pregnenolone (Painter & Moore, 2005). In-vitro experiments revealed that placental tissue will rapidly clear P4 and corticosterone (CORT) from culture media, indicating the ability of the placenta to convert P4 and even corticosteroids to other metabolites (Painter & Moore, 2005). Painter and Moore (2005) suggested that steroid hormone metabolism by the squamate chorioallantoic placenta could play an important role in the regulation of maternal and embryonic hormone concentrations by preventing steroid hormones from freely diffusing between the developing embryo and mother. Aside from its endocrine role in the maintenance of pregnancy, P4 also acts as an immunosteroid in eutherian mammals and plays a role in local immunosuppression of the uterus (Arck, Hansen, Jericevic, Piccinni, & SzekeresBartho, 2007). These effects of P4 are mediated by cytokines, which are immunoregulatory peptides that regulate immune cell activity and are involved in the immunological protection of the mammalian fetus. A number of immune cytokines have been identified in mammalian placentae and it appears these compounds could have a role in the chorioallantoic placenta of squamates as well. Paulesu et al. (1995) were the first to show the secretion of a cytokine crucial in mammalian pregnancy, interleukin1 (IL-1), in the chorioallantoic placenta of a squamate species. In C. chalcides, IL-1 was localized to the chorial epiblast and uterine epithelial cells of the placenta (Paulesu et al., 1995). Another important cytokine in mammalian reproduction, transforming growth factor b (TGF-b), also is secreted by the chorioallantoic placenta in this species (Paulesu, 1997). More recently, IL-1 was investigated in the reproductively bimodal species, L. vivipara. The uterine tissues of both oviparous and viviparous females expressed IL-1 and its corresponding receptor. This suggests that immunological mechanisms that facilitate maternal tolerance of the fetus were in place prior to the evolution of viviparity in this species (Paulesu et al., 2005). The extraembryonic membranes of oviparous species and placentae of viviparous species perform similar functions to meet the gas, water, and nutrient demands of the embryo (Figure 9.3). This indicates that very little specialization of extraembryonic membranes is required in a transition to viviparity and explains why the majority of viviparous species exhibit a relatively simple chorioallantoic placenta that appears to predominately function in gas exchange. Yet, evidence indicates that the chorioallantoic placenta has additional roles in endocrine and immunemediated processes in squamates. Are these specializations unique to the chorioallantoic placentae of viviparous mammals and squamates or could the CAM of oviparous species also perform similar functions?
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We suggest that the endocrine properties of the chorioallantoic placenta are not novel features of viviparous mammals and squamates. Rather, we hypothesize that the oviparous CAM has endocrine properties, which gave rise to an endocrine placenta. The basis for this hypothesis comes from studies investigating mammalian (Petraglia et al., 1996; Giannoulias et al., 2005; Pasqualini, 2005) and lizard (Girling & Jones, 2003) placentae, which revealed that both extraembryonic membranes and maternal tissues contribute to hormone synthesis and metabolism. Given that extraembryonic membranes share numerous similarities in their basic structure and function, which are conserved across amniotes (reptiles, birds, and mammals) (Kluge, 1977), we hypothesize that the oviparous CAM is an endocrine organ that has the capability to synthesize and receive signaling steroid hormones (see Albergotti, Hamlin, McCoy, & Guillette (2009) and discussion in Section 9).
9. MATERNAL RECOGNITION OF PREGNANCY In placental mammals, the maternal recognition of pregnancy is defined as the prevention of luteolysis (deterioration of the corpus luteum) by biochemical signals of embryonic origin (Senger, 2005). These embryonic signals vary in mammals. Depending on the species, maternal recognition of pregnancy can be provided by a number of embryonic factors, such as chorionic gonadotropins (CGs), interferons (IFNs), steroids, and peptide hormones (Senger, 2005). Chorionic gonadotropins are glycoprotein hormones produced by the trophoblast of the placenta in some mammals (e.g., humans and horses) that prevent the disintegration of the CL and prolong P4 secretion (Johnson & Everitt, 2000; Senger, 2005). Interferons are cytokines that have antiviral, antiproliferative, and immunosuppressive properties and are classified by the cells from which they originate: IFN-a (leucocytes), IFN-b (fibroblasts), IFN-g (lymphocytes), and IFN-s (trophoblast cells). Interferon-s is antiluteolytic in ruminant mammals and has a major role in the early maintenance of pregnancy in ovine and bovine species (Demmers, Kaluz, Deakin, Jabbour, & Flint, 1999; Senger, 2005; Roberts, 2007). Steroids and peptide hormones also act as agents in mammalian maternal recognition of pregnancy. For example, estrogens appear to be of primary importance in the maternal recognition of pregnancy in the pig (Bogacki, Wasielak, & Ziecik, 2005; Wollenhaupt, Brussow, Tiemann, & Tomek, 2007). Specifically, E2 synthesized and released by the early embryo seems to be a crucial signaling mechanism in this process and estrogens of embryonic origin are able to mediate maternal responses by interactions with ERs present in the uterus. 17b-estradiol synthesized by the early llama embryo (Lama glama) has
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also been posited as the primary embryonic signal in the recognition of pregnancy in this species (Powell, Smith, Timm, & Menino, 2007). In conjunction with estrogens, a lipid signaling system of prostaglandins, specifically the ratio of PGE2 and PGF2a, is considered crucial for the establishment of pregnancy in the pig (Ziecik, Waclawik, & Bogacki, 2008). In rodents, maternal recognition of pregnancy is dependent on semicircadian surges of prolactin (PRL), which appear to be under the control of the protein puromycin-sensitive aminopeptidase (Osada, Watanabe, Sakaki, & Takeuchi, 2001). The point here is that many steroid hormones and peptide factors of embryonic origin play a role in the mammalian recognition of pregnancy. Essentially, nothing is known about the maternal recognition of pregnancy in viviparous reptiles, but a few studies suggest that this phenomenon could occur in viviparous lizards (Guillette, 1989; 1991). If maternal recognition of pregnancy occurs in reptiles, chemical signals of embryonic origin could function to prevent CL deterioration, modify the uterine environment, and increase the length of gestation. These embryonic chemical signals could be compounds similar to those found in mammals, suggesting that the early embryonic expression of cytokines, PGs, estrogens, and other steroids as well as peptide hormones should be investigated in oviparous and viviparous squamates (Guillette, 1991). The discovery of the cytokines IL-1a, IL-1b, and TGFb in the reptilian chorioallantoic placenta as well as in the uterine epithelium of viviparous and oviparous squamates (Paulesu et al., 1995; Paulesu, 1997; Paulesu et al., 2005) suggests that other cytokines important in the mammalian recognition of pregnancy could be present in these tissues. Although we are not aware of any study investigating the presence of IFN compounds in reptiles, IFNs have been identified in at least one avian species, the chicken. Interestingly, IFNs are not only present in chicken embryonic and adult tissues but also are synthesized in the CAM (Ho, 1964). In fact, the chick CAM was key to the discovery of IFNs by Issacs and Lindenmann (1957). Due to the shared evolutionary history of amniotes, in particular the homology of extraembryonic membranes, it seems likely that IFNs could also be present in the CAMs of reptiles, even potentially in the reptilian chorioallantoic placenta. We are beginning to explore the role of extraembryonic membranes as potential sources of such embryonic signaling agents. We hypothesized that the CAM of oviparous squamates is capable of producing embryonic signaling factors, such as steroid hormones. These signaling factors would have functions other than maternal recognition of pregnancy in oviparous species and could likely be involved in tissue proliferation, vascularization, and embryonic growth. However, these factors would be useful embryonic signals of pregnancy as viviparity
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evolves. As squamates retain the egg in utero for longer time periods and eggshell thickness is reduced, signaling factors synthesized by the extraembryonic membranes would be free to communicate with the maternal uterus and could play a role in maintaining the life of the CL. We have shown recently that endocrine activity of extraembryonic membranes extends beyond placental amniotes by documenting steroidogenesis and the in-vitro synthesis of P4 in the CAM of the chicken (Gallus gallus) (Albergotti et al., 2009). Our study revealed the expression of key steroidogenic enzymes involved in P4 biosynthesis, such as 3b-HSD, as well as the PRs in the CAM (Figure 9.4). We also discovered that the CAM synthesized P4 in vitro in the presence of the steroid precursor, cholesterol (Figure 9.5). Further, we showed mRNA expression and protein immunolocalization of PR in the oviparous CAM (Albergotti et al., 2009). This work indicates that endocrine activity of extraembryonic membranes is not a novel characteristic of viviparity, but rather could be an ancestral condition of amniotes. Endocrine properties of oviparous species could become co-opted in a novel environment such as the uterus, thus facilitating the use of embryonic signaling factors in the maternal recognition of pregnancy in viviparous squamates. Although this hypothesis has not been tested directly in squamates, we have observed mRNA expression of steroidogenic enzymes and receptors in the reptilian CAM of the American alligator (Alligator mississippiensis) (Albergotti, unpublished data). Given the dramatic increase in literature documenting the presence of steroid hormones in the yolk of many species of reptile and their mobilization during embryonic development, we now hypothesize that the oviparous yolk sac membrane also plays an active role in the mobilization, degradation, and conversion of maternal steroid hormones deposited in that yolk (yolk steroids) (Figure 9.6). The oviparous amniote thus has an elaborate system for steroid synthesis and metabolism, allowing regulation of the steroid milieu of the developing embryo (Figure 9.6), and this evolved long before the evolution of viviparity. This system has been co-opted and modified to serve a viviparous mode in reproduction, but did not evolve as a response to the evolution of this parity mode. For example, the avian yolk sac membrane functions in the transport and conversion of lipids deposited in the yolk to the developing embryo (Noble, Connor, & Smith, 1984; Speake, Murray, & Noble, 1998). We suggest that maternally deposited yolk lipids and yolk steroid hormones also could be mobilized and either modified by the yolk sac or delivered to the CAM, providing substrates for steroidogenesis in this tissue (Figure 9.6). Likewise, we suggest that the yolk sac membrane itself could be involved in the biotransformation of steroid hormones, and this action is likely older than even amniotes, as maternally derived steroid and thyroid
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FIGURE 9.4 Relative mRNA expression of 3b-hydroxysteroid dehydrogenase and progesterone receptor (ab) in the chicken chorioallantoic membrane. Real time quantitative PCR (RTq PCR) analysis of mRNA coding for 3b-hydroxysteroid dehydrogenase (3b-HSD) and PR(ab) on chick embryonic days 8, 10, 12, 14, 16, and 18. Data represent mean normalized mRNA transcript number in copies/mL SEM. 3b- hydroxysteroid dehydrogenase (p < 0.0001) and PR(ab) (p < 0.0001) increased significantly between embryonic days 8 and 18. Data from Albergotti, Hamlin, McCoy, and Guillette (2009).
hormones have been found in the egg yolk of fishes (Kobuke, Specker, & Bern, 1987; Hwang, Wu, Lin, & Wu, 1992; Lam, 1994; Brooks, Tyler, & Sumpter, 1997; McCormick, 1999; Greenblatt, Brown, Lee, Dauder, & Bern, 2005) and maternally deposited thyroid hormones have been found in amphibian eggs (Fujikara & Suzuki, 1991 as cited by Hanken, Jennings, & Olsson, 1997; Morvan Dubois et al., 2006). We suspect that a combination of these pathways is acting in the extraembryonic membranes and embryos of all amniotes (Figure 9.6).
10. CONCLUSIONS In squamates, the transition from oviparity to viviparity involves changes in the timing of egg retention, thickness of the eggshell, and the development of a placenta. These physiological and morphological requirements of viviparity have occurred more than 100 times in squamates alone. Therefore, we should not be surprised to find that many of the required molecular-, cellular-, and organ-level characteristics of viviparity, including endocrine signaling, were likely already present in the oviparous ancestors, allowing them to be modified for a viviparous mode of reproduction. Differences in the use of various pathways and mechanisms and timing associated with these processes would likely occur.
From the above discussion, we consider the following to have evolved concurrently with live birth and to be requirements of viviparity: intrauterine retention of the egg for the duration of embryonic development; reduction of the eggshell; and formation of a fully functioning placenta that facilitates not only the exchange of gases, waste, and nutrients but also endocrine signals. In light of our recent findings that steroidogenesis occurs in the CAM of an avian and crocodilian species, it is likely that embryonic endocrine signals from the extraembryonic membranes play a key role in the maternal recognition, establishment, and maintenance of gestation in viviparous squamates. Given that the CAM is present in all birds, reptiles, and mammals, why has viviparity not evolved in birds or in the other reptiles (tuatara, crocodilians, and turtles)? Simply put, the presence of a steroidogenic CAM is not enough in itself to facilitate a transition from oviparity to viviparity in the absence of extended egg retention in conjunction with continued embryonic development and decreased eggshell thickness. Egg retention is brief in birds and crocodilians, whereas egg retention is extended in some species of turtle and in the tuatara (Andrews & Mathies, 2000). However, embryonic development is arrested in turtles and presumed to be so in the tuatara as little embryonic development occurs in utero and embryos are at the gastrula stage at the time of oviposition (Andrews & Mathies, 2000). Thus, oviposition occurs at early stages of development prior to
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FIGURE 9.5 Progesterone (P4) synthesis in vitro by the chicken chorioallantoic membrane (CAM). Chorioallantoic membrane sections were incubated in culture media for two, four, or eight hours either with (circles) or without (diamonds) cholesterol (plus cAMP) as a precursor. Concentration of P4 in the culture medium is represented as pg/ml of P4 per g of CAM tissue (pg/ml/g). The addition of precursor significantly increased the concentration of P4 in the culture media (p < 0.0001) with a significant interaction between time of incubation and addition of precursor (p ¼ 0.0305). Data from Albergotti, Hamlin, McCoy, and Guillette (2009).
development of the CAM in birds, crocodilians, turtles, and the tuatara. (Andrews, 2000; 2004). In contrast, oviparous squamates typically retain eggs in utero past the time of CAM development, on average for one third to one half of embryonic development (Andrews, 2000; 2004). Moreover, if the thickness of the eggshell is not reduced in utero, embryonic or extraembryonic signals that might play a role in further extending egg retention will not be in communication with the maternal uterus.
11. RESEARCH QUESTIONS & FUTURE DIRECTIONS With the over 100 independent evolutions of viviparity in squamates reptiles, it seems equally likely that the transition in reproductive mode (1) may not be an exceedingly difficult one to make and (2) may occur in response to various selective forces but likely involves similar molecular, cellular, and morphogenic pathways. We currently understand much about the environmental selective forces
and the morphological and physiological attributes of viviparity but, despite a century of inquiry, many major questions remain. The molecular mechanisms involved in the suite of physiological (especially endocrine), anatomical, and developmental modifications associated with the evolution of viviparity have only begun to be explored. Investigations of the proximate mechanisms involved in the reduction of the eggshell, development of the placenta, maintenance of the CL, and timing of parturition from a molecular perspective are needed. By elucidating the genetic and developmental regulation of these processes we could begin to understand how the evolution of viviparity might have occurred within squamata in particular, and perhaps use this information to look for similarities in patterns across other non-mammalian vertebrates exhibiting viviparity, especially within fishes and amphibians. The synthesis of methodologies from genetics, embryology, and reproductive biology can stimulate fresh perspectives on evolutionary questions. Such an approach could prove useful in uncovering genetic and
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FIGURE 9.6 Model for the evolution of endocrine function in the reptilian yolk sac and chorioallantoic membrane (CAM). We propose that initially the yolk sac mobilized yolk steroids and delivered them to the developing embryonic circulation (pathway 1). Later, the yolk sac itself acquired the ability to modify yolk steroids to ‘protect’ the developing embryo or provide ‘developmental signaling’ (pathway 2). Subsequent to or coincident with the evolution of a steroid modifying yolk sac, we propose that the CAM evolved the ability to convert yolk steroids (pathway 3) as well as synthesize steroid hormones de novo (pathway 3) prior to the evolution of viviparity. The ability to synthesize steroid hormones would be a conserved ability of the amniote CAM.
developmental mechanisms involved in the evolution of viviparity, provide insights into the evolutionary steps involved in a transition of parity mode, and investigate the effects of genetic and environmental interactions in terms of genetic variation and response to selection. Molecular techniques are fairly new to questions concerning the evolution of viviparity in squamates; however, a number of studies have used a molecular approach to elucidate reptilian phylogenetic relationships (Benabib, Kjer, & Sites, 1997; Fairbairn, Shine, Moritz, & Frommer, 1998; Keogh, Shine, & Donnellan, 1998; Schulte, Macey, Espinoza, & Larson, 2000; Smith, Austin, & Shine, 2001; Townsend & Larson, 2002; Townsend et al., 2004; Vidal & Hedges, 2005). A possible strategy for identifying genetic and developmental mechanisms associated with parity mode in squamates involves the use of tissue, cell, and gene expression studies. For instance, comparative studies of gene expression associated with environmental sex determination (ESD) in reptiles is providing important new insights (Crews, 2003). We have recently shown sexually dimorphic expression of several heat shock proteins in the American alligator with temperature-dependent sex determination (TSD) using such an approach, including Hsp27,
which is known to alter ER activity (Kohno et al., in press). Such a comparison between oviparous and viviparous squamate species, using tools such as in-situ hybridization and real-time quantitative PCR, could reveal modifications in the timing of gene expression or where genes are expressed. Past and current work in developmental genetics continues to demonstrate how the same genes expressed in different temporal and spatial patterns create an immense amount of evolutionary diversity and how small developmental modifications can create major phenotypic changes (Jacob, 1977; Duboule & Wilkins, 1998). In the preliminary stages of investigating a genetic and developmental basis for viviparity, comparative gene expression studies in closely related oviparous and viviparous species could be particularly useful in identifying candidate genes of interest, which could then be explored in a broader phylogenetic context. For example, closely related sister taxa such as are found in the genus Sceloporus or in reproductively bimodal species such as L. bougainvillii, L. vivipara, and Saiphos equali (Smith & Shine, 1997) could be used to compare tissue-specific spatial and temporal expression patterns of genes in the steroidogenic pathway, such as steroidogenic enzymes and steroid receptors. Using a comparative molecular approach in this
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way could help us answer the question of whether there are general patterns of molecular mechanisms associated with evolution of viviparity, or if it occurs differently in each lineage. In identifying candidate genes of interest, another approach involves looking for the expression of homologous genes in squamates that play a role in the pregnancy of mammals. Expression studies of genes associated with the development and maintenance of mammalian gestation could be performed in squamates to compare gene expression patterns in oviparous and viviparous species. This approach has been applied recently with expression studies of the transcription factor, HoxA10 in squamates. In general, homeobox or Hox genes are key players in embryonic development, and HoxA10 is required for the formation of uterodomes, which are projections of the plasma membrane in the mammalian uterine epithelium associated with uterine receptivity and implantation in mice and humans (Thomson, 2006; Zhao, Koon, & Bethin, 2006). Thomson, Herbert, Murphy, and Thompson (2005) investigated the expression of HoxA10 in the viviparous lizard Eulamprus tympanum, which forms uterodomes, and compared that to the oviparous lizard Lampropholis guichenoti, which does not (Thomson et al., 2005). This study found evidence of oviductal HoxA10 expression in both species, but also reported differences in the molecular size of HoxA10-like proteins between species. This suggests that the oviparous and viviparous species express different isoforms of this protein. Although this study was not able to test the biological significance of different isoforms, it is possible that this difference in HoxA10-like proteins could mediate different functions in uterine development associated with gestation in oviparous and viviparous squamates. It is also noteworthy that estrogens (and other sex steroids) regulate HoxA10 expression in humans and mice, and in humans this is mediated by an estrogen response element in the promoter of HoxA10 (Eda Akbas, Song, & Taylor, 2004). Therefore, it would be interesting to know whether estrogens regulate HoxA10 expression in squamates and how this could play a role in uterodome development. There are numerous genes that could be investigated in this manner. For example, mammalian placental development is dependent on a number of transcription factors such as HoxA13, HAND1, FOXF1, and DLX3. HoxA13 was recently found to be crucial in the vascular patterning of the mouse placenta, where it is expressed in the vascular endothelia of the umbilical artery, chorionic plate, and placental labyrinth (Shaut, Keene, Sorensen, Li, & Stadler, 2008). Does HoxA13 also play a role in the placental development of squamates? If so, how does the expression pattern compare between the oviparous CAM and the viviparous chorioallantoic placenta?
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Although we have stressed the importance of examining genetic mechanisms associated with the evolution of viviparity, this discussion would not be complete without mentioning the likely role of epigenetics in this process. Epigenetics refers to heritable changes in phenotype that do not involve changes in genotype; i.e., phenotypic changes resulting from mechanisms that do not alter the nucleotide sequence of DNA. These mechanisms are typically the result of DNA methylation and histone modifications (e.g., methylation, acetylation, phosphorylation, ubiquitination). These processes affect not only which genes are expressed, but also how and when these genes are expressed (Jaenisch & Bird, 2003; Jirtle & Skinner, 2007). Epigenetics is a fundamental aspect of embryonic development and differentiation. However, at least in mice and humans, epigenetic processes can also generate changes in adult gene expression patterns in response to environmental factors, such as ageing, nutrition, and exposure to environmental contaminants (Toyota & Issa, 1999; Dolinoy, Huang, & Jirtle, 2007). By altering the expression of genes, epigenetics provides a connection between environment and genome. Epigenetics has a role in reptilian reproduction. An obvious example of this occurs in ESD of crocodilians, turtles, and some squamates, where incubation temperature determines the sex of the offspring (TSD). The bipotential gonad of the developing embryo has the same set of genes, regardless of whether they will become male or female, but the environment (in this case, temperature) determines the levels, timing, and location of genes to be expressed. Temperature has been found to regulate the activity of aromatase, the steroidogenic enzyme that converts androgens to estrogens. Regulation of aromatase expression during the thermosensitive period appears to play a role in the TSD of some reptilian species (Sarre, Georges, & Quinn, 2004; Manolakou, Lavranos, & Angelopoulou, 2006). In mammals, the expression of other steroidogenic enzymes, as well as the ERs and PRs, are regulated by epigenetic mechanisms; e.g., DNA methylation (Yan, Yang, & Davidson, 2001; Xue et al., 2007). Because many of the morphological and physiological processes we have discussed in this chapter are influenced by steroid hormones, it is possible that some of these processes in squamates also are regulated by epigenetic factors. Evidence from rodent studies indicates that environmental factors experienced by the embryo in utero can result in an altered epigenetic program with trans-generational effects (Anway, Cupp, Uzumcu, & Skinner, 2005; Skinner & Anway, 2005; Dolinoy et al., 2007; Jirtle & Skinner, 2007). Therefore, it is also possible that squamate embryos may be susceptible to epigenetic reprogramming by environmental factors while in utero. We find this possibility both exciting and
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overwhelming in its implications. On the one hand, epigenetics might help to explain variation in the pathways and mechanisms invoked in a transition of parity mode. However, it also greatly increases the complexity of how and in what environmental conditions viviparity has evolved in squamates.
ABBREVIATIONS 3b-HSD AVT CAM CG CL CORT E2 EGF ER ESD IFN IGF-1 IL-1 P4 PDGF PG PGE2 PGF2a PR PRL TGF-a TGF-b TSD
3b-hydroxysteroid dehydrogenase Arginine vasotocin Chorioallantoic membrane Chorionic gonadotropin Corpus luteum Corticosterone 17b-estradiol Epidermal growth factor Estrogen receptor Environmental sex determination Interferon Insulin-like growth factor 1 Interleukin 1 Progesterone Platelet-derived growth factor Prostaglandin Prostaglandin E2 Prostaglandin F2a Progesterone receptor Prolactin Transforming growth factor a Transforming growth factor b Temperature-dependent sex determination
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Chapter | 9
Viviparity in Reptiles: Evolution and Reproductive Endocrinology
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Chapter 10
Hormones and Reproductive Cycles in Turtles Gae¨lle Blanvillain,* David Wm. Owens,* and Gerald Kuchlingy *
College of Charleston, Charleston, SC, USA, y The University of Western Australia, Crawley, WA, Australia
SUMMARY Reproductive cycles of cryptodire turtles and species from the northern hemisphere have been investigated extensively compared to those of pleurodire species from the southern hemisphere. Radiography, laparoscopy, and ultrasonography have been used on a few species of turtle to describe a diversity of ovarian and testicular cycles. Radioimmunoassays (RIAs), on the other hand, have provided evidence of the dynamic cycling of peptides and steroid hormones within a reproductive season. All turtles are cyclic breeders and show a range of annual and multiannual reproductive patternsdregulated by nutritional status and environmental conditionsdwhich are most often described as prenuptial or postnuptial. These cycles may be seasonally coordinated by an active pineal gland in the brain via melatonin secretion affecting the hypothalamus. Moreover, the steroid hormone cycles can be rapid and dynamic, and steroid hormones stimulate a carefully orchestrated mating receptivity period: a series of rapid ovulations in the case of multiclutched turtles, egg production, and oviposition events.
1. INTRODUCTION The study of reproductive cycles in turtles began in earnest in the early 1970s (Moll, 1979; Licht, 1984) and has rapidly progressed (reviews by Owens, 1997; Kuchling, 1999; Hamann, Limpus, & Owens, 2003; Miller & Dinkelacker, 2008). An improved knowledge of turtle reproductive cycles is of increasing value for many reasons. Firstly, their very highly conserved basic ‘life in a shell’ format with ‘eggs for all’ is of particular evolutionary interest due to its long-term success. Secondly, from an ecological perspective, few taxa have been as resourceful as turtles in so many different habitats, from high temperate regions to tropical forests, from deserts to estuaries to oceans to lakes and streams. They actually seem to be pre-adapted for many of the more difficult environments of the planet. Thirdly, a special urgency is now needed for an improved understanding of their reproduction due to the clear declines in Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
many turtle species around the world. As their essential habitats have been destroyed or populations depleted by exploitation, a focus on reproduction in the many small remaining populations has become critical. Ironically, several turtle species are still of distinct economic importance: certainly for food and medicine in many regions, for craft materials and artisan work in others, and as pets nearly everywhere that they occur. Finally, as invasive species, some turtles are adding to the problems of some of their fellow chelonians. Regardless of the etiology, there are many reasons to want to improve our understanding of turtle reproductive cycles. Despite the well-deserved perception that turtles are a conservative lot (Bickham, 1981), with their over 200-million-year-old box (shell) to live in and their pervasive terrestrial egg-laying habit, turtles have shown some very interesting and successful modifications on this basic theme. Various adaptations and flexibilities of reproductive patterns have evolved to allow them to reproduce successfully from oceans to deserts and from tropical to temperate zones throughout the world. As will be described below, the nesting patterns of the leatherback sea turtle (Dermochelys coriacea) with vitellogenesis, ovulation, fertilization, albumin coating, shelling, and precise oviposition of 600 or more eggs in up to 9 or 10 clutches is a tour de force of reproductive dynamics (Hirth, 1980; Tucker & Frazer, 1991) (Figure 10.1). At the other extreme, the pancake tortoise (Malacochersus tornieri) lays only a single egg each year, and that is also reflective of very specific adaptations. This species also sports a flat shell and soft plastron, allowing it to hide in rocky crevices when escaping predators (Bonin, Devaux, & Dupre´, 1998). Nevertheless, several single-egg-laying turtles are critically endangered today and their low reproductive output can be seen to be seriously compromising speedy recovery efforts (Bonin et al., 1998). The major life-history challenge for a turtle is to survive to adulthood, which in these late-maturing animals may 277
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FIGURE 10.1 Leatherback sea turtle (Dermochelys coriacea) covering her nest. Note the white spot on the skull (arrow), indicating the location of the well-developed pineal complex. See color plate section.
take several years or even decades, depending on the species (Gibbs & Amato, 2000). Once they are adults, however, they are typically hardy and iteroparous in their reproductive habit; i.e., they repeatedly cycle for reproduction for up to several decades. The anatomical, behavioral, endocrinological, and regulatory organization of the many forms these cycles take is both fascinating and complicated.
2. METHODOLOGIES 2.1. Gonadal Cycles The methodologies used to understand turtle reproductive cycles have improved considerably in recent years (Kuchling, 1999). Originally, research focused mainly on gonadal cycles, often by sacrificing turtles or by using stranded or locally slaughtered animals, looking at gross morphology and histology of the gonads (e.g., Owens, 1980). Over time, the killing of animals, which is now inappropriate for protected species and is best avoided whenever possible, was replaced by less invasive techniques including radiography, endoscopy (sometimes associated with histology), and ultrasound scanning (Gibbons & Greene, 1979; reviews by Kuchling, 1999; Owens, 1999). Each technique has its own set of advantages and disadvantages, and researchers are now able to choose the best methods based on their training, equipment, and the funding available, as well as the appropriateness of the technique for the question they are investigating.
Briefly, radiography represents a powerful technique used on females to determine clutch size while shelled eggs are still present in the oviduct; however, this technique does not allow visualization of other structures (e.g., ovarian follicles, testes, or epididymides) and concerns exist on the effects of irradiation on embryos after multiple exposures, especially when dealing with endangered species (Hinton, Fledderman, Lovich, Congdon, & Gibbons, 1997; Keller, 1998; Kuchling, 1998; Zuffi, Citi, Giusti, & Teti, 2005). Endoscopy, or laparoscopy, is a more invasive technique involving a surgical procedure allowing one to look directly at the gonads and therefore to obtain information on gender, maturity status, and reproductive state of the turtle through direct gonadal visualization and tissue biopsy (Figure 10.2) (e.g., Wood, J., Wood, F., Critchley, Wildt, & Bush, 1983; Limpus & Read, 1985; Wibbels, Owens, Limpus, Reed, & Amoss, 1990; Limpus, 1992; Plotkin, Owens, Byles, & Patterson, 1996; Blanvillain et al., 2008). Laparoscopy has been used extensively over the last few decades, with the main downside being that only highly trained scientists can perform this surgery, as death of the turtle can occur if it is not performed correctly (Owens, 1999). Ultrasonography can replace the techniques described above in many instances, as it allows researchers to visualize and measure follicles and eggs (e.g., Kuchling, 1989; Rostal, Robeck, Owens, & Kraemer, 1990; Kuchling & Bradshaw, 1993; Rostal, Grumbles, Byles, Ma´rquez, & Owens, 1997; Rostal, Owens, Grumbles, MacKenzie, & Amoss, 1998; Kuchling & Razandrimamilafiniarivo, 1999; Shelby, Mendonc¸a, Horne, & Seigel, 2000), as well as testes and
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FIGURE 10.2 Laparoscopy on an adult male loggerhead sea turtle (Caretta caretta) following proper surgical procedures. See color plate section.
epididymides of some adult male turtles (Figure 10.3) (Lee, 2003; Blanvillain et al., 2008). This technique has the advantage of being non-invasive; however, it does not allow the visualization of the corpus luteum, and therefore researchers can not know if a female has laid a clutch of eggs in the recent past. In addition, ultrasonography cannot distinguish the inactive male gonadal structures. Finally,
histology of the gonads is still performed today as it can answer basic questions on a male’s reproductive status and spermatogenic stages by looking at the cells lining the seminiferous tubules. This technique can be undertaken during endoscopy by taking a small biopsy sample (e.g., Wibbels et al., 1990; Limpus, 1992; Plotkin et al., 1996; Lee, 2003; Blanvillain et al., 2008).
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FIGURE 10.3
Ultrasound examination of the gonads in an adult male loggerhead sea turtle (Caretta caretta). See color plate section.
2.2. Hormonal Cycles Hormonal cycles were first elucidated using direct methods to evaluate plasma hormone levels by RIA (radioimmunoassay) or more recently ELISA (enzyme-linked immunosorbent assay). With these methods, hormonal cycles of steroids (testosterone (T), estradiol (E2), progesterone (P4), and corticosterone (CORT)), and gonadotropins (GTHs) (follicle-stimulating hormone (FSH) and luteinizing hormone (LH)) can be evaluated using a simple blood sample and relatively straightforward laboratory techniques. Nowadays, ELISA kits are available to analyze most of the steroid hormones without the downside of using radioactive chemicals. The relatively large size of some turtles, including sea turtles and large tortoises, has made it somewhat easier to obtain repeated blood samples (with a larger volume) than with smaller or more dangerous reptiles (Owens & Ruiz, 1980; Jacobson, Shumacher, & Green, 1992). This fact has provided a distinct advantage to those interested in studying the regulatory physiology of reptiles. On the other hand, turtles can be very hard to find at times, especially during aestivation or hibernation when they understandably are less well studied.
3. DIFFERENT TYPES OF CYCLES AND TIMING OF REPRODUCTION IN TURTLES 3.1. Cyclicity As Licht (1984) discusses most convincingly, there is strong evidence of seasonal breeding in reptiles, especially
turtles. Even the tropical sea turtle species, in which nesting can occur in all months, have a clear peak of nesting during specific seasons. Most importantly, each individual has a very clear seasonal reproductive cycle.
3.1.1. Prenuptial and postnuptial reproductive cycles One of the most useful paradigms developed for reptilian reproductive cycle descriptions is that originally described by Volsoe on Vipera berus (in Saint Girons, 1963), who noticed two primary patterns that he called prenuptial and postnuptial (also referred to as ‘associated’ and ‘dissociated’). These two patterns are based on the timing of spermatogenesis (peak testis weight) and ovulation relative to the mating season (Figure 10.4). The prenuptial pattern (type I pattern of Lance (1998)), which is more typically seen in tropical and subtropical breeding turtles, is characterized by a steady and somewhat synchronized development of the ovary and testis continuing through the winter months (or dry season) and culminating with maximal sperm production and epididymal recrudescence just prior to the spring mating period and simultaneously to the initiation of a single or multiple ovulation and nesting sequence in the female. In terms of parsimony, this would be the simpler and more basic cyclic form. One can envision the more complicated and derived condition in the postnuptial cycle (type II pattern of Lance (1998)) of more temperate regions in which testicular and gonadal growth commence full-scale in the late summer (or late rainy season) and move to full gonadal development in the late fall, at which time cold weather, or
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FIGURE 10.4 Schematic description of the two types of gonadal cycle in turtles based on gametogenesis and mating. Reproduced from Licht (1984), with permission.
other extreme climatic conditions, cause a full interruption in the sequence. Instead of continuing to breed, these species then go into hibernation or aestivation for up to several months. Then, in the spring, under warming or hydrating conditions, these fully developed or nearly fully recrudesced individuals emerge and soon begin their courtship and mating phase to permit nesting as early as possible in the spring. After the completion of the reproductive phase, the postnuptial cyclers begin an intense foraging period to permit nutritional accumulations for full gonadal growth in the late fall prior to the next hibernation. The critical point is that the postnuptial species must be able to store their gonadal products (sperm and ova) through the hibernation or aestivation period. Essentially, however, the regrowth phase after the breeding season starts off again in a similar way in both types of cycle. This dichotomic categorization is very useful but many variations exist. For example, some species of reptile have
been shown to commence spermatogenesis/vitellogenesis in the late summer/fall but to complete gamete production (spermiation, preovulatory follicles) only in spring of the following year. This has been termed a ‘mixed’ cycle or combination of prenuptial and postnuptial patterns (Saint Girons, 1963). In this case, mixed and prenuptial cycles only differ through the interruption of the cycle by winter hibernation in the first instance. One other pattern is that many species of turtle from temperate climates breed both in the fall and the spring, and their testicular cycle could be described as both prenuptial and postnuptial (Licht, 1984).
3.1.2. Sexual maturation Turtles require years or decades to reach sexual maturity. Good foraging opportunities and an optimal temperature history will minimize the time required to reach sexual maturity. For example, in captive green sea turtles Chelonia
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mydas (normally herbivores) fed a high-protein diet, sexual maturity was reached in 8–9 years (Wood & Wood, 1980) compared to 20–40 years in the wild (Seminoff, Rosendiz, Nichols, & Jones, 2002; Balazs & Chaloupka, 2004). These captive conditions would appear to reflect the maximum reproductive potential for the species. Similarly, in poor foraging habitat or poor forage years, or when climatic conditions are sub-optimal such as during El Nin˜o Southern Oscillation events (associated with periods of warmer waters, therefore less productive), sea turtles will be delayed in maturation or skip a year or more of reproduction as adults (Limpus & Nicholls, 1988; Chaloupka, 2001; Solow, Bjorndal, & Bolten, 2002; Saba et al., 2007).
3.1.3. Nesting cyclicity Most turtles appear capable of annual breeding when in appropriate habitats and under ideal environmental conditions. In particular, most cryptodire and pleurodire freshwater turtles (except Erymnochelys madagascariensis and Carettochelys insculpta, in which females show biennial ovarian cycles, as described in Section 4.2.2) and tortoises seem to breed annually. Interestingly, some adult females may not reproduce in a given year (see references in Kuchling, 1999; Miller & Dinkelacker, 2008). However, as Kuchling (1999) discusses, this may not reflect a multiannual cycle but rather the capability of the females to skip reproduction if the environmental conditions are suboptimal, or even to abort ovulation and egg production despite showing a normal vitellogenic cycle, as in the western Australian freshwater turtle, Pseudemydura umbrina (Kuchling & Bradshaw, 1993). Female sea turtles of the genus Lepidochelys (the ridleys) often breed annually (average of 1.8 years for the Kemp’s ridley) (Witzell, Salgado-Quintero, & Gardun˜oDionte, 2005; Tripathy & Pandav, 2008), whereas female sea turtles of the other genera will normally reproduce every 2–4 years, as apparently do the large river turtles Podocnemis expansa from the Amazon Basin (Junk & Silva, 1997). However, recent studies of P. expansa found individual females returning to nest on an annual basis (R. Vogt, personal communication). With sea turtles, as Kuchling discusses (1999), ‘capital’ breeding in the sense of Drent and Daan (1980) is the basic plan: when the animal has gained enough nutritional reserves (fat, probably) to allow both the energetically demanding migration and a full reproductive series, then and only then do they initiate the reproductive migration. In essence, selection must have favored an all or none reproductive effort for these migratory capital breeders. In contrast, the female giant Aldabran tortoise Dipsochelys dussumieri could be described as an ‘income’ breeder (Kuchling, 1999) in that she will simply reduce the number of eggs she is producing for the season to compensate for a poor foraging year. As Kuchling further
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notes, these two species, the green sea turtle and the Aldabran tortoise, represent the two extremes of capital and income breeders, with all other turtles showing intermediate adaptations tailored to their historic environments. Interestingly, in some areas, adult male sea turtles C. mydas and Caretta caretta both show clear signs of annual reproductive cycles (thus much shorter than those of females); however, the presence of a small number of reproductively inactive males, often remaining near the mating grounds, has been noted in a few studies (Wibbels, Owens, & Amoss, 1987; Limpus, 1993; Jessop, Limpus, & Whittier, 2004; Blanvillain et al., 2008). Limpus (1993) determined the remigration interval of adult male C. mydas to be 2.08 years (range 1–5 years), whereas 10–15% of adult male C. caretta captured in the Cape Canaveral shipping channel were found to be inactive in two separate studies (Wibbels et al., 1987; Blanvillain et al., 2008). These males, captured during the mating season, showed basal T levels, regressed epididymides, and inactive testes based on laparoscopy and/or histology (Figure 10.5). Moreover, it appears that inactive males do not show the soft plastra exhibited by reproductively active males of several sea turtle species during the breeding season (Figure 10.6) (Blanvillain et al., 2008). In contrast to adult males C. mydas and C. caretta, the single study on the reproductive physiology of adult male Eretmochelys imbricata in Australia reveals that hawksbill males might reproduce annually (Jessop et al., 2004), although this study may not have sampled sufficient numbers of males, which might have remained in deeper-water foraging grounds. Clearly, more studies are needed on the questions of male mating cyclicity. The arribada, or synchronous nesting behavior, is well documented in the two ridley sea turtle species (Plotkin, 2007). Thousands or even hundreds of thousands of individuals have been known to nest at specific mainland beaches in brief periods of from a few hours to a few days. The most endangered sea turtle, the Kemp’s ridley (Lepidochelys kempii), averages about three nests per season, nests synchronously in the daytime, and breeds primarily in the western Gulf of Mexico (Rostal, 2007). In contrast, the sister species, the olive ridley (Lepidochelys olivacea), is the most abundant sea turtle on the planet, averages about two clutches per season, nests synchronously at night only, and breeds in the rest of the tropical oceans except for the Gulf of Mexico (Plotkin, 2007). While the overall organization of the arribada does not appear to be socially coordinated (for a review see Bernardo & Plotkin, 2007), the way females seem to resist the stress of high-density interaction while the massing is occurring certainly suggests a physiological facilitation of the unusual behavior through the hypothalamus–pituitary–adrenal (HPA) axis (Valverde, Stabenau, & Mackenzie, 2007). All sea turtles ovulate the next clutch of follicles within hours of nesting (Owens &
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FIGURE 10.5 Laparoscopic (1, testis; 2, epididymis) and histological images (3) of reproductively active (a) and inactive (b) adult male loggerhead sea turtles (Caretta caretta) collected in Cape Canaveral, Florida. E, epididymis; T, testis; ST, seminiferous tubules. Reproduced from Blanvillain et al. (2008), with permission. See color plate section.
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FIGURE 10.6 Soft plastron in a reproductively active male Caretta caretta with contour lines from the PLASTron software, as described in Blanvillain et al. (2008). Reproduced from Blanvillain et al. (2008), with permission. See color plate section.
Morris, 1985). What is unique about the ridleys is that they will prepare the next clutch of eggs in the typical 10- to 14-day cycle as do other sea turtles (and they do occasionally nest on this ‘short cycle’); however, most ridleys typically wait for about a month for another lunar/tidally cued arribada event (Bernardo & Plotkin, 2007). In fact, they usually hold the fully calcified eggs in the oviducts for an extra two weeks for the next arribada. Plotkin, Rostal, Byles, and Owens (1997) documented a surprising but apparently not unusual event when a heavy multi-day rain (a possible natural stress-inducing event) occurred at Nancite in Costa
Rica in which large numbers (thousands) of olive ridleys that were physiologically prepared to nest were turned away by the rains only to return after a second lunar cycle to nest synchronously and successfully after a full 60-day waiting period. The eggs were successfully retained in the oviducts for more than 50 days. Other species of turtle are known to exhibit aggregative nesting. For example, the Amazon River turtle P. expansa has been shown to nest in groups of about 20 individuals, for a total of 200 to 500 individuals nesting on the beach in one night (Alho & Pa´dua, 1982). These turtles (both adult
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males and females) also exhibit reproductive migrations from lakes to rivers once the water level of the rivers has declined enough to expose sandy beaches on the riverbanks used as nesting grounds. This distinct aggregative behavior is not displayed by the other five species (much smaller) of Podocnemis from South America, and it has been hypothesized that the unique nesting pattern of P. expansa could be due to their large size and density (at least in the past), resulting in few adequate nesting beaches and group nesting sites (Pritchard & Trebbau, 1984). Another explanation could be the predator swamping effect as originally described in olive ridley turtles (Eckrich & Owens, 1995; Bernardo & Plotkin, 2007). Historic records of large Asian river turtles (Batagur baska, Batagur trivittata) also report aggregative nesting events on sandbanks (Maxwell, 1911), but today these populations are extirpated or too depleted for any mass nesting to take place.
3.1.4. Variation in the number of clutches The number of clutches deposited per season varies greatly depending on the species. Some species are known to lay only one clutch, as in the common snapping turtle Chelydra serpentina (White & Murphy, 1973); the painted turtle Chrysemys picta (Gibbons, 1968); the desert and gopher tortoises Gopherus agassizii (Rostal, Lance, Grumbles, & Alberts, 1994) and Gopherus polyphemus (Landers, Garner, & McRae, 1980); the Australian snapping turtles Elseya dentata (Kennett, 1999); and even the Amazon river turtle P. expansa, despite its large size (Alho & Pa´dua, 1982). The sea turtles, however, are all known to deposit from 2 (in L. olivacea) to up to 10 clutches (in D. coriacea) with a renesting interval varying from 9–10 days (in D. coriacea) to a typical 30 days and a maximum of 66 days (in L. olivacea arribada) (Plotkin et al., 1997; Kalb, 1999; Rostal et al., 2001; Reina, Mayor, Spotila, Piedra, & Paladino, 2002; Tripathy & Pandav, 2008). Other species of turtle are multiclutched (e.g., Stenotherus odoratus, Testudo hermanni, Testudo horsfieldii, Geochelone nigra; discussed in Section 4.1). However, for many additional species, the nesting patterns in the wild simply are not known, due to the requirement of long-term mark recapture or ultrasound studies, which are difficult to implement in many places.
3.2. Timing of Reproduction The timing of mating, mating receptivity, and the effectiveness of individual mounting activities are not at all well understood in turtles (Kuchling, 1999). In wild green sea turtles, Booth and Peters (1972) described a very definite ‘receptivity’ period of female choice on Wreck Island off the Great Barrier Reef. Similarly, Crowell Comuzzie and Owens (1990), in captive green turtles, present more
quantitative evidence of a distinct receptive period about 30 days prior to the first nesting, with females permitting no additional mating after this time despite the multiple clutch nesting sequence that follows. Generally speaking, spring mating, whether prenuptial or postnuptial, is well documented, with some species showing both fall and spring mating. The musk turtle (S. odoratus) and the desert and gopher tortoise (G. agassizii and G. polyphemus), discussed in Section 4.1, seem to mate primarily in the fall (McPherson & Marion, 1981; Mendonc¸a & Licht, 1986; Ott, Mendonc¸a, Guyer, & Michener, 2000; Lance & Rostal, 2002). However, in the case of the diamondback terrapin (Malaclemys terrapin centrata), Estep (2005) showed a pronounced period of courtship in the spring with a much shorter and presumably less effective period in the fall. This, and similar observations of shorter fall courtship episodes, does raise the important question of the actual significance or functionality of the fall mating period, described in many turtle species. Gist, Michaelson, and Jones (1990) showed that insemination in C. picta occurred only during fall mating as sperm in the oviducts were recovered only during October, and electroejaculation in the males yielded sperm only during the fall. Therefore, it appears that spermatozoa can be stored in the female reproductive tract until the following spring, when ovulation occurs (see Chapter 5, this volume). Finally, a few turtles have their peak mating activity in winter, e.g., Mauremys rivulata in Israel (Gasith & Sidis, 1985) and Chelodina oblonga and P. umbrina in southwestern Australia (Kuchling, 1999). Since turtles have the potential to store spermatozoa in the male as well as in the female reproductive tract and, in many species, to mate at any time of the year, it would be more precise to use the time of ovulation and fertilization, rather than the time of mating and insemination, as defining parameters to differentiate between prenuptial and postnuptial gonadal cycles.
4. HORMONAL AND GONADAL REPRODUCTIVE CYCLES 4.1. Cryptodira Cryptodire turtles represent the majority of living turtles (10 out of 13 families) and flex their neck in the vertical plane to pull it inside their shell, between the shoulder girdles.
4.1.1. Freshwater turtles The musk, or stinkpot, turtle, S. odoratus, has been studied extensively and represents a good example of what typical gonadal and hormonal cycles look like for both male and female freshwater turtles (McPherson & Marion, 1981; 1982; McPherson, Boots, MacGregor, & Marion, 1982;
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Mendonc¸a & Licht, 1986). Musk turtles hibernate during the winter months, emerging in late February to early March, depending on the geographical location (with southern populations having a reduced period of hibernation). Spermatogenesis in males begins in April and is maximal in August until early October, at which time testis weight is also maximal and most mating occurs (throughout the fall). Epididymal duct diameters are maximal in September during spermiation and testes then regress in late October. Levels of T and FSH (measured using an antibody derived from sea turtles) are higher from August until the end of October (Mendonc¸a & Licht, 1986). Concentrations of these hormones were correlated throughout the entire year, but LH was undetectable. The peak of FSH and T in late summer and fall occurred concomitantly with spermiogenesis and spermiation; however, the peak of FSH occurred slightly earlier (in August) than that of T (prior to October when testes started regressing) (Figure 10.7). Mendonc¸a and Licht (1986) hypothesized that FSH was the primary GTH regulating androgen secretion and gonadal growth in turtles, although differences across species may exist. However, spermatogenesis started in the spring when FSH levels were low, suggesting that elevated FSH at lower temperatures is not necessary to stimulate spermatogenesis.
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In S. odoratus females, LH and FSH (assayed by RIAs developed from C. mydas) remained undetectable throughout the year (Mendonc¸a & Licht, 1986). Ovarian weights were high from March until July, when preovulatory follicles and oviductal eggs were present, and in November and December again, when preovulatory follicles were present. The first oviductal eggs were visible at the beginning of April until mid-July. In August and September, ovarian weight was low because females had just finished laying their last clutches of eggs, and vitellogenesis started again in October or November. Estradiol levels were high in the spring (and higher in females carrying eggs), during follicular growth of the next clutches of eggs, then decreased in July and remained low until November (Figure 10.8). Estradiol then increased in November during follicular growth for the next reproductive season (McPherson et al., 1982; Mendonc¸a & Licht, 1986). This rise in E2 stimulates the liver to synthesize vitellogenin (Vtg), a precursor protein of egg yolk, which explains the rise in ovarian weight at this time. Females hibernate with preovulatory follicles, which are ovulated during the next spring. Testosterone levels in females were highest in April just prior to ovulation of the first clutch of eggs and remained low thereafter until vitellogenesis in the fall, at which time a clear T increase was seen but not statistically evaluated (McPherson et al., 1982).
FIGURE 10.7 Monthly mean levels of testicular gonadosomatic index (GSI) and plasma testosterone (T) and follicle-stimulating hormone (FSH) for male musk turtles, Sternotherus odoratus, from South Carolina. Vertical lines are 1 standard error; numbers are sample sizes. Reproduced from Mendonc¸a and Licht (1986).
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FIGURE 10.8 Seasonal variation in sex steroid concentrations (testosterone (T), estradiol (E2), and progesterone (P4), in ng/mL) of the female musk turtle, Sternotherus odoratus from Alabama. Three clutches are represented with ovulation (OV) and nesting (N) being hypothesized to occur around the time indicated by vertical arrows during the nesting season. SS, soft-shelled eggs in the oviduct; HS, hard-shelled eggs in the oviduct; Preov, preovulatory follicles but no eggs in the oviduct. Adapted from McPherson, Boots, MacGregor, and Marion (1982).
One reason why T does not fluctuate with the next nesting cycle (in this multiclutch turtle) could be that T, a known inhibitor of vitellogenesis (Ho, Danko, & Callard, 1981), acts as a precursor for E2, allowing vitellogenesis to proceed between clutches. Progesterone, on the other hand, increases during the periovulatory period and just after ovulation, and then decreases quickly during the shelling of the eggs in the oviduct (Figure 10.8). This quick peak in P4 is observed for each subsequent clutch of eggs and has been described in several other species of turtle. Progesterone levels also increase during the fall ovarian growth period. Ho et al. (1981) suggested that P4 synthesized by the corpora lutea inhibit vitellogenesis, as usually no change in follicular size is observed during ovulation, when P4 levels are elevated. Progesterone also may have a role in the final maturation of the follicles, the ovulation process (Callard, Lance, Salhanick, & Barad, 1978), and the stimulation of enzyme production involved in follicular rupture (McPherson et al., 1982). This hormone possibly decreases myometrial contractility in the oviduct in order to retain the eggs so that normal shelling may occur (Callard & Hirsch, 1976; Giannoukos & Callard, 1996). Much of this pattern of gonadal cycles remains constant for other species of freshwater turtle. Hormonal cycles, however, show considerable variations, and one notable difference is the existence of a second T peak in the spring
for some male turtles, sometimes associated with mating, although the exact timing of the mating activity is often not well known or described. This was first shown in the painted turtle, C. picta (Callard, I., Callard, G., Lance, & Eccles, 1976; Licht, Breitenbach, & Congdon, 1985) and later on in the common snapping turtle C. serpentina (Mahmoud, Cyrus, Bennett, Woller, & Montag, 1985). As in the musk turtle, FSH and T levels in C. picta are highly correlated and are low in animals captured just after coming out of hibernation. However, FSH and T rise quickly in April, coinciding with mating activity and a period of gonadal quiescence (no spermatogenetic activity) (Licht et al., 1985a). These authors suggested that water and air temperatures in early April might still be too low to induce testicular growth, even though FSH levels are elevated at this time. As in the musk turtle, FSH and T also peak in the fall (but not as high as in the spring), just after summer testicular recrudescence is maximal. This circumstance of T levels being at their highest during a time of gonadal quiescence (in the spring) and after the peak of spermiogenesis (in summer) differs from S. odoratus (and many other turtle species), as discussed by Kuchling (1999). Spermatogenesis in C. picta is described as being postnuptial (spermatozoa are produced in the fall and stored over the winter until the spring mating season) with high levels of T possibly associated with mating activity.
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However, many species exhibit mating activity when T levels are low, including Chrysemys dorbigni (Silva, Moraes, & Wassermann, 1984); Graptemys flavimaculata (Shelby et al., 2000); and G. agassizii for the spring mating period (Rostal et al., 1994; see under ‘terrestrial turtles’). Female painted turtles and common snapping turtles (single clutch turtles) have very similar hormonal and gonadal cycles to those of musk turtles (multiclutch turtle): high but transient levels of T, E2, and P4 just prior to ovulation in the spring, and smaller peaks of E2 and T during vitellogenesis in the fall (Gibbons, 1968; Callard et al., 1978; Mahmoud & Licht, 1997). Gapp, Ho, and Callard (1979) also detected peaks of Vtg in the spring and the fall concurrent with elevated steroid hormones in C. picta. However, other species show variations on this general pattern. For example, females of G. flavimaculata undergo vitellogenesis in the spring, when a small rise in E2 is visible, and not in the fall (Shelby et al., 2000). In addition, T levels did not seem to be related to other steroid hormones throughout the study period. Similarly, a single peak in E2 was measured in Lissemys punctata punctata during the final growth of ovarian follicles (late summer) in preovulatory females during the mating season (Sarkar, Sarkar, & Maiti, 1996). The diamondback terrapin Malaclemys terrapin is the only truly obligatory estuarine turtle. They range widely from near tropical areas in the southern Florida Keys and Bermuda to high temperate regions in Massachussetts (Brennessel, 2006). In general, their reproductive patterns and endocrinology are very similar to the other emydids, to which they are closely related (postnuptial) (Lee, 2003), although there is some inconsistency in the literature on the question of multiple clutches in some of the subspecies.
4.1.2. Sea turtles The reproductive physiology of sea turtles has been studied extensively, both in the wild and in captivity; however, most of what we have learned is based on nesting females, due to their large size and ease of sampling during nesting events. In contrast, few studies of adult males exist, especially from the wild. The only species for which hormonal cycles of adult males have been investigated in the wild are the loggerhead, C. caretta; the hawksbill, E. imbricata; and the green sea turtle, C. mydas (Wibbels et al., 1987; 1990; Jessop et al., 2004; Blanvillain et al., 2008), while Kemp’s ridley, L. kempii, and C. mydas have been studied in captivity at the Cayman Turtle Farm, Grand Cayman, BWI (Licht, Wood, Owens, & Wood, 1979; Licht, Wood, & Wood, 1985; Rostal et al., 1998a). No data on reproductive physiology of adult male leatherbacks, D. coriacea; olive ridleys, L. olivacea; or flatbacks, Natator depressus, exist,
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and research should be carried out to better understand their reproductive physiology. Typically, adult males display a prenuptial rise in T levels in which T rises in the fall, coincident with spermatogenesis and testicular recrudescence, peaks in the winter, and starts decreasing around the time of the reproductive migration and at the onset of mating in the spring (Figure 10.9) (Licht et al., 1985b; Wibbels et al., 1987; 1990; Rostal et al., 1998a). Generally, male T levels have returned to baseline during the female’s nesting period, when males are departing on the return migration to the foraging grounds (Wibbels et al., 1990; Rostal et al., 1998a). Inactive males, however, show much lower T levels (< 5ng/mL) and no peak in winter and spring, as seen in C. mydas from Australia (Jessop et al., 2004) and C. caretta from the southeast USA (Wibbels et al., 1987; Blanvillain et al., 2008). Interestingly, males of E. imbricata (Jessop et al., 2004) did not show the same T pattern as found in the other species of sea turtles, in that T levels were significantly higher in December (austral spring/ summer) when compared to August (austral winter). Therefore, it appears that the prenuptial rise in T is not as clear in male hawksbills; however, the authors suggested that a delayed phase shift in the T cycle might take place, as the main nesting period usually occurs later in the season in E. imbricata (December–March; peak in January and February) when compared to C. mydas (peak in December and January) (Dobbs, Miller, Limpus, & Landry, 1999; Limpus, Miller, Parmenter, & Limpus, 2003; Dobbs, Miller, Owens, & Landry, 2007). However, samples were collected at only two time points and more work needs to be done to clearly understand adult male hawksbill reproductive cycles. Unlike that of adult males, the reproductive physiology of adult female sea turtles, in particular during the nesting period, has been studied extensively, both in the wild and in captivity, and we have a fairly good understanding of the hormonal changes that happen during the reproductive cycle (Licht et al., 1979; Licht, Owens, Cliffton, & Penaflores, 1982; Wibbels et al., 1990; Rostal, Paladino, Patterson, & Spotila, 1996; Rostal et al., 1997; Whittier, Corrie, & Limpus, 1997; Rostal et al., 1998a; 2001; Dobbs et al., 2007). Most species have been studied to some extent, with the exception of the Australian flatback N. depressus. Typically, T levels rise in the winter to reach a peak in March, coinciding with migration and mating activity (Figure 10.10). Testosterone levels then decrease slowly, following a stepwise progression, as the nesting season advances. Perhaps the granulosa cells of the follicles stop producing T every time a new set of follicles is ovulated, thus explaining the sequential dips in T that occur with each new ovulation (Figure 10.10). Estradiol levels tend to follow the same general pattern; however, the more gradual increase of this steroid hormone in the fall is
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FIGURE 10.9 Seasonal variation in testosterone (T) levels of reproductively active and inactive adult male loggerhead sea turtles, in relation to their reproductive behavior. Data are adapted from Wibbels, Owens, and Amoss (1987) and Blanvillain et al. (2008), with permission.
FIGURE 10.10 General model for endocrine fluctuations in adult female sea turtles, in relation to their reproductive behavior. In this example, the female would ovulate and nest three times. M, mating; O, ovulation; N, nesting; RM, return migration. Model adapted from Owens (1997), with permission.
correlated to long-term (several months) vitellogenesis. This was confirmed by ultrasound imaging of the growing follicles, as well as with the presence of elevated concentrations of serum calcium, indicative of vitellogenesis, from June to December. Free calcium ions readily bind to Vtg, causing dissolution of bone and a rise in total circulating
calcium whenever Vtg is present. Low, more constant levels of E2 were observed during the nesting season (Rostal et al., 1997; 1998a; 2001). Low levels of serum calcium during nesting also indicate that vitellogenesis might essentially be completed before females start laying their first clutch of eggs. However, other studies on
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C. caretta and E. imbricata have reported peak levels of E2 only after the first or second clutches of eggs have been laid, and this could suggest that vitellogenesis, at least at a lower rate, might continue into the nesting season, until all follicles are fully grown (Wibbels et al., 1990; Dobbs et al., 2007). Progesterone levels showed some contradictory results, possibly due to different sampling protocols. While L. kempii (Rostal et al., 1997) and D. coriacea (Rostal et al., 2001) showed highly variable levels of P4 throughout the nesting period, and therefore no correlation between this hormone and the reproductive condition of the females, Whittier et al. (1997) in C. caretta and Rostal et al. (1998a) in L. kempii showed higher levels of P4 at the beginning of the nesting season. More detailed studies of hormonal changes around the time of ovulationdabout 1–3 days following oviposition for the current clutch of eggs, until all clutches have been laid (Licht et al., 1979; 1982)dshowed a rapid surge in P4 concentrations at the time of ovulation, quickly returning to basal levels before shell deposition started (Licht et al., 1982; Wibbels et al., 1992). This surge was associated with similar transient high levels of LH in both studies, and of T in C. caretta and C. mydas only (Figure 10.10) (Wibbels et al., 1992). Estradiol levels, on the other hand, were low and did not exhibit significant changes in both studies. However, in an earlier study in C. mydas, total estrogens were measured in the middle of the internesting cycle at the Grand Cayman Turtle Farm. Estrogens significantly increased during this time, compared to the actual time of nesting four to six days later (Lance, Owens, & Callard 1979). Interestingly, an FSH surge seen at the time of ovulation described in Wibbels et al. (1992) suggests a distinct function for this GTH in the slower and less dramatic stimulation of E2 during the internesting period (Lance et al., 1979; Wibbels et al., 1992). As mentioned above, P4, secreted by the corpora lutea after stimulation by LH from the pituitary, has been linked to the ovulation process, functioning by acting on the maturation of follicles and reducing oviductal movement during the shelling of the eggs. Owens and Morris (1985) also suggested that the P4 surge at the time of ovulation may have an important role in oviductal albumin production as the fertilized ova are processed in the oviduct to form eggs. Indeed, the common phenomenon of ‘spacer’ or ‘yolkless’ eggs seen at the end of the leatherback and other sea turtle clutches is likely to be leftover albumin being shelled as it follows the real clutch through the shelling region of the oviduct. In addition, a short cycle or spike of both arginine vasotocin (AVT) and neurophysin (NP) has been shown during the nesting period of olive ridleys and loggerhead sea turtles by Figler, MacKenzie, Owens, Licht, and Amoss (1989) (Figure 10.11). These hormones are secreted by the neurohypophysis and act to regulate oviductal contractions in sea turtles and probably in other species of
Hormones and Reproduction of Vertebrates
turtle as well. In this study, both plasma AVT and NP increased drastically from the time of emergence from the ocean to reach a peak just prior to oviposition of the first egg. Arginine vasotocin then declined to low levels as the turtle returned to the water; however, NP levels stayed relatively elevated after oviposition, possibly due to a longer halflife of the much larger protein. Similarly, levels of prostaglandin F (PGF) and prostaglandin E2 (PGE2) were measured at nine different time points during nesting and oviposition in the loggerhead sea turtle (Guillette et al., 1991). The results showed an increase in both PGF and PGE2 during nest digging, reaching a peak during midoviposition for PGE2 and during nest covering for PGF. Concentrations returned to basal levels during body pit covering. The authors suggested that, as in other species of reptile, AVT in turtles might induce the synthesis of PGF. However, the stimuli for release for both PGE2 and AVT themselves are still unknown. Finally, Guillette et al. (1991) hypothesized that PGE2 might be important for cloacal relaxation, whereas PGF, in concert with AVT, could stimulate the oviductal contraction necessary for the expulsion of the eggs through the cloaca.
4.1.3. Terrestrial turtles (tortoises) There are four species of tortoise in North America, and of these the desert tortoise (G. agassizii) has been the most extensively studied, providing detailed insight into both male and female gonadal and hormonal cycles (Rostal et al., 1994; Lance & Rostal, 2002). The desert tortoise exhibits mating activity twice a year (in the spring and in the fall) and its cycle is described as being postnuptial, much like freshwater turtles such as S. odoratus (McPherson et al., 1982; Mendonc¸a & Licht, 1986). Nesting typically occurs in the spring to early summer. In Rostal et al. (1994), the male testicular cycle was elucidated using sacrificed individuals. In May, the testes are regressed, then spermatogenesis progresses until it reaches a peak in October (prior to hibernation), when the seminiferous tubule diameters are maximal. Mature spermatozoa migrate into the epididymides prior to hibernation. Plasma T rises significantly from May to August to a peak of ~244 ng/ mL, concomitantly to gonadal recrudescence, and declines prior to hibernation. Upon emergence from hibernation, T levels are relatively low (~18 ng/mL), despite the spring mating period. Female T levels rise from mid to late summerdduring fall matingdto a presumed peak in April following emergence from hibernation and prior to ovulation. At this time, P4 levels increase and then rapidly decline to baseline levels once the eggs are laid. Ultrasonography was used to investigate ovarian follicular growth in summer and fall, after nesting. Follicles had reached ovulatory size prior to hibernation, and shelled
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FIGURE 10.11 Summary of relative changes in plasma arginine vasotocin (AVT), neurophysin, prostaglandin F (PGF), and prostaglandin E2 (PGE2) in nesting female loggerhead sea turtles. E, emerging; BP, body pit construction; N, nest chamber construction; EO, early oviposition; LO, late oviposition; C, covering body pit; R, returning to the ocean. * Data from Figler, MacKenzie, Owens, Licht, and Amoss (1989). Reproduced from Guillette et al. (1991).
eggs were observed in the oviducts in mid-April. Nesting started in early May, after which atretic follicles were observed. Estradiol, lipids, and plasma calcium levels (indicative of Vtg) were high in the fall during follicular growth and started decreasing after emergence in April. Most females laid only one clutch. Similar hormonal and gonadal cycles were observed in a wild population of gopher tortoises (G. polyphemus) in the southeast USA by Ott et al. (2000), with the only difference from Gopherus gopherus being that mating occurred only during the fall, before hibernation. Oviposition typically occurred from April–July and females only laid one clutch, like most desert tortoises.
In Hermann’s tortoise, T. h. hermanni, from Mediterranean Europe, spermatogenesis starts in April to May, just after hibernation and during the main mating season for the eastern subspecies (Testudo h. boettgeri) collected in the former Socialist Federal Republic of Yugoslavia (SFRY) (Kuchling, Skolek-Winnisch, & Bamberg, 1981; Kuchling, 1982). Spermatogenesis continued through the summer, during which time T peaked in the eastern (August) as well as in the western (July/August) subspecies T. h. hermanni, and declined in September and October (HuotDaubremont, Bradshaw, Bradshaw, Kuchling, & Grenot, 2003). In the eastern subspecies, slightly elevated T was observed in April (compared to October/November), with
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FIGURE 10.12 Schematic representation of testosterone (T) levels and the ovarian and testicular cycles in relation to behavior in the Steppe’s tortoise, Testudo horsfieldii. Plain lines represent T levels based on Lagarde et al. (2003); dashed lines are hypothetical.
lower levels in May and June prior to a steep rise in summer. In the western subspecies, slightly elevated T was observed in March (compared to October), with lower levels in April through June, prior to a steep rise in summer. In both subspecies, the post-hibernation T increased to approximately the same levels as in September. The difference between the studies (post-hibernation spring elevation either in March or April) may reflect an earlier termination of hibernation in the west. In both subspecies, the secondary post-hibernation spring peak does not seem to be directly related to mating: in the former SFRY the main mating activity in the wild occurs in May and June, with again a slight increase in September (Kuchling, 1982), whereas in France the main mating period for the western subspecies was supposed to be in August/September when T levels peak. However, this mating pattern in France was observed in a semi-captive population (Huot-Daubremont & Grenot, 1997) and may have been skewed by a constant provision of water and food. During hibernation, testes are regressed and spermatozoa are stored in the epididymides for the following mating season. Testosterone levels in females were high after hibernation, suggesting that follicles are mature by April. Testosterone then fell to low levels in June and July, during oviposition, and rose again in August during renewed vitellogenesis and follicular growth. Progesterone levels were highest during the periovulatory period, in April and May, and peaked prior to each nesting event (Huot-Daubremont et al., 2003).
In contrast to this somewhat general pattern, the Steppe’s tortoise, T. horsfieldii, provides an excellent example of a tortoise that has to accomplish all of its tasks (i.e., feeding, mating, and nesting) in an extremely short amount of time (three months) due to environmental and climatological constraints. Indeed, the Steppe’s tortoise spends nine months in dormancy underground, during which spermatogenesis and vitellogenesis must occur. Lagarde et al. (2003) studied plasma T and P4 cycles during the active period (mid-March to mid-June) and found that T levels in males were highest in March, just after hibernation and during the mating season. Brushko (1981) showed that sperm production occurs during aestivation (dormancy), in July–August, when tortoises are buried underground and when peak levels of T might be expected. Female T and P4 levels peaked in mid-April, prior to ovulation of the first clutches. Plasma calcium levels were higher in mid-April and May, indicative of vitellogenesis and/or egg production at this time. We suspect that vitellogenesis and ovarian growth starts during aestivation, in the summer, so that the females are physiologically preparing for the next breeding season before hibernation. A summary of the hormonal pattern and hypothetical gonadal growth pattern in the Steppe’s tortoise is represented in Figure 10.12. Different but also extreme breeding patterns and gonadal cycles can be found in arid areas of South Africa. Psammobates tentorius tentorius in the Karoo of southern
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Africa starts vitellogenesis in fall and females produce several small clutches (1–3 eggs) from spring to fall (Leuteritz & Hofmeyr, 2007). The distribution of the tortoise Chersina angulata extends further to the arid west, and females lay several single egg clutches per year. They initiate egg production in February (late summer) and individual females can be gravid for eight to nine months per year. They can be gravid in all months except January (summer) despite living in a highly seasonal Mediterranean climate (Hofmeyr, 2004). Unfortunately, the hormonal regulation underlying this pattern has not yet been studied. The smallest tortoise, Homopus signatus signatus, also produces single egg clutches in the extremely arid southwestern Africa, but only in spring (August to October (Loehr, Henen, & Hofmeyr, 2004)). Aridity and unpredictable rainfall determine which tortoises lay single egg clutches, with taxa in these less predictable environments also producing larger eggs. H. s. signatus produces the largest egg relative to body size of any turtle, probably to enhance offspring survival in its harsh environment. These females produce rigid-shelled eggs larger than their pelvic canal and use pelvic kinesis (i.e., the bony suture of the kinetic bony elements of the shell and plastron are replaced with fibrous connections) to pass eggs at oviposition (Hofmeyr, Henen, & Loehr, 2005). Finally, the Gala´pagos tortoises (G. nigra) present an interesting example of a reproductive cycle characteristic of tropical climates. These giant tortoises do not hibernate and therefore exhibit a prenuptial cycle, like sea turtles, as presented by Rostal, Robeck, Grumbles, Burchfield, and Owens (1998) in captive animals from a zoo in Texas, USA. In this scenario, T levels are high during the mating period (August– November) for both sexes, and the rise in T just prior to mating is thought to coincide with spermatogenesis in males. Estradiol and calcium levels in females are highest in July, during which time vitellogenesis occurs, as confirmed by ultrasonographic images of growing follicles. Progesterone levels in females peaked in March and October, most likely linked to the ovulation of subsequent clutches of eggs. During nesting (November–April), T levels are low in both sexes. Schramm, Casares, and Lance (1999) confirmed this pattern using tortoises kept in semi-natural conditions at the Charles Darwin Center in Santa Cruz, Gala´pagos. The mating season occurred during the months of January through June, which corresponded to the hot and rainy season. Corticosterone concentrations were correlated to T levels in both sexes.
4.2. Pleurodira Pleurodire turtles, also called ‘side-necked’ turtles, represent an ancient group of turtles that originated in the late Triassic. Three families are extant and occur mainly in the southern hemisphere: the Chelidae in South America,
Australia, and New Guinea; the Pelomedusidae in Africa south of the Sahara and on a few islands in the Indian Ocean; and the Podocnemididae (with Pelomedusidae in the hyperfamily Pelomedusoides) in South America and Madagascar. The reproductive cycles of pleurodire turtles have been much less studied than those of cryptodire turtles; however, the limited data show some interesting differences compared to the cycles of a typical freshwater turtle of the northern hemisphere.
4.2.1. Chelidae Gonadal and hormonal cycles of several species of sidenecked turtles belonging to the family Chelidae have been studied in Australia, and in many cases they resemble closely those of cryptodire turtles, as discussed in Section 4.1. Legler (1985) defined a tropical reproductive pattern with nesting occurring during the cool dry season (fall and winter) and a temperate pattern with nesting during spring and summer. Male Chelodina steindachneri and C. oblonga, two species of western Australian long-necked turtle showing a temperate pattern as defined by Legler (1985), exhibit similar gonadal cycles (Kuchling, 1988a). Spermatogenesis begins in the austral spring (September–October)dwhen T levels are lowdand continues through the summer until June (late fall), at which time T slowly increases. Testes reach their maximal size in January and February for C. steindachneri (occurs mainly in temporary streams in arid subtropical climate with unpredictable rainfall) and in April for C. oblonga (inhabits more permanent wetlands in Mediterranean and temperate climate) (Kuchling, 1988a). Testosterone levels in C. oblonga reach elevated levels in April and remain so until mid-July (Figure 10.13) (Kuchling, 1999). Epididymides are maximally engorged with spermatozoa from May to November, and most mating occurs in the winter when T is low. In the female of C. oblonga, follicular growth starts in late summer, when temperatures are declining, and follicles reach preovulatory size at the beginning of winter (May). Because this turtle does not hibernate during winter, follicles continue to grow and oviductal eggs, along with different-sized follicles, are found as early as September (in the spring). Several clutches of eggs are laid during spring and summer. In C. steindachneri, follicular growth starts in fall and progresses until spring. If water is available in early spring (September), ovulation occurs, but under dry conditions when turtles have to stay in aestivation, no further follicular development seems to take place until summer (Kuchling, 1988a). Rainfall and the availability of water are unpredictable, and ovulation and nesting may still take place after summer rains (mainly connected to cyclones) at least until January. On the other hand, if this turtle has to undergo aestivation due to the lack of rainfall while gravid, the
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FIGURE 10.13 Annual cycle of testis volume (open circles), spermatogenetic activity, and mean plasma testosterone (T) concentration (solid line) in Chelodina oblonga. X-axis indicates the months of the year from July to June. Data adapted from Kuchling (1988a; 1999), with permission.
oviductal period can be extended to several months until rainfall again allows nesting to occur (Kuchling, 1999). Testosterone levels rise in females during May and reach a peak in mid-July, just prior to ovulation. Curiously, E2 levels are low throughout the year and do not show the common periovulatory peak, as seen in all other turtle species studied to date (Kuchling, 1999). This could be a problem relative to the laboratory technique employed to detect this hormone (RIA specific to E2, but other estrogens may be present), or due to the possibility that very low levels are sufficient to stimulate vitellogenesis in this species. Moreover, the somewhat atypical ovarian cycle of C. steindachneri, with the possibility of a stable vitellogenic phase without follicular growth in late spring and summer, seems to be unique in the turtle world, along with an extremely reduced period of ovarian regression. This might be the result of unpredictable environmental conditions pushing females to aestivate during dry periods and to have preovulatory follicles ready for long periods until water availability allows ovulation and nesting. The
potentially long periods of aestivation along with the small size of this turtle might also explain why only one clutch of eggs is typically laid each year. Female western swamp tortoises (P. umbrina) from Western Australia were studied by Kuchling and Bradshaw (1993) in an attempt to better understand their ovarian cycle and egg production. The ultimate goal was to improve captive breeding techniques in order to reintroduce this critically endangered species. Using both captive and wildcaught tortoises, ultrasonography was effectively used to evaluate follicular growth and oviductal eggs over a period of approximately four years. Like many other chelonians, including other Australian chelids such as Emydura krefftii (Georges, 1983), C. oblonga, and C. steindachneri (Kuchling, 1988a), as discussed above in this section, vitellogenesis starts in the summer, during the long aestivation period, and follicles continue to grow in the fall and winter, while the animals are active and feeding, until they reach preovulatory size in spring. Interestingly, in this study, maturing females were shown to start vitellogenesis
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several years before mature follicles were finally ovulated. Another peculiarity in P. umbrina is that, despite being a single-clutch tortoise, different-sized follicles grow in one reproductive season, with the smaller ones always becoming atretic after the nesting season. The evolutionary significance of this observation is not well understood and may suggest that the single-clutch pattern of this tortoise might be derived from multiple clutch capabilities. This theory was supported with the observation that animals held in captivity occasionally double-clutch under optimal conditions (Kuchling, unpublished data).
wild adult females are reproductively active during the reproductive season (Table 10.1). The only other freshwater turtle for which a biennial ovarian cycle has been demonstrated is the large cryptodire river turtle C. insculpta in tropical northern Australia (Doody, Georges, & Young, 2003). Spermatogenesis in E. madagascariensis appears to start at the end of the nesting season, in April, with spermiogenesis and spermiation taking place in the cool dry season until it comes to an end in September, therefore showing a prenuptial pattern (Kuchling & Garcia, 2003). E. madagascariensis shows several reproductive patterns that are similar to those of sea turtles, rather than to other better documented cryptodire freshwater turtles.
4.2.2. Podocnemididae The Podocnemididae, primarily large river turtles inhabiting tropical South America and Madagascar, are less well studied and deserve more attention, especially the Podocnemis and Peltocephalus genera from South America. Interestingly, E. madagascariensis, a large turtle endemic to Madagascar, can lay several clutches of eggs per season, and nesting occurs over an extended period of time, from the first rains in the late dry season (or spring: October) to the late wet season (or fall: May) (Kuchling, 1988b; Kuchling & Garcia, 2003). In addition, E. madagascariensis seems to have a biennial ovarian cycle, based on a number of assumptions after carefully evaluating the ovaries and oviducts of a dozen freshly slaughtered carcasses (Kuchling, 1993) and on ultrasound scanning and endoscopic data: although no longitudinal studies have been done on individual females, about 50% of
4.2.3. Pelomedusidae The pelomedusid turtles Pelusios castanoides and Pelomedusa subrufa, which occur sympatrically with Erymnochelys in Madagascar, show annual ovarian cycles as well as postnuptial spermatogenesis, which takes place during the hot wet season (Kuchling, unpublished data), like most cryptodire freshwater turtles. The first clutches are laid in September (late dry season). One interesting finding in females of P. castanoides and P. subrufa is that, despite laying several clutches of eggs in each season (up to February in the wet season), follicles are not present in different size classes at one time, as is usually the case in other multiclutch turtles. Instead, it appears that follicles for the next nesting event only start growing once the
TABLE 10.1 Breeding and non-breeding females of Erymnochelys madagascariensis at Ankarafantsika National Park assessed by ultrasound scanning and in some cases by endoscopy during the breeding season (September to May). Females assumed to be breeding had vitellogenic follicles > 10 mm and/or oviductal eggs and corpora lutea/albicantia > 2 mm. Females lacking these features were assumed to be non-breeding Vitellogenic follicles > 10 mm and/or oviducal eggs
Corpora lutea/C. albicantia > 2 mm
Sample size (n)
present
absent
present
absent
October 1999
1
0
1
–
–
April/May 2000
10
4
6
4
6
October 2001
4
4
0
–
–
Jan/Feb 2002
8
4
4
4
4
December 2002
2
1
1
–
1
All years combined
25
13
12
8
11
Total breeding females: n ¼ 13 Total of non-breeding females: n ¼ 12
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previous clutch of eggs has been laid (Kuchling, unpublished data). From February/March onwards, vitellogenesis commences again for the first clutch of the next breeding season in September. P. subrufa is also widely distributed, from drier areas in sub-Saharan Africa to wet/dry tropical areas, to the Mediterranean climate in South Africa where its reproduction also has been studied in the Western Cape, the species’ southernmost distribution (Van Wyk, Strydom, & Leslie, 2005). Vitellogenic recrudescence begins in summer (December) and continues through winter, with ovulation occurring in spring (September–October). Reportedly, females lay only one clutch of eggs, but nesting occurs from September to January and early vitellogenic follicles are also noted during November. These observations and incongruous interpretations suggest that Van Wyk et al. (2005) may have misinterpreted their data and overlooked multiple clutching due to the fact that this species does not show concurrent different-size classes of follicles as, based on the commonly accepted wisdom, the authors may have expected for a multi-clutch turtle. Plasma E2 and Vtg peaked during summer and plasma T in females varied throughout the year. Progesterone was elevated prior to oviposition in summer (December). Unfortunately, the interpretation of these steroid data likely also suffered from the misinterpretation of the ovarian cycle. As in Madagascar, spermiogenesis and spermiation took place in summer and fall and plasma T peaked once from February to March.
5. OTHER HORMONAL CYCLES 5.1. Metabolic and Stress Hormones 5.1.1. Thyroid hormones and their possible role in reproduction Thyroid hormones (thyroxine (T4) and triiodothyronine (T3)) are very conserved within the vertebrates, and their function has been linked to the regulation of many energydemanding activities, including development, growth, reproduction, and metabolism (Eales, 1979; Norris, 2007). It has been suggested that thyroid hormones therefore should exhibit distinct annual cycles in temperate-zone animals, including turtles, during periods of metabolic activity (Kohel, MacKenzie, Rostal, Grumbles, & Lance, 2001). Levels of T3 are much lower than those of T4 in a number of turtle species, and are often undetectable. Seasonal changes in hormone levels were investigated over the course of two years in the desert tortoise (Kohel et al., 2001; Lance & Rostal, 2002). Triiodothyronine was undetectable; however, T4 exhibited distinct cycles for both male and female G. agassizii. Higher levels of thyroid
Hormones and Reproduction of Vertebrates
hormones were associated with increased activity and mating behavior. For both males and females, T4 levels were at their lowest during hibernation (0.3 ng/mL) and rose to reach a peak in early spring (2.8 ng/mL), coinciding with feeding and mating behavior. When tortoises were fasted for two weeks, T4 levels decreased and were significantly lower than in control tortoises fed a normal diet. Once tortoises were fed again, T4 levels increased significantly, suggesting that thyroxine is directly linked to feeding activity. In reproductively active males only, T4 concentrations exhibited a second peak in the summer, coinciding with high T levels and a period of intense aggression between males. This second peak was not observed in juvenile and subadult males, although they showed the same feeding behavior as reproductively active males at this time of year. Thus, the peaks of T4 were correlated to behavioral activity patterns including increased movement, feeding, and mating. These results are somewhat in contrast to earlier work from Licht et al. (1985a) showing a peak in plasma T4 in male painted turtles in the summer when Twas at its nadir. In further contrast, plasma T4 remained fairly constant throughout the year in male green sea turtles held captive (and constantly well fed) at the Cayman Turtle Farm (Licht et al., 1985b), as well as in wild adult green turtles from Australia captured during winter and spring (Moon, Mackenzie, & Owens, 1998). However, wild male L. kempii exhibited a rise in T4 at the onset of mating, in February– March (Rostal et al., 1998a), whereas Moon et al. (1998) did not see any trend in captive adult male L. kempii from the Sea Arama Marineworld (Galveston, TX). Both studies (Moon et al., 1998; Rostal et al., 1998a) found elevated T4 levels in adult female L. kempii during the winter, coinciding with vitellogenesis, and in the spring, at the onset of mating. In addition, Wibbels, Mackenzie, Owens, Amoss, & Limpus (1986) detected higher T4 levels in C. caretta during the prebreeding season. In wild immature sea turtles from Australia, T4 levels were low but tended to increase with increased water temperature in green turtles (this increase was statistically significant only in females); however, levels remained low at all times in loggerheads (Moon et al., 1998). Finally, Moon et al. (1998) noted that T4 levels were higher in captive turtles (up to 13 ng/mL in L. kempii) than in wild ones (up to 3 ng/mL in wild immature C. mydas), possibly related to the higher nutritional content of their diet. However, Rostal et al. (1998a) reported T4 levels in wild adult female L. kempii as high as 11 ng/mL. These results suggest that the thyroid system might be activated by various factors including temperature, reproductive state, and nutritional status, but it is not well understood how the changes in environmental stimuli and endogenous conditions affect thyroid activity, and which factors, if any, might be most important in influencing reproductive activities.
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5.1.2. Corticosterone (CORT) The role of CORT during the reproductive cycle of reptiles is not well understood but results from various studies suggest that glucocorticoids are secreted during periods of increased activity and metabolism in reptiles. Lance, Grumbles, and Rostal (2001) measured CORT and T levels in male and female G. agassizii throughout the reproductive period, finding that males had higher levels of CORT than females and that the levels peaked in late summer and fall, corresponding to peaks of T, intense male–male combat, and mating. A female CORT cycle was less clear, with a peak in May during mating and nesting but no association with E2 levels later in the season. Similarly, CORT (and T) levels were higher in male and female G. nigra during the mating season (Schramm et al., 1999). However, in G. polyphemus, no seasonal variation was observed for either sex, and no correlation was found with sex steroids (Ott et al., 2000). Corticosterone levels are commonly thought to reflect stress level in reptiles, including turtles, especially related to capture or long-term captivity (Ott et al., 2000; Chapter 7, this volume). Stress in captivity has been suggested to result in drastic sex steroid decreases, in particular for T. For example, Mendonc¸a and Licht (1986) noted a 35–60% decrease in T levels in male musk turtles within 24 hours of capture, which might have been correlated with increased levels of CORT (unfortunately not measured). Given these results, it is not surprising that T levels are often much lower in captive animals when compared to wild populations, although seasonal cycles are often exhibited none the less (Mahmoud & Licht, 1997; Huot-Daubremont et al., 2003). Corticosterone measured in female leatherback sea turtles during the nesting period showed no significant variation over time. In addition, there was no correlation between CORT and T (Rostal et al., 2001). These results are in agreement with other studies showing, for example, no significant elevation in CORT levels in female L. olivacea nesting in an arribada (high density) nesting aggregation in Costa Rica or in C. mydas on an exceptionally high-density nesting beach in Australia, when compared to a low-density nesting population (Jessop, Limpus, & Whittier, 1999; Valverde, Owens, Mackenzie, & Amoss, 1999). Further, green sea turtles exposed to high daytime heat for several hours while nesting on the beach did not exhibit the expected high levels in CORT due to this potentially lethal environmental stressor. In fact, they exhibited lower CORT levels when compared to levels in non-breeding females held captive for the same amount of time in the shade (Jessop, Hamann, Read, & Limpus, 2000). These authors proposed that the low adrenocortical response to various stressors while nesting on these unusual but very remote beaches might be part of a generalized nesting adaptation to prevent stress from compromising
reproduction along with its high investment. This inhibited stress response may be adaptive in female sea turtles. This theory was further confirmed by the study of Jessop (2001), in which female C. mydas and E. imbricata in different reproductive states (non-breeding, pre-breeding, and breeding) showed different levels of CORT, with breeding females exhibiting the lowest concentrations. Corticosterone levels increased in all three categories as time after capture increased. Adult males in the three reproductive conditions did not show the pattern observed in females. Other studies in adult female sea turtles also failed to show stereotypical stress responses due to capture (Gregory, Gross, Bolten, Bjorndal, & Guillette, 1996; Valverde et al., 1999), corroborating the studies of Jessop et al. (1999; 2000) and Jessop (2001).
5.1.3. Catecholamines Catecholamines, in particular epinephrine and norepinephrine, have been studied very little in turtles, and showed no particular trend or association with glucose levels in nesting female green sea turtles (Jessop & Hamann, 2004). In the soft-shelled turtle, L. p. punctata, both epinephrine and norepinephrine levels of the adrenal gland were elevated following administration of FSH (although no mention was made of its source) and high doses of estrogens, but not by LH (Ray, Chaudhuri, & Maiti, 2002). Progesterone seemed to have the opposite effect to that of estrogens. These results were supported by adrenal histology.
5.2. Pineal Hormones and Annual Cycles Turtles have a large secretory pineal gland, which is the primary source of the hormone melatonin (Owens & Ralph, 1978; Owens, Gern, & Ralph, 1980). In addition, many turtles exhibit a melatonin diurnal cycle with a nocturnal high and a distinct responsiveness to both light (i.e., a dosimeter) and temperature (Figure 10.14) (Owens and Gern, 1985; Owens & Morris, 1985; Vivien-Roels, Pe´vet, & Claustrat, 1988). These authors suggested that the very active pineal system of turtles could serve as a photoperiod-based annual clock, which would function as a day length measurer to coordinate the annual cycles. Thus, when both the nutritional and temperature history of the individual are good, melatonin would facilitate a coordinated seasonal gonadal maturation. For a more complete discussion see Kuchling (1999). A recent review by Ubuka, McGuire, Calisi, Perfito, and Bentley (2008) proposes a mechanism wherein melatonin may act centrally in the hypothalamus to, in turn, regulate GTH-releasing and release-inhibiting factors and thus control the release of pituitary GTHs. A curious and potentially relevant observation in the temperate-ranging leatherback turtle is
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FIGURE 10.14 Diurnal rhythm in cerebrospinal fluid and serum melatonin in green sea turtles. Each turtle was sampled simultaneously for blood and cerebrospinal fluid at only one of the eight times. Four turtles were sampled at each time. From Owens, Gern, and Ralph (1980).
a striking white spot on top of the skull just above the brain where the pineal is located (Figure 10.1).
6. CONCLUSIONS Several researchers have observed that a full understanding of endocrine regulation in turtles has been limited because of the old habit of taking too few and infrequent blood samples in turtles (Licht, 1984; Kuchling, 1999). This problem has recently been addressed in a few species; however, many gaps exist, particularly with males, hibernating or aestivating turtles, and the endangered and threatened species. Coulson and Hernandez (1964) once described reptilian physiology (alligators) as ‘studies of physiology in slow motion.’ Whereas this may be true for turtles in situations where temperatures are sub-optimal, it is definitely not true all of the time and especially not during peak reproductive periods. Indeed, some of the most striking examples of very rapid physiological changes are seen with the steroid hormone P4. Although the baseline circulating P4 level is what is regularly observed (e.g., Ott et al., 2000), many studies have shown that, during ovulation and the albumin deposition phase of the nesting cycle, which may only last from 10 to 48 hours, a tremendous surge in P4 will occur to facilitate the rapid movement of follicles into the oviducts and the concurrent secretion of large volumes of albumin around each fertilized ovum. Just as this rapid surge facilitates nearly simultaneous
ovulations of 1 to 100 or more follicles, a similar sudden drop in this steroid hormone is required to terminate the albumin deposition phase of egg formation. Since it is important for efficiency and speed in the oviduct for the entire clutch to be at the same stage of egg production, selection would favor a rapid rise and fall in P4 secretion. The dynamics of CORT are rather unclear in some studies where infrequent sampling protocols have been used. Moreover, many early studies were done while animals were held in captivity, with blood samples being taken several hours or even days after capture. Many studies have shown that the stress of capture in wild turtles, or stress from long periods of time in captivity, suppresses hormone levels (Kuchling et al., 1981; Licht et al., 1985a; Mendonc¸a & Licht, 1986; Mahmoud & Licht, 1997; HuotDaubremont et al., 2003), and studies of hormonal cycles based on captive animals should be evaluated with caution (see Chapter 7, this volume). Studying the reproductive cycles of turtles can lead to an improved knowledge of the reproductive potential for the species, specifically how many eggs are being produced in each clutch, how many clutches are laid per year, and what is the periodicity of the reproductive activity. As described above, some turtles reproduce every year, while othersdmainly the sea turtles, but other species as welldhave been found to only reproduce every two to four years, or less frequently. For example, Limpus, Eggler, and Miller (1994) reported that breeding green sea turtles of
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Eastern Australia have an average remigration interval of about 5.8 years, with many females nesting after an 8–9 year interval. Adult male sea turtles cycle faster than females do; however, resident and migratory breeding males may have differential reproduction success. More work needs to be done to answer questions on cyclicity and turtle reproductive potential, as well as on the developmental onset of cycling and reproduction. Further, for how long during their long lives can turtles reproduce? We know nothing of this in the wild. An improved understanding of turtle reproduction is critical to developing better lifehistory and population dynamics models. In turn, such models are urgently needed for improving conservation strategies, especially when applied to threatened or endangered species. Indeed, a good understanding of the reproductive potential of a species, and how it cycles over time, can also be used in captive breeding programs for the reintroduction of endangered species. There are too many turtle species on the verge of extinction in many countries around the globe about which nothing is known of their reproductive biology. Finally, because of the well-documented importance of ambient temperature in all phases of turtle reproduction, including sex determination (see Chapter 1, this volume), the current global warming crisis adds special urgency to future research on the reproductive cycles of turtles.
ABBREVIATIONS AVT CORT E2 ELISA FSH GSI GTH HPA LH NP P4 PGE2 PGF RIA T T3 T4 Vtg
Arginine vasotocin Corticosterone Estradiol Enzyme-linked immunosorbent assay Follicle-stimulating hormone Gonadosomatic index Gonadotropins Hypothalamus–pituitary–adrenal Luteinizing hormone Neurophysin Progesterone Prostaglandin E2 Prostaglandin F2a Radioimmunoassay Testosterone Triiodothyronine Thyroxine Vitellogenin
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Mendonc¸a, M. T., & Licht, P. (1986). Seasonal cycles in gonadal activity and plasma GTH in the musk turtle, Sternotherus odoratus. Gen. Comp. Endocrinol., 62, 459–469. Miller, J. D., & Dinkelacker, S. A. (2008). Reproductive structures and strategies of turtles. In J. Wyneken, M. H. Godfrey, & V. Bels (Eds.), ‘‘Biology of Turtles’’ (pp. 225–278). Boca Raton: CRC Press. Moll, E. O. (1979). Reproductive cycles and adaptations. In M. Harless, & H. Morlock (Eds.), ‘‘Turtles: Perspectives and Research’’ (pp. 305–331). New York: John Wiley & Sons. Moon, D.-Y., Mackenzie, D. S., & Owens, D. W. (1998). Serum thyroid hormone levels in wild and captive sea turtles. Korean J. Biol. Sci., 2, 177–181. Norris, D. O. (2007). Vertebrate Endocrinology (4th ed.). San Diego: Academic Press. Ott, J. A., Mendonc¸a, M. T., Guyer, C., & Michener, W. K. (2000). Seasonal changes in sex and adrenal steroid hormones of gopher tortoises (Gopherus polyphemus). Gen. Comp. Endocrinol., 117, 299–312. Owens, D. Wm. (1980). The comparative reproductive physiology of sea turtles. Am. Zool., 20, 549–563. Owens, D. W. (1997). Hormones in the life history of sea turtles. In P. L. Lutz, & J. A. Musick (Eds.), ‘‘The Biology of Sea Turtles’’, Vol. 1 (pp. 315–341). Boca Raton: CRC Press. Owens, D.W. (1999). Reproductive cycles and endocrinology. In ‘‘Research and Management Techniques for the Conservation of Sea Turtles’’ K.L. Eckert, K.A. Bjorndal, F.A. Abreu-Grobois, and M. Donnelly, (Eds.), (pp. 119–123). IUCN/SSC Marine Turtle Specialist Group Publication No. 4. Owens, D. W., & Gern, W. A. (1985). The pineal gland and melatonin in sea turtles. In B. Lofts, & W. N. Holmes (Eds.), ‘‘Current Trends in Comparative Endocrinology’’ (pp. 645–648). Hong Kong: Hong Kong University Press. Owens, D. W., & Morris, Y. A. (1985). The comparative endocrinology of sea turtles. Copeia, 1985, 723–735. Owens, D. W., & Ralph, C. L. (1978). The pineal–paraphyseal complex in sea turtles, I. Light microscopic description. J. Morph, 158, 169–180. Owens, D. W., & Ruiz, G. J. (1980). New methods of obtaining blood and cerebrospinal fluid from marine turtles. Herpetologica, 36, 17–20. Owens, D. W., Gern, W. A., & Ralph, C. L. (1980). Melatonin in the blood and cerebrospinal fluid of the green sea turtle. Gen. Comp. Endocrinol., 40, 180–187. Plotkin, P. T. (2007). Biology and Conservation of Ridley Sea Turtles. Baltimore: Johns Hopkins University Press. Plotkin, P. T., Owens, D. W., Byles, R. A., & Patterson, R. (1996). Departure of male olive ridley turtles (Lepidochelys olivacea) from a nearshore breeding ground. Herpetologica, 52, 1–7. Plotkin, P. T., Rostal, D. C., Byles, R. A., & Owens, D. W. (1997). Reproductive and developmental synchrony in female Lepidochelys olivacea. J. Herpetology, 31, 17–22. Pritchard, P. C. H., & Trebbau, P. (1984). The Turtles of Venezuela. Oxford: Society for the study of Amphibians and Reptiles. Ray, P. P., Chaudhuri, S., & Maiti, B. R. (2002). Adrenomedullary hormonal and glycemic modulation following GTH and sex hormone administration in the soft-shelled turtle Lissemys punctata punctata. Endocr. Res., 28, 83–90.
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Reina, R. D., Mayor, P. A., Spotila, J. R., Piedra, R., & Paladino, F. V. (2002). Nesting ecology of the leatherback turtle, Dermochelys coriacea, at Parque Nacional Marino Las Baulas, Costa Rica: 1988–1989 and 1999–2000. Copeia, 2002, 653–664. Rostal, D. C. (2007). Reproductive physiology of the ridley sea turtle. In P. T. Plotkin (Ed.), ‘‘Biology and Conservation of Ridley Sea Turtles’’ (pp. 151–165). Baltimore: Johns Hopkins University Press. Rostal, D. C., Lance, V. A., Grumbles, J. S., & Alberts, A. C. (1994). Seasonal reproductive cycle of the desert tortoise (Gopherus agassizii) in the Eastern Mojave Desert. Herpetological Monographs, 8, 72–82. Rostal, D. C., Paladino, F. V., Patterson, R. M., & Spotila, J. R. (1996). Reproductive physiology of nesting leatherback turtles (Dermochelys coriacea) at Las Baulas National Park, Costa Rica. Chel. Conserv. Biol., 2, 230–236. Rostal, D. C., Robeck, T. R., Owens, D. W., & Kraemer, D. C. (1990). Ultrasound imaging of ovaries and eggs in Kemp’s ridley sea turtles (Lepidochelys kempi). J. Zoo. Wildl. Med., 21, 27–35. Rostal, D. C., Grumbles, J. S., Byles, R. A., Ma´rquez-M., R., & Owens, D. W. (1997). Nesting physiology of Kemp’s ridley sea turtles, Lepidochelys kempi, at Rancho Nuevo, Tamaulipas, Mexico, with observations on population estimates. Chel. Conserv. Biol., 2, 538–547. Rostal, D. C., Owens, D. W., Grumbles, J. S., MacKenzie, D. S., & Amoss, M. S., Jr. (1998a). Seasonal reproductive cycle of the Kemp’s ridley sea turtle (Lepidochelys kempi). Gen. Comp. Endocrinol., 109, 232–243. Rostal, D. C., Robeck, T. R., Grumbles, J. S., Burchfield, P. M., & Owens, D. W. (1998b). Seasonal reproductive cycle of the Gala´pagos tortoise (Geochelone nigra) in captivity. Zoo Biology, 17, 505–517. Rostal, D. C., Grumbles, J. S., Palmer, K. S., Lance, V. A., Spotila, J. R., & Paladino, F. V. (2001). Changes in gonadal and adrenal steroid levels in the leatherback sea turtle (Dermochelys coriacea) during the nesting cycle. Gen. Comp. Endocrinol., 122, 139–147. Saba, V. S., Santidria´n-Tomillo, P., Reina, R. D., Spotila, J. R., Musick, J. A., Evans, D. A., & Paladino, F. V. (2007). The effect of the El Nin˜o Southern Oscillation on the reproductive frequency of eastern Pacific leatherback turtles. J. Applied Ecol., 44, 395–404. Saint Girons, H. (1963). Spermatoge´ne`se et e´volution des caracte`res sexuels secondaires chez les squamata. Annales des Sciences Naturelles, 5, 461–478. Sarkar, S., Sarkar, N. K., & Maiti, B. R. (1996). Seasonal pattern of ovarian growth and interrelated changes in plasma steroid levels, vitellogenesis, and oviductal function in the adult female soft-shelled turtle Lissemys punctata punctata. Can. J. Zool., 74, 303–311. Schramm, B. G., Casares, M., & Lance, V. A. (1999). Steroid levels and reproductive cycle of the Gala´pagos tortoise, Geochelone nigra, living under seminatural conditions on Santa Cruz Island (Gala´pagos). Gen. Comp. Endocrinol., 114, 108–120. Seminoff, J. A., Rosendiz, A., Nichols, W. J., & Jones, T. T. (2002). Growth rates of wild green turtles (Chelonia mydas) at a temperate foraging area in the Gulf of California, Mexico. Copeia, 2002, 610–617. Shelby, J. A., Mendonc¸a, M. T., Horne, B. D., & Seigel, R. A. (2000). Seasonal variation in reproductive steroids of male and female yellow-blotched map turtles, Graptemys flavimaculata. Gen. Comp. Endocrinol., 119, 43–51.
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Silva, A. M. R., Moraes, G. S., & Wassermann, G. F. (1984). Seasonal variations of testicular morphology and plasma levels of T in the turtle Chrysemys dorbigni. Comp. Biochem. Physiol., 78A, 153–157. Solow, A. R., Bjorndal, K. A., & Bolten, A. B. (2002). Annual variation in nesting numbers of marine turtles: the effect of sea surface temperature on re-migration intervals. Ecol. Lett., 5, 742–746. Tripathy, B., & Pandav, B. (2008). Beach fidelity and internesting movements of olive ridley turtles (Lepidochelys olivacea) at Rushikulya, India. Herp. Conserv. Biol., 3, 40–45. Tucker, A. D., & Frazer, N. B. (1991). Reproductive variation in leatherback turtles, Dermochelys coriacea, at Culebra National Wildlife Refuge, Puerto Rico. Herpetologica, 47, 115–124. Ubuka, T., McGuire, N. L., Calisi, R. M., Perfito, N., & Bentley, G. E. (2008). The control of reproductive physiology and behavior by GTH-inhibitory hormone. Integr. Comp. Biol., 48, 560–569. Valverde, R. A., Owens, D. W., Mackenzie, D. S., & Amoss, M. S. (1999). Basal and stress-induced CORT levels in olive ridley sea turtles (Lepidochelys olivacea) in relation to their mass nesting behavior. J. Exp. Zool., 284, 652–662. Valverde, R. A., Stabenau, E. K., & Mackenzie, D. S. (2007). Respiratory and endocrine physiology. In P. T. Plotkin (Ed.), ‘‘Biology and Conservation of Ridley Sea Turtles’’ (pp. 119–149). Baltimore: Johns Hopkins University Press. Van Wyk, J. H., Strydom, A. V., and Leslie, A. J. (2005). Sexual dimorphism and seasonal reproduction in the South African fresh water turtle, Pelomedusa subrufa (Chelonia: Pelomedusidae). Abstract, 5th World Congress of Herpetology Stellenbosch 19-24 June 2005, p. 104. Vivien-Roels, B., Pe´vet, P., & Claustrat, B. (1988). Pineal and circulating melatonin rhythms in the box turtle, Terrapene carolina triunguis: effect of photoperiod, light pulse, and environmental temperature. Gen. Comp. Endocrinol., 69, 163–173. White, J. B., & Murphy, G. G. (1973). The reproductive cycle and sexual dimorphism of the common snapping turtle. Chelydra serpentina serpentina. Herpetologica, 29, 240–246.
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Whittier, J. M., Corrie, F., & Limpus, C. (1997). Plasma steroid profiles in nesting loggerhead turtles (Caretta caretta) in Queensland, Australia: relationship to nesting episode and season. Gen. Comp. Endocrinol., 106, 39–47. Wibbels, T., Mackenzie, D. S., Owens, D. W., Amoss, M. S., & Limpus, C. J. (1986). Aspects of thyroid physiology in loggerhead sea turtles, Caretta caretta. Am. Zool., 26, 106A. Wibbels, T., Owens, D. W., & Amoss, M. (1987). Seasonal changes in serum T of loggerhead sea turtles captured along the Atlantic coast of the United States. In W. Witzell (Ed.), ‘‘Ecology of East Florida Sea Turtles’’ (pp. 59–64). U.S. Dept. Commer., NOAA Tech., Rep. NMFS 53. Wibbels, T., Owens, D. W., Limpus, C. J., Reed, P. C., & Amoss, M. S., Jr. (1990). Seasonal changes in serum gonadal steroids associated with migration, mating, and nesting in the loggerhead sea turtle (Caretta caretta). Gen. Comp. Endocrinol., 79, 154–164. Wibbels, T., Owens, D. Wm., Licht, P., Limpus, C., Reed, P. C., & Amoss, M. S., Jr. (1992). Serum GTHs and gonadal steroids associated with ovulation and egg production in sea turtles. Gen. Comp. Endocrinol., 87, 71–78. Witzell, W. N., Salgado-Quintero, A., & Gardun˜o-Dionte, M. (2005). Reproductive parameters of the Kemp’s ridley sea turtle (Lepidochelys kempii) at Rancho Nuevo, Tamaulipas, Mexico. Chel. Conserv. Biol., 4, 781–787. Wood, J. R., & Wood, F. E. (1980). Reproductive biology of captive green sea turtles Chelonia mydas. Am. Zool., 20, 499–505. Wood, J. R., Wood, F. E., Critchley, K. H., Wildt, D. E., & Bush, M. (1983). Laparoscopy of the green sea turtle, Chelonia mydas. Brit. J. Herp., 6, 323–327. Zuffi, M. A. L., Citi, S., Giusti, M., and Teti, A. (2005). Assessment of reproductive frequency in the European pond turtle (Emys orbicularis) using manual palpation, ultrasonography, and radiography. In (N. Ananjeva, and O. Tsinenko, Eds.), ‘‘Proceedings of the 12th Ordinary General Meeting of the Societas Europaea Herpetologica’’ (pp 245–246). Herpetologia Petropolitana: Russia, Saint-Petersburg.
Chapter 11
Hormones and Reproductive Cycles in Crocodilians Matthew R. Milnes Zoological Society of San Diego, Escondido, CA, USA
SUMMARY Hormonal influence upon sexual differentiation in crocodilians begins during embryonic development and continues for years until sexual maturation is attained. Shortly after sex determination, estrogen production in the embryonic ovary increases and promotes proliferation of the Mu¨llerian ducts, whereas the testis produces anti-Mu¨llerian hormone, which results in its regression. Following hatching, gonadal production of sex steroids increases gradually with body size, and secondary sex structures become increasingly responsive to steroidal stimulation. As crocodilians approach one meter in length, males and females begin showing subdued seasonal variations in androgens and estrogens, respectively. Mature crocodilians exhibit distinct annual cycles that are primarily dictated by temperature and photoperiod in temperate species or by seasonal rainfall patterns in tropical species. The onset of gametogenesis begins approximately four months prior to the peak of copulation, as gonadal and reproductive tract recrudescence is stimulated by increases in sex steroids. Following ovulation, active corpora lutea produce progesterone (P4) during gravidity, stimulating uterine secretions involved in the production of albumen and egg shelling. Secretions of prostaglandins from the uterus are greatest in postoviposition females, suggesting a role in egg laying and luteolysis. The most recent advances in crocodilian reproduction include the cloning of genes involved in hormone signaling. Further efforts are needed to place the expression of these genes within the context of ontogenetic development and reproductive cycles of crocodilians.
1. INTRODUCTION The order Crocodilia shares a position with birds and dinosaurs on the archosaurian branch of the diapsid amniotes (Laurin & Gauthier, 1996). Evolutionarily, crocodilians are unique in that they share many reproductive traits homologous to those found in birds and presumably dinosaurs, yet also have retained some ancestral characteristics common to lepidosaurian and chelonian reptiles. For
Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
instance, crocodilians and birds share similar regional specialization of the oviducts for the secretion of albumen, the formation of non-calcified eggshell membranes, and the shelling of eggs, whereas oviparous squamates and chelonians exhibit a temporal rather than spatial separation of the secretory and shelling processes (Palmer & Guillette, 1992). On the other hand, crocodilians, like other reptiles, ovulate and process an entire clutch simultaneously, whereas birds ovulate and process individual eggs sequentially. Crocodilians are a taxonomic group of aesthetic and commercial interest in many countries. Applied conservation measures, which include captive propagation of endangered crocodilians and ecotourism, as well as financial interests in farming or ranching of crocodilians, have promoted many of the advances in our understanding of crocodilian reproductive physiology. Many species of crocodilian must reach a total length of nearly two meters or more before they are capable of successful reproduction. This can take more than a decade in the wild; thus, key events related to sexual differentiation and maturation span many years. During embryogenesis and neonatal development, egg incubation temperature and hormones influence the formation of primary and secondary reproductive traits. As juvenile crocodilians mature, they transition into a peripubescent period during which they experience attenuated annual hormonal cycles that parallel those of adults. Upon reaching sexual maturity, most crocodilian species exhibit highly seasonal and synchronous reproduction. Reproductive activity within a species typically corresponds to seasonal changes in temperature or rainfall, depending on whether that species inhabits a temperate, subtropical, or tropical environment. Energetic investment in reproduction for female crocodilians is relatively high, with clutches of 12 to 50 or more megalecithal eggs, weighing 45 to 160 g each (Thorbjarnarson, 1996). Many species also exhibit nest-guarding behavior throughout egg incubation and provide varying 305
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degrees of parental care following hatching. Although the maximal life span of crocodilians is not known, a conservative estimate would be greater than 50 years. They are generally thought to continue to reproduce and grow, albeit more slowly as they age, throughout much of their lives; however, the cessation of growth and reproductive senescence have been noted anecdotally in exceptionally large individuals. With only three recognized families (Alligatoridae, Crocodylidae, and Gavialidae) and 23 to 26 species (Willis, McAliley, Neeley, & Densmore, 2007; Eaton, Martin, Thorbjarnarson, & Amato, 2008), crocodilians are less diverse than squamates and turtles, and their basic reproductive anatomy and physiology appear highly conserved. It should be noted, however, that the primary literature examining crocodilian reproduction is heavily biased towards Alligator mississippiensis, followed distantly by Crocodylus niloticus, Crocodylus johnstoni, Crocodylus palustris, and Crocodylus porosus. Recent studies focused on central and South American crocodilians, in particular Crocodylus moreletii, Caiman latirostris, Caiman crocodilus, and Caiman yacare, are beginning to fill in the gaps concerning interspecies variation and further confirm the conservation of many traits within this order. For each topic covered within this chapter, every attempt will be made to point out characteristics generally considered to be representative of all crocodilians. Similarly, special attention
will be paid to interspecific variation and how it relates to phylogenetic or environmental influences.
2. DEVELOPMENT Early embryonic development of the reproductive system in crocodilians is quite similar to that of birds, with a key exception being the absence of heteromorphic sex chromosomes. All crocodilians studied to date exhibit temperature-dependent sex determination. Data that include incubation temperatures and resulting sex ratios in 11 species suggest that all crocodilians follow the same female–male–female pattern with increasing incubation temperature (Lang & Andrews, 1994). Considering the latitudinal range and environmental variation that exist across species, it is interesting to note that the viable range (approximately 28–34 C) and male-producing range (approximately 31–33 C) are relatively consistent among species from all three families. Key events related to sexual differentiation and maturation in crocodilians are summarized in Table 11.1. In addition to sex determination, development and differentiation of secondary sex traits and hormone-dependent tissues occur in ovo. Upon hatching, crocodilians exhibit markedly male or female characteristics; however, neonates continue to undergo significant morphological differentiation of the gonads and reproductive tract over the ensuing monthsdmuch of which is under
TABLE 11.1 Key events related to sexual differentiation and maturation in crocodilians Embryonic Stagea /Sizeb
Description
References
12
Genital ridge composed of geminal epithelium and medullary sex cords
Forbes, 1940; Smith and Joss, 1993
21–23
Thermosensitive period of sex determination. Significant decrease in yolk steroid concentrations, and sexual differentiation of gonad
Lang and Andrews, 1994; Conley et al., 1997
24
Morphologically and steroidogenically distinct testis and ovary
Smith and Joss, 1994b; Smith et al., 1995; Gabriel el al., 2001; Milnes et al., 2002a
25–28 (hatching)
Males: Sertoli cells and germ cells enclosed in seminiferous tubules. Mu¨llerian duct begins to regress. Females: Cortical proliferation and development of lacunae in ovary. Oogonial nests and primary oocytes present at hatching
Austin, 1989; Smith and Joss, 1993; 1994a; Moore et al., 2008
z 40 cm snout-to-vent Males and females begin to show an attenuated seasonal length (SVL) cycle with regard to testosterone and 17b-estradiol (E2), respectively
Coutinho et al., 2000; Rooney et al., 2004
z 90 cm SVL
Thorbjarnarson, 1996
a
Sexual maturity
Embryonic stages according to Ferguson (1985) and Iungman, Pina, and Siroski (2008). Sizes are approximate for most species and not necessarily applicable to the genus Paleosuchus (dwarf caiman) or Osteolaemus (dwarf crocodile).
b
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Hormones and Reproductive Cycles in Crocodilians
hormonal control. Juvenile crocodilians exhibit an extended period of time analogous to puberty, a developmental stage not well studied in non-mammalian vertebrates. During these years they exhibit increasingly pronounced cyclic patterns of hormone production as they grow larger, and show signs of seasonal responsiveness in the gonads and reproductive tract.
2.1. Sexual Differentiation 2.1.1. Gonadal development The duration of egg incubation in crocodilians averages about 70 days, and varies from two to four months depending on the species. The rate of embryonic development is proportional to incubation temperature, and incubation time within a species can vary by three weeks under constant incubation temperatures that span the viable range (Ferguson, 1985; Deeming & Ferguson, 1989; Lang & Andrews, 1994; Pina, Larriera, & Cabrera, 2003). As a result of interspecific variation in size and the effect of temperature on rate of development, embryo size and age are not reliable bases for comparison of development across species or under different incubation conditions. In order to accommodate this variability, a standardized series of 28 embryonic stages of development in A. mississippiensis was presented by Ferguson (1985). Each stage is based upon several diagnostic morphological features originally present at that point in development, rather than on morphometrics. The series was cross-referenced against C. johnstoni and C. porosus development (Ferguson, 1985), and more recently against C. latirostris (Iungman, Pina, & Siroski, 2008) (Table 11.1). Although some morphological discordance exists between consecutive stages among species, the series is generally accepted as applicable to other crocodilians, and will be used to define embryonic events discussed in this chapter. Several detailed accounts of the differentiation of the bipotential gonad in A. mississippiensis have been published (Forbes, 1940; Joss, 1989; Smith & Joss, 1993; 1994a). Nothing is known concerning germ cell migration in crocodilians, but the primordial gonad is first seen as a thickening of the coelomic epithelium along the ventromedial surface of the mesonephric kidney. By stage 12, the genital ridge is composed of an overlying germinal epithelium that becomes increasingly distinct from the medullary sex cords as development progresses. The embryonic gonad remains morphologically indistinguishable at male- and female-producing temperatures through stage 20, at which time it consists of a distinct cortex and medulla. Between stages 21 and 23, enlarged pre-Sertoli cells begin to appear in the medulla of the putative testis,
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while the putative ovary shows signs of medullary cord fragmentation and proliferation of germ cells in the cortex (Smith & Joss, 1993). By stage 24 at male-producing temperatures, seminiferous cords are widespread and the germinal epithelium is greatly reduced, whereas oogonial nests and prefollicular cells are forming in the cortex of the embryonic ovary. Temperature shift experiments indicate that the thermosensitive period (TSP) of sex determination in A. mississippiensis (stages 21 to 24) corresponds to the appearance of morphological differences in the embryonic gonad (Lang & Andrews, 1994). Throughout the TSP, de novo steroidogenic activity, measured by enzyme histochemistry of 3b-hydroxysteroid dehydrogenase (3b-HSD) activity in C. porosus, is present in adrenal tissue but nonexistent in the adjacent embryonic gonad (Smith & Joss, 1994b). Aromatase enzyme activity, measured by the tritiated water assay in C. porosus and A. mississippiensis, is present in the gonad–adrenal–mesonephros complex throughout the TSP, but does not increase in a sex-specific manner until the end of the TSP, when signs of morphological differentiation in the gonad are already present (Smith & Joss, 1994b; Smith, Elf, Lang & Joss, 1995; Milnes, Roberts, & Guillette, 2002). Extragonadal aromatase activity follows a similar pattern, increasing in the brain throughout the TSP but failing to exhibit sexual dimorphism until stage 24 (Milnes et al., 2002a). It is not known whether adrenal enzyme activity provides the substrate for aromatase activity in the embryonic gonad or brain, but the undifferentiated gonad is capable of binding 17b-estradiol (E2) throughout the TSP (Smith & Joss, 1994c). Although no difference in the uptake of 3H-E2 was observed between male- and female-producing temperatures, the occurrence of E2-binding in the undifferentiated gonad provides a possible mechanism for the temperature-overriding effects of exogenous estrogens on gonadal sex determination. Maternally derived yolk steroids provide additional potential sources of aromatizable substrates in the form of androstenedione and testosterone (T). These C19 steroids, along with E2, are found in ng/g concentrations in the yolk prior to the TSP. The concentrations of all three steroids decline dramatically at male- and female-producing temperatures throughout the period of sex determination, with the most significant decrease occurring between stages 21 and 23 (Conley et al., 1997). The decline in yolk androstenedione concentration is inversely proportional to incubation temperatures; however, this is not necessarily linked directly to sex determination as a large percentage of females are produced at both the lower and upper range of viable incubation temperatures. The fact that aromatase activity does not increase until the end of the TSP at female-producing temperatures, and that the decline in yolk steroids is more closely related to temperature as opposed to presumptive sex, suggests that embryonic
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steroidogenesis and yolk steroids do not direct gonadal sex determination. On the other hand, the presence of measurable 3b-HSD in the adrenal gland, the dramatic decline in yolk steroid concentrations throughout the TSP, the uptake of E2 in the medulla of the embryonic gonad at male- and female-producing temperatures, and the presence of sexually dimorphic aromatase activity following the TSP suggest that steroids have a role in directing embryonic development and sexual differentiation in crocodilians. At this point, the mechanism for preferential uptake of yolk steroids during the TSP, the ontogeny of tissue-specific steroid receptor expression, and the role of these hormones in embryonic gonadal organization remain subject to further investigations. Significant differentiation of gonadal cell types and structures continue from late embryogenesis through neonatal and early juvenile development. From stage 25 to hatching, the testes are distinguished from previous stages by increasing basal nuclei and apical endoplasmic reticulum in Sertoli cells enclosed in seminiferous tubules (Smith & Joss, 1994a). At hatching, clearly identifiable germ cells are present within the seminiferous cords, and the germinal epithelium consists of only a squamous cell monolayer (Smith & Joss, 1993). Over the ensuing months of neonatal development, a compact tunica albuginea encapsulates the testis and the germinal epithelium continues to regress so that only remnant patches remain (Forbes, 1940). By six months posthatching, wellorganized seminiferous tubules, Sertoli cells, and Leydig cells are present in the testis, and germ cells are undergoing mitotic divisions (Guillette et al., 1994). Although it is probably several years under natural environmental conditions until complete spermatogenesis occurs, Forbes (1940) noted the presence of a narrow seminiferous tubule lumen and occasional spermatocytes at 18 months after hatching. Over the final stages of development in female embryos, cortical proliferation continues as lacunae, or fluid-filled spaces, develop within the fragmented cords of the medulla (Smith & Joss, 1993). Oogonia and primary oocytes are scattered throughout the germinal epithelium at hatching, and prefollicular cells, adjacent to spherical germ cells containing meiotic chromosomes, are present by day seven in A. mississippiensis (Moore, UribeAranzabal, Boggs, & Guillette, 2008). A dramatic increase in thickness of the lacunar system separating the germinal epithelium from the mesonephros develops over the next few months while folliculogenesis proceeds. By three months posthatching, some oocytes have advanced to the diplotene stage of meiosis, marked by the appearance of lampbrush chromosomes and a single layer of cuboidal follicular cells surrounded by a fibrous theca (Moore et al., 2008). Detailed ontogenetic studies of germ
Hormones and Reproduction of Vertebrates
cell maturation in older juvenile and subadult crocodilians have yet to be published.
2.1.2. Secondary sex characteristics Following the initial stages of gonadal differentiation, sex steroids and other hormones begin directing the development and differentiation of secondary sex characteristics in crocodilians. The Mu¨llerian duct, precursor to the oviduct, is present in all early embryos, but begins to regress approximately two weeks prior to hatching (stage 24/25) at male-producing temperatures in A. mississippiensis. The regression of the Mu¨llerian duct following the grafting of testicular explants into newly hatched and ovariectomized females implicates antiMu¨llerian hormone (AMH) (also termed Mu¨llerianinhibiting substance (MIS)) secreted from the testis as the cause (Austin, 1989). Ovariectomized females lacking testis grafts but supplemented with T implants do not exhibit Mu¨llerian duct regression, supporting the role of AMH as opposed to T as the causal agent for regression (Austin, 1990). More recently, cloning of the AMH gene from A. mississippiensis, the observation that AMH mRNA expression substantially increases from stages 23 to 25 in putative males, and the lack of expression in females confirm AMH as the cause of Mu¨llerian duct regression (Western, Harry, Graves, & Sinclair, 1999). Additional experimental data suggest that the presence of the Mu¨llerian duct is independent of estrogen signaling, but proper growth and differentiation may be dependent upon it. For example, in ovo exposure to an aromatase inhibitor does not disrupt the formation of the Mu¨llerian duct (Lance & Bogart, 1992), but in ovo exposure to exogenous E2 does result in hypertrophy (Crain, Spiteri, & Guillette, 1999). Finally, ovariectomized female hatchlings receiving testis grafts and supplemented with E2 do not exhibit Mu¨llerian duct regression, thus indicating that E2 antagonizes the effects of AMH (Austin, 1990). Less is known about hormonal influences on the development of male secondary sex characteristics compared to those of female crocodilians. The Wolffian duct develops parallel to the dorsal margin of the mesonephros and becomes continuous with medullary sex cords that extend into the mesonephros as rete cords (Forbes, 1940). It has a similar appearance in both sexes throughout embryonic and early posthatching growth, functioning as the excretory duct of the active mesonephros and emptying into the cloaca (Forbes, 1940; Ferguson & Joanen, 1983). At one year posthatching, the Wolffian duct is only slightly larger in males compared to females (Ferguson & Joanen, 1983), but is unresponsive to exogenous androgens from early posthatching through two years of age (Forbes, 1938; Ramaswani & Jacob, 1965). The onset of regional
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Hormones and Reproductive Cycles in Crocodilians
differentiation of the Wolffian duct and hormonal responsiveness has yet to be studied in detail. Growth of the clitero-penis during embryonic development is somewhat temperature- and sex-dependent. The length and width of the clitero-penis of neonatal A. mississippiensis increases with egg incubation temperature in both sexes, and there is some overlap in size of this homologous structure in males and females incubated at transitional temperatures (Allsteadt & Lang, 1995). In contrast to the Wolffian duct, the clitero-penis of hatchling crocodilians exhibits species-specific degrees of sexual dimorphism. Males and females can usually be distinguished by examination of externalized genitalia within months of hatching in Crocodilus porosus and C. johnstoni (Webb, Manolis, & Sack, 1984); C. niloticus (Hutton, 1987); C. palustris (Lang, Andrews, & Whitaker, 1989); and C. siamensis, C. moreletti, and Caiman crocodilus (Lang & Andrews, 1994). A noted exception is Gavialis gangeticus, in which males cannot be distinguished from females based on clitero-penis morphology until one meter or more in total length is reached (Lal & Basu, 1982). Increasing dimensions of the male phallus relative to that of females as body size increases suggests that the male phallus responds to ontogenetic increases in androgens (Allsteadt & Lang, 1995). This is consistent with findings that hatchling male crocodilians respond to exogenous T with pronounced growth of the penis, whereas the structural homolog in females does not respond (Forbes, 1938; Ramaswani & Jacob, 1965). Further, a positive relationship exists between plasma androgen concentration and penis size in juvenile A. mississippiensis. More variation in this relationship is explained by plasma dihydrotestosterone than plasma T, suggesting that 5a-reductase activity is particularly important in phallus development in crocodilians, as it is in mammals (Guillette et al., 1999; Pickford, Guillette, Crain, Rooney, & Woodward, 2000).
2.2. Juvenile Growth and Peripubertal Seasonality Puberty refers to the period during which the immature hypothalamus–pituitary–gonadal (HPG) axis of a juvenile transitions into that of a sexually mature adult capable of producing offspring. It is a complex process that involves the resetting of endocrine homeostasis and concomitant changes in reproductive anatomy, physiology, and behavior. As crocodilians mature, gonadal activity and magnitude of response along the HPG axis to hormonal stimulation increases. For example, plasma T and E2 concentrations increase in juvenile males and females, respectively, with increasing body size (Crain, Guillette, Pickford, Percival, & Woodward, 1998; Milnes, Woodward, Rooney, & Guillette, 2002). Further, although
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pituitary activity in juvenile crocodilians has not been studied directly, experimental evidence suggests that responsiveness to gonadotropin (GTH) stimulation is correlated with size. Injections of ovine pituitary extract cause hypertrophy of the testes, ovaries, and the female reproductive tract in immature A. mississippiensis, and the degree of this response increases with the size of the animal (Forbes, 1934; 1937). In juvenile males, plasma T concentrations increased following injections of ovine follicle-stimulating hormone (FSH). Larger males produced more T, and, although still immature, the largest alligators in the study exhibited a more pronounced seasonal variation in response to FSH stimulation than smaller males (Edwards, Gunderson, Milnes, & Guillette 2004). Crocodilians mature at a rate somewhat analogous to humans and the transition from juvenile to sexual maturity occurs over several years. The synchronized and seasonal nature of crocodilian reproduction has enabled researchers to characterize the onset of seasonal cyclicity and maturation by sampling across size classes throughout the year. Seasonal variation in plasma sex steroids has been characterized in wild-caught juvenile A. mississippiensis (Figure 11.1), and a size threshold of approximately 38 cm snout-to-vent length (SVL) was observed (Rooney, Crain, Woodward, & Guillette, 2004). Animals below this size show little discernable pattern with regard to seasonal variation in plasma E2 or T concentrations, whereas males and females above 38 cm SVL begin to show pronounced seasonal variation in T and E2, respectively, suggesting the onset of puberty. Similarly, male C. yacare show signs of spermatogenesis at ~40 cm SVL, and subadults (70–90 cm SVL) exhibit a seasonal rise in plasma T that coincides with peak spermatogenetic activity (Coutinho, Campos, Cardoso, Massara, & Castro, 2000). At 40 cm SVL, wild alligators and caimans are approximately three to five years of age (Coutinho et al., 2000; Milnes et al., 2002b) and are less than half the size at which both species are generally considered fully mature (~90 cm).
3. REPRODUCTIVE CYCLES 3.1. Seasonality All species of crocodilian examined show a pattern of annual reproductive activity. The first published descriptions of reproductive seasonality in crocodilians came from naturalists’ accounts of A. mississippiensis in the late nineteenth and early twentieth centuries. Clarke (1891) noted that male alligators in Florida, USA, were most frequently on the move and bellowing in May and June, and newly laid eggs in nests were found only in early to mid June. McIlhenny (1935) offered the first detailed accounts
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FIGURE 11.1 Seasonal variation in plasma steroid concentrations of juvenile Alligator mississippiensis 38 cm snout-to-vent length (SVL). Monthly variation is observed in (a) plasma 17b-estradiol (E2) in juvenile females and (b) plasma testosterone in juvenile males. Error bars represent 1 standard error. Redrawn from Rooney, Crain, Woodward, and Guillette (2004).
of mating, nest building, and egg laying from his observations of alligators maintained in semi-natural enclosures on Avery Island, LA, USA. He remarked on the increased interest and bellowing carried on by males in late spring and early summer towards females in adjacent enclosures. He also noted that all egg deposition took place between May 20 and June 25, and egg incubation lasted approximately nine weeks. American alligators are among the most temperate crocodilian species and exhibit highly synchronous and seasonal reproductive activity. The initiation of reproductive events in temperate species corresponds to increasing photoperiod and ambient temperatures in early spring (Figure 11.2). Warmer temperatures and increasing photoperiod trigger HPG activity and gametogenesis, which culminates with copulation and oviposition in late spring and early summer. The window of reproductive
activity in temperate species is limited by the onset of cooler temperatures in the late fall, which bring about a period of metabolic dormancy that lasts through the winter months. Tropical species also exhibit patterns of annual seasonality, but the timing corresponds to local rainfall patterns rather than temperature or photoperiod (Figure 11.2). In these species, ambient temperatures are permissive of year-round metabolic activity, and periods of reproductive activity tend to be more protracted compared to those of their temperate counterparts. Many tropical environments have distinct wet and dry seasons that affect population density and food availability. As water begins to recede during the dry season, food resources, such as fish, initially become more concentrated. Eventually, however, the crocodilian population
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FIGURE 11.2 Relationship between environmental factors and reproductive cycles in crocodilians from tropical and temperate environments. In tropical environments, the lack of rainfall during the dry season leads to a reduction in wetlands area covered by water and a subsequent increase in crocodilian population density. Reproductive activity is suppressed during periods of high population density, resumes with the onset of seasonal rains, and culminates with egg laying near the time of peak water levels. In temperate environments, reproductive activity is suppressed during periods of cooler temperatures and shortened day length. The resumption of reproductive activity corresponds to increasing temperature and photoperiod in early spring.
density also increases to a point where food and suitable habitat become limiting resources. Studies of captive crocodilians have shown that plasma corticosterone (CORT) increases with population density, even when food is not a limiting factor, and females with the greatest plasma CORT concentrations had the poorest nesting success (Elsey, Joanen, McNease, & Lance, 1990). Interspecific competition for food and high population density leading to elevated plasma glucocorticoids might inhibit HPG activity. This hypothesis has yet to be specifically tested in wild crocodilians, but is supported by observational data. Tropical species that experience the most conspicuous annual wet and dry seasons, such as C. crocodilus, exhibit gonadal recrudescence and initiate reproductive behaviors only after water levels begin to rise and animals disperse from areas of high density (Thorbjarnarson, 1994). Nesting takes place when water levels peak, possibly an adaptation to avoid flooding and limit accessibility by terrestrial nest predators. No species are known to produce more than one clutch per year in the wild, but multiple clutches deposited by individuals in a single season have been documented in
C. palustris in captivity, a species that ranges from equatorial to temperate central Asia (Whitaker & Whitaker, 1984). These observations indicate that the frequency of reproduction in the wild is likely dictated by environmental factors rather than inherent physiological limitations. Very little is known about the variability in timing and length of reproductive cycles in species with extensive latitudinal ranges. Crocodylus acutus and C. porosus, for instance, inhabit tropical and semitropical regions and are adapted to brackish and saltwater environments. These coastal areas are less prone to dramatic water level changes, but food availability is probably affected by inland rainfall patterns and the resulting freshwater and nutrient runoff. Abiotic factors such as photoperiod, temperature, and rainfall patterns are variable within these species’ ranges, and almost nothing is known concerning their reproductive endocrinology in the wild.
3.2. Male Reproductive Cycles A consistent pattern of hormonal and/or testicular cyclicity corresponding to male reproductive seasonality has been
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documented in A. mississippiensis (Lance, 1989), C. crocodilus (Thorbjarnarson, 1994), C. yacare (Coutinho et al., 2000), and C. niloticus (Kofron, 1990). Gonadal recrudescence in male crocodilians begins approximately four months prior to nesting. An increase in plasma T coincides with an increase in testicular and seminiferous luminal volume over the ensuing months. Peak concentrations of plasma T are correlated with body size and gonadosomatic index, but a great deal of variation exists among large males during this time. Spermatogonia are the most prevalent germ cell type during the first month of testicular activity, and are undergoing a proliferative phase consisting of mitotic divisions followed by meiosis I (Gribbins, Elsey, & Gist, 2006). Spermatocytes then progress as a cohort to spermatids and then through spermiogenesis. Mature spermatozoa are abundant in the seminiferous tubules for about two months encompassing the peak of plasma T, courtship, and copulation. Plasma T concentration decreases abruptly at the end of the breeding season, after which the testes begin to regress and enter a period of relative quiescence. In A. mississippiensis, a second, but smaller, rise in plasma T concentration occurs in the fall. The purpose of this increase in T is unknown, but active spermatogenesis is not present at this time (Lance, 1989).
3.3. Female Reproductive Cycles 3.3.1. Vitellogenesis and oocyte maturation Similarly to male crocodilians, annual hormonal and ovarian cycles in females have only been described in a few species, but these limited data generally support the postulate that reproductive endocrinology is conserved among crocodilians. The most notable difference among species is the variation in the onset, length, and continuity of ovarian activity that corresponds to differences in temperature, photoperiod, and/or rainfall patterns. In A. mississippiensis, a postnuptial period of elevated plasma E2 and follicular recruitment occurs in the fall, eight months prior to ovulation (Guillette et al., 1997). The timing of this increased ovarian activity corresponds to elevated plasma T reported in male alligators (Lance, 1989). The enlarged follicles resulting from this first phase of vitellogenesis may serve as a ready source of E2 after a period of ovarian quiescence in the winter. This hypothesis is supported by the simultaneous presence of large atretic follicles and smaller vitellogenic follicles in reproductively active females examined in early spring (Guillette et al., 1997). In C. niloticus in Zimbabwe, plasma E2 and calcium, both indicative of vitellogenesis, begin to rise as temperatures cool in April and May. Unlike A. mississippiensis, these animals continue to feed throughout the coolest months, and plasma E2 and calcium concentrations continue to increase in reproductively active females until ovulation in early August (Kofron, 1990).
Hormones and Reproduction of Vertebrates
In tropical species such as C. crocodilus and C. yacare, vitellogenic follicles and elevated plasma E2 concentrations have only been observed in the months following the dry season and immediately preceding ovulation (Thorbjarnarson, 1994; Coutinho et al., 2000). In spite of diversity in the environments that different species inhabit, reproductively active female crocodilians typically exhibit an increase in circulating E2 four to five months prior to oviposition (Figure 11.3). This stimulates vitellogenin production and eventually an approximate tenfold increase in the size of pre-ovulatory follicles. As the follicles increase in size, the thecal layer composed of fibroblasts begins to form and the zona pellucida develops and thickens into two layers consisting of the striated zona radiata and an outer hyaline layer (Uribe & Guillette, 2000; Calderon, De Perez, & Pinilla, 2004). As vitellogenesis advances, small yolk platelets begin to appear inside vacuoles and differences develop in the animal and vegetal poles based on peripheral placement of the nucleus and the morphology and size of the yolk platelets. The granulosa cells gradually transform from a single squamous to cuboidal to columnar layer throughout vitellogenesis (Uribe & Guillette, 2000; Calderon et al., 2004) and show intense staining for 3b-HSD activity, indicating active steroidogenesis (Lance, 1989). The theca continues to thicken and by late vitellogenesis consists of large blood vessels, fibroblasts, collagen fibers, flattened lacunae, and smooth muscle cells. Prior to ovulation, the oocyte reaches its greatest diameter, which ranges from 35–45 mm. The preovulatory increase in plasma E2 also corresponds to the rapid and dramatic growth of the oviduct that occurs during the four months prior to ovulation. The length of the oviduct in a non-reproductive female alligator averages 45 cm, whereas reproductive tracts of vitellogenic females reach 1.5–2.5 m in length by the time of ovulation (Guillette et al., 1997). During late vitellogenesis, an increase in plasma T in females corresponds to the onset of courtship and copulatory behavior (Guillette et al., 1997; Lance, 1989). It is unclear what role T has in female crocodilians, but in songbirds it has been linked to territorial aggression towards intruding same-sex individuals and breeding pairs (Gill, Alfson, & Hau, 2007). Copulation in crocodilians can precede ovulation by a month or more, and females may breed with multiple males during this time. This has been substantiated by the confirmation of multiple paternity through microsatellite DNA comparisons within clutches from A. mississippiensis and C. moreletii (Davis, Glenn, Elsey, Dessauer, & Sawyer, 2001; McVay et al., 2008).
3.3.2. Ovulation, gravidity, and oviposition A sharp decrease in plasma E2 and a rise in plasma progesterone (P4) are observed at the time of ovulation, and
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Hormones and Reproductive Cycles in Crocodilians
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FIGURE 11.3 Mean ( 1 standard error) plasma steroid concentrations in relation to the annual reproductive cycle of adult female Alligator mississippiensis. Data taken from Guillette et al. (1997).
elevated plasma P4 concentration is maintained throughout the period of gravidity. In A. mississippiensis, granulosa cells associated with the newly formed corpora lutea show intense 3b-HSD staining (Lance, 1989), and plasma P4 concentrations are positively correlated with corpora luteal volume (Figure 11.4) (Guillette et al., 1995). Elevated P4 is also closely associated with insulin-like growth factor 1 (IGF-1) and oviductal secretions during the three weeks of gravidity (Figure 11.5). Plasma concentrations of IGF-I are greatest at this time and both IGF-I and IGF-II have been
identified in the albumen of A. mississippiensis (Guillette & Williams, 1991; Guillette, Cox, & Crain, 1996). Immunolocalization of IGF-I to tubal glands of the oviduct that secrete albumen indicates that this hormone is synthesized locally for incorporation into the egg, and suggests a role for maternal growth factors in embryonic development (Cox & Guillette, 1993). The bifurcated oviduct of crocodilians consists of the anterior infundibulum, posterior infundibulum, tube, utero–tubular junction, anterior and posterior uterus,
FIGURE 11.4 Mean ( 1 standard error) plasma progesterone and corpora luteum volume during early (E), mid (M), and late (L) vitellogenesis (V), gravidity (G), and postoviposition (P) in female Alligator mississippiensis. Data taken from Guillette et al. (1995) and Guillette, Cox, and Crain (1996).
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FIGURE 11.5 Mean ( 1 standard error) plasma insulin-like growth factor I (IGF-I) during early (E), mid (M), and late (L) vitellogenesis (V), gravidity (G), and postoviposition (P) in female Alligator mississippiensis. Data taken from Guillette, Cox, and Crain (1996).
and vagina (Palmer & Guillette, 1992; Gist, Bagwill, Lance, Sever, & Elsey, 2008). The anterior infundibulum is funnel-shaped, opens to the coelom, and has thin, transparent walls. The posterior infundibulum is tubular and consists of muscular walls with mucosal foldings and secretory cells. Immunocytochemical data and de novo synthesis in tissue explants suggest that both the posterior infundibulum and tube function in albumen formation (Palmer & Guillette, 1991; Buhi, Alvarez, Binelli, Walworth, & Guillette 1999). Thin, translucent walls and a lack of mucosal glands distinguish the utero–tubular junction. Both functionally and ultrastructurally, the anterior uterus of the alligator resembles the avian isthmus, in which eggshell membrane formation takes place. Endometrial glands in the anterior uterus secrete proteinaceous fibers similar in structure and diameter to those of the eggshell membrane (Palmer & Guillette, 1992). The posterior uterus is histologically similar to the avian shell gland, which secretes the calcareous eggshell. Eggs are retained in this portion of the crocodilian uterus for most of gravidity, where eggshell calcification takes place simultaneously on the entire clutch. In recently mated females, large quantities of sperm are found in the lumen of the vagina near the opening of deep mucosal folds. It is unclear exactly where fertilization takes place or when in temporal relation to ovulation, but stored sperm can be found in mucosal folds and glands that open into the lumen of the oviduct,
extending from the vagina to the utero–tubular junction (Gist et al., 2008). Oviposition in crocodilians is marked by a sharp decline in plasma P4 concentration and corpora luteal volume. The rate of luteolysis slows after the first month postoviposition in A. mississippiensis, and persistent remnants of the corpora lutea in the fall can be used as an indicator that ovulation occurred the previous spring (Guillette et al., 1995). Hormonal control of oviposition is poorly understood, but limited experimental data suggest it is similar to that of other vertebrates. For instance, injection of oxytocin has been used to treat dystocia and induce oviposition in C. porosus (Carmel, 1991). Arginine vasotocin (AVT), the homolog of mammalian oxytocin, stimulates inositol trisphosphate (IP3) production in A. mississippiensis uterine endometrium in vitro. Stimulation of IP3, a secondary messenger that stimulates prostaglandin (PG) production in mammals, was greatest in uterine endometrium from late gravid females (Mirando & Guillette, 1991). In birds, PGs inhibit P4 production in corpora lutea, stimulate uterine contractions, and relax the utero–vaginal sphincter near the time of oviposition (Hertelendy et al., 1984). Circulating PG concentrations have not been reported in any crocodilian, but PGF2a and PGE2 are secreted by the uterus, and secretions are greatest in postoviposition animals (Dubois & Guillette, 1992). Oddly, AVT inhibited PG secretion in uterine explants, indicating that more work is required to fully understand this aspect of crocodilian reproduction.
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Hormones and Reproductive Cycles in Crocodilians
4. CURRENT AND FUTURE RESEARCH DIRECTIONS 4.1. Molecular Endocrinology in Crocodilians Seminal advances in reproductive endocrinology relied heavily on the ablation of endocrine tissues and the subsequent rescue of function by replacement with tissue explants or whole-gland extract. While effective, these techniques are highly invasive and not particularly desirable for use in endangered or threatened species, such as 10 of the 23 extant crocodilians listed by the International Union for Conservation of Nature (IUCN) (2008). In the latter half of the twentieth century, antibody development made it possible to track changes in hormone concentrations that coincide with changes in reproductive physiology and immunolocalize endocrine-related proteins to specific tissues and cell types. These techniques offered an improvement in specificity, but, from a comparative perspective, have been somewhat limited by the availability of antibodies that will cross-react with homologous antigens in non-mammalian and non-laboratory species such as crocodilians. As an emerging discipline, molecular endocrinology is rapidly advancing our understanding of endocrine signaling in crocodilians. The ever-growing database of nucleic acid sequences, simplification of molecular cloning protocols, and development of sensitive techniques such as quantitative, real-time PCR (Q-PCR) and in situ hybridization are making it progressively easier to investigate reproductive endocrinology at the subcellular level, independent of antibody production. Ideally, the information obtained from studies of molecular endocrinology in crocodilians will be assimilated into sections organized principally by physiological topic rather than presented in a separate section based on a laboratory technique. Unfortunately, most of the molecular endocrinology data currently available are too disparate to be fully understood within the context of our overall understanding of crocodilian biology.
4.1.1. Steroidogenesis and steroid metabolism Steroid hormone concentrations in relation to sexual development and reproductive cycles have been well characterized in some species of crocodilian; however, the underlying mechanisms of steroid homeostasis are only partially understood. Circulating concentrations of these hormones are dictated by a combination of gonadal steroidogenesis and hepatic catabolism, and the genes coding for several key regulators of these processes have recently been cloned or partially cloned in A. mississippiensis and their expression measured by Q-PCR. For example, mRNA expression of steroidogenic acute regulatory protein, steroidogenic factor 1, and several
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steroidogenic enzymes involved in the conversion of cholesterol to biologically active steroids has been examined in the context of environmental contaminant exposure (Kohno, Bermudez, Katsu, Iguchi, & Guillette, 2008; Milnes et al., 2008). Similarly, hepatic expression of cytochrome P450 3A77, an enzyme involved in catabolism of steroid hormones, has been measured following injections of xenobiotic compounds (Gunderson, Kohno, Blumberg, Iguchi, & Guillette, 2006). Both studies found changes in expression related to chemical exposure, but the significance of the expression levels is not completely clear. Analysis of mRNA abundance is often predictive of cellular activity, but results must be interpreted with caution as gene transcripts are subject to considerable modification en route to the expression of functional proteins. Future work should examine temporal expression patterns of these genes following GTH stimulation. Corresponding measures of steroid hormone production and excretion of steroid metabolites would resolve how closely mRNA expression patterns reflect enzyme activity. As additional gene expression patterns are characterized in more instances and species in relation to phenotypic effects, these data will become increasingly meaningful.
4.1.2. Peptide hormones and hormone receptors Current understanding of the regulation of crocodilian reproduction by peptide hormones is lacking relative to our knowledge of sex steroids. This is due, in part, to the difficulty in obtaining purified proteins and/or the limited availability of useful antibodies. For instance, chemical purification of A. mississippiensis pituitary extracts and their action in lizard (Anolis) and anuran (Xenopus) bioassays suggest that crocodilians produce two FSH-like and one LH-like glycoprotein (Licht, Farmer, & Papkoff, 1976), but no direct measure of GTHs has been made. Cloning of A. mississippiensis pre-proopiomelanocortin (prePOMC) has identified the POMC-derived subunits of this gene (Kobayashi et al., 2007). Complementary MALDI-TOF mass spectrometry has further characterized post-transcriptional modifications involved in the production of melanotropin (MSH), b-endorphin, and corticotropin (ACTH). Molecular cloning of the genes encoding the alpha and beta glycoprotein subunits of LH and FSH would allow similar characterization of the GTHs. In addition, the cloning of these genes and synthesis of recombinant proteins can circumvent the need for whole gland extracts to obtain purified peptides or generate species-specific antibodies. The difficulties of studying peptide hormones in nonmodel species, such as the lack of available antibodies, are similar to the challenges related to understanding the
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role of hormone receptors in reproductive endocrinology. Fortunately, recent progress in the cloning of crocodilian steroid receptors has been made, and complete mRNA and derived peptide sequences for estrogen and/or P4 receptors in C. crocodilus (Sumida, Ooe, Saito, & Kaneko, 2001), A. mississippiensis (Katsu et al., 2004), and C. niloticus (Katsu et al., 2006) are available. As in other vertebrates, crocodilian steroid receptors can be divided into five domains, suggesting functional correspondence, and their sequence homologies reflect consensus evolutionary relationships. The next step will be to characterize the expression of these and other receptors in possible target tissues in response to changing reproductive status. It will also be useful to develop in-vitro binding and activation assays to quantify the sensitivity of these receptors to endogenous ligands, similar to what has been done in the freshwater turtle, Pseudemys nelsoni (Katsu et al., 2008).
4.2. Research Gaps Significant advances in our understanding of crocodilian reproductive endocrinology have been made over the past 20 years, but significant gaps still exist. For instance, egg incubation temperature is known to affect posthatching growth rates (Pina, Larriera, Medina, & Webb, 2007), but how? Does the embryonic environment lead to nongenomic effects such as changes in DNA methylation patterns that affect the expression of thyroid hormones, growth factors, hormone receptors, synthetic or degrading enzymes, etc.? Ontogenetic changes related to posthatching sexual differentiation, puberty, and the onset of reproductive maturation and senescence are also only partially understood. The characterization of GTHs and their production in relation to steroid concentrations and how these correspond to various life-history stages is needed. As a long-lived taxonomic group, a better understanding of changes in peripheral steroid receptor expression related to responsiveness to GTHs and steroid stimulation would make crocodilians an interesting model for reproductive ageing. Finally, what environmental stimuli have the greatest impact on the frequency and success of reproduction? As environmental factors are altered in response to pollution and global climate change, an understanding of how this will affect reproduction and evolution in crocodilians will enhance future conservation efforts.
ABBREVIATIONS 3b-HSD ACTH
3b-hydroxysteroid dehydrogenase Corticotropin
AMH AVT CORT E2 FSH GSI GTH HPG IGF-I IGF-II IP3 LH MIS MSH P4 PCR PG PGE2 PGF2a POMC prePOMC Q-PCR SF-1 StAR SVL T TSP
Anti-Mu¨llerian hormone Arginine vasotocin Corticosterone 17b-estradiol Follicle-stimulating hormone Gonadosomatic index Gonadotropin Hypothalamus–pituitary–gonad Insulin-like growth factor I Insulin-like growth factor II Inositol triphosphate Luteinizing hormone Mu¨llerian-inhibiting substance Melanotropin Progesterone Polymerase chain reaction Prostaglandin Prostaglandin E2 Prostaglandin F2a Preopiomelanocortin Pre-proopiomelanocortin Quantitative, real-time polymerase chain reaction Steroidogenic factor 1 Steroidogenic acute regulatory protein Snout-to-vent length Testosterone Thermosensitive period
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Coutinho, M., Campos, Z., Cardoso, F., Massara, P., & Castro, A. (2000). Reproductive biology and its implication for management of Caiman Caiman yacare in the Pantanal Wetland, Brazil. In G. C. Grigg, F. Seebacher, & C. E. Franklin (Eds.), ‘‘Crocodilian Biology and Evolution’’ (pp. 229–243). Chipping Norton, NSW, Australia: Surrey Beatty & Sons. Cox, C., & Guillette, L. J., Jr. (1993). Localization of insulin-like growth factor-I-like immunoreactivity in the reproductive tract of the vitellogenic female American alligator, Alligator mississippiensis. Anat. Rec., 236, 635–640. Crain, A. D., Spiteri, I. D., & Guillette, L. J., Jr. (1999). The functional and structural observations of the neonatal reproductive system of alligators exposed in ovo to atrazine, 2,4-D, or estradiol. Toxicol. Ind. Health, 15, 180–185. Crain, D. A., Guillette, L. J., Jr., Pickford, D. B., Percival, H. F., & Woodward, A. R. (1998). Sex-steroid and thyroid hormone concentrations in juvenile alligators (Alligator mississippiensis) from contaminated and reference lakes in Florida, USA. Environ. Toxicol. Chem., 17, 446–452. Davis, L. M., Glenn, T. C., Elsey, R. M., Dessauer, H. C., & Sawyer, R. H. (2001). Multiple paternity and mating patterns in the American alligator, Alligator mississippiensis. Mol. Ecol., 10, 1011–1024. Deeming, D. C., & Ferguson, M. W. J. (1989). Effects of incubation temperature on growth and development of embryos of Alligator mississippiensis. J. Comp. Physiol. B, 159, 183–193. Dubois, D. H., & Guillette, L. J., Jr. (1992). Secretion of prostaglandin F2a and E2 in vitro by the uterus of the American alligator (Alligator mississippiensis). J. Exp. Zool., 264, 253–260. Eaton, M. J., Martin, A., Thorbjarnarson, J., & Amato, G. (2008). Species-level diversification of African dwarf crocodiles (Genus Osteolaemus): A geographic and phylogenetic perspective. Mol. Phylogenet. Evol. in press. Edwards, T. M., Gunderson, M. P., Milnes, M. R., & Guillette, L. J., Jr. (2004). Gonadotropin-induced testosterone response in peripubertal male alligators. Gen. Comp. Endocrinol., 135, 372–380. Elsey, R. M., Joanen, T., McNease, L., & Lance, V. A. (1990). Stress and plasma corticosterone levels in the American alligatordrelationships with stocking density and nesting success. Comp. Biochem. Physiol., 95A, 55–63. Ferguson, M. W. J. (1985). In C. Gans, P. Maderson, & F. Billet (Eds.), ‘‘Biology of the Reptilia’’. Reproductive biology and embryology of the crocodilians, Vol. 14 (pp. 329–491). New York: J. Wiley and Sons. Ferguson, M. W. J., & Joanen, T. (1983). Temperature-dependent sex determination in Alligator mississippiensis. J. Zool. Lond., 200, 143–177. Forbes, T. R. (1934). Effect of injections of pituitary whole gland extract on immature alligator. Proc. Soc. Exp. Biol. Med., 31, 1129–1130. Forbes, T. R. (1937). The effects of prolonged injections of pituitary whole gland extract in the immature alligator. Anat. Rec., 70, 113–137. Forbes, T. R. (1938). The action of testosterone on the accessory sex structures of recently hatched female alligators. Anat. Rec., 72, 87– 95. Forbes, T. R. (1940). Studies on the reproductive system of the alligator. IV. Observations on the development of the gonad, the adrenal cortex, and the Mu¨llerian duct. Contrib. Embryol., 174, 131–163.
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Iguchi, T. (2004). Molecular cloning of the estrogen and progesterone receptors of the American alligator. Gen. Comp. Endocrinol., 136, 122–133. Katsu, Y., Ichikawa, R., Ikeuchi, T., Kohno, S., Guillette, L. J., Jr., & Iguchi, T. (2008). Molecular cloning and characterization of estrogen, androgen, and progesterone nuclear receptors from a freshwater turtle (Pseudemys nelsoni). Endocrinology, 149, 161–173. Katsu, Y., Myburgh, J., Kohno, S., Swan, G. E., Guillette, L. J., Jr., & Iguchi, T. (2006). Molecular cloning of estrogen receptor alpha of the Nile crocodile. Comp. Biochem. Physiol. A Mol. Integr. Physiol., 143, 340–346. Kobayashi, Y., Sakamoto, T., Iguchi, K., Imai, Y., Hoshino, M., Lance, V. A., Kawauchi, H., & Takahashi, A. (2007). cDNA cloning of proopiomelanocortin (POMC) and mass spectrometric identification of POMC-derived peptides from snake and alligator pituitaries. Gen. Comp. Endocrinol., 152, 73–81. Kofron, C. P. (1990). The reproductive cycle of the Nile crocodile (Crocodylus niloticus). J. Zool. Lond., 221, 477–488. Kohno, S., Bermudez, D. S., Katsu, Y., Iguchi, T., & Guillette, L. J., Jr. (2008). Gene expression patterns in juvenile American alligators (Alligator mississippiensis) exposed to environmental contaminants. Aquat. Toxicol., 88, 95–101. Lal, S., & Basu, D. (1982). Sexing and sex ratios of gharial (Gavialis gangeticus) raised in captivity. J. Bombay Nat. Hist. Soc., 79, 688–691. Lance, V. A. (1989). Reproductive cycle of the American alligator. Am. Zool., 29, 999–1018. Lance, V. A., & Bogart, M. H. (1992). Disruption of ovarian development in alligator embryos treated with an aromatase inhibitor. Gen. Comp. Endocrinol., 86, 59–71. Lang, J. W., & Andrews, H. V. (1994). Temperature-dependent sex determination in crocodilians. J. Exp. Zool., 270, 28–44. Lang, J. W., Andrews, H., & Whitaker, R. (1989). Sex determination and sex ratios in Crocodylus palustris. Am. Zool., 29, 935–952. Laurin, M., & Gauthier, J. A. (1996). The Tree of Life Web Project. Amniota. Mammals, reptiles (turtles, lizards, Sphenodon, crocodiles, birds) and their extinct relatives. Version 01 January 1996. In: ‘‘The Tree of Life Web Project’’, http://tolweb.org/. Licht, P., Farmer, S. W., & Papkoff, H. (1976). Further studies on the chemical nature of reptilian gonadotropins: FSH and LH in the American alligator and green sea turtle. Biol. Reprod., 14, 222– 232. McIlhenny, E. A. (1935). The Alligator’s Life History. Boston, MA: Christopher Publishing House. McVay, J. D., Rodriguez, D., Rainwater, T. R., Dever, J. A., Platt, S. G., McMurry, S. T., Forstner, M. R., & Densmore, L. D., 3rd (2008). Evidence of multiple paternity in Morelet’s Crocodile (Crocodylus moreletii) in Belize, CA, inferred from microsatellite markers. J. Exp. Zool. A Ecol. Genet. Physiol., 309, 643–648. Milnes, M. R., Bryan, T. A., Katsu, Y., Kohno, S., Moore, B. C., Iguchi, T., & Guillette, L. J., Jr. (2008). Increased posthatching mortality and loss of sexually dimorphic gene expression in alligators (Alligator mississippiensis) from a contaminated environment. Biol. Reprod., 78, 932–938. Milnes, M. R., Roberts, R. N., & Guillette, L. J., Jr. (2002a). Effects of incubation temperature and estrogen exposure on aromatase activity in the brain and gonads of embryonic alligators. Environ. Health Perspect, 110, 393–396.
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Milnes, M. R., Woodward, A. R., Rooney, A. A., & Guillette, L. J., Jr. (2002b). Plasma steroid concentrations in relation to size and age in juvenile alligators from two Florida lakes. Comp. Biochem. Physiol. A., 131, 923–930. Mirando, M. A., & Guillette, L. J., Jr. (1991). Inositol phosphate formation in uterine tissue from two species of reptiles is stimulated by arginine vasotocin and influenced by stage of reproduction. Gen. Comp. Endocrinol., 83, 481–486. Moore, B. C., Uribe-Aranzabal, M. C., Boggs, A. S., & Guillette, L. J., Jr. (2008). Developmental morphology of the neonatal alligator (Alligator mississippiensis) ovary. J. Morph., 269, 302–312. Palmer, B., & Guillette, L. J., Jr. (1991). Oviductal proteins and their influence on embryonic development in birds and reptiles. In D. Deeming, & M. Ferguson (Eds.), ‘‘Egg Incubation: Its Effects on Avian and Reptilian Embryonic Development’’ (pp. 29–46). Cambridge: Cambridge University Press. Palmer, B. D., & Guillette, L. J., Jr. (1992). Alligators provide evidence for the evolution of an archosaurian mode of oviparity. Biol. Reprod., 46, 39–47. Pickford, D. B., Guillette, L. J., Jr., Crain, D. A., Rooney, A. A., & Woodward, A. R. (2000). Phallus size and plasma dihydrotestosterone concentrations in juvenile American alligators (A. mississippiensis) from contaminated and reference populations. J. Herp., 34, 233–239. Pina, C. I., Larriera, A., & Cabrera, M. R. (2003). Effects of incubation temperature on incubation period, sex ratio, hatchling success, and survivorship in Caiman latirostris (Crocodylia, Alligatoridae). J. Herp., 37, 199–202. Pina, C. I., Larriera, A., Medina, M., & Webb, G. J. W. (2007). Effects of incubation temperature on the size of Caiman latirostris (Crocodylia: Alligatoridae) at hatching and after one year. J. Herp., 41, 205–210. Ramaswani, L. S., & Jacob, D. (1965). Effect of testosterone proprionate on the urinogenital organs of immature crocodile Crocodylus palustris Lesson. Experientia, 21, 206–207. Rooney, A. A., Crain, A. D., Woodward, A. R., & Guillette, L. J., Jr. (2004). Seasonal variation in plasma sex steroid concentrations in juvenile American alligators. Gen. Comp. Endocrinol., 135, 25–34. Smith, C. A., & Joss, J. M. P. (1993). Gonadal sex differentiation in Alligator mississippiensis, a species with temperature-dependent sex determination. Cell Tissue Res., 273, 149–162. Smith, C. A., & Joss, J. M. P. (1994a). Sertoli cell differentiation and gonadogenesis in Alligator mississippiensis. J. Exp. Zool., 270, 57–70. Smith, C. A., & Joss, J. M. P. (1994b). Steroidogenic enzyme activity and ovarian differentiation in the saltwater crocodile, Crocodylus porosus. Gen. Comp. Endocrinol., 93, 232–245. Smith, C. A., & Joss, J. M. P. (1994c). Uptake of 3H-estradiol by embryonic crocodile gonads during the period of sexual differentiation. J. Exp. Zool., 270, 219–224. Smith, C. A., Elf, P. K., Lang, J. W., & Joss, J. M. P. (1995). Aromatase enzyme activity during gonadal sex differentiation in alligator embryos. Differentiation, 58, 281–290. Sumida, K., Ooe, N., Saito, K., & Kaneko, H. (2001). Molecular cloning and characterization of reptilian estrogen receptor cDNAs. Mol. Cell. Endocrinol., 183, 33–39. Thorbjarnarson, J. B. (1994). Reproductive ecology of the spectacled caiman (Caiman crocodilus) in the Venezuelan Llanos. Copeia, 4, 907–919.
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Thorbjarnarson, J. B. (1996). Reproductive characteristics of the order Crocodylia. Herpetologica, 52, 8–24. Uribe, M. C., & Guillette, L. J., Jr. (2000). Oogenesis and ovarian histology of the American alligator Alligator mississippiensis. J. Morph., 245, 225–240. Webb, G. J. W., Manolis, S. C., & Sack, G. C. (1984). Cloacal sexing of hatchling crocodilians. Austra. Wildl. Res., 10, 607–637. Western, P. S., Harry, J. L., Graves, J. A. M., & Sinclair, A. H. (1999). Temperature-dependent sex determination in the
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American alligator: AMH precedes SOX9 expression. Dev. Dyn., 216, 411–419. Whitaker, R. H., & Whitaker, Z. (1984). Reproductive biology of the mugger (Crocodylus palustris). J. Bombay Nat. Hist. Soc., 81, 297–317. Willis, R. E., McAliley, L. R., Neeley, E. D., & Densmore, L. D., 3rd (2007). Evidence for placing the false gharial (Tomistoma schlegelii) into the family Gavialidae: inferences from nuclear gene sequences. Mol. Phylogenet. Evol., 43, 787–794.
Chapter 12
Hormones and Reproductive Cycles in Lizards Matthew B. Lovern Oklahoma State University, Stillwater, OK, USA
SUMMARY Lizard species, of which there are more than 5000, occupy all continents except Antarctica, in habitats ranging from desert to rainforest, and exhibit arboreal, terrestrial, fossorial, and even semi-aquatic life histories. Detailed knowledge of reproductive cycles and their endocrine mediation, although increasing, is available for only a small percentage of these speciesdeven less when both males and females are considered. From a review of available data, it is clear that gonadal steroid profiles across the reproductive cycle and their hypothalamic–pituitary regulation have remained generally consistent across lizard species regardless of reproductive mode (e.g., oviparity vs. viviparity, genotypic vs. temperature-dependent sex determination (TSD)). Thus, evolutionary conservation is demonstrated in that gonadal steroids play critical roles in the physiology and behavior necessary for reproduction. Evolutionary innovation is demonstrated in the variety of organismal responses to these gonadal steroids. Targeted research with established model species along with underrepresented taxa, including work on the role of endogenous rhythms and seasonal control of reproduction, will greatly advance our understanding of lizard, and likely vertebrate, reproduction.
1. INTRODUCTION 1.1. Why Study Lizards? It is common for researchers to extol the virtues of their particular model organism, and those of us who work with lizards are no exception. In fact, we may engage in this positivism more frequently than researchers working with other taxa for at least two reasons aside from the biological and experimental justifications discussed below. First, less attention has been paid to the biology of lizards and reptiles overall compared to other vertebrate groups. For example, from 2001–2008, studies on reptiles comprised just 5% of the total number of studies using vertebrate models (Science Citation Index Expanded database on Web of Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
Science; Thomson Reuters). Second, a recent analysis demonstrates that it is common for researchers to demonstrate a citation bias towards their model organism and its taxonda so-called ‘taxonomic parochialism’dand that this problem may be more widespread in studies on mammals and birds than studies on reptiles, amphibians, and fishes (Taborsky, 2009). Taxonomic parochialism can be a problem for all of us to the extent that we become blinded to the broader context in which our work is relevant. Increased research effort using reptiles and greater attention paid to the existing studies of reptiles will promote a strong comparative approach to our respective disciplines. Why study lizards? In reviews of then-current states of knowledge and prospects for future work, numerous authors have cogently advocated conducting research on reptiles to gain insight into a variety of disciplines including, for example, physiological ecology, behavioral neuroendocrinology, biomedical science, and ecotoxicology (e.g. Gans & Dawson, 1976; Greenberg et al., 1989; Crews & Gans, 1992; Moore & Lindzey, 1992; Hopkins, 2000; Godwin & Crews, 2002; Talent, Dumont, Bantle, Janz, & Talent, 2002; Lovern, Holmes, & Wade, 2004; Crews & Moore, 2005). Regardless of the specific discipline, the advantages of working with lizards in particular, and the benefits that come from studies on their reproductive cycles, fall into one of three general categories: practical considerations, evolutionary considerations, and environmental and conservation considerations.
1.1.1. Practical considerations There are numerous practical considerations that make lizards good study organisms. Many species are relatively easy to observe repeatedly in the field because of their conspicuous reproductive morphology and behavior, comparatively restricted space use and fidelity, and terrestrial lifestyle. Additionally, many species are relatively 321
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small and pose little to no threat to the investigator (e.g., in terms of bites or other injury-causing behaviors, use of venoms or toxins, etc.), thus making them amenable to capture and handling for measurement and manipulation. One practical tradeoff to relatively small body size, however, is that it often is not feasible to conduct repeatedmeasures designs on hormone profiles in the blood, given the volume of blood drawn vs. total blood volume and presumed effects on subsequent hormone profiles. However, sufficiently large lizards, sufficiently small samples (possible due to increased sensitivity of many hormone assays), time between samples, or other empirically based justifications may allow for it (e.g., Smith & John-Alder, 1999; Rhen, Sakata, Zeller, & Crews, 2000; Wack, Fox, Hellgren, & Lovern, 2008; Husak et al., 2009). Many species also can be maintained in the laboratory in naturalistic enclosures that promote the full range of reproductive behaviors seen in the field, permitting integrative studies using both field and laboratory approaches. Finally, due to comparatively high population densities for many lizard species, laboratory colonies can be maintained and supplemented with easily obtained wild stock as necessary, to prevent unwanted genetic separation over successive generations in the laboratory. Overall, these practical considerations give us the ability to examine endogenous hormone profiles, manipulate hormone levels, and examine hormone–reproductive cycle relationships while maintaining both high internal and external validity of study design and ecological relevance (Miller, 1994).
1.1.2. Evolutionary considerations ‘Taken in its entirety, the evolutionary history of lizards is among the most fascinating examples of natural selection in action among vertebrates’ (Pianka & Vitt, 2003, p. 257). Reptiles are a diverse and ancient assemblage of species that yield insight into present-day adaptations and historical traits that made terrestrial living possible over 300 million years ago. The extant amniotesdtaxa possessing extraembryonic membranes that form early in development and allowed the transition from an aquatic to a terrestrial environmentdare a monophyletic clade comprised of reptiles (including birds, which are nested within Reptilia) and mammals that arose from a common reptile-like ancestor 200–300 million years ago (Pough et al., 2004). Lizards and snakes (which arose within the lizard lineage) comprise order Squamata. As of February 2008, there were 8396 extant squamate species recognized, including 5079 lizards (JCVI/TIGR Reptile Database, 2008), comparable to the total number of mammalian species (~5000). Lizards traditionally are divided into two clades based on a hypothesized split occurring early in evolutionary history in foraging mode and associated tongue morphology and function: (1) the Iguania are fleshy-tongued lizards that use
Hormones and Reproduction of Vertebrates
the tongue for feeding and (2) the Scleroglossa are ‘hardtongued’ lizards that use their jaws for feeding and tongues primarily for chemoreception (Pianka & Vitt, 2003; Pough et al., 2004). However, the taxonomy of squamates currently is undergoing a reorganization due to recent molecular phylogenies that do not support this traditional split and instead demonstrate that Iguanians are highly nested within species traditionally grouped separately as Scleroglossans (e.g., Townsend, Larson, Louis, & Macey, 2004; Vidal & Hedges, 2005; 2009) (Figure 12.1; Table 12.1). Finally, lizards are nearly global in distribution, occurring on all continents except Antarctica (Pough et al., 2004). Given high species diversity and widespread geographic distribution, it should not be surprising that lizards show tremendous variety in their reproductive biology and associated life histories; the following considerations illustrate a small part of this variety. First, although most lizard species are oviparous, approximately 20% of species are viviparous (Blackburn, 1982; 1985; see also Chapter 9, this volume). Viviparity has evolved independently in approximately half of all lizard families (reviewed in Pianka & Vitt, 2003), even multiple times within the same genus (Sceloporus) (Benabib, Kjer, & Sites, 1997; Me´ndezDe La Cruz, Villagra´n-Santa Cruz, & Andrews, 1998). Second, typical clutch sizes can vary tremendously across oviparous lizard species. Some species exhibit oneegg (e.g., Anolis (Smith, Sinelnik, Fawcett, & Jones, 1973)) or two-egg clutches (e.g., many geckos including Eublepharis (LaDage, Gutzke, Simmons, & Ferkin, 2008)) produced repeatedly over the course of a breeding season, whereas other species produce clutches averaging approximately 20–30 eggs produced once per breeding season (e.g., Phrynosoma (Ballinger, 1974; Endriss, Hellgren, Fox, & Moody, 2007)). Within species, female body size can play a significant role in determining clutch size. For example, Nile monitor lizards (Varanus niloticus) average 21 eggs per clutch overall, but small females average less than 15 eggs per clutch whereas large females average over 50 (de Buffre´nil & Rimblot-Baly, 1999). Third, there is considerable diversity in how reproductive effort is allocated over the lifespan. Males and females of some lizard species take several years to become reproductively mature but then reproduce across multiple years over a comparatively long lifespan (20þ years in some cases), whereas in other species maturation occurs within the year of birth or hatching and reproduction is limited to one or a few breeding events (Tinkle, Wilbur, & Tilley, 1970; Shine & Charnov, 1992; Pianka & Vitt, 2003). In general, high probability of annual adult survival is related to slow reproductive maturation and low annual fecundity, whereas low probability of annual adult survival is related to fast maturation and high annual fecundity. In an extreme case that appears to represent the shortest lifespan for a tetrapod, the chameleon Furcifer labordi lives only
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FIGURE 12.1 Molecular phylogeny of lizard families and associated divergence time estimates. Reproduced with permission from Vidal and Hedges (2009).
four to five months outside of the egg (in contrast to eight to nine months as an embryo), during which time growth to sexual maturity, mating, and oviposition by females must occur before senescence and death take place in the entire adult population (Karsten, Andriamandimbiarisoa, Fox, & Raxworthy, 2008). Fourth, sex determination is genetically controlled in most species of lizard, similarly to the large majority of vertebrate species overall. However, temperature-dependent sex determination (TSD), in which the incubation temperature experienced by the egg is the trigger for sexual differentiation into male (typically intermediate temperatures) or female (typically low and high
temperatures), occurs in many lizard species, distributed across at least five lizard familiesdIguanidae, Agamidae, Eublepharidae, Gekkonidae, and Lacertidae (Viets, Ewert, Talent, & Nelson, 1994; see also Chapter 1, this volume). Fifth and even more fundamental than variation in sexdetermining systems, lizard species differ in whether they reproduce sexually or asexually. Although most lizards reproduce sexually, there are approximately 30 all-female species that reproduce asexually by parthenogenesis (Pianka & Vitt, 2003). Parthenogenetic species occur in at least seven lizard families (Agamidae, Chameleonidae, Gekkonidae, Gymnophthalmidae, Lacertidae, Teiidae, and
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TABLE 12.1 Classification, description, and approximate number of genera and species of lizarda Family Sub-family
Description
No. genera (species)
Worm lizards
16 (150)
Shorthead worm lizards
4 (6)
Two-legged worm lizards
1 (3)
Worm lizards
1 (4)
Worm lizards
1 (2)
Worm lizards
1 (1)
Wall lizards
32 (275)
Whiptails and tegus
9 (85)
Microteiids, spectacled lizards
41 (180)
Glass lizards, alligator lizards
9 (65)
American legless lizards
1 (2)
Galliwasps
3 (50)
Helodermatidae
Gila monster, beaded lizard
1 (2)
Xenosauridaed
Knob-scaled lizards
1 (6)
Earless monitors
1 (1)
Monitor lizards
1 (68)
Crocodile lizard
1 (1)
b
Amphisbaenidae b
Trogonophidae b
Bipedidae b
Blanidae
b
Cadeidae
b
Rhineuridae c
Lacertidae c
Teiidae
c
Gymnophthalmidae d
Anguidae
Anniellidaed d
Diploglossidae
d
d
Lanthanotidae d
Varanidae
d
Shinisauridae e
50 (950)
Iguanidae
Corytophaninae
Basilisks, casqueheaded lizards
Crotaphytinae
Collared lizards, leopard lizards
Hoplocercinae
Wood lizards, clubtails
Iguaninae
Iguanas
Leiocephalinae
Curly-tailed lizards
Leiosaurinae
Anole-like lizards
Liolaeminae
Ground lizards
Oplurinae
Madagascar ground lizards
Phrynosomatinae
Earless lizards, tree lizards, side-blotched lizards, spiny lizards, horned lizards
Polychrotinae
Anoles
Tropidurinae
Neotropical ground lizards
Chamaeleonidaee
Chameleons
9 (180)
Agamidaee
Agamas, common lizards, frill-necked lizards, bearded dragons, flying ragons, jacky dragons
55 (440)
Scincidaec
Skinks
134 (1300)
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Hormones and Reproductive Cycles in Lizards
TABLE 12.1 Classification, description, and approximate number of genera and species of lizardadcont’d Family Sub-family c
Xantusiidae
c
Gerrhosauridae c
Cordylidae
f
Sphaerodactylidae f
Gekkonidae
f
Phyllodactylidae f
Eublepharidae
f
Diplodactylidae
f
Carphodactylidae f
Pygopodidae g
Dibamidae
Description
No. genera (species)
Night lizards
3 (30)
Plated lizards
5 (34)
Girdled lizards
3 (55)
Geckos
5 (160)
Geckos
70 (690)
Geckos
8 (100)
Geckos
6 (28)
Geckos
16 (120)
Geckos
2 (30)
Geckos (legless lizards)
7 (40)
Blind lizards
2 (21)
a
Family listings based on molecular phylogenies summarized in Vidal and Hedges (2009). Sub-families listed under Iguanidae from Pianka and Vitt (2003). Genera and species totals mostly are from JCVI/TIGR Reptile Database (2008). b Placed in Scleroglossa (Amphisbaenia, Incertae sedis) based on morphological characters (Pianka & Vitt, 2003). c Placed in Scleroglossa (Scincomorpha) based on morphological characters (Pianka & Vitt, 2003). d Placed in Scleroglossa (Anguimorpha) based on morphological characters (Pianka & Vitt, 2003). e Placed in Iguania based on morphological characters (Pianka & Vitt, 2003). f Placed in Scleroglossa (Gekkota) based on morphological characters (Pianka & Vitt, 2003). g Placed in Scleroglossa (Incertae sedis) based on morphological characters (Pianka & Vitt, 2003).
Xantusiidae) and have arisen as the result of sexual reproduction between two parental species (Godwin & Crews, 2002; Pianka & Vitt, 2003). Lizards are a rich taxonomic group for integrative and comparative studies, in part due to the diversity of lifehistory and reproductive traits outlined above. Adding to their appeal is the fact that both shared and novel hormonal targets and mechanisms have been documented in the process of investigating the endocrine regulation of these traits. Many of the hormones, e.g., the sex steroids testosterone (T) and 17b-estradiol (E2), are identical in structure and importance across amniotes, and they modulate activity of homologous central and peripheral targets via structurally similar or identical receptors; this conservation of endocrine mechanisms is amply supported in Section 2.3. However, it is not the case that ‘one should feel sorry for works on [reptiles], condemned, it seems, to follow in the footsteps of those more preoccupied with rodents’ (Herbert, 1979, p386; cited in Crews & Moore, 2005)! Rather, work on lizards frequently has revealed novel relationships between hormones, their targets and mechanisms, and association (or lack thereof) with specific behavioral and physiological events. For example, the study of lizards in the laboratory and field has helped to reveal that large taxonomic differences exist
in hormone concentrations found in the plasma and associated responsiveness to these hormones; that hormones and the behaviors or processes they regulate do not have to be temporally associated; and that hormones previously thought to be primarily important in one sex often play an equally important role in the opposite sexde.g., the role of progesterone (P4) not only in female but also in male sexual behavior (reviewed in Godwin & Crews, 2002; Crews & Moore, 2005). Indeed, such phenomena first documented in lizards often lead to the identification of previously unrecognized but fundamental regulatory pathways and endocrine relationships in the other amniotes (Godwin & Crews, 2002).
1.1.3. Environmental and conservation considerations A frequently quoted passage from the familiar ‘Golden Guide’ series of field books states the following about amphibians and reptiles: ‘As a group [they] are neither ‘good’ nor ‘bad’, but are interesting and unusual, although of minor importance. If they should all disappear, it would not make much difference one way or the other’ (Zim & Smith, 1953, p. 9). In contrast to this (hopefully) outdated view, it has since been found that amphibians and reptiles
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are integral components of many ecosystems and our study of these groups contributes much to our understanding of organismal biology overall. Unfortunately, there is good evidence that many amphibian and reptile populations, including lizards, are indeed declining, a striking fact considering how little is known about the status of the majority of species to begin with (Pianka & Vitt, 2003). Further, the decline in reptile populations appears to be at least on par with that of the more widely reported amphibian declines (Gibbons et al., 2000; Whitfield et al., 2007). Numerous factors are involved: habitat alteration and loss (e.g., due to deforestation, conversion to agricultural land, development); human population growth; release of pollutants and agrichemicals into the environment; negative impacts of non-native species; disease and parasitism; and overcollecting all have been implicated, depending on the taxon in question (reviewed in Gibbons et al., 2000; Pianka & Vitt, 2003). Additionally, several recent studies have supported the idea that global warming has already or will begin to negatively impact amphibian and reptile populations across a wide variety of geographic locations and habitats (Pounds et al., 2006; Whitfield et al., 2007; Huey et al., 2009; Kearney, Shine, & Porter, 2009; but see Arau´jo, Thuiller, & Pearson, 2006; Rohr, Raffel, Romansic, McCallum, & Hudson, 2008). Efforts to monitor and protect reptilian populations (including lizards) are important simply for the reason that preserving biodiversity is a worthy goal. However, as a strictly practical consideration, continued study and monitoring of these organisms may allow us to use them as indicators of environmental stability and health in the face of anthropogenic disturbance leading to environmental change. In this light, the study of reproductive cycles and their endocrine regulation will become increasingly important as many amphibians and reptiles are particularly sensitive to environmental cues, such as temperature, for determining the onset of breeding or even the resulting sex ratios of offspring in the case of species with TSD. Indeed, effects of warming on the onset of breeding and offspring sex ratios already have been demonstrated in amphibians and reptiles including lizards (e.g., Kusano & Inoue, 2008; Mitchell, Kearney, Nelson, & Porter, 2008; Schwanz & Janzen, 2008; Wapstra et al., 2009). Additionally, amphibians and reptiles are susceptible to pollutants such as endocrine disruptors that interfere with hormonal and developmental processes, thus rendering breeding a particularly affected stage of the life cycle (e.g., Willingham & Crews, 2000; Hayes et al., 2006; Orlando & Guillette, 2007; see also Chapter 14, this volume). Knowledge of ‘baseline’ breeding patterns and their hormonal regulation will allow us to identify when disturbance is present as well as to take advantage of potentially unique opportunities for uncovering novel hormone–reproduction pathways.
Hormones and Reproduction of Vertebrates
1.2. Overview for Remainder of Chapter The remainder of this chapter reviews the reproductive cycles of lizards and their endocrine regulation. First, the seasonality of reproduction, which times breeding events with environmental conditions most amenable to reproductive success, will be discussed. This will be followed by review of the general patterns of reproductive cycles that have been revealed through studies of hormones, physiology, and behavior. The role of hypothalamic, pituitary, and gonadal hormones in regulating reproductive cycles in some detail, as well as our current understanding of how endogenous biological rhythms influence reproduction, will then be considered. Throughout this review, the emphasis will be on information that has become available in the last twenty or so years, and reliance will be placed on several excellent reviews of lizard (or more generally reptilian) reproductive physiology for older information (primarily Crews, 1979; 1980; Duvall, Guillette, & Jones, 1982; Licht, 1984; Whittier & Crews, 1987; Moore & Lindzey, 1992; Whittier & Tokarz, 1992). Finally, some topics closely related to any consideration of hormones and reproductive cycles in lizards are minimally discussed here but treated in considerable detail elsewhere in this volume; e.g., neuroendocrinology of reproduction (Chapter 2), stress and reproduction (Chapter 7), and reproductive behaviors (Chapter 8).
2. REPRODUCTIVE CYCLES 2.1. Seasonality of Reproduction Classic life-history theory describes variation in growth, reproduction, survival, and aging with the goal of understanding how energy is invested to maximize reproductive success (Stearns, 1992; 2000). Critically, energy is limiting, such that energy used for reproduction cannot be used for growth or maintenance and vice versa; thus, tradeoffs can exist between growth, maintenance, and reproduction in that energy allocation that benefits one represents energy loss to the detriment of the others (Stearns, 1989), although not all tissues need be maintained at a constant cost yearround. For example, hibernation can greatly reduce energetic demands both overall and targeted towards specific organ systems (e.g., Naya, Veloso, and Bozinovic (2008) for the Andean ground lizard, Liolaemis bellii). Finally, reproduction is costly; the nature of the costs can vary widely across species and may include increased mortality due to injury or predation as well as direct metabolic costs of reproduction (Shine, 1980; Whittier & Crews, 1987; Crews, 1998). Typically, males expend energy on reproduction via higher activity levels during times of breeding, in an attempt to secure reproductive access to females (e.g., territory maintenance, direct aggressive interactions with
Chapter | 12
Hormones and Reproductive Cycles in Lizards
other males). For example, experiments using a field population of mountain spiny lizards (Sceloporus jarrovi) have demonstrated that T implants given to breeding males lead to increased aggressive activity as well as mortality (Marler & Moore, 1988; 1989). Further work demonstrated that this increased mortality was most likely due to associated high energy demands rather than predation, as food supplementation caused T-implanted males to survive as well as control males (Marler & Moore, 1991). In contrast to males, energy expenditure by females is primarily in the form of allocation to eggs or developing young in the case of viviparous species. For example, whereas male green anoles (Anolis carolinensis) expend approximately 48% of their total metabolizable energy on reproduction, females expend approximately 66% on reproduction including 36% specifically on egg production (Orrell, Congdon, Jenssen, Michener, & Kunz, 2004). Other studies examining energy allocation by females to their eggs have found that it ranges from approximately 10–25% of the energy budget per clutch (reviewed in Whittier & Crews, 1987). Although data are limited for lizards overall, females in general allocate more energy to reproduction than do males (reviewed in Orrell et al., 2004). As discussed above, reproduction is costly and utilizes resources that could be allocated to other necessary functions such as growth and maintenance. Therefore, it is not surprising to find that reproduction typically occurs when the environment is most amenable to offspring survival and when parents are able to take on the physiological demands of reproduction at the least cost to themselves. In many cases, this leads to seasonal reproduction; i.e., a restricted time of year when individuals are breeding. Seasonal reproduction is widespread for lizard species in temperate zones (Fitch, 1970; Licht, 1984; Pianka & Vitt, 2003), in which climate produces well-defined seasons and lizards typically mate in the spring and offspring hatch or are born in the summer. This coordinates reproduction with the time of year providing the necessary sunlight, heat, moisture, and/or availability of food resources necessary for offspring production and survival (Duvall et al., 1982; Whittier & Crews, 1987; Rubenstein & Wikelski, 2003). Although most temperate zone lizards reproduce in the spring, nearly all lizards of the genus Sceloporus that are viviparous (approximately 40% of the genus) breed in the fall and give birth in the late spring or early summer; this timing is presumably associated with increased survival and reproductive opportunities for offspring (reviewed in Guillette & Me´ndez-De La Cruz, 1993; Me´ndez-De La Cruz et al., 1998). Even within-species variation in the seasonality of reproduction can occur, as is the case for the brown anole, Anolis sagrei, in which Florida populations show a strong seasonality in reproduction consistent with temperate zone climate (Lee, Clayton, Eisenstein, & Perez, 1989), but tropical Caribbean (Licht & Gorman, 1970; Sexton &
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Brown, 1977) and Hawaiian (Goldberg, Kraus, & Bursey, 2002) populations show a less pronounced seasonality in which reproductively active individuals can be found throughout the year. The fact that most lizard species in temperate zones have restricted time periods of reproduction does not mean that lizards living in tropical environmentsdin which moisture, day length, and temperature may be amenable to reproduction year-rounddreproduce continuously. In fact, the diversity of reproductive patterns seen in tropical lizards nicely demonstrates that generalizations from temperate zone species do not necessarily apply to lizards in other habitats. For example, among species of tropical lizards in Australia (genera Carlia, Cryptoblepharus, Ctenotus, Morethia, Sphenomorphus, Diporiphora, and Gemmatophora), some breed during the wet season, some during the dry season, and other species breed across both seasons (James & Shine, 1985). Embryonic moisture tolerance and phylogenetic constraint appear to be more likely factors than embryonic thermal tolerance or resource availability contributing to these broad differences in breeding (James and Shine, 1985). Additional factors may complicate the determination of the seasonal nature of breeding, or lack thereof, and the environmental correlates, for a given species. First, males and females may have evolved to respond to different environmental features to cue reproductive activity. For example, in the Mexican gecko Phyllodactylus lanei, the male gonadal cycle cues to photoperiod, but the female gonadal cycle cues to mean monthly temperature, producing overlapping but not identical durations of reproductive activity for males and females (Ramı´rezSandoval, Ramı´rez-Bautista, & Vitt, 2006). Second, although some populations of lizards may be described as breeding continuously, this description does not necessarily apply to the individual nor does it necessarily apply equally across the sexes. This is because no individual within the population breeds year-round, although at any time within the population breeding individuals can be found. Such is the case for many tropical lizard species, e.g., the anoles Anolis acutus (Ruibal, Philibosian, & Adkins, 1972), Anolis limifrons (Sexton, Ortlet, Hathaway, Ballinger, & Licht, 1971), and Anolis opalinus (Jenssen & Nunez, 1994), as well as the gecko Cyrtodactylus malyanus and the flying lizard Draco melanopogon (Inger & Greenberg, 1966), and the parthenogenetic, oviparous whiptail lizard Cnemidophorus nativo (Menezes, Rocha, & Dutra, 2004). In these examples, the proportions of breeders are not equal across all twelve months or between the sexes (when two sexes exist). In general, slightly more breeders are present during the wet than during the dry season, and males are more likely to be gametogenic during the dry season than females, largely due to moisture requirements for eggs (e.g., Jenssen &
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Nunez, 1994). Reproductive patterns in these species can be described as cyclic for the individual but asynchronous for the population (Crews, 1998). Third, some species may breed seasonally, but not necessarily annually, as is the case when breeding occurs at a particular time each year for a population but individualsdtypically femalesdwithin the population do not breed each year, a phenomenon known as multiennial reproduction. This pattern may result when individuals skip a breeding season, or because it takes longer than a year to complete egg production or gestation. Multiennial reproduction occurs in a variety of lizards, including the viviparous New Zealand gecko, Hoplodactylus maculatus (Cree & Guillette, 1995); the oviparous Argentinean gecko, Homonota darwini (Ibargu¨engoytı´a & Casalins, 2007); viviparous ground lizards, Phymaturus patagonicus (Ibargu¨engoytı´a, 2004), Phymaturus antofagastensis (Boretto & Ibargu¨engoytı´a, 2006), Phymaturus punae (Boretto et al., 2007), and Liolaemus pictus (Ibargu¨engoytı´a & Cussac, 1996); and the viviparous blue-tongued skink, Tiliqua nigrolutea (Edwards, Jones, & Wapstra, 2002). The species in the examples above occur on different continents but share the constraint of living in relatively cool environments that result in short activity periods and a suite of lifehistory traits adapted to survival and successful reproduction in such a climate (e.g., Adolph & Porter, 1996). In fact, populations of the geographically widespread H. maculatus that live in warmer regions produce offspring annually rather than biennially (Cree & Guillette, 1995). Fourth, although there are many examples of phenotypic flexibility, it is clear that phylogenetic history of lizard species can be a critical influence, even constraint, on reproductive pattern, such that species sharing a close evolutionary history but living in very different environments may have more similar reproductive patterns than species sharing a common environment but that are more evolutionarily distant. Whether breeding occurs in the wet season, dry season, or both differs based on phylogeny rather than converges based on habitat in Australian and Brazilian tropical skinks, agamids, and teiids (reviewed in Pianka & Vitt, 2003). Thus, environmental cues frequently are critical to successful reproduction, but environmental considerations alone will not explain the full range of reproductive patterns seen in lizards. Lizards from one taxon may reproduce very differently to lizards from a different taxon, even within a common environment.
2.2. Types of Reproductive Cycle Reproductive cycles, regardless of the extent to which they are seasonal, can be described in a variety of ways. Work by
Hormones and Reproduction of Vertebrates
Fitch and colleagues extensively cataloged reproductive cycles in lizards and snakes based upon mode and degree of seasonality of reproduction, size at reproductive maturity, fecundity, and other life history-based assessments (e.g., Fitch, 1970). Licht, Saint Girons, and their respective colleagues were among the first to systematically characterize reproductive cycles based upon relationships between mating behavior, sex steroid production, and gametogenesis (summarized in Licht, 1984; see also Saint Girons, 1963). Although it was acknowledged that not all species fit neatly into such categorizations, two general types of reproductive cycle were recognized: prenuptial and postnuptial cycles (type I and type II reproductive cycles, respectively, as reviewed in Lance (1998)). Prenuptial reproductive cycles are those in which gonadal recrudescence, sex steroid production, and gametogenesis occur in advance of mating, whereas in postnuptial cycles they occur following mating. The potential relationships between mating behavior, sex steroids, and gamete production were further generalized by Crews (1984). Associated reproductive patterns were defined as those for which maximal sex steroid secretion and gametogenesis immediately preceded or coincided with mating behavior, roughly equivalent to the prenuptial cycles discussed above. In contrast, dissociated reproductive patterns were defined as those for which mating behavior is uncoupled from maximal sex steroid secretion and gamete production, similar to postnuptial cycles discussed above. Around the time of publication of Crews (1984), the large majority of work in reproductive endocrinology had been conducted on mammalian species, principally laboratory rodents, which have an associated reproductive pattern. This resulted in what was perhaps a too narrowly restricted view of the relationship between hormones and reproduction. The paper by Crews (1984) has been highly influential, cited over 100 times by research and review articles, and these ideas have stimulated investigation of reproductive patterns in reptiles, amphibians, and other ‘non-traditional’ vertebrate model organisms. With respect to the framework proposed by Crews (1984), the large majority of lizard species studied to date exhibit an associated reproductive pattern, as the available data support a positive relationship between peak gametogenesis, sex steroid production, and mating behavior for both males and females. This is true for virtually all of the widely studied species (Table 12.2). In contrast to lizards, snakes, which together with lizards form the order Squamata, demonstrate more diversity in reproductive pattern with respect to the associated/dissociated framework (Chapter 13, this volume). Although most lizards show a positive relationship between gametogenesis, sex steroids, and mating, there are exceptions. Several species of viviparous sceloporines (e.g., Sceloporus formosus, Sceloporus malachiticus, and
Chapter | 12
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Hormones and Reproductive Cycles in Lizards
TABLE 12.2 Examples of well-studied lizard species in which both males and females exhibit a positive association between gametogenesis, sex steroid production, and mating behavior Common name
Scientific name
Supporting reference(s)
Green anoles
Anolis carolinensis
Reviewed in Crews (1980); Lovern et al. (2004)
Brown anoles
Anolis sagrei
Tokarz (1986; 1987a); Lee et al. (1989); Tokarz (1995); Tokarz et al. (1998; 2002)
Eastern fence lizards
Sceloporus undulatus
Marion (1970); McKinney and Marion (1985); Klukowski and Nelson (1998); Smith and John-Alder (1999); Cox et al. (2005)
Mountain spiny lizards
Sceloporus jarrovi
Moore (1986; 1987); Woodley and Moore (1999a; 1999b)
Tree lizards
Urosaurus ornatus
Moore et al. (1998); French and Moore (2008)
Wall lizards
Podarcis sicula
Ando` et al. (1990); Carnevali et al. (1991); Ando` et al. (1992); Putti et al. (2009)
Common lizards
Lacerta vivipara
Courty and Dufaure (1982); Uller and Olsson (2006); Heulin et al. (2008); Vercken and Clobert (2008)
Little striped whiptail lizards
Cnemidophorus inornatus
Lindzey and Crews (1986); Moore and Crews (1986); Lindzey and Crews (1988); Sakata et al. (2003); Crews (2005)
Garden lizards
Calotes versicolor
Reviewed in Shanbhag (2003)
Leopard geckos
Eublepharis macularius
Tousignant et al. (1995); Rhen et al. (1999; 2000; 2005)
some populations of Sceloporus grammicus and Sceloporus mucronatus) exhibit dramatic sex differences in the onset of gametogenesis, in which testicular recrudescence occurs in the spring but ovarian recrudescence does not begin until the fall and the timing of mating determines which sex is associated/dissociated (Guillette & Me´ndez-De La Cruz, 1993; Me´ndez-De La Cruz et al., 1998). Timing of mating is highly variable among species and even among populations of the same species. In a high elevation population of S. grammicus in Parque Nacional de Zoquiapan in central Mexico, breeding occurs in the early fall, and thus is dissociated from testicular recrudescence in males but associated with the initiation of ovarian recrudescence in females (Guillette & Casas-Andreu, 1980; 1981; Zu´n˜igaVega, Me´ndez-De La Cruz, & Cuellar, 2008). This is in contrast to S. grammicus from Teotihuaca´n, Mexico, in which testicular recrudescence and breeding occur in the summer and fall, at the onset of female ovarian recrudescence (Jime´nez-Cruz, Ramı´rez-Bautista, Marshall, LizanaAvia, & Nieto-Montes De Oca, 2005). In S. mucronatus from Valle de la Cantimplora, Mexico, peak testicular recrudescence and mating occur during the summer, prior to ovarian recrudescence, which does not occur until several months later (Ortega-Le´on, Villagra´n-Santa Cruz, Zu´n˜iga-Vega, Cueva-del Castillo, & Me´ndez-De La Cruz, 2009); this is distinct from many fall-breeding populations elsewhere (Me´ndez-De La Cruz et al., 1994; Villagra´nSanta Cruz, Me´ndez-De La Cruz, & Pa´rra-Gamez, 1994). The examples above demonstrate that gonadal activity and
mating behavior clearly are variable, but hormone analyses have not been performed in these species so endocrine relationships cannot be assessed at this point. More complete data are available for the viviparous Tasmanian skink Niveoscincus ocellatus. This species has a primary breeding period in the austral fall, associated with peak testis volume (and therefore spermatogenesis) and plasma T concentrations in males but dissociated from ovulation, fertilization, and pregnancy in females, which occur the following spring (Jones et al., 1997). Additionally, some animals mate a second time in the austral spring, associated with ovulation in females but when testis volume and T concentrations are basal in males (Jones et al., 1997). A similar reproductive pattern is seen in another viviparous skink found in Tasmania, Niveoscincus metallicus (Swain & Jones, 1994; Jones & Swain, 1996), and likely Niveoscincus coventryi, Pseudemoia entrecasteauxii, and Hemiergis decresiensis as well (Murphy, Hudson, & Shea, (2006); hormones were not measured in this study). Thus, males and females in these skink species possess asynchronous gonadal cycles that are neither strictly associated nor dissociated. Species that possess an associated reproductive pattern nevertheless exhibit flexibility in hormone–behavior relationships. For example, in the fall-breeding mountain spiny lizard, S. jarrovi, castration (and consequent removal of gonadal sex steroids such as T) does not completely abolish sexual and territorial behavior (Moore, 1987), nor does exogenous T administered during the
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summer nonbreeding season increase territorial and reproductive behavior to levels typical of the fall breeding season (Moore & Marler, 1987). In green anoles, A. carolinensis, headbobbing display behavior is critical to communication during courtship and, not surprisingly, display rates are highest during the April–July breeding season, associated with peak T (Jenssen, Greenberg, & Hovde, 1995; Jenssen, Lovern, & Congdon, 2001). We also know that experimental manipulation of T directly affects display behavior; castration diminishes it and replacement T, or T given to gonadally intact juveniles, increases display behavior (Mason & Adkins, 1976; Adkins & Schlesinger, 1979; Winkler & Wade, 1998; Lovern, McNabb, & Jenssen, 2001). Yet, display behavior still is expressed during the nonbreeding season, when reproduction is absent and T concentrations are basal (Jenssen et al., 1995; 1996; 2001). Given our current understanding of relationships between breeding, gonadal state, and sex steroids, what can we say of the utility of using the associated/dissociated framework for understanding reproductive cycles and their physiological correlates? Numerous authors have noted that, while the majority of amphibian and reptile species studied to date fit within this framework, many clearly do not; some have viewed this situation as one in which the associated/dissociated model for describing mating patterns has general utility but numerous exceptions (e.g., Woolley, Sakata, & Crews, 2004), whereas others view the situation as one in which the model has been useful for stimulating interest in mating patterns but generally has led to confusion and obfuscation of potentially revealing mechanisms because they do not fit within the framework (e.g., Schuett, Harlow, Rose, Van Kirk, & Murdoch, 1997; Schuett et al., 2006; Benner & Woodley, 2007). Over the last 25 years it has become clear that reproductive physiology and behavior can be functionally, or at least temporally, uncoupled, that males and females of the same species may express different reproductive patterns, and that studying a diversity of species with different reproductive patterns can lead to a better understanding of the evolutionary determinants of hormone–behavior relationships. These advances in our understanding are at least partly due to the interest in reproductive physiology and behavior stimulated by the associated/ dissociated framework. However, it is possible that such advances have peaked, acting now as a constraint on the recognition of additional reproductive patterns that do not fit neatly within. Phylogeny, environment, and differing strategies of males and females for reproductive success have led to the evolution of even more diversity in gonad–steroid–behavior relationships than previously envisioned by authors who were trying to promote diversity in a world dominated by mammalian studies!
Hormones and Reproduction of Vertebrates
2.3. Endocrine Regulation 2.3.1. Overview of the hypothalamus–pituitary– gonadal (HPG) axis regulation of reproductive cycles The hypothalamus–pituitary–gonadal (HPG) axis is the main regulatory pathway for reproduction in male and female vertebrates, including reptiles (reviewed in Godwin & Crews, 2002; see also Chapters 3 and 4, this volume). Gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates the release of gonadotropins (GTHs) from the anterior pituitary (adenohypophysis). In mammals, these GTHs are follicle-stimulating hormone (FSH), which primarily influences gamete development, and luteinizing hormone (LH), which primarily influences sex steroid production and gamete release (Norris, 2007). In addition to GnRH, gonadotropin-inhibiting hormone (GnIH) has recently been described in birds (Tsutsui et al., 2000) and subsequently this peptide has been identified in mammals, amphibians, and fishes (reviewed in Tsutsui et al., 2007; Tsutsui & Osugi, 2009). Gonadotropininhibiting hormone also has been documented in one nonavian reptile to date: the garden lizard, C. versicolor (Singh, Krishna, Sridaran, & Tsutsui, 2008). Additional research should reveal the extent to which GnIH functions similarly in avian vs. non-avian reptiles. The reproductive hormones of the hypothalamus and adenohypophysis show structural diversity across taxonomic groups. Gonadotropin-releasing hormone comes in multiple forms including salmon (sGnRH), mammalian (mGNRH), chicken-I (cGnRH-I), and chicken-II (cGnRH-II), named for the group/species in which it was first isolated, not for the distribution across groups in which it is found (Norris, 2007). It is generally the case that species possess more than one molecular form of GnRH: cGnRH-II is the most widespread and it has been found in lizards along with cGnRH-I, sGnRH, and several uncharacterized GnRH-like molecules (Powell, Ciarcia, Lance, Millar, & King, 1986; Lescheid et al., 1997; Montaner, Gonzalez, Paz, Affanni, & Somoza, 2000; Ikemoto & Park, 2007). Similarly, FSH and LH can be variable in structure and function across taxa. While both FSH-like and LH-like hormones have been found in turtles and crocodilians, only FSH-like variants are detectable in squamate reptiles (Licht, 1974; Gist, 1998; Norris, 2007). This is in spite of the fact that some squamates possess at least a small proportion of LHproducing cells in the adenohypophysis (e.g., wall lizards, Podarcis sicula campestris (Desantis, Labate, & Corriero, 1998)) and may respond to exogenous treatment with FSH or LH in similar fashion (e.g., male western whiptails, Cnemidophorus tigris, respond to both FSH and LH with increased androgen production in the
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Hormones and Reproductive Cycles in Lizards
testis (Tsui, 1976)). Overall, squamate reptiles appear to be unique among vertebrate taxa in possessing an evolutionarily derived, FSH-like GTH that carries out the functions performed in other vertebrates by both FSH and LH (Licht, 1983; Gist, 1998). Different cell populations in the gonads are responsible for different aspects of reproductive function. Similarly to the mammalian testis, in the reptilian testis Sertoli cells of the seminiferous tubules play a direct role in spermatogenesis whereas Leydig cells outside of the seminiferous tubules are responsible for androgen production (Gist, 1998; Norris, 2007; Chapter 3, this volume). However, unlike what is seen in mammals, in at least some reptiles it is likely that both Leydig and Sertoli cells are capable of significant androgen production and that one or the other may be the primary source of androgen, depending on the time of year (e.g., Lofts, 1987; Mesner, Mahmoud, & Cyrus, 1993; Gist, 1998). This may help to explain how testosterone production in some species is maximal and associated with reproductive behavior at a time of year when testes are regressed and spermatogenically inactive. The Sertoli cells may produce sufficient androgens, along with upregulation of androgen receptor and production of androgen-binding proteins, to maintain spermatogenesis when the testes are spermatogenetically active, whereas during testicular regression it may be the Leydig cells that are producing androgens that result in a rise in plasma androgen concentrations associated with reproductive behavior (Gist, 1998; Benner & Woodley, 2007). In the mammalian ovary, granulosa cells of the follicular wall are responsible for egg maturation, whereas thecal cells, more exterior in the follicular wall, are responsible for the initiation of sex steroid production (Norris, 2007). Thecal cells primarily synthesize androgens, most of which subsequently are converted to estrogens by the granulosa cells; granulosa cells also form the bulk of the corpus luteum following ovulation and produce P4 (Norris, 2007). The reptilian ovary functions very similarly to that described above for mammals, although squamate reptiles possess multiple granulosa cell types, one of which is unique to the group: pyriform cells, which form cytoplasmic connections to the egg in previtellogenic follicles that degenerate once follicular development begins (Chieffi & Pierantoni, 1987; Lance, 1998; Norris, 2007; Chapter 4, this volume). In the discussion that follows, the taxonomic breadth of studies that have yielded insight into the relationship between hormones and reproductive cycles in lizards are reviewed. The focus is on the role that gonadal steroids play in initiating and/or maintaining reproductive conditiondprimarily T, E2, and P4. The androgen dihydrotestosterone (DHT), which results from the biochemical reduction of T, also is considered when possible. In vertebrates, plasma DHT profiles generally mirror those of T but
at lower concentrations (Meisel & Sachs, 1994; Norris, 2007). Details about the role of the central nervous system in the regulation of reproduction in reptiles are considered elsewhere (Godwin & Crews, 2002; Wade, 2005; Beck, O’Bryant, & Wade, 2008; Chapter 2, this volume). In taxa lacking studies on hormones and reproductive cycles, the little (if any) information available on reproductive cycles more generally, including related information on species traits that could be of interest for future studies, is reviewed. The order of presentation generally follows Figure 12.1 and Table 12.1, except that Iguanidae is presented first and in comparatively more detail, because most of our information comes from this family. 2.3.1.1. Iguanidae By far, most of the research on hormones and reproductive cycles has been conducted on lizards in this family, and in particular from the genera Anolis (subfamily Polychrotinae) and Sceloporus (sub-family Phrynosomatinae). In Anolis, details of the relationship between hormones and reproduction generally are well known. Reproduction is seasonal in most anoles, including the most well-studied members of the genusdthe green anole (A. carolinensis) and the brown anole (A. sagrei). Males and females transition from an overwintering to reproductive state during spring, showing increased locomotor and behavioral activity, sex steroid concentrations (T for males; E2 and P4 for females), gonadal recrudescence, and ultimately courtship and copulation (Crews, 1980; Jones, Guillette, Summers, Tokarz, & Crews, 1983; Tokarz, McMann, Seitz, & John-Alder, 1998; Jenssen et al., 2001). Levels of reproductive activity and steroids remain high for the duration of the four-month breeding season, after which they decline rapidly prior to the transition into fall and overwintering (Jenssen et al., 1995; 1996; Tokarz et al., 1998; Lovern & Wade, 2001; Lovern et al., 2001). Plasma T levels in males are maximal during breeding; even GnRH injections failed to increase concentrations further in a field population of breeding male green anoles (Husak et al., 2009). Increasing temperature along with day length are both important environmental inputs for initiating reproduction (Licht, 1967; 1971; 1973). In the laboratory under high temperatures and long days, breeding can be initiated earlier and maintained for longer than environmental conditions permit in the field; however, females and eventually males will undergo gonadal regression and will stop reproductive activity regardless of photothermal regime (Licht 1971; 1973; Lovern et al., 2004). Reproductive activity in anoles is mediated by headbobbing displaysdcommunication signals that are
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species-specific and stereotyped (Jenssen, 1977; Crews, 1980), similar to the acoustic signals of songbirds. Headbobbing displays involve vertical movement of the head associated with extension of a flexible piece of skin under the throat called a dewlap. In A. carolinensis, adult males and females possess a common display type repertoire, but the sexes differ substantially in the use of these displays. Male dewlaps are substantially larger than those of females, and they use them more often (Jenssen, Orrell, & Lovern, 2000). During courtship, both males and females headbob, but male displays almost always include dewlap extension whereas female displays almost always do not (Orrell & Jenssen, 2003). The male continues to approach the female while displaying as courtship progresses. Female receptivity typically is indicated by stationary posture, headbobbing without dewlap extension, and, sometimes, a posture in which the neck is bent forward. The male then grasps the female on the back of the neck as he everts one of his two bilateral and independently controlled hemipenes and intromits. The duration of copulation is variable, lasting anywhere from under five minutes to near sixty minutes (Greenberg & Noble, 1944; Jenssen & Nunez, 1998). Sex differences in reproductive behavior, as described above, are paralleled by sex and seasonal differences in endogenous steroid levels. Plasma T levels are elevated in males during the breeding season as compared to the nonbreeding season (20–25 ng/ml breeding vs. < 10 ng/ml nonbreeding) for both A. carolinensis (Lovern et al., 2001) and A. sagrei (Tokarz et al., 1998). These breeding T concentrations are relatively low when compared to values obtained in some other lizards (200 to > 500% higher; cf. Courty and Dufaure (1982) for L. vivipara; Moore (1986) for S. jarrovi; Ando` et al. (1990) for P. sicula; Klukowski and Nelson (1998) for S. undulatus; and Wack et al. (2008) for Phrynosoma cornutum). Experiments manipulating steroid exposure have demonstrated that T is critical for stimulating male reproduction in A. carolinensis and A. sagrei. Castration or treatment with the antiandrogen cyproterone acetate reduces reproductive activity, and exogenous T prevents or reverses the effect (A. carolinensis (Mason & Adkins, 1976; Crews, Traina, Wetzel, & Muller, 1978; Adkins & Schlesinger, 1979; Winkler & Wade, 1998; Rosen & Wade, 2000); A. sagrei (Tokarz, 1986; 1987a; 1995)). In many vertebrates, T is a prohormone for male sexual behaviors, which are facilitated following conversion of T into E2 by the enzyme aromatase and/or into DHT by the enzyme 5a-reductase (Meisel & Sachs, 1994). In contrast, T conversion into E2 is not necessary for the expression of male sexual behavior. Treatment of male A. carolinensis with the aromatase inhibitor fadrozole has no effect on their sexual behavior (Winkler & Wade, 1998). Similarly, exogenously applied E2 following castration has little or
Hormones and Reproduction of Vertebrates
no effect on male sexual behavior in A. carolinensis (Mason & Adkins, 1976; Crews et al., 1978; Winkler & Wade, 1998) or A. sagrei (Tokarz, 1986). The data on DHT, however, are more complicated. Adkins and Schlesinger (1979) found that DHT stimulates reproductive behavior in castrated male A. carolinensis and Tokarz (1986) found the same for A. sagrei, whereas Crews et al. (1978) found for A. carolinensis that it is ineffective when given alone in stimulating masculine reproductive behavior, but in combination with E2 it can promote behavior in some individuals. Systemic inhibition of 5areductase indicates that T conversion into DHT can promote full expression of masculine sexual behaviors, but DHT alone does not appear to do so (Rosen & Wade, 2000). Males housed under nonbreeding conditions do not exhibit reproductive behavior, even when exogenous androgens are given, although androgen-treated males will extend their dewlaps with increased frequency (O’Bryant & Wade, 2002). Female anoles ovulate a single follicle at a time, generally alternating between the left and right ovary, leading to the production of multiple, single-egg clutches spaced out over the course of the breeding season (Smith et al., 1973). In A. carolinensis, FSH stimulates ovarian growth, vitellogenesis, and follicle recruitment, although even at high doses females still produce just one egg at a time (Jones, Tokarz, LaGreek, & Fitzgerald, 1976; Roth & Jones, 1992). Control of ovulation involves sensory input from the ovaries to the hypothalamus, asymmetric amine production in the hypothalamus (dopamine, serotonin, norepinephrine; initially associated with the ovary containing the smaller yolking follicle but switching to the ovary containing the preovulatory follicle a couple of days prior to ovulation) and neurohormonal output that maintains a pattern of alternation (Desan, Lopez, Austin, & Jones, 1992; Jones, Wapstra, & Swain, 1997). Once ovulated, the egg is fertilized and shelled in the ipsilateral oviduct prior to oviposition (Conner & Crews, 1980). As at any one time it is common to have a corpus luteum present, plasma P4 levels remain consistently high in the breeding season (across multiple ovulatory cycles) as compared to the nonbreeding season (Jones et al., 1983; Jenssen et al., 2001). In contrast, E2 varies during the breeding season across the ovulatory cycle (Crews, 1980; Jones et al., 1983). Although plasma T concentrations are a fraction of those seen in males, they do vary in females as well. Plasma T concentrations are higher in breeding females than in nonbreeding females, and they are higher in breeding females specifically when they are likely to be receptive than at other reproductive cycle stages (Lovern & Wade, 2001; Lovern et al., 2001). Feminine reproductive behavior also is mediated by gonadal steroids. 17b-estradiol clearly is the most potent activator of the behavior for A. carolinensis (Winkler &
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Hormones and Reproductive Cycles in Lizards
Wade, 1998); this effect can be maximized in the presence of P4 (McNicol & Crews, 1979) and inhibited by administration of an estrogen receptor blocker (Tokarz & Crews, 1980). Testosterone also facilitates reproduction in females (Adkins & Schlesinger, 1979), an effect that is at least partially induced by its conversion into E2 (Winkler & Wade, 1998). Interestingly, during the breeding season androgens readily facilitate masculine sexual behaviors in gonadectomized female A. carolinensis, as for males described above, and the reproductive behaviors are qualitatively similar in individuals of both sexes when tested with receptive stimulus females. However, manipulated females consistently display the behaviors at lower frequencies than do males, although results vary across studies as to whether this sex difference in androgen responsiveness reaches statistical significance (Mason & Adkins, 1976; Adkins & Schlesinger, 1979; Winkler & Wade, 1998). Similarly, T implants in juvenile males as well as females can stimulate adult male-typical, breeding season behavior such as dramatically increased display rates, territoriality, and aggressive and courtship-like interactions not otherwise seen in juveniles (Lovern et al., 2001), suggesting high flexibility of the sexes to respond with male-typical reproductive behaviors when the underlying hormonal regime supports them. Sceloporus has been the most widely studied genus of lizards from sub-family Phrynosomatinae. Fence lizards (S. undulatus) have a reproductive cycle very similar to anoles, with spring emergence from overwintering dormancy followed by increased activity, territoriality, breeding, and oviposition completed in the summer before a fall inactive period preceding winter dormancy. The primary difference is that S. undulatus females lay one or two large clutches of eggs during the reproductive season, whereas anoles lay single-egg clutches. In males, plasma T begins to increase in advance of the breeding season and remains high before declining post-breeding (McKinney & Marion, 1985; Klukowski & Nelson, 1998). A similar pattern is seen in the striped plateau lizard (Sceloporus virgatus), a congener that also is oviparous, in which not only T but also DHT, androstenedione (AND), and E2 attained peak mean concentrations during the breeding season in males; P4 was also measured in these males but did not differ from breeding to post-breeding (Abell, 1998). Whether plasma T concentrations are interpreted as maximal (e.g., as for A. carolinensis following GnRH injections (Husak et al., 2009)) or sub-maximal during breeding may depend on the timing of measurement or the population studied. Klukowski and Nelson (1998) studied a field population of Northern fence lizards (S. undulatus hyacinthinus) in Indiana, USA, and found that plasma T concentrations in males that were presented
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with tethered, conspecific intruders for 3–15 minutes were not elevated compared to values obtained from control males when blood samples were collected 30 minutes following intrusion. In contrast, Smith and John-Alder (1999) collected males from a population of Eastern fence lizards (S. undulatus) in New Jersey and housed them in outdoor enclosures for trials. They found that repeated exposure to intruder males over nine consecutive days caused elevated plasma T in territorial males after four days of exposure to intruders, but that this effect was eliminated by day ten. No differences in plasma T were observed at day four or day ten when females were introduced into the enclosures, and no differences were observed in response to either sex in trials conducted during the postbreeding season (Smith & John-Alder, 1999). Plasma T concentrations clearly are higher in these males during the breeding season than during the non-breeding season, but whether they are maximal is not clear. Such information has implications for hypothesized relationships between T, mating system (higher T predicted in polygynous systems), parental care (higher T predicted when paternal care is absent), and male–male aggression (higher T predicted when male–male aggression is common), as described in the Challenge Hypothesis (Wingfield, Hegner, Dufty, & Ball, 1990). Female S. undulatus have received less attention than have males, although females of other sceloporine species have been studied in some detail. In S. undulatus, plasma T concentrations are significantly lower in females than males (Cox, Skelly, Leo, & John-Alder, 2005), but whether plasma T cycles with reproductive state in females is not known. Plasma P4 does cycle; as is common in other species, it is higher when females are gravid than when they are non-reproductive, vitellogenic, or post-reproductive (Masson & Guillette, 1987). In contrast to males, Abell (1998) found that female S. virgatus did not differ in plasma T, DHT, AND, or E2 concentrations from breeding to postbreeding, although there was a marginally significant reduction in plasma E2 and, like males, there was no difference in P4. Mean values of E2 were higher in females than in males; P4 was comparable; and T, DHT, and AND were higher in males (Abell, 1998). Another study of S. virgatus females (Weiss, Jennings, & Moore, 2002) examined endocrine profiles across the reproductive cycle (rather than from breeding to post-breeding) and found a different pattern. In this study, conducted on females from southeastern Arizona, similar to Abell (1998), plasma P4 and to a lesser extent T increased following vitellogenesis and then declined following oviposition, whereas plasma E2 was highest during vitellogenesis and declined steadily through the gravid and then post-oviposition stages (Weiss et al., 2002). Consistent with these results, in another oviparous congener, the Mexican boulder spiny lizard
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(Sceloporus pyrocephalus), both plasma E2 and T concentrations were found to be higher during vitellogenesis than when females were gravid (Calisi & Hews, 2007). Overall, plasma T was much lower in female S. pyrocephalus than in males measured in the same study (plasma E2 was not measured in males (Calisi & Hews, 2007)). Sexually dimorphic, dorsal, and especially ventral color patterns are important components of communication displays during the breeding season in many sceloporine lizards, and the hormonal regulation of this reproductive trait has received considerable attention. In S. undulatus, this sex difference is dependent on plasma T and can be diminished in males by castration and restored in males, as well as masculinized in females, by exogenous T (Rand, 1992; Cox et al., 2005). As discussed in Section 1.1.2, the genus Sceloporus is comprised of both oviparous and viviparous species, and viviparity has evolved independently multiple times within this genus alone (Pianka & Vitt, 2003; see also Chapter 9, this volume). S. jarrovi, the mountain spiny lizard, is a viviparous species that has been studied in some detail. This species breeds in the fall, after which gonads regress in both sexes; females are pregnant over winter and spring and subsequently give birth in early summer, prior to initiating gonadal recrudescence and mating behavior once again (Ruby, 1978; Moore, 1986). Aside from the seasonal shift in timing of reproduction, hormone–reproduction patterns in S. jarrovi are similar to those found in oviparious congeners and other spring-/summer-breeding lizards. Males have peak plasma T concentrations in the fall breeding season compared to other times of year, but no changes in plasma P4 (detectable throughout the year) or plasma E2 (not detectable in any sample) (Moore, 1986). Consistent with a role for T in maintaining reproductive behavior, castration diminishes behaviors related to both territory maintenance and courtship (Moore, 1987). Females, like males, show peak plasma T concentrations in the fall breeding season but, unlike males, plasma E2 also peaks at this time (Moore, 1986; Woodley & Moore, 1999a). Plasma P4 concentrations in females are higher during pregnancy than at other times of the year (Moore, 1986). Plasma T concentrations are approximately an order of magnitude higher in males than in females; the reverse is true for plasma E2, and plasma P4 concentrations are approximately equivalent between the sexes (Moore, 1986). In one of the few studies to experimentally manipulate sex steroids in a field population of females, Woodley and Moore (1999b) demonstrated a likely role for plasma E2 and possibly to a lesser extent plasma T in promoting female territorial aggression. In females, as in males of this species, territories are set up in the summer prior to breeding and maintained through the breeding season, and thus are important for successful reproduction (Ruby, 1978; Woodley & Moore, 1999b).
Hormones and Reproduction of Vertebrates
Other Phrynosomatine lizards in the family Iguanidae for which there is information on hormones and reproduction are tree lizards (Urosaurus), sideblotched lizards (Uta), keeled earless lizards (Holbrookia propinqua), and horned lizards (Phrynosoma). U. ornatus and Uta stansburiana have been particularly useful models for examining the relationships between hormones and environment, and specifically the alternative reproductive phenotypes expressed by males of these species (U. ornatus (Moore, Hews, & Knapp, 1998; Knapp, Hews, Thompson, Ray, & Moore, 2003; and reviewed in Crews & Moore, 2005); U. stansburiana (Sinervo, Miles, Frankino, Klukowski, & DeNardo, 2000; Mills et al., 2008)). Collectively this work has revealed, not surprisingly, that hormones such as T are important for reproduction and associated behaviors. More intriguingly, this work has revealed that the ways in which hormones such as T influence reproduction can be extremely variable. For example, plasma T concentrations may differ between adult male morphs (Uta) or it may not (Urosaurus), and morphs may differ in their T (and other hormone) responses to social and environmental stimuli (Uta and Urosaurus). Work with keeled earless lizards, H. propinqua, has focused on females and the relationship between reproductive coloration, hormones, and behavior in females (Cooper & Crews, 1987; 1988). In this species, the development of bright coloration signals non-receptivity; bright females reject male courtship whereas plain females do not. Females with bright reproductive coloration have higher plasma concentrations of P4, androgens, and E2 than do plainly colored females, and these steroids are associated with the latter stages of follicle growth, ovulation, and shelling of eggs (Cooper & Crews, 1988). Further, treatment with exogenous P4 or T stimulates this color development along with the associated aggressive rejection behavior of females towards males; treatment with E2 partially stimulates color development but does not produce rejection behavior (Cooper & Crews, 1987). In comparison to females, male H. propinqua have much higher plasma T concentrations and plasma P4 concentrations that are roughly equivalent to plain females but substantially lower than those for bright females (Cooper & Crews, 1988). Plasma steroids have been measured across the reproductive season for both males and females in freeranging Texas horned lizards, P. cornutum (Wack et al., 2008). Males have higher plasma T as well as, intriguingly, plasma E2 concentrations during the breeding than nonbreeding season but show no difference in plasma P4 across seasons. Similarly, females have higher plasma E2 and marginally significantly higher plasma T during the breeding season prior to gravidity, after which time
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Hormones and Reproductive Cycles in Lizards
concentrations of both steroids decline. Male plasma T is over an order of magnitude higher than female plasma T; plasma E2 and P4 actually are comparable between the sexes. The results for plasma T in males and plasma E2 in females parallel those documented for the San Diego coast horned lizard, P. coronatum blainvillei (plasma T was not measured in females, plasma E2 was not measured in males, and plasma P4 was not measured in either sex (Alberts et al., 2004)). In collared lizards (Crotaphytus collaris), sub-family Crotaphytinae, previous research has investigated the role of hormones in affecting reproductive coloration in females (Cooper & Ferguson, 1972; 1973) and the association between breeding season territoriality and hormones in males (Baird & Hews, 2007). Similar to work with H. propinqua discussed above, treatment with exogenous T, P, or especially P4 following E2 priming for several days stimulates development of reproductive coloration in females (Cooper & Ferguson, 1972; 1973). Plasma P4 concentration is highest when females are gravid compared to non-reproductive, vitellogenic, or post-gravid (Masson & Guillette, 1987). Although likely, it is unknown whether endogenous plasma T or E2 profiles peak at times that support a natural role for these hormones in mediating changes in color and behavior across the reproductive cycle in females. Unlike coloration in female H. propinqua, in C. collaris coloration appears to signal receptivity rather than non-receptivity (Baird, 2004). In male collared lizards, first-year, non-territorial subordinates have higher concentrations of plasma T than do older, dominant territory holders (Baird & Hews, 2007). Plasma concentrations of DHT are lower than those for T and not different between subordinates and territory holders. In sub-family Iguaninae, a variety of hormone– reproduction studies have been performed, primarily using the Gala´pagos marine iguana, Amblyrhynchus cristatus. In this lekking species, some males aggressively defend mating areas and court females with headbobbing displays whereas other males do not defend mating areas but rather attempt to intercept females for mating opportunities using a satellite or sneaker-male tactic (Vitousek, Rubenstein, Nelson, & Wikelski, 2008). Females can be highly aggressive towards males during the mating period after they have copulated, signaling non-receptivity, and highly aggressive towards other females during the nesting period in competition for limited nesting sites (Rubenstein & Wikelski, 2005). In males, those that defend mating areas have higher plasma T concentrations than do either satellite or sneaker males (Wikelski, Steiger, Gall, & Nelson, 2005), there is a positive relationship between headbobbing frequency and copulations, and males that headbob more also have higher plasma T concentrations (Vitousek et al., 2008). Interestingly, Vitousek et al. (2008) demonstrated annual variation within males such that hotshots in the first
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year of the study (i.e., those with high plasma T concentrations and a high number of copulations) were less likely to be hotshots the following year, having lower plasma T concentrations and a lower number of copulations. In female A. cristatus, plasma T and E2 are significantly higher during the mating period than during the nesting period, and plasma P4 declines across the mating period but then increases sharply and again declines sharply across nesting, reaching minimal concentrations following oviposition (Rubenstein & Wikelski, 2005). Plasma T concentrations in males are over an order of magnitude higher than those for females (cf. Rubenstein & Wikelski, 2005; Wikelski et al., 2005; Vitousek et al., 2008). 2.3.1.2. Amphisbaenidae, Trogonophidae, Bipedidae, Blanidae, Cadeidae, and Rhineuridae These six families of lizard are commonly known as worm lizards and comprise approximately 170 species. Worm lizards are fossorial and, in association with this environment, they possess reduced vision, elongated bodies, and all but Bipedidae have lost their limbs (Bipedidae retains front limbs) (Pianka & Vitt, 2003). Most species studied thus far are oviparous (Pianka & Vitt, 2003). No endocrine studies have been conducted to date, although some work on reproductive ecology and behavior has been done (e.g., in Anops kingii (Vega, 2001) and Monopeltis anchietae and Zygaspis quadrifrons (Webb, Shine, Branch, & Harlow, 2000)). In the Iberian worm lizard, Blanus cinereus, it has been shown that chemical communication allows individuals to discriminate between males and females and that precloacal secretions of both sexes contain numerous lipophilic compounds, including steroids (Cooper, Lo´pez, & Salvador, 1994; Lo´pez & Martı´n, 2005). 2.3.1.3. Lacertidae A considerable amount of work has been done with lacertids, in particular wall lizards of the genus Podarcis and the common lizard, L. vivipara. Information on hormones and the reproductive cycle also is available for Psammodromus algirus, an oviparous lacertid common throughout the Mediterranean. The Italian wall lizard, P. sicula, is an oviparous seasonal breeder with springtime emergence from overwintering sites followed by courtship and mating in late spring and early summer. Consistent with this type of reproductive cycle, in males plasma T concentrations are elevated when courtship and breeding are occurring but not at other times of the year; plasma DHT shows a similar, but lower amplitude, seasonal profile (Ando` et al., 1990; 1992; Panno et al., 1992). Plasma T shows the same pattern in P. algirus (Dı´az, Alonso-Go´mez, & Delgado, 1994). Interestingly, plasma E2 and P4 in males are elevated postbreeding in P. sicula, suggesting a role for these steroids in
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males in initiating testicular regression and the onset of the post-reproductive period during the late summer (Ando` et al., 1990; 1992; Panno et al., 1992). In support of this relationship, experimental injection of the aromatase inhibitor fadrozole during the fall nonreproductive period causes an increase in plasma T, a decrease in plasma E2, and a resumption of spermatogenesis (Cardone, Comitato, Bellini, & Angelini, 2002). However, the generality of increased plasma E2 and P4 concentrations in males following the breeding season is difficult to evaluate because so few studies measure E2 or P4 in males. Recent work in P. sicula additionally has begun to explore the potential role of leptin in the regulation of reproductive cycles in males; thus far it appears that, similar to work in mammals, leptin can have a direct effect on the reproductive axis at multiple locations including the gonads. In P. sicula, leptin causes a delay in summer regression of the testes (Putti, Varricchio, Gay, Elena, & Paolucci, 2009). Less work, both descriptively and experimentally, has been carried out in females. However, work thus far is consistent with what is seen in other oviparous females; plasma E2 peaks prior to ovulation and plasma P4 follows closely behind, peaking after ovulation (Carnevali et al., 1991; Ciarcia, Paolucci, & Di Fiore, 1993). Plasma E2 and P4 concentrations show the same pattern in P. algirus (Dı´az et al., 1994). Most, but not all, populations of the common lizard, L. vivipara, are viviparous (Uller & Olsson, 2006; Heulin, Garnier, Surget-Groba, & Deunff, 2008). Breeding is seasonal, occurring in late spring/early summer, with birth of young occurring towards the end of summer. In males, hormone relationships to the reproductive cycle are the same as for oviparous lacertids; males show peak plasma T and DHT associated with breeding, and the DHT profile mirrors that of T but at a much lower amplitude (Courty & Dufaure, 1979; 1980; 1982). The values seen for plasma Tdpeaking at 200–500 ng/mldmay be the highest for any lizard measured thus far. In females, plasma E2 is higher during the latter stages of vitellogenesis than it is after ovulation, and this is true of females of both viviparous and oviparous populations (Heulin et al., 2008). 2.3.1.4. Teiidae Whiptail lizards of the genus Cnemidophorus have been studied extensively and have yielded tremendous insights into hormone–reproduction relationships. This genus has multiple parthenogenetic species, but the best studied by far is the desert-grassland whiptail, Cnemidophorus uniparens, a triploid, all-female species that arose from the hybridization of the little striped whiptail, C. inornatus, and, most likely, the rusty-rumped whiptail, Cnemidophorus burti (reviewed in Godwin & Crews, 2002; Crews & Moore, 2005; Dias & Crews, 2008). All species in this genus are
Hormones and Reproduction of Vertebrates
oviparous, regardless of whether reproduction is sexual or parthenogenetic. In the sexually reproducing C. inornatus, males have high plasma T and especially high DHT concentrations when reproductively active and detectable but not significantly different plasma P4 concentrations across the breeding season; plasma E2 is not detected (Moore & Crews, 1986). Castration diminishes male courtship and copulatory behavior, and implants of T, DHT, and P4 (alone or synergistically with T) restore these behaviors in previously castrated males (Lindzey & Crews, 1986; Sakata, Woolley, Gupta, & Crews, 2003). In females, plasma E2 is highest before ovulation, and plasma P4 is highest after ovulation and before oviposition; plasma androgens are not detectable (Moore & Crews, 1986). In both males and females, the presence of the opposite sex is important for stimulating gonadal recrudescence (Lindzey & Crews, 1988). In the parthenogenetic C. uniparens, there is no opposite sex to stimulate behavior; rather, individuals alternate in expressing male-typical mounting behavior and female-typical receptive behavior of the ancestor sexual species. As for C. inornatus, plasma E2 concentration is higher prior to ovulation and plasma P4 concentration is higher following ovulation in C. uniparens (Moore, Whittier, & Crews, 1985). Thus, this endocrine profile is conserved, but the response to it has changed. Prior to ovulation (high E2, low P4), individuals are more likely to express female-typical receptive behavior during pseudosexual interactions; following ovulation and until oviposition (low E2, high P4), individuals are more likely to express male-typical mounting behavior during pseudosexual interactions (Moore, Whittier, Billy, & Crews 1985). As seen for C. inornatus, experimentally elevated plasma T or P4 (alone or in synergism with T) promotes male-typical mounting behavior in C. uniparens as well (Sakata et al., 2003). This sensitivity to T exists in spite of the fact that plasma T is undetectable throughout the breeding season in C. uniparens (Crews, Grassman, & Lindzey, 1986). Further, although male and female C. inornatus express sex differences in brain regions associated with reproduction and typical of other vertebrates (males have a larger anterior hypothalamus-preoptic area (AH-POA) and females have a larger ventromedial region of the hypothalamus (VMH)), in the all-female C. uniparens, both brain regions are comparable in size to those of female C. inornatus, regardless of reproductive state (Wade & Crews, 1991). Thus, like endocrine profiles, size relationships in brain regions associated with reproduction are conserved. Recent work by Dias and Crews (2008) suggests that the brain neurotransmitter serotonin links hormones and behavior in C. uniparens, as in many other taxa, by playing an inhibitory role in the expression of reproductive behavior. Serotonin presence in the AH-POA suppresses mounting, whereas in the VMH it suppresses receptivity.
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Hormones and Reproductive Cycles in Lizards
2.3.1.5. Gymnophthalmidae Also known as microteiids, this family is comprised of 41 genera and approximately 180 species, mostly in the tropical latitudes. These lizards are comparatively small, as their name suggests, and they often live together in community assemblages of up to ten species (Pianka & Vitt, 2003). As for Cnemidophorus in family Teiidae, some genera in family Gymnopthalmidae are parthenogenetic, including the best-known species of this group, Gymnophthalmus underwoodi (Hardy, Cole, & Townsend, 1989). No information is available on hormones and reproductive cycles for lizards in this family.
2.3.1.6. Anguidae, Anniellidae, and Diploglossidae These families are comprised of 13 genera and approximately 117 species of lizard generally known as glass lizards, alligator lizards, and legless lizards. Some of these lizards are oviparous and some viviparous; some are terrestrial and some fossorial; some are legless or possess very reduced limbs; and some are crocodilian in appearance (although not size), largely due to scalation (Pianka & Vitt, 2003). Species of several genera including Anguis, Ophisaurus, and Diploglossus are known to be comparatively very late-maturing and long-lived for lizards (over 40 years for one captive male in the genus Anguis); this is probably the case for many more (Pianka & Vitt, 2003). Few studies exist on the reproductive biology of these taxa, in part because of the secretive or cryptic nature of many of the species. Reproduction is seasonal in the species studied thus far (e.g., the alligator lizard, Gerrhonotus coeruleus principis (Vitt, 1973) and the glass snake, Ophiodes fragilis (Pizzatto, 2005)); in the California legless lizard, Aniella pulchra, breeding is seasonal but it appears that not all females breed annually (Goldberg & Miller, 1985). In one study examining hormones and reproduction, plasma P4 concentrations were positively associated with corpus luteum development, peaking during early pregnancy and declining slowly for the remainder of gestation in the viviparous imbricate alligator lizard Barisia imbricata imbricata (Martı´nez-Torres, Herna´ndez-Caballero, Alvarez-Rodriguez, Luis-Dı´az, & Ortı´z-Lo´pez, 2003). 2.3.1.7. Helodermatidae This family is comprised of one genus and two species, the gila monster (Heloderma suspectum) and the Mexican beaded lizard (Heloderma horridum). These are large (> 300 mm total length) venomous lizards and until recently were thought to be the only extant venomous lizards (Pianka & Vitt, 2003); recent work on Komodo dragons (Varanus komodoensis; Varanidae) suggests that this species is venomous as well (Fry et al., 2009). In spite of their striking appearance and ‘charismatic species’ status, surprisingly
little is known about reproductive cycles in these species. Gila monsters are oviparous, showing summer gonadal recrudescence and oviductal eggs in the late summer, with oviposition apparently occurring in the fall, after which eggs incubate overwinter and hatch in the spring of the following year; Mexican beaded lizards also are oviparous but the timing of reproductive activity appears shifted to the fall and winter (Goldberg & Lowe, 1997). Hormone data related to reproduction are not available. 2.3.1.8. Xenosauridae This family is comprised of one genus (Xenosaurus) and six species of knob-scaled lizard found in Mexico and Guatemala. All are viviparous and give birth in the summer (Pianka & Vitt, 2003; Zamora-Abrego, Zu´n˜iga-Vega, & Nieto-Montes De Oca, 2007); there is a possibility that neonates receive maternal care following birth through an extended time period of association (Lemos-Espinal, Smith, & Ballinger, 1997). Some populations are distributed over a wide range of environments and show potential local adaptation in reproductive traits, for example a population of Xenosaurus platyceps invests more in relative litter mass in a high altitude, temperate forest than in a low altitude, tropical forest (Rojas-Gonza´lez, Zu´n˜iga-Vega, & Lemos-Espinal, 2008). Hormone data related to reproduction are not available. 2.3.1.9. Lanthanotidae This family, the sister taxon to Varanidae, is thought to be comprised of a single species: the earless monitor, Lanthanotus borneensis, found only in Borneo. Virtually nothing is known about this rarely encountered, nocturnal, fossorial species (Maisano, Bell, Bauthier, & Rowe, 2002; Pianka & Vitt, 2003). No data on hormones or reproduction are available. 2.3.1.10. Varanidae This family is comprised of one genus (Varanus) and nearly 70 species of monitor lizard. Compared to Lanthanotidae, much more is known about varanid lizards, although endocrine data generally are lacking. Varanids are longlived; geographically widespread across Africa, Asia, and Australia; exceptionally diverse in habitats (desert to swamp, arboreal to terrestrial to aquatic); and highly variable in adult body size (e.g., the pygmy monitor, Varanus brevicaudus, weighs less than 20 g as an adult whereas the Komodo dragon, V. komodoensis, can weigh 150 kg) (Pianka & Vitt, 2003). All are oviparous, and at least two species appear to show facultative parthenogenesis: the Argus monitor lizard, Varanus panoptes (Lenk, Eidenmueller, Staudter, Wicker, & Wink, 2005) and the Komodo dragon, V. komodoensis (Watts et al., 2006). Clutch size can be highly variable within and between species (e.g., de
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Buffre´nil & Rimblot-Baly, 1999), but generally reproduction occurs during warm, dry months, oviposition occurs shortly thereafter, and incubation duration (~90–270 days across species) times hatching to coincide with the wet season and/or prey abundance (Jacob & Ramaswami, 1976; Wikramanayake & Dryden, 1988; Phillips & Millar, 1998). Plasma T and total estrogens have been measured in the white-throated savannah monitor, Varanus albigularis (Phillips & Millar, 1998). Plasma T concentration is higher in males than in females, but it peaks in both sexes, associated with mating. Plasma estrogens also peak at this time in females (they were not measured in males). 2.3.1.11. Shinisauridae This family is comprised of one species found in southern China, the crocodile lizard, Shinisaurus crocodilurus. This species is long-lived and viviparous, like Xenosauridae, the knob-scaled lizards with which S. crocodilurus was historically grouped prior to elevation to independent family status (Pianka & Vitt, 2003). S. crocodilurus is threatened with extinction due to habitat loss and degradation, although captive breeding and husbandry is widely carried out due to the popularity of this species in zoos and in the pet trade (Pianka & Vitt, 2003). No data are available on hormones and reproductive cycles for this species. 2.3.1.12. Chamaeleonidae In spite of the charismatic nature of chameleons and the intense popularity of several species in zoos and the pet trade, nothing yet is known about hormonal influences on reproduction in this group of about 180 species. The opportunities for interesting hormone–reproduction relationships seem compelling given that, across species, there is considerable diversity in the degree of sexual dimorphism and reproductive lifespan (e.g., Pianka & Vitt, 2003; Karsten et al., 2008). Further, in recent years reproductive behavior has been well-studied for both males and females of several species (e.g., the Madagascan chameleons Furcifer labordi and Furcifer verrucosus (Karsten, Andriamandimbiarisoa, Fox, & Raxworthy, 2009) and the veiled chameleons, Chamaeleo calyptratus (Kelso & Verrell, 2002)), revealing dramatic and in some cases sex-atypical behaviors for lizards such as high sexual aggression in females (dwarf chameleons, Bradypodion pumilum (StuartFox & Whiting, 2005)) and active mate guarding by males of recently mated females in a serially polygynous system (common chameleons, Chameleo chamaeleon (Cuadrado, 2000; 2001)). Most chameleons are oviparous although a few give live birth; across oviparous species, clutch sizes can vary tremendously, from 2–4 eggs in smaller species such as leaf chameleons of the genus Brookesia to 40 or more in species such as C. calyptratus (Pianka & Vitt, 2003;
Hormones and Reproduction of Vertebrates
Andrews, 2008). Finally, our understanding of how to maintain chameleons successfully in captivity has improved considerably (e.g., Ferguson et al., 1996; Ferguson, Gehrmann, Chen, Dierenfeld, & Holick, 2002; Andrews, 2008; Karsten, Ferguson, Chen, & Holick, 2009) making them increasingly amenable to laboratory study as well. 2.3.1.13. Agamidae Agamids form the sister group to Chamaeleonidae and are a diverse group of lizards classified into 55 genera with over 400 species. Like Chamaeleonidae, most agamid lizards are oviparous but a few are viviparous (Pianka & Vitt, 2003). Unlike in the case of Chamaeleonidae, there has been considerable work on hormones and reproduction in one agamid: the tropical or oriental garden lizard, C. versicolor. This lizard is widespread throughout India and southern Asia and is an oviparous, multiclutching, seasonal breeder (Shanbhag, Radder, & Saidapur, 2000; Shanbhag, 2003). In males, plasma T is highest during the breeding season, correlated with testis mass and reproductive behavior (Radder, Shanbhag, & Saidapur, 2001). In females, plasma E2 and P4 are higher during the breeding season than during the nonbreeding season, and within the breeding season E2 and P4 are inversely related (Radder et al., 2001). Plasma E2 peaks before ovulation and plasma P4 peaks after ovulation. Interestingly, prior to oviposition, when vitellogenesis is initiated for a subsequent clutch of eggs, plasma E2 again is elevated and plasma P4 concentrations decline, demonstrating that P4 is not necessary for oviductal egg retention under normal conditions (Radder et al., 2001). However, under unfavorable environmental conditions, a secondary rise in plasma P4 of adrenal rather than luteal origin appears to promote a cessation of vitellogenesis and extended oviductal egg retention (Shanbhag, Radder, & Saidapur, 2001). Recent work in female C. versicolor has demonstrated the presence of GnRH, GnRH receptor, GnIH, and bradykinin in ovarian tissue (Singh et al., 2008). Although not experimentally demonstrated at this point, Singh et al. (2008) provide support for the hypothesis that local ovarian production of GnRH is stimulated by bradykinin and influences follicle and oocyte maturation through interaction with the GnRH receptor. Such a stimulatory role for bradykinin is consistent with work from studies on mammalian reproduction (reviewed in Singh et al., 2008). In their study, locally produced GnIH was, interestingly, positively correlated with GnRH but negatively correlated with GnRH receptor, suggesting that receptor but not hormone production is inhibited by GnIH. Hormone relationships with reproduction also have been studied in bearded dragons, Pogona barbata (Amey & Whittier, 2000); jacky dragons, Amphibolurus muricatus (Watt, Forster, & Joss, 2003); and mountain lizards,
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Hormones and Reproductive Cycles in Lizards
Japalura mitsukurii formosensis (Chen, Lin, Yu, & Cheng, 1987). In males of each species, plasma T is positively associated with reproduction. In male bearded dragons, plasma P4 also was measured and was consistently detectable but did not vary with season. Surprisingly, in female bearded dragons plasma E2 may be unimportant in the reproductive cycle, as it was low or non-detectable in females across all reproductive states (nonreproductive, vitellogenic, gravid) and all months of the year. Plasma P4 is higher during breeding than nonbreeding, with peak levels during vitellogenesis that slowly decline following ovulation. This may indicate an earlier peak in plasma P4 than is typical for other lizards, in which plasma P4 is highest after ovulation; such a pattern could be important, particularly if E2 is not necessary to stimulate vitellogenesis (Amey & Whittier, 2000).
2.3.1.14. Scincidae This is the largest lizard family, with over 100 genera and 1300 species. Skinks form a cosmopolitan group, occurring on all continents except Antarctica as well as on many oceanic islands. Coupled with their diversity of daily activity rhythms (diurnal and nocturnal), habitats (desert to rainforest; terrestrial, arboreal, fossorial, semi-aquatic niches), diet (insectivory/carnivory to herbivory), and body size and morphology (under 4 cm snout-to-vent length for adults of the genus Menetia to over 40 cm snout-to-vent length for the Solomon Island skink, Corucia zebrata, along with varying degrees of limb reduction or even absence), it is difficult to stereotype a ‘typical’ skink (Pianka & Vitt, 2003). Reproductive mode and ecology have been studied in some detail in Scincidae, revealing oviparous and viviparous species, some of which breed annually and some of which breed multiennially, and some of which time breeding with the wet season, some with the dry season, and some can initiate breeding year-round (e.g., James & Shine, 1985; Edwards et al., 2002). Among the viviparous species, most show complete lecithotrophy, in which eggs contain all the nutrients that will be provided to offspring prior to live birth, whereas others such as the Brazilian skink Mabuya heathi have highly developed placentae through which nutrients are transferred from mother to embryo (Blackburn, Vitt, & Beauchat, 1984). Populations of the same species can even vary in reproductive mode from oviparity to viviparity, as is the case for some Australian skinks (Pianka & Vitt, 2003). Although skink reproduction has been studied in several species, their reproductive endocrinology has been particularly under-described when the diversity of skinks is considered. Hormones and reproduction have been studied only in species of Niveoscincus and Tiliqua in
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detail, with limited information available for Eumeces and Oligosoma as well. As discussed in Section 2.2, Tasmanian skinks (N. ocellatus) are viviparous and have a primary breeding period in the austral fall, when plasma T of males is highest and is associated with peak testis volume and spermatogenesis but dissociated from ovulation, fertilization, and pregnancy in females, which occur the following spring (Jones et al., 1997). Plasma T generally is low throughout the rest of the year, including the austral spring when some individuals mate again (Jones et al., 1997). In females, similarly to other studies reviewed above, plasma E2 is highest during vitellogenesis whereas plasma P4 is highest during gestation, although both steroids are detectable throughout the reproductive cycle (Jones et al., 1997). A similar reproductive pattern is seen in another viviparous Tasmanian skink, N. metallicus (Swain & Jones, 1994; Jones & Swain, 1996), including elevated plasma P4 concentrations during gestation but not sooner (Bennett & Jones, 2002). In Niveoscincus microlepidotus, reproduction is biennial, largely due to a gestation period lasting a year or more (Girling, Jones, & Swain, 2002). In this species, plasma P4 is high during gestation, but it also shows a significant peak upon emergence from hibernation prior to ovulation, earlier in the reproductive cycle than is seen in N. ocellatus and N. metallicus and most other vertebrate females. Girling et al. (2002) suggest that high P4 at emergence could be a mechanism for preventing additional follicle development during a gestation-only year. Plasma E2 is high during vitellogenesis, as would be expected, but a secondary peak is seen during the latter stages of gestation and several months before vitellogenesis is initiated (Girling et al., 2002). Although the reasons presently are not known, it is clear that reproductive timing and its relationship to hormone profiles can be variable, even within a genus. In the case of N. microlepidotus, the hormone profiles for plasma P4 and E2 suggest that each hormone shows a similar pattern annually but with different amplitudes in alternating years in response to, or to allow for, an extended reproductive cycle. Plasma steroids also have been measured across the reproductive cycle of males and females of another viviparous species: blue-tongued skinks (T. nigrolutea). In males, plasma T as well as E2 and to a lesser extent P4 are higher during the breeding than nonbreeding season (Edwards & Jones, 2001a). This is similar to the androgen profiles found across the annual cycle in the congener Tiliqua rugosa (Bourne, Taylor, & Watson, 1986). Elevated E2 and/or P4 for males have been reported in a few other studies, as discussed above (e.g., S. virgatus, P. cornutum) as well as the reverse case in which E2 and P4 are elevated in post-breeding males (P. sicula); either these endocrine profiles are uncommon for males or, more likely, especially for E2, not measured often enough for us to know how
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common they may be. Female blue-tongued skinks are multiennial such that, during any breeding season, some females are reproductively active and some are quiescent (Edwards & Jones, 2001b). Reproductively active females show a rise in plasma E2 and T leading up to and during the mating and ovulatory periods, followed by a decline after ovulation; plasma P4 increases around the time of mating and ovulation and peaks during gestation (Edwards & Jones, 2001b). Interestingly, reproductively quiescent females show a similar but muted pattern for plasma E2 and T, while plasma P4 in quiescent females remains basal throughout the year (Edwards & Jones, 2001b). This suggests that factors other than reproductive competence (e.g., environmental factors) contribute to annual steroid profiles and/or that reproductively quiescent females retain some possibility to reproduce if conditions change during a quiescent year. Plasma P4 also has been measured in females of the oviparous great plains skink, Eumeces obsoletus (Masson & Guillette, 1987) and females of the viviparous McCann’s skink, Oligosoma maccanni (Holmes & Cree, 2006). In both of these species, plasma P4 is highest during gravidity/gestation. 2.3.1.15. Xantusiidae Lizards in this family commonly are known as night lizards, and they are found in North and Central America and Cuba. They are small in body size (typically 0.5 cm snout-to-vent length or less), long-lived (around 10 years for some species), and viviparous (Pianka & Vitt, 2003). Although known as night lizards, they can be active during the day as well as night, but tend to be difficult to detect, moving about in rock crevices and under leaf litter and other organic ground matter (Pianka & Vitt, 2003). Xantusiids mate in the fall, with gestation and birth occurring in late summer or early fall of the following year (Aguilar Corte´s, Camarillo, & Bezy, 1990; Pianka & Vitt, 2003; Ramı´rez-Bautista, Vitt, Ramı´rez-Hernandez, Quijano, & Smith, 2008). Reproductive hormone profiles have not been published for any lizard in this family, although one study found that exogenous P4 did not reduce vitellogenin production stimulated by exogenous E2 in females, but it did reduce ovarian weight and follicle size, presumably by inhibiting vitellogenin uptake (Yaron & Widzer, 1978). 2.3.1.16. Gerrhosauridae Plated lizards occur in Africa (three genera) and Madagascar (two genera); all are small- to medium-bodied (approximately 5–30 cm adult snout-to-vent length), diurnally active, and oviparous (Pianka & Vitt, 2003; JCVI/ TIGR Reptile Database, 2008). Most are terrestrial, although at least one species is arboreal (Zonosaurus boettgeri) and one is semi-aquatic (Zonosaurus maximus)
Hormones and Reproduction of Vertebrates
(Pough et al., 2004). Reproductive endocrinology has not been studied in this group, although pheromonal communication has been documented in Gerrhosaurus nigrolineatus, in which males can discriminate between other males and females based on cloacal chemical signals (Cooper & Trauth, 1992). 2.3.1.17. Cordylidae Girdled lizards are found predominantly in southern Africa; like their sister taxon, Gerrhosauridae, they are small- to medium-bodied and diurnally active. Members of the genus Platysaurus are oviparous, but the other two genera (Cordylus and Chamaesaura) are viviparous (Pianka & Vitt, 2003; JCVI/TIGR Reptile Database, 2008). Hormones and reproductive cycles have been studied to some extent in the giant girdled lizard (Cordylus giganteus) and the Karoo girdled lizard (Cordylus polyzonus). C. giganteus give birth in late summer to one to three young, but individual females only reproduce biennially or possibly triennially (van Wyk, 1994). Plasma E2 and P4 concentrations follow the typical patterndE2 is highest during vitellogenesis and P4 is highest during gestation; plasma T also was measured but not detected in this study (van Wyk, 1994). C. polyzonus, in contrast to C. giganteus, breeds annually (Flemming, 1993; 1994). In males, testicular recrudescence is initiated in the fall and is maximal in the spring, when mating commences; plasma T as well as AND concentrations exhibit a bimodal profile, peaking at the onset of recrudescence and then again in the spring during maximal recrudescence and mating (Flemming, 1993). In females, plasma E2 and P4 values are similar to those reported for C. giganteus above (Flemming, 1994). Although hormones related to reproductive cycles have not specifically been reported, work on reproductive tactics and male aggression in the Augrabies flat lizard (Platysaurus broadleyi) suggests interesting opportunities for future work. This lizard shows dramatic sexual color dimorphism in which males but not females possess green, orange, or yellow front legs, ventral orange or yellow ventral coloration, blue-turquoise flanks, and blue-violet throats that reflect highly in the UV range (Whiting, Nagy, & Bateman, 2003; Whiting et al., 2006). As a result of temporally restricted, locally abundant food resources (black fly plumes), these lizards can be found in highdensity groups where mating territories established and defended by males are at a premium (e.g., around 50% of males do not acquire territories) (Whiting et al., 2003; 2006). Testosterone may influence the development of the UV signal, and animals with an experimental reduction in UV are aggressively challenged by other males more frequently (Stapley & Whiting, 2006; Whiting et al., 2006). Finally, non-territorial males may mimic females visually in an attempt to avoid detection by territorial males and
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thereby gain access to reproductively active females (Whiting, Webb, & Keogh, 2009). 2.3.1.18. Sphaerodactylidae, Gekkonnidae, Phyllodactylidae, Eublepharidae, Diplodactylidae, Carphodactylidae, Pygopodidae Lizards commonly known as geckos (or legless lizards for the Australian family Pygopodidae, due to their loss of front limbs and vestigial hindlimbs (Patchell & Shine, 1986)) are distributed in seven families and number nearly 1200 species. They are distributed worldwide, though predominantly in desert, tropical, and subtropical regions (Pianka & Vitt, 2003; JCVI/TIGR Reptile Database, 2008). Most but not all are nocturnal, unlike the majority of other lizard taxa, and species range in adult size from under 3 to over 30 cm snout-to-vent length (Pianka & Vitt, 2003). Nearly all gecko species are oviparous, with clutch sizes of one or two eggs produced multiple times per season, but three genera from family Diplodactylidae (Naultinus, Hoplodactylus, and Rhacodactylus) are viviparous (Pianka & Vitt, 2003). Reproductive mode is diverse; most reproduce sexually and possess genotypic sex determination, but a few species are parthenogenetic (some species of the Gekkonid genera Hemidactylus, Hemiphyllodactylus, Heteronotia, Lepidodactylus, and Nactus), and a few exhibit TSD (e.g., Eublepharis from the family Eublepharidae and some Phelsuma from the family Gekkonidae (Pianka & Vitt, 2003)). Similarly to Scincidae, for such a large group of lizards there is very little information on hormones and reproductive cycles, although reproductive cycles more generally have been studied, e.g., for the Gekkonid species Gekko japonicus (Ikeuchi, 2004), Hemidactylus flaviviridis (Khan & Rai, 2004), and Phyllodactylus lanei (Ramı´rezSandoval et al., 2006). However, one species from the family Eublepharidae, the leopard gecko (Eublepharis macularius), has become a model organism in lizard reproduction due to its ease of maintenance in captivity and its reproductive mode of TSD (e.g., Crews & Moore, 2005). This species is native to southwestern Asia, although most if not all studies on its reproduction have occurred in laboratories using animals from captive-bred stock. Females are produced at low and high incubation temperatures, whereas mixed-sex ratios are produced at intermediate temperatures (female-biased at low–intermediate and male-biased at high–intermediate temperatures) (Crews & Moore, 2005). Adult females lay two-egg clutches (or, rarely, one-egg clutches) repeatedly across the breeding season (Rhen et al., 2000; LaDage et al., 2008). Females readily mate with multiple males both within and between clutches although they can store sperm and continue egg production in the absence of males (LaDage et al., 2008). Most of the work on reproduction in leopard geckos has
been on females, specifically concerning the effects of incubation temperature on reproductive physiology, morphology, and behavior (see Crews & Moore, 2005; Chapter 2, this volume). Hormonal changes across the reproductive cycle also have been assessed, however. In females, plasma concentrations of the androgens T and DHT increase markedly during late vitellogenesis and then decline sharply after ovulation; plasma E2 shows the same pattern and is higher in concentration than T and DHT except during late vitellogenesis, when T is nearly twice as high as E2 (Rhen et al., 2000). Unlike most lizards, plasma P4 peaks during early vitellogenesis but then declines to pre-vitellogenic levels following ovulation (Rhen et al., 2000); this is opposite to the pattern typically observed. Interestingly, incubation temperature influences plasma steroid concentrations in adult females; females from eggs incubated at high temperatures had higher T and DHT concentrations and lower P4 concentrations (they did not differ in E2 concentrations) (Rhen et al., 2000). During juvenile development the pattern is the opposite, as juvenile females from eggs incubated at different temperatures did not differ in plasma T or DHT concentrations, but females at high incubation temperatures had lower plasma E2 concentrations than females incubated at lower incubation temperatures (Rhen, Sakata, & Crews, 2005). In general, incubation temperature effects on male plasma steroid concentrations were absent. 2.3.1.19. Dibamidae Little is known about blind lizardsdthe 21 species comprising this family of two genera (Dibamus of southeastern Asia and Anelytropsis of Mexico). These lizards are fossorial; males have reduced hindlimbs only, whereas females are limbless, eyes are vestigial, and there is no external ear opening (Pianka & Vitt, 2003). Available data suggest that all members of this family are oviparous with single-egg clutches (Pianka & Vitt, 2003). Nothing is known about the endocrinology of reproduction in these species.
2.3.2. Other aspects of endocrine regulation of reproductive cycles 2.3.2.1. Hypothalamus–pituitary–adrenal (HPA) axis Hormones of the HPG axis play the central role in stimulating reproduction and reproductive cycling. However, hormones of the hypothalamus–pituitary–adrenal (HPA) axis can have a significant impact on reproduction as well. This topic is covered in much more detail elsewhere in this volume (Chapter 7). Briefly, the hypothalamus produces corticotropin-releasing factor (CRF), stimulating release of corticotropin (ACTH) from the adenohypophysis, which in turn targets the adrenal glands (specifically, the adrenal cortices) and stimulates release of glucocorticoid steroids
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(Norris, 2007). The primary glucocorticoid produced in lizards is corticosterone (CORT) (Norris, 2007). Glucocorticoids cause sustained elevation of blood glucose, playing a critical role in energy mobilization. Hypothalamic–pituitary–adrenal axis activation along with initial rapid autonomic stimulation of catecholamine release form the bases of the stress responseda suite of neuroendocrine and behavioral mechanisms that are activated to re-establish homeostasis in response to unpredictable, potentially harmful stimuli or circumstances (e.g., Sapolsky, 2002; Romero, 2004). In vertebrates including reptiles, the HPA axis can also play an important role in life stage transitions such as birth or hatching and dispersal (reviewed in Wada, 2008). As a result of a rising interest in the interactions between stress and reproduction and associated potential energetic constraints imposed, CORT profiles and their relationship to reproduction and other life history variables increasingly are being studied in reptiles including lizards (e.g., Moore & Jessop, 2003). At least two related generalities have become apparent from this work. First, although it is often the case that experimental elevation of CORT leads to a reduction in reproductive steroid concentrations in lizards (e.g., Tokarz, 1987b; Moore, Thompson, & Marler, 1991; Husak et al., 2009), other studies suggest modulation of the stress response during the reproductive season, minimizing either elevation in plasma CORT or apparent responsiveness to such an elevation that could disrupt reproductive activity (Moore & Jessop, 2003; Preest, Cree, & Tyrrell, 2005; Cartledge & Jones, 2007; Wack et al., 2008). Second, baseline concentrations of plasma CORT often are higher during the breeding season than the nonbreeding season, are positively associated with reproductive steroids, and are presumably related to the energy mobilization required for successful breeding (reviewed in Moore & Jessop, 2003; see also MacDonald, Czekala, Gerber, & Alberts, 2007; Wack et al., 2008). Such observations are counter to the general idea that CORT necessarily inhibits reproduction. Rather, social or reproductive context, energetic demands associated with reproduction, and species as well as individual variation all contribute to whether a ‘stressor’ is stressful to the point of inhibiting reproduction (Sapolsky, 2002; Romero, 2004). 2.3.2.2. Endogenous rhythms The most direct mechanism for timing seasonal events such as reproduction would be for seasonal cues, such as the especially reliable cue of photoperiod, to directly regulate necessary physiological and behavioral parameters (‘type 3 rhythm,’ sensu Zucker, Lee, and Dark (1991)). However, the relationship typically is not this simple, as biological rhythms persist even when the relevant environmental cues
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are removed (reviewed in Gorman & Lee, 2002; Kriegsfeld, LeSauter, Hamada, Pitts, & Silver, 2002; Nelson, 2007; Paul, Zucker, & Schwartz, 2008). Indeed, such endogenous rhythms are ubiquitous across vertebrate and invertebrate taxa. In mammals, circadian (daily) rhythms are regulated by activity of the suprachiasmatic nuclei (SCN) of the hypothalamus, which receive information about photoperiod via neural input from extra-retinal photoreceptors (Gorman & Lee, 2002; Kriegsfeld et al., 2002; Nelson, 2007; Paul et al., 2008). The SCN communicate with, among many other tissues, the pineal gland; lack of input during the photoperiod dark phase leads to elevated production of the hormone melatonin (MEL) during nighttime hours. This endocrine signal is an important communicator of photoperiod that synchronizes many daily and seasonal activities of cells and tissues throughout the body. Interestingly, MEL release is highest at night for both diurnal and nocturnal mammals (summarized in Kriegsfeld et al., 2002); thus, target tissue responses rather than MEL profiles have changed in response to these different activity cycles. Although neural regulation of circannual (seasonal) rhythms remains less understood in comparison to circadian rhythms, endocrine mediation via the pineal gland and MEL secretion clearly is important (Nelson, 2007; Paul et al., 2008). Pinealectomy abolishes seasonal rhythms and causes expression of long-day-typical traits, whereas exogenous MEL restores rhythmicity (Goldman, 2001). In general, increased duration of MEL exposure, as occurs under short-day conditions when nights are longer, inhibits reproduction. Underwood (1992) reviewed our understanding of endogenous rhythms in non-avian reptiles and pointed out that far less was known than for birds and mammals. Although additional work has been done, this discrepancy largely remains true today (e.g., Paul et al., 2008), particularly for potential effects on reproduction (Mayer, Bornestaf, & Borg, 1997). Regarding circadian rhythms in lizards, there are parallels with studies on mammalian vertebrates. For example, MEL secretion from the pineal gland shows a circadian rhythm with the highest output occurring at night (Mayer et al., 1997; Tosini, Bertolucci, & Foa`, 2001). Pinealectomy drastically alters circadian rhythms, as does exogenous MEL, depending on timing, dose, and duration (Hyde & Underwood, 2000; Tosini et al., 2001). In contrast to mammals, however, most reptiles including lizards (but not snakes) studied thus far possess pineal glands that contain photoreceptors, thus allowing direct light input (Underwood, 1992; Mayer et al., 1997). The SCN may not communicate photoperiodic information to the pineal gland of lizards as it does in mammals, although it does itself receive input from the retina (Tosini et al., 2001). Instead, it appears that the SCN contain circadian oscillators that are subordinate to the circadian
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pacemaker(s) in the pineal gland as communicated by MEL release. Also in contrast to mammals, temperature in addition to photoperiod can affect MEL synthesis and release in several reptiles including lizards, with an inverse relationship between temperature and amplitude of MEL release (Underwood, 1992; Tosini et al., 2001; Paul et al., 2008). Temperature may be more important than photoperiod per se in seasonal MEL profiles (Mayer et al., 2007). With respect to annual rhythms, work in lizards predominantly has focused on locomotor activity, thermoregulatory behavior, and reproduction, each of which shows seasonal rhythmicity (Underwood, 1992; Ellis, Firth, & Belan, 2008 and references therein; Paul et al., 2008). Regarding reproductive cycles, work in lizards has shown that, under the appropriate environmental conditions, pinealectomy can stimulate the onset of reproduction whereas MEL administration can suppress it (Underwood, 1992; Mayer et al., 2007; Paul et al., 2008). However, neither pinealectomy nor MEL administration completely override photoperiodic influences on reproduction (Mayer et al., 2007), suggesting additional and possibly independent inputs outside of the pineal melatonin system. Interestingly, while MEL secretion in reptiles is highest during the night, as it is for mammals, it is not clear that the duration of secretion mirrors length of night as it does for mammals. Hyde and Underwood (1993) found that manipulating photoperiod had no effect on the duration of MEL secretion in green anoles, A. carolinensis. Overall, understanding of the neuroendocrine correlates of seasonal rhythms in reptiles isdsimilarly to mammalsdrelatively poor.
3. CONCLUSIONS AND FUTURE DIRECTIONS Hormones and their relationship to reproductive cycles have been well-described for relatively few lizard species, representing only 8 of 32 families (Figure 12.1; Table 12.1) – Lacertidae, Teiidae, Varanidae, Iguanidae, Agamidae, Scincidae, Cordylidae, and Eublepharidae. Given the number of species represented by these families (over 3200; 63% of total) and the diversity of life histories and reproductive strategies found within them, even in these groups there is considerably more room for research. For example, Scincidae contains approximately 1300 species in 134 genera, but in only Niveoscincus and Tiliqua have endocrine profiles and reproduction been well-characterized. This distribution of data on hormones and reproductive cycles in lizards leads to several conclusions and points to fruitful areas for future study. First, the wealth of information available for genera such as Podarcis, Lacerta, Cnemidophorus, Anolis, Sceloporus, Calotes, Niveoscincus, and Eublepharis and
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reviewed above gives us a firm foundation for understanding hormones and reproductive cycles in lizards. From a review of available data, it is clear that gonadal steroid profiles across the reproductive cycle and their hypothalamic–pituitary regulation have remained generally consistent across lizard species regardless of reproductive mode (e.g., sexual vs. asexual reproduction, oviparity vs. viviparity, genotypic vs. temperature-dependent sex determination). Roles for E2 and P4 in females and T in males in initiating and maintaining reproductive condition further are similar to those seen in mammalian species. Additionally, work on androgen roles in females and E2 and P4 roles in males demonstrate some potentially novel or at minimum not widely recognized steroid effects on reproduction. More studies that characterize all of these steroids in both males and females will be useful. Second, in conjunction with work on the ‘traditional non-traditional’ model species in the genera listed above, additional targeted work on under-represented lizard taxa would yield a tremendous comparative perspective. For example, there are taxa such as Chamaeleonidae and Gekkonidae that contain hundreds of lizard species and for which there is good information on reproduction and behavior but no data on underlying endocrine regulation. Chameleons in particular generate widespread interest and appeal, and they exhibit considerable diversity in sexual dimorphism, lifespan, and reproductive strategies and modes (e.g., Karsten et al., 2008), providing excellent justification and preliminary data for tests of hormonal effects. There are also individual well-studied species within taxa that have otherwise received little attention but seem to be prime targets for productive research. For example, the Augrabies flat lizard (Cordylidae: P. broadleyi) has dramatic sexual color dimorphism, highdensity mating territories, and multiple reproductive strategies in males, but limited work on female reproductive strategies and potential seasonal and strategy-based hormonal correlates in either sex (e.g., Whiting et al., 2003; 2006; 2009). Finally, there also are atypical lizard taxa for which almost nothing is known and therefore any contribution would be valuable. One example of an area in need of attention is the hormonal underpinnings of the ecologically similar but phylogenetically very distant worm lizards (Families Amphisbaenidae, Trogonophidae, Bipedidae, Blanidae, Cadeidae, and Rhineuridae); glass, alligator, and legless lizards (Families Anguidae, Anniellidae, and Diploglossidae); and blind lizards (family Dibamidae) (Figure 12.1). These groups apparently have experienced similar selection pressures, leading to convergent evolution of limb reduction or loss and various degrees of fossoriality, although reproductive mode has not converged, as some show oviparity and some viviparity, with long reproductive lifespans and generally few offspring per clutch/litter (Pianka & Vitt, 2003). Another
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example is the crocodile lizard (S. crocodilurus), the only species in family Shinisauridae. This lizard is long-lived, viviparous, and exhibits low fecundity. It is also threatened due to loss of necessary habitat, but popular and widespread among zoos and in the pet trade, making laboratory, if not field, investigations feasible. Third, given advances in our understanding of the neuroendocrine and molecular expression of endogenous rhythms seen in other taxonomic groups (Kriegsfeld et al., 2002; Paul et al., 2008) and the data thus far known for lizards, opportunities for advances in reptiles are strong (e.g., Underwood, 1992; Tosini et al., 2001). Because neuroendocrine regulation of seasonal rhythms is generally not understood in either mammalian or non-mammalian vertebrates, work on endogenous rhythms and reproductive cycles in lizards would be of wide interest and utility. In attempting to describe and understand the bases of seasonal rhythms, two research directions in particular seem promising. One direction would be to investigate species- and population-level variation in a genus such as Sceloporus, which exhibits widespread differences in reproductive mode and seasonality of reproduction, as reviewed in Sections 2.1. and 2.2. To what extent, if any, have changes in pineal function, MEL sensitivity, and/or extra-pineal rhythms contributed to reproductive flexibility in this genus? A second promising direction would be to investigate pineal function and peripheral response in nocturnal species (e.g., most geckos) as compared to the diurnal species studied thus far. Do pineal activity, MEL release, and/or extra-pineal rhythms exhibit an opposite pattern to that seen in diurnal species (e.g., peak daytime release of MEL), suggesting a change in neuroendocrine response to the environment but no change in organismal response to MEL? Or is it that, like in nocturnal mammals, MEL and related parameters remain the same as in diurnal species (e.g., peak nighttime release of MEL), suggesting a similar neuroendocrine response to the environment but a changed organismal response to MEL? How do these relationships compare across reproductive cycles and between reproductive and nonreproductive periods? The evolutionary position and life-history diversity of reptiles make them an excellent group in which to examine such questions. Fourth, efforts to monitor and protect lizard populations are worthy for biodiversity as well as practical considerations. Increasingly, physiological and neuroendocrine approaches are being used to address conservation goals by informing both causes and consequences of population change or decline as well as providing useful tools for assessment of the effectiveness of actions taken (Cockrem, 2005; Wikelski & Cooke, 2006; Germano & Bishop, 2009). Given documented effects of endocrine disruptors, habitat alteration or loss, the invasion of non-native species, and global warming, as reviewed above, there is ample reason to initiate new as well as to continue existing monitoring
Hormones and Reproduction of Vertebrates
programs of lizard populations. Monitoring of reproductive cycles and hormonal regulation in particular allow for an integrative assessment of individual responses to environmental inputs. Thus, documenting ‘baseline’ population conditions will greatly inform us regarding whether changes in environmental inputs lead to changes in reproduction and, if so, to what extent endocrine modulation is involved.
ABBREVIATIONS ACTH AND cGnRH-I cGnRH-II CORT CRF DHT E2 FSH GnIH GnRH GTH HPA HPG LH MEL mGnRH P4 SCN sGnRH T TSD
Corticotropin Androstenedione Chicken-I gonadotropin-releasing hormone Chicken-II gonadotropin-releasing hormone Corticosterone Corticotropin-releasing factor Dihydrotestosterone 17b-estradiol Follicle-stimulating hormone Gonadotropin-inhibiting hormone Gonadotropin-releasing hormone Gonadotropin Hypothalamus–pituitary–adrenal Hypothalamus–pituitary–gonadal Luteinizing hormone Melatonin Mammalian gonadotropin-releasing hormone Progesterone Suprachiasmatic nuclei Salmon gonadotropin-releasing hormone Testosterone Temperature-dependent sex determination
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Chapter 13
Hormones and Reproductive Cycles in Snakes Emily N. Taylor* and Dale F. DeNardoy *
California Polytechnic State University, San Luis Obispo, CA, USA, y Arizona State University, Tempe, AZ, USA
SUMMARY The study of the hormonal regulation of reproduction in snakes is in its infancy. Studies have disproportionately examined temperate zone viperid and colubrid snakes, especially the redsided garter snake (Thamnophis sirtalis parietalis). Indeed, extensive observational and experimental studies on T. s. parietalis form the basis for our understanding of the hormonal regulation of reproduction in snakes. This review focuses on seasonal hormone concentrations in snakes in relation to events in the reproductive cycle; the limited data available on hypothalamic hormones, gonadotropins (GTHs), hormone receptors, and binding globulins; the neuroendocrinology of reproduction; and the environmental, social, physiological, and embryonic aspects of hormonal regulation of reproduction in snakes. The review ends with suggestions for future research, including studies of a more diverse ecological and taxonomic representation of snakes, experimental studies on the effects of hormones on reproductive tissues and behaviors, and further research into the neuroendocrinology of reproduction in this highly diverse group of animals.
1. INTRODUCTION Despite there having been a fair number of studies conducted on snake reproduction, these studies have predominantly been conducted on a relatively small number of temperate species. Further, up until the past few decades, studies of snake reproduction have been mostly anatomical, being limited to the histology of the male reproductive organs to quantify the timing of spermatogenesis and the examination of female reproductive tracts for evidence of vitellogenesis. This review focuses on current knowledge of the hormonal regulation of reproduction in snakes. Because so many studies have been published on the hormonal regulation of various aspects of reproduction in snakes, especially the red-sided garter snake (Thamnophis sirtalis parietalis), we attempt to provide a broad overview of the effects of hormones on reproduction in snakes, focusing on seminal studies and referring to review articles Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
when possible. First, we describe what is known about the relationship between sex steroids and reproduction in snakes. In this section, we discuss T. s. parietalis separately from all other snake species. Although such a distinction is not phylogenetically justified, it enables us to provide a thorough presentation of the most well-studied species without biasing the review of reproductive endocrinology of snakes in general with data from a single species. We then discuss the limited literature on hypothalamic hormones, gonadotropins (GTHs), hormone receptors, binding globulins, and neuroendocrinology for all snakes. We follow this with a discussion of environmental, behavioral, and non-reproductive physiological influences on hormones and reproduction in snakes, ending with suggestions for future research.
2. HORMONES AND REPRODUCTIVE CYCLES IN SNAKES Saint Girons (1982) reviewed the reproductive cycles of male snakes in relation to female cycles and climate. In doing so, Saint Girons described four patterns of male reproduction. First, the postnuptial (dissociated) or estival type, which occurs in many temperate and subtropical species, is a pattern in which males undergo spermatogenesis during the summer, sperm are stored over the winter (in the vasa deferentia and sometimes also in female oviducts if fall mating occurs), and the principal mating season occurs in spring. This postnuptial pattern of spermatogenesis is predominant among temperate colubrids and crotalines. Second is the prenuptial (associated) or vernal type, which typically occurs in warm climates. In this type, spermatogenesis begins in the fall and is completed by the following spring or early summer, at which time mating occurs. Third is the ‘mixed type,’ which is characterized by spermatogenesis beginning in spring and being completed one year later; there can be one (spring) or two (spring and fall) mating seasons. As with 355
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the postnuptial pattern, the mixed type typically occurs in temperate and subtropical species. Finally, the continuous type describes species in environments where there is little seasonal variation in temperature (e.g., most tropical areas). As the name implies, species that express continuous male reproduction exhibit spermatogenesis and mating behavior throughout the year. Although distinct in definition, these four types of male reproduction in snakes should be viewed as noteworthy points along a cline from distinctly seasonal (displaying estival spermatogenesis) to aseasonal (displaying continuous spermatogenesis), as exceptions certainly exist (Saint Girons, 1982). Reproductive cycles in female snakes are less variable than in males. Females typically produce, at most, a single clutch or litter per year. Many species exhibit less-thanannual reproduction, in that not all adult females reproduce each year (Duvall, Arnold, & Schuett, 1992). In most species studied, the timing of reproductive events is such that neonates are born or hatched when resources are abundant and climatic conditions are favorable. Typically, ovulation occurs in late spring or early summer, and hatching or parturition occurs in summer or early fall (Saint Girons, 1982). Of course, exceptions exist, especially in tropical species in which reproduction may occur at any time of year, and there may be multiple clutches per year. In fact, aseasonal reproduction may well be the dominant reproductive pattern in female snakes, considering the higher diversity of snakes in the tropics (Stevens, 1989; Reed, 2003), but these species are mostly unstudied. Whereas the timing of reproduction is most frequently assessed according to the traditional seasons (spring, summer, fall, and winter in the northern or southern hemispheres), other critical factors including annual weather patterns, altitude, phylogeny, foraging mode, body size, and body condition also regulate the timing of reproduction (Dunham, Miles, & Reznick, 1988; Vitt, 1992). To more fully understand reproductive timing, it is essential to understand reproductive physiology, especially the relationships between hormones and events in reproduction (e.g., mating, spermatogenesis, vitellogenesis, gestation, parturition). Such studies have been conducted relatively recently in snakes. Snakes are notoriously secretive animals, but advances in techniques for following individual snakes in the wild (e.g., radiotelemetry) as well as in methods of quantifying hormones (e.g., radioimmunoassay) in the 1970s and 1980s allowed researchers to begin examining the hormonal regulation of reproduction in snakes. Some of the earliest studies examined snake GTHs and sex steroid hormones, in general confirming that the structure and function of these hormones in snakes are similar to those of their better-studied mammalian counterparts. In particular, T. s. parietalis emerged as a model organism for studies of hormonal regulation of reproduction in snakes (Krohmer, 2004), largely because
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a population in Manitoba, Canada that dens communally by the thousands provided accessibility to large numbers of snakes for both field and laboratory experiments. Most of our knowledge of advanced topics in this field comes from studies of T. s. parietalis. These studies have provided extremely valuable insights into the hormonal regulation of reproduction in snakes, due to the ease of sampling multiple individuals as well as their ready display of mating behaviors in captivity. However, the extremely high latitude and the unique mating behavior of this population call into question the applicability of these results to snakes in general. Recent studies by several laboratories have provided data on the hormonal regulation of reproduction in other snake species. These comparative data have proven quite valuable, but the universality of existing knowledge on snake reproductive physiology must remain uncertain as these additional studies still disproportionately represent temperate species, especially viperids. Crews (1984) reviewed studies examining the temporal association of mating and gonadal activity (defined as gametogenesis and/or secretion of sex steroid hormones) in seasonally breeding organisms. He differentiated between species showing associated and dissociated reproductive tactics (Figure 13.1). Species showing associated reproductive tactics exhibit mating that is coincident with elevated gonadal activity, whereas species showing the dissociated reproductive pattern mate when gonadal activity is basal. These terms can be applied to both males and females; sometimes both sexes show the same tactic, while at other times the sexes differ. Considering the patterns of spermatogenesis reviewed by Saint Girons (1982), the estival or postnuptial pattern most closely resembles the dissociated tactic, and the vernal or prenuptial pattern most closely resembles the associated tactic. Although the dissociated and associated patterns clearly represent extremes, and many species show tactics intermediate between these extremes, the distinction provides a useful framework for examining the dependence of mating behaviors upon gonadal hormones. In this section, we describe how these paradigms have been applied to snakes. With notable exceptions, it appears that the majority of male snakes of those few species examined indeed exhibit mating behaviors when plasma testosterone (T) concentrations are high. Females of these same species, on the other hand, will often mate at times when circulating sex steroid concentrations are basal.
2.1. Steroids and Reproduction in Snakes other than Thamnophis sirtalis parietalis Studies of plasma steroid hormone concentrations in freeranging snakes (other than T. s. parietalis) have mostly
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Figure 13.1 Associated and dissociated reproductive patterns. The shaded bar represents mating activity. Gonadal activity is usually defined as sex steroid hormone secretion, although it can also refer to production of gametes. Modified from Crews (1984).
been conducted on vipers, with several studies on colubrids and other families. These studies typically examined seasonal variation in hormone concentrations in relation to reproductive events, including mating, spermatogenesis, vitellogenesis, and gestation. In many viperid species, reproduction is seasonal, but not all females reproduce in a given year. Thus, it is possible to collect blood samples for hormone quantification simultaneously from nonreproductive and reproductive females in a single population. This is advantageous for studying the relationship between hormones and reproduction because differences in hormone concentrations between these groups can more likely be attributed to reproduction (although correlated variables such as body condition may contribute to such differences). In addition, intra-specific and inter-specific variation in the association between mating and follicular activity allows hormonal changes responsible for mating behaviors to be at least partially uncoupled from those responsible for physiological events.
2.1.1. Females In general, studies of the relationship between steroid hormones and reproduction in female snakes show that plasma 17b-estradiol (E2) (and in some studies, T) is elevated during vitellogenesis, reflecting its role in stimulating production of vitellogenin by the liver (Ho, KleisSan Francisco, McPherson, Heiserman, & Callard, 1982). Plasma progesterone (P4) is often elevated during gestation in viviparous snakes, reflecting its role in the maintenance of pregnancy (Mead, Eroschenko, & Highfill, 1981; reviewed in Custodia-Lora & Callard, 2002). Taylor, DeNardo, and Jennings (2004) described the following patterns in female western diamondback rattlesnakes
(Crotalus atrox). Plasma concentrations of E2, T, and P4 were low throughout the year if females did not reproduce in that year. If females did reproduce, E2 was elevated from March through June, and especially in April and May. This timing coincides with vitellogenesis in C. atrox (Taylor & DeNardo, 2005) and in many other temperate species. Testosterone concentrations are low throughout the year in reproductive females, with a slight elevation in spring that may be due to the production of E2 (T being a precursor to E2 in steroid biosynthesis). Increased plasma T during vitellogenesis was observed in the Chinese cobra (Naja naja) (Bona-Gallo, Licht, MacKenzie, & Lofts, 1980) and three species of marine snake (Acrochordus granulatus, Cerberus rhynchops, Laticauda colubrina) (Gorman, Licht, & McCollum, 1981). Saint Girons, Bradshaw, and Bradshaw (1993) found that females displaying mating behaviors had significantly higher plasma dihydrotestosterone (DHT) concentrations, but not T concentrations, than females that were not displaying mating behaviors; however, sample size was very low in this analysis. In C. atrox, P4 is elevated in reproductive females in May through August, and peaks in June/July, corresponding with the gestation period (Taylor & DeNardo, 2005). In the oviparous N. naja, P4 concentrations similarly are elevated from ovulation until oviposition (Bona-Gallo et al., 1980). In C. atrox, plasma corticosterone (CORT) concentrations are more variable annually than the other steroid hormones, but increase dramatically in reproductive females in July and August, during late gestation. Plasma CORT, along with P4, concentrations return to baseline by September (after parturition) in reproductive females (Taylor et al., 2004). Plasma E2 and P4 concentrations also have been quantified in viperids from Europe, Asia, and South America.
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Female aspic vipers (Vipera aspis) in France have the same seasonal mating patterns as C. atrox (spring vitellogenesis and late summer parturition), and also show elevated E2 during spring (Saint Girons et al., 1993). Interestingly, reports of plasma P4 concentrations in reproductive V. aspis are inconsistent. Saint Girons et al. (1993) concluded that plasma P4 is highly variable among snakes, with no clear difference between reproductive and non-reproductive females. However, in other studies, plasma P4 concentrations of reproductive female V. aspis are elevated in May through August (gestation), decrease in September (after parturition), and rise again during winter (Naulleau & Fleury, 1990; Bonnet, Naulleau, Bradshaw, & Shine, 2001). Bonnet et al. (2001) hypothesized that the winter increase in P4, which is especially marked in post-reproductive snakes, may act to block vitellogenesis in emaciated snakes, as P4 may inhibit hepatic synthesis of vitellogenin (Callard et al., 1992; Callard, Giannoukos, CharnockJones, Benson, & Paolucci, 1994). In Chinese green tree vipers, Viridovipera (Trimeresurus) stejnegeri, E2 concentrations are slightly elevated during vitellogenesis and P4 concentrations are dramatically elevated during gestation (Tsai & Tu, 2001). Almeida-Santos et al. (2004) found similar relationships between sex steroid concentrations and reproductive states in female Neotropical rattlesnakes (Crotalus durissus terrificus) in southeastern Brazil. In this species E2 peaks during vitellogenesis (winter) and P4 is high during gestation (late winter, spring, and summer), whereas both hormones are low throughout the year in nonreproductive females.
2.1.2. Males As with females, the intra-specific and inter-specific variation in the number of mating periods per year and timing of the mating periods relative to gonadal activity among temperate male snakes provides a potential opportunity to uncouple the relationship between T and breeding behavior from that of T and other physiological events, including spermatogenesis. Several studies have shown that T is elevated during the breeding season(s), providing a strong indication that T stimulates reproductive behaviors in male viperids. In viperid species with a single annual breeding season, plasma T concentrations are high when spermatogenesis and breeding behaviors are occurring. In the cottonmouth (Agkistrodon piscivorus), T peaks in late summer, at the same time that males show spermiogenic activity, hypertrophy of the sexual segment of the kidney, and breeding behavior (Johnson, Jacob, & Torrance, 1982; Zaidan, Kreider, & Beaupre, 2003; Graham, Earley, Hoss, Schuett, & Grober, 2008). Similarly, the black-tailed rattlesnake (Crotalus molossus) has a single mating season (late summer) and shows a single annual peak in
Hormones and Reproduction of Vertebrates
T concentrations during this time (Schuett et al., 2005). The Chinese cobra (N. naja) and European adder (Vipera berus) show a similarly coordinated single peak in T at the same time as spermiogenesis and mating, although these occur in the spring rather than late summer (Lofts, Phillips, & Tam, 1966; Bona-Gallo et al., 1980; Naulleau & Fleury, 1984). Many snake species show two annual mating seasons, usually in spring and fall (separated by summer and winter periods of reproductive inactivity). Water snakes (Nerodia sipedon); rough greensnakes (Opheodrys aestivus); C. atrox; Mojave rattlesnakes (Crotalus scutulatus); and V. aspis breed in both spring and fall, and males show elevated T in both of these seasons (Aldridge, Greenhaw, & Plummer, 1990; Weil & Aldridge, 1981; Naulleau, Fleury, & Boissin, 1987; Saint Girons et al., 1993; Schuett et al., 2002; Taylor et al., 2004; Schuett et al., 2005). C. atrox and C. scutulatus show bimodal peaks in circulating DHT at the same time as the peaks in T (Schuett et al., 2002; 2005), although DHT concentrations are much lower than T concentrations. In a laboratory study, T concentrations of copperheads (Agkistrodon contortrix) peak in spring and late summer, corresponding to their breeding seasons (Schuett, Harlow, Rose, Van Kirk, & Murdoch, 1997). These species show the estival or postnuptial pattern of spermatogenesis: T concentrations are high during spermatogenesis and mating in the late summer and fall, but are also high during the spring when mating is occurring in the absence of spermatogenesis. Testosterone concentrations are lowest in summer, when breeding activity does not occur. In the Sonoran Desert, C. atrox sampled while basking during the winter, when the species is mostly inactive, exhibit T concentrations intermediate between those of summer (lowest) and of the spring and fall breeding seasons (Schuett et al., 2006). The reason for elevated T concentrations in winter is unknown, but it is possible that basking behavior during mild winter days prepares the snakes for the abrupt onset of the spring mating season. Interestingly, absolute plasma steroid hormone concentrations of many viperids appear to be much higher than those of most other snakes and other vertebrates. For example, peak T concentrations in A. contortix and rattlesnakes are in the region of 100 ng/mL (Schuett et al., 1997a; 2002; Taylor et al., 2004; Schuett et al., 2005), which is 1e3 orders of magnitude higher than concentrations observed in most other snakes. However, the closely related A. piscivorus has much lower plasma T concentrations than A. contortix (Johnson et al., 1982; Zaidan et al., 2003; Graham et al., 2008), and the congeneric C. molossus has substantially lower steroid concentrations than C. atrox (Schuett et al., 2005). The reasons for and implications of differences in absolute steroid concentrations in snakes, whether closely or distantly related, are unknown.
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In snakes with a single annual breeding season, spermatogenesis, maximal T production, and mating behaviors usually occur simultaneously (e.g., the ‘associated’ reproductive pattern (Crews, 1984; Whittier & Crews, 1987)), thereby making it difficult to assess the importance of T in each process. However, studies on those species with bimodal mating seasons suggest that T is responsible for mating behaviors, since T is elevated during both mating periods regardless of whether the testes are spermatogenically active or regressed. Some exceptions are evident: male V. aspis sometimes have very low androgen concentrations during mating (Naulleau et al., 1987; Saint Girons et al., 1993). It is important to note that species with two mating seasons are intermediate in the associated/dissociated dichotomy of reproductive patterns (Crews, 1984; Whittier & Crews, 1987), as they have associated reproduction in one season (late summer/fall) and dissociated reproduction in another (spring) with respect to spermatogenesis. In this context, it is evident that the associated/ dissociated paradigm should be viewed as describing two extremes along a continuum in which many snakes fall somewhere in the middle (Moore & Lindzey, 1992). Most research on steroid hormones in snakes has focused on terrestrial snakes from temperate regions, in which reproduction is highly seasonal. A noteworthy exception, however, deserves comment. Gorman et al. (1981) described the seasonal steroid hormone concentrations in relation to reproductive events in three marine snakes (A. granulatus, C. rhynchops, and L. colubrina) from the Philippines. These three species show varying degrees of seasonality in their reproductive patterns. A. granulatus is the most seasonal, with mating activity, spermatogenesis, and peak T concentrations occurring in the fall, shortly after vitellogenesis in females. In contrast, C. rhynchops, and especially L. colubrina, show spermatogenesis throughout the year. Although spermatogenesis and T concentrations in C. rhynchops peak in the fall, there is evidence for year-round gonadal activity. In L. colubrina, males show no seasonal trends in spermatogenic activity or T concentrations. These data suggest that species inhabiting more thermally constant environments, such as the ocean and the tropics, may show year-round reproduction. However, the paucity of data on tropical snakes highlights the need for research in this area.
2.2. Steroids and Reproduction in Red-sided Garter Snakes (Thamnophis sirtalis parietalis) 2.2.1. Females Unlike our limited knowledge regarding steroids and reproduction in other species of snake, studies of T. s. parietalis go beyond a basic correlational examination between plasma hormone concentrations and the
reproductive cycle. Studies of the reproductive endocrinology of T. s. parietalis have employed manipulative designs to more closely examine their reproductive endocrinology. Female T. s. parietalis exhibit a rather unusual relationship between reproductive events and hormones in comparison to other species of snake. Notably, they do not show elevated P4 during gestation (Whittier, Mason, & Crews, 1987). Like many other snakes studied, plasma T is elevated during vitellogenesis (Whittier et al., 1987a). Spring mating occurs when E2 concentrations are low (Garstka, Camazine, & Crews, 1982), but Mendonc¸a and Crews (1996) have shown through ovariectomy and hormone replacement therapy that even low E2 concentrations appear to be important in making female snakes attractive and receptive to males. Additionally, the physical act of mating induces a surge in E2 in females, but plasma E2 concentrations are not necessarily elevated during vitellogenesis (Garstka, Tokartz, Diamond, Halpert, & Crews, 1985; Whittier et al., 1987a; Whittier & Crews, 1989; Mendonc¸a & Crews, 1990). Copulation, but not courtship, stimulates release of prostaglandin F2a (PGF2a) by female T. s. parietalis (Whittier & Crews, 1989). It was hypothesized that this prostaglandin, possibly along with the mating-induced surge in plasma E2, causes females to become unreceptive and unattractive to males. This decline in receptivity is effected by cloacal distention during mating, which transmits a neural signal to the brain, and the decline in attractivity is mediated possibly by a pheromone present in copulatory plugs deposited by males (Mendonc¸a & Crews, 2001). Whittier (1992) examined the effects of steroid hormones on ovarian and liver tissue in adult female T. s. parietalis. Treatment of non-reproductive females with E2 stimulates an increase in oviductal mass and oviductal cell size, but does not affect ovarian mass. 17b-estradiol also stimulates an increase in liver mass and hepatocyte size and number. Treatment with T and DHT stimulates an increase in oviductal mass and oviductal cell size and a slight increase in hepatocyte size and number. These data suggest that E2 and androgens, even when not aromatizable (i.e., DHT), can affect the oviduct and to a lesser extent the liver of female T. s. parietalis.
2.2.2. Males Male T. s. parietalis emerge from communal hibernacula in spring and initiate courtship behavior that lasts several weeks (Crews & Garstka, 1982). Hawley and Aleksiuk (1975; 1976) showed that increased temperatures experienced during spring stimulate reproductive behaviors. Krohmer and Crews (1987a) suggest a possible role for the anterior preoptic area of the hypothalamus (APOA) in this process, as lesions to this area disrupted both courtship behavior and thermoregulation. During spring mating,
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males utilize sperm produced the previous summer and stored through the winter in the vasa deferentia (Krohmer, Grassman, & Crews, 1987); according to the paradigms described earlier, they therefore exhibit the postnuptial, dissociated reproductive tactic (Crews, 1976). However, studies suggest that T. s. parietalis also mate in the late summer and fall (Mendonc¸a & Crews, 1989; Whittier & Crews, 1989). It was initially reported that plasma androgen concentrations are low in the spring, when mating occurs (Camazine, Garstka, Tokarz, & Crews, 1980), again highlighting the postnuptial, dissociated pattern of reproduction in this species. However, later studies demonstrated that androgen concentrations are in fact elevated upon emergence from hibernation but drop rapidly after emergence (Krohmer et al., 1987; Moore, LeMaster, & Mason, 2000). In accordance with this, the sexual segment of the kidney, which is known to be stimulated by androgens in squamate reptiles (Bishop, 1959), was hypertrophied in the late summer and the spring, but regressed during the summer when androgen concentrations are low (Krohmer et al., 1987). Since there is now evidence for both spring and late summer mating and elevated T during both fall and emergence from hibernation in spring, this species does not exhibit a strictly dissociated pattern of reproduction. However, mating behavior is not dependent upon T in male T. s. parietalis (Camazine et al., 1980; Crews, 1984; Crews et al., 1984). Courtship behavior is independent of androgens because castration, adrenalectomy, or hypophysectomy fails to prevent courtship behavior in males, whereas treatment with hypothalamic hormones, GTHs, arginine vasotocin, or sex steroid hormones does not stimulate courtship behavior in adult males (Garstka et al., 1982; Crews et al., 1984). It is possible that prior exposure to androgens organizes brain regions involved in reproduction, and then low temperatures in winter followed by a spring warming activate reproductive behaviors. In support of this hypothesis, T implants elicit courtship behavior in neonatal and yearling, but not adult, males and females (Crews, 1985). Alternatively, it is possible that androgens play a typical role in gametogenesis in this species but do not play a role in mating behavior, or that their role in mating behavior is yet to be discovered.
2.3. Hypothalamic Hormones, Gonadotropins, Hormone Receptors, and Binding Globulins in Snakes Data on the structure and distribution of gonadotropinreleasing hormone (GnRH) in snakes are scarce and contradictory. Licht et al. (1984) found that chicken and mammalian GnRH failed to stimulate GTH release in
Hormones and Reproduction of Vertebrates
cobras (N. naja). However, other studies have provided evidence that snakes possess a hormone resembling either mammalian or chicken GnRH. Nozaki, Tsukahara, and Kobayashi (1984) found cell bodies in the hypothalamus and hippocampus of rat snakes (Elaphe climacophora) that were immunoreactive for mammalian GnRH. Sherwood and Whittier (1988) used high performance liquid chromatography to show that T. s. parietalis has a single form of GnRH that most closely resembles chicken-I GnRH (cGnRH-I). Smith, Moore, and Mason (1997) examined the distribution of GnRH in the brains of male T. s. parietalis and confirmed that this species has a GnRH resembling the cGnRH-I, with GnRH-like immunoreactivity distributed in many brain regions. Only a single study has measured concentrations of GTHs in snakes. Bona-Gallo et al. (1980) found that plasma GTH concentrations (detected using a radioimmunoassay with a labeled cobra GTH of unspecified structure) peaked in male N. naja during winter, when testicular recrudescence begins. In females, plasma GTH concentrations also peaked in winter, but showed a second peak during spring vitellogenesis. In general, the relationship between GTH concentrations, steroid hormone secretion, and events in the reproductive cycle is not straightforward in this species, leading to the hypothesis that functional changes in gonadal tissue rather than in concentrations of circulating GTHs are responsible for changes in circulating steroid hormone concentrations. The structure of snake GTHs has yet to be identified, and studies have indirectly assessed the actions of GTHs by evaluating the effects of mammalian GTHs on androgen secretion and testicular development in snakes. Both mammalian follicle-stimulating hormone (FSH) and luteinizing hormone (LH) stimulate gametogenesis, steroid production, and ovulation in snakes (reviewed in Licht, 1974). Tsui and Licht (1977) showed that LH and FSH from various species of vertebrate, including other reptiles, stimulated androgenesis in C. atrox and T. s. parietalis, but to varying degrees (depending both on the source species of the GTH and the species being tested). This study also suggested that snake GTHs may be structurally divergent from those of many other vertebrates, since snake GTHs failed to elicit androgen production in turtle gonads. Gartska et al. (1985) showed that ovine FSH stimulated hepatic yolk synthesis and yolk deposition into eggs of T. s. parietalis. Krohmer (1986) found evidence that ovine FSH stimulates testicular development (e.g., increased testis mass, seminiferous tubule diameters, and epithelial cell size), and ovine LH stimulates androgenesis in immature water snakes (N. sipedon). Interestingly, however, these effects may depend on season, as bovine LH (but not FSH) stimulates androgenesis in the spring, and bovine FSH (but not LH) stimulates androgenesis in the summer and
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Hormones and Reproductive Cycles in Snakes
fall in adult water snakes (Weil, 1982). Alternatively, ovine and bovine GTHs may be divergent enough in structure to elicit different responses in water snakes, or the differences in the two studies may have to do with the maturity of the snakes. Studies utilizing radioiodinated human FSH showed that this hormone binds readily to snake gonads, and that LH may act at the same binding site as FSH (Licht & Midgley, 1976; 1977; Licht et al., 1977; Licht, Farmer, Gallo, & Papkoff, 1979). Collectively, these studies raised the question as to whether snakes actually have two GTHs, or a single GTH with the actions attributed collectively to FSH and LH in mammals. Although this question has yet to be answered definitively, a recent study using tissues from the viper Bothrops jaracara successfully cloned the snake FSH receptor (FSHR) but failed to identify a receptor in the LH receptor family (Bluhm et al., 2004). It is therefore possible that snakes have a single GTH that stimulates gametogenesis, steroid production, and ovulation, or conversely that the snake FSHR possesses moieties distinct from mammal FSHR that account for its apparent lack of discrimination between FSH and LH. Like studies of GTH receptors, studies of steroid hormone receptors and binding proteins in snakes are limited. Kleis-San Francisco and Callard (1986) identified P4 receptor in the oviduct of Nerodia sp. The presence of a P4 receptor in the oviducts of this viviparous snake may reflect its role in the inhibition of oviductal motility during pregnancy (Callard et al., 1992). Riley and Callard (1988) described a high-affinity, nuclear estrogen receptor in the liver of female Nerodia sp., reflecting the role of this hormone in vitellogenesis in snakes. Only a single study exists on steroid hormone binding proteins (SHBP) in snakes. Riley, Kleis-San Francisco, and Callard (1988) identified a SHBP in Nerodia sp. with a broad binding specificity, binding to E2, P4, T, DHT, and CORT, with medium-high affinity and limited capacity.
2.4. The Neuroendocrinology of Reproduction in Snakes Most of our current knowledge of the neuroendocrinology of reproduction in snakes comes from studies on T. s. parietalis. This species has a distribution of sex steroidresponsive brain neurons similar to other vertebrates (Crews & Silver, 1985). Researchers have utilized histological techniques, hormone implants, lesioning, and surgical removal techniques to study the roles of the APOA, the ventromedial hypothalamus (VMH), the nucleus sphericus (NS, or amygdala), the septum, and the pineal gland (see Section 3.1) in the regulation of reproduction in T. s. parietalis and a few other species.
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Crews, Robker, and Mendonc¸a (1993) found evidence for sexual dimorphism in two brain regions in laboratoryhoused T. s. parietalis: hibernating females have a smaller APOA than hibernating males, and females prevented from hibernating have a smaller NS than males prevented from hibernating. Otherwise, there are no sexual dimorphisms in the brain regions studied. Males show no seasonal or hormone implant-induced variation in APOA, VMH, or NS volume, whereas females show significantly smaller APOA and VMH regions during hibernation than during the rest of the year. Implants of E2 stimulated an increase in the size of the preoptic area (POA) of females (Crews et al., 1993). The APOA is integral in the coordination of stimuli and sexual behaviors in this species (Friedman & Crews, 1985a; 1985b; Krohmer & Crews, 1987a), which is typical of vertebrates in general (Crews & Silver, 1985; Panzica, Viglietti-Panzica, & Balthazart, 1996). Lesions to the APOA in male T. s. parietalis at various times in the annual cycle abolish courtship behavior (Friedman & Crews, 1985; Krohmer & Crews, 1987a). These lesions also disrupt thermoregulatory behavior (Krohmer & Crews, 1987a), and, since exposure to cold winter temperatures followed by heating in spring is essential to the activation of courtship behavior in males (Hawley & Aleksiuk, 1975; 1976), it is probable that the APOA integrates temperature cues with efferent networks important in courtship behavior. The NS and the septum appear to play inhibitory roles in courtship behavior in male T. s. parietalis, as lesions to these areas facilitate courtship behavior in males (Krohmer & Crews, 1987b). Lesions to the NS, but not the septum, lead to significant increases in circulating androgens, and lesions to both areas increase the quantity of sexual granules in the sexual segment of the kidney (Krohmer & Crews, 1987b). The enzyme aromatase is responsible for converting T to estrogens, and it is E2 rather than T that actually stimulates many male sexual behaviors in vertebrates (Roselli, Horton, & Resko, 1985; Vagell & McGuiness, 1997). Therefore, the distribution of aromatase in the male brain can be informative about areas of the brain important in sexual behavior. Krohmer, Bieganski, Baleckaitis, Harada, and Balthazart (2002) found evidence for aromatase immunoreactivity throughout the brain of T. s. parietalis, but it was highly concentrated only in the APOA, the NS, and the septum. Since these are the brain areas associated with courtship behavior, local conversion of androgens to E2 may be important in the activation of sexual behavior. This is surprising, considering that studies have repeatedly shown that mating behavior in male T. s. parietalis is not dependent on androgens (Camazine et al., 1980; Garstka et al., 1982; Crews, 1984; Crews et al., 1984). This discrepancy has yet to be resolved.
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3. INFLUENCES ON THE REPRODUCTIVE CYCLE 3.1. Environmental Influences Environmental stimuli are critical to seasonal reproduction in most animals. In mammals and birds, photoperiod is the primary proximate factor that regulates reproductive activity (reviewed in Dawson, King, Bentley, & Ball, 2001). However, in snakes and other reptiles, temperature appears to be the primary cue that controls reproductive activity. In T. s. parietalis, the duration of exposure to cold temperatures is critical for stimulating reproduction, whereas photoperiod has inconsistent effects (reviewed in Whittier, Mason, Crews, & Licht, 1987). Although the relationship between temperature and reproduction has been known for decades, the hormonal mechanisms by which the interaction operates remain unclear. In cobras (N. naja), hibernation reduces plasma concentrations of numerous hormones, and concentrations return to elevated levels in spring (El-Deib, 2005). Thyroid hormone warrants specific attention as a potential regulator between temperature and reproduction. There is a direct relationship between temperature and iodine uptake by the thyroid (Turner & Tipton, 1972a), and cold temperatures can also reduce target tissue response to thyroid hormones (Turner & Tipton, 1972b). As in other taxa, thyroxine (T4) increases tissue metabolism in snakes (Thapliyal, Kumar, & Oommen, 1975), but it may also have a direct effect on reproduction. In the most thorough study of T4 and reproduction in snakes, Bona-Gallo et al. (1980) demonstrated a pronounced annual T4 cycle with plasma concentrations lowest in winter and substantially elevated during late spring. In males, plasma T4 concentrations were elevated when testes mass and plasma T concentrations peaked in May (when females were vitellogenic), but plasma T4 concentrations did not actually peak until June, which led the authors to suggest that T4 may play a role in testis regression. In females, T4 concentrations peak at the height of ovarian activity (May). Similarly, in male European adders (V. berus), circulating concentrations of T4 are greatest during the period of sexual activity (L. Kelleway, reported in Garstka et al., 1982). However, acutely treating sexually inactive male T. s. parietalis with either T4 or triiodothyronine (T3) led to no changes in reproductive activity within 24 hours (Garstka et al., 1982). Interestingly, thyroid-stimulating hormone (TSH) treatment of hypophysectomized male glossy snakes (Arizona elegans) restored spermatogenesis (Chiu & Lynn, 1971), but the mechanism of this effect was not determined and the effect may simply be a result of cross-reactivity of TSH with GTH receptors. While limited, the data on thyroid hormones hint of a possible role of thyroid hormones in reproduction, including a possible
Hormones and Reproduction of Vertebrates
mechanistic link between temperature and reproductive activity. The pineal gland also deserves specific attention as a potential mediator between environmental conditions and reproduction (Tamarkin, Baird, & Almeida, 1985; Pang et al., 1998). In reptiles, the pineal gland, through the secretion of melatonin, communicates changes in photoperiod and environmental temperature to the body. In turn, melatonin affects circadian cycles, thermoregulation, and reproductive activity (reviewed in Tosini, 1997). In T. s. parietalis, melatonin shows a diel cycle with elevated plasma concentrations during scotophase; however, during hibernation at cold temperatures in total darkness, plasma melatonin is undetectable (Mendonc¸a, Tousignant, & Crews, 1995). Within hours of emergence, circulating melatonin concentrations peak. In the water snake Nerodia rhombifera, cool temperatures reduce plasma melatonin concentrations and remove the difference in plasma concentration between samples taken during the scotophase and those taken during the photophase despite controlling photoperiod at 12 : 12 L : D (light : dark) (Tilden & Hutchison, 1993). Regarding melatonin’s effect on reproductive activity, pinealectomy disrupts gonadal development and regression in Indian chequered water snakes (Natrix piscator) (Haldar & Pandey, 1989). In T. s. parietalis, males that court during the spring have normal melatonin diel cycles (with increased concentrations during the scotophase), whereas males that do not court have reversed diel cycles (Mendonc¸a, Tousignant, & Crews, 1996a). Pinealectomizing males prior to hibernation reduces courtship behavior the following spring (Nelson, Mason, Krohmer, & Crews, 1987; Crews, Hingorani, & Nelson, 1988; Mendonc¸a et al., 1996a). Further, treatment with exogenous melatonin inhibits courtship behavior (Lutterschmidt, LeMaster, & Mason, 2004). Together, these results demonstrate that gonadal development and courtship behavior depend on normal diel cycles of melatonin. However, pinealectomy in spring does not alter existing courtship behavior, suggesting that melatonin cycles are critical for providing the initial stimulus for courtship but not for modulating courtship once initiated (Mendonc¸a, Tousignant, & Crews, 1996b). Seasonal melatonin cycles vary among Thamnophis sirtalis populations with differing climate conditions and courting cycles, although the relationship between the inter-population variations in melatonin and courtship is unclear (Lutterschmidt & Mason, 2008).
3.2. Social Influences Although descriptive studies on seasonal hormonal cycles in snakes have been conducted in numerous species, it is remarkable that studies that manipulate sex steroid profiles are almost completely limited to studies of T. s. parietalis.
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Hormones and Reproductive Cycles in Snakes
As a result, knowledge of how specific reproductive behavioral components (e.g., attractivity, courtship, receptivity, copulation) are regulated and how these events influence the hormonal milieu of the individuals experiencing them comes almost exclusively from this species. In T. s. parietalis, mating occurs at the time of emergence when both males and females have low plasma concentrations of sex steroids (reviewed in Mendonc¸a & Crews, 1996). The onset of male courtship behavior seems to be independent of T, and, instead, dependent on warming upon post-hibernation emergence (Hawley & Aleksiuk, 1975; 1976). In fact, males that have been castrated for as long as three years will still court females after emergence from hibernation. Courting by males is enhanced by the presence of other males (Joy & Crews, 1985). In females, attractivity is not affected by ovariectomy during either the fall or the hibernation period preceding emergence, but is abolished when females are ovariectomized the spring prior to emergence. Receptivity, on the other hand, is inhibited by ovariectomy at any of the three time points. Receptivity was reinstated in ovariectomized females that were given exogenous E2 one hour prior to emergence, but not in females treated approximately one week after emergence (Mendonc¸a & Crews, 1996). Thus, reproductive behavior of the two sexes seems to be regulated by different stimuli. Male courting is hormonally independent and driven by environmental cues (both thermal and social) while female reproductive behavior is affected by a combination of environmental conditions and the presence of at least low levels of E2. It is important to reiterate that the seasonal hormonal patterns of other snake species can differ greatly from those of T. s. parietalis (see Section 2), and thus the hormonal and social effects on reproductive behavior in this discussion, which is based predominantly on T. s. parietalis, should not be broadly applied to snakes in general. Whereas environmental stimuli tend to regulate the seasonality of reproduction, social cues often fine tune the timing of reproduction. Snakes are mostly solitary animals, and thus locating a mate is not a foregone conclusion. To facilitate mate acquisition, it has been demonstrated in many species that females produce pheromones that enable males to trail them (LeMaster, Moore, & Mason, 2001; Chapter 6, this volume). Methyl ketones secreted from the skin of the female’s dorsum are also critical to female attractivity and induce intensive courtship by males (reviewed in Mason 1993; Mason et al., 1989). These sexual signals are highly specific and thus can contribute both to species isolation (Ford & O’Bleness, 1986; Mason, 1993; Shine, Reed, Shetty, LeMaster, & Mason, 2002; Shine, Phillips, Waye, LeMaster, & Mason, 2004) and population isolation (LeMaster & Mason, 2003), as well as enable males to distinguish female body size (LeMaster & Mason, 2002) and female mating history (O’Donnell, Ford,
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Shine, & Mason, 2004), and orient copulatory posture (Shine, O’Connor, & Mason, 2000a). Additionally, male snakes possess skin lipids that inhibit female pheromoneinduced courtship by other males (Mason et al., 1989). Brown treesnakes (Boiga irregularis) are atypical in that both males and females show courtship behavior (Greene & Mason, 2000). In this species, females use a combination of an attractiveness pheromone and an inhibitory pheromone, the latter of which is present in cloacal secretions, to regulate courtship by males; however, cloacal secretions from males or females do not inhibit female courtship (Greene & Mason, 2003). The act of copulation leads to numerous physiological changes. As sperm have been delivered during copulation, future copulations are of much less value to the female and would negatively affect the reproductive success of the first male (but see review on multiple paternity in reptiles by Uller and Olsson (2008)). The ejaculate of male T. s. parietalis contains secretory granules from the renal sex segment that combine with cloacal secretions from the female to form a gelatinous copulatory plug at the oviductalecloacal junction. Copulatory plugs are relatively rare among snakes, and may reflect costs associated with high density mating aggregations (reviewed in Uribe, Gonza´lez-Porter, Palmer, & Guillette, 1998). The copulatory plug provides a temporary physical barrier against future matings. Inhibition of attractivity is apparently a result of chemical cues in the semen, as females smeared on the back with semen, but not ones smeared with a copulatory plug, failed to elicit courting responses from males (Shine, Olsson, & Mason, 2000). Although the seminal chemical cues have not yet been identified, it has been proposed that PGF2a may play a role. Prostaglandin F2a is present in the semen of other vertebrates. Female plasma concentrations of PGF2a are elevated immediately after copulation in T. s. parietalis, and injection of PGF2a decreases attractivity and receptivity in unmated females. Mechanical stimulation of the reproductive tract can stimulate endogenous release of prostaglandins, and thus the female may provide an alternate or additional source of PGF2a (reviewed in Mendonc¸a & Crews, 2001). Regardless of whether PGF2a is involved, physical stimulation of the cloaca is important in altering attractivity and receptivity at copulation in T. s. parietalis (reviewed in Mendonc¸a & Crews, 2001). More recent work has demonstrated that sensory input from the cloaca at copulation alters patterns of metabolism in the VMH, an area of the brain often associated with female sexual behavior (Mendonc¸a, Daniels, Faro, & Crews, 2003). In addition to playing a role in altering attractivity and receptivity, physical cues during mating stimulate progression of reproductive activity in the ovary. Shortly after copulation, female T. s. parietalis have a surge in plasma E2 and undergo ovarian recrudescence. These
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responses can be prevented by removal of cloacal sensation via spinal transection or the application of local anesthetic prior to mating (Mendonc¸a & Crews, 1990). Similarly, female blood pythons (Python curtus) will not undergo vitellogenic growth of follicles without the presence of, and likely copulation by, a male (DeNardo & Autumn, 2001). It is intriguing that, at least in these two distantly related species, males are involved in promoting early follicular growth despite the fact that sperm is not needed for fertilization until weeks later, at ovulation. However, some female T. s. parietalis undergo ovarian recrudescence without mating in spring, at least in some years (reviewed in Mendonc¸a & Crews, 1990). The predominant social influences on reproduction are a result of maleefemale interactions, but maleemale interactions can also alter reproductive activity. Although snakes neither form social groups nor defend established territories, encounters between two males can lead to agonistic interactions. Maleemale combat is widespread among snakes (Shine, 2003), with the combat of viperid snakes being the best documented. In V. berus, chemical signals from the female elicit courtship by males, whereas other skin-derived chemical cues from the male appear to induce combat behavior (Andre´n, 1986). Among crotaline snakes, combat is very ritualistic. In the copperhead (A. contortrix), individuals that lose combats not only flee the site, but courtship remains inhibited for at least 24 hours (Schuett, 1996). These combat losers have increased plasma CORT concentrations, which may provide a mechanistic link between combat loss and courtship suppression (Schuett, 1996; Schuett & Grober, 2000). A fascinating maleemale interaction exists in the unique mating system of T. s. parietalis. This species shows no maleemale aggression, but instead some males act as ‘she-males’ that exhibit altered courtship behavior and are attractive to other males (Mason & Crews, 1985). The significance of this reproductive strategy is unclear. It was initially postulated that ‘she-maleness’ is relatively permanent and may serve as a distractant to other males present in the mating ball and thus enhance the mating success of the she-male (Mason & Crews, 1985). More recently, Shine, Harlow, LeMaster, Moore, & Mason (2000) described the scenario where she-maleness is a transient stage of recently emerged males, since they have poor locomotory performance and thus would not be competitive against other males in accessing copulations. By being she-males, newly emerged snakes would not waste energy on fruitless courting and they would also induce other males to waste energy by courting the shemales (Shine et al., 2000c). Regardless of the ultimate strategy, the she-male phenomenon, including both the inhibition of courting behavior and the attractiveness of she-males to other males, is driven by male production of a pheromone similar to that of females (Mason & Crews,
Hormones and Reproduction of Vertebrates
1985; Shine et al., 2000c). The regulatory mechanism for the production of this pheromone is not known, but it may involve T, since she-males have higher plasma T concentrations than do normal males (Mason & Crews, 1985). Additionally, the involvement of brain aromatase should be considered, since snakes collected from the den in early winter show high concentrations of aromatase in brain regions involved in the control of courtship and mating (Krohmer et al., 2002).
3.3. Physiological Influences Beyond environmental and social influences, reproductive activity is also influenced by non-reproductive aspects of the individual. Because of the high energy demand associated with reproduction, body condition is especially important in influencing the reproductive activity of snakes. Many snakes use a capital breeding strategy rather than one of income breeding (reviewed in Gregory, 2006). That is, in capital breeders, reproductive activity is based on energy reserves more so than current food intake (Drent & Daan, 1980). In capital breeders, females need to attain a minimum threshold of body condition prior to committing to a reproductive event, although this threshold may vary among years (Naulleau & Bonnet, 1996; Madsen & Shine, 1999). The relationship between body condition and reproductive activity is well-established; however, little is known about the hormonal regulation of this relationship in snakes. In V. aspis, females in poor body condition have low circulating E2 concentrations and are not sexually receptive, but once a female is beyond a critical threshold of body reserves, she exhibits elevated plasma E2 concentrations and is receptive (Aubret, Bonnet, Shine, & Lourdais, 2002). Males, however, do not show a threshold response, as all males had detectable plasma T concentrations and displayed courtship behavior, but both plasma T and courtship behavior increased proportionately with body condition (Aubret et al., 2002). The hormone leptin might be considered an important link between energy state and reproduction. Leptin occurs in a diverse group of vertebrates and is known, at least in mammals, to be involved in the energy storesereproduction relationship (reviewed in Paolucci, Rocco, & Varricchio, 2006) and might play a similar role in snakes. In addition to body reserves influencing reproduction, many snake species show size-dependent fecundity with larger females producing a greater clutch mass (Ford & Seigel, 1989; Madsen & Shine, 1994, but see Lourdais et al., 2002). This relationship can lead to sexual size dimorphism with females being larger than males. Many factors including age, food intake, and possibily hormones can affect growth rate and body size (reviewed in Taylor & DeNardo, 2005) and thus influence sexual size dimorphism.
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There is some evidence that, in T. sirtalis, where females are larger than males, estrogens promote growth while T suppresses it (Crews, Diamond, Whittier, & Mason, 1985; Lerner & Mason, 2001). Stress is also known to have a dramatic effect on reproduction in reptiles, including snakes (reviewed in Moore & Jessop, 2003; Chapter 7, this volume). Commonly, stress negatively influences reproduction, either through direct or indirect effects of elevated CORT on reproductive hormones or other components of the reproductive system (Wingfield et al., 1998). In brown treesnakes (B. irregularis), capture and placement in outdoor enclosures under ambient conditions terminates spermatogenesis and development of the sexual segment of the kidney (Aldridge & Arackal, 2005). Plasma steroids were not assessed in this study; however, trapping and confinement of B. irregularis leads to in an increase in plasma CORT concentrations (Mathies, Felix, & Lance, 2001). Further, elevated plasma CORT was thought to explain, at least in part, low reproductive activity in a freeliving population of B. irregularis on Guam (Moore et al., 2005). Apparently, however, B. irregularis can acclimate to captivity, as individuals held long-term in captivity are reproductively active and have low plasma CORT concentrations (Moore et al., 2005). The susceptibility of an animal to stress can vary seasonally. Unlike B. irregularis, which has an extended breeding season, T. s. parietalis in Manitoba, Canada, has an extremely short reproductive period. During the nonbreeding summer, males respond to capture stress with elevated CORT and reduced T. However, during the primary reproductive period in spring and the secondary mating and pre-hibernation period in fall, the hormonal response to stress is variable but, in general, reduced (Moore, Greene, & Mason, 2001; Lutterschmidt & Mason, 2005; but see Moore et al., 2000). In the conspecific redspotted garter snake (Thamnophis sirtalis concinnus), which has an extended breeding season in Oregon, capture during the reproductive season induces the classical stress response of increasing CORT while decreasing T (Moore et al., 2001; Lutterschmidt et al., 2004). Interestingly, in the one study in which capture induced an elevated CORT response in the Manitoba population of T. s. parietalis during the breeding season, there was no effect on courtship behavior (Moore et al., 2000). However, in separate studies, exogenous treatment with CORT inhibits courting (Moore and Mason, 2001; Lutterschmidt et al., 2004). Although inconsistencies exist, the overall conclusion from these studies is that, like other vertebrates, snakes haves a classical stress response, but the hormonal and or behavioral response may be attenuated during the reproductive season for populations in which reproductive opportunities are limited. Surprisingly, the shift from non-responsiveness to responsiveness to capture stress can be abrupt, as the
classical hormonal response to capture stress is intact in male T. s. parietalis that are dispersing from the mating sites to feeding sites (Cease, Lutterschmidt, & Mason, 2007). Analyzing the effects of CORT as a modulator for the stress response (and thus an inhibitor of reproduction) is complicated by CORT’s most basic function as an energy mobilization hormone. As such, elevated CORT may augment reproduction (reviewed in Moore & Jessop, 2003). In male T. s. parietalis, baseline plasma CORT concentrations are elevated during the spring breeding season (Moore et al., 2001), and it is thought that this elevated CORT facilitates reproduction by mobilizing much-needed energy stores to sustain costly courtship activity during this period of aphagia (Moore & Jessop, 2003).
3.4. Embryonic Influences Nearly all studies of snake reproductive endocrinology have examined the relationships between behavior, physiological state, and hormones in adults, but it is important to at least briefly consider the embryonic snake. Many reptiles, including lizards, are known to have environmental sex determination in which environmental conditions, particularly temperature, influence sex by altering embryonic hormone exposure. Environmental sex determination has not been identified in any snake (reviewed in Janzen & Paukstis, 1991). However, exposure of embryos to steroids may alter other embryonic traits. Corticosterone treatment of gravid female Thamnophis elegans led to reduced embryonic and early offspring survivorship as well as changes in morphological and behavioral characteristics of the offspring (Robert, Vleck, & Bronikowski, 2009). Also in T. elegans, prenatal sex ratio affects subcaudal scale counts of female offspring in one of two populations (Osypka & Arnold, 2000). Although limited, these studies together suggest that snakes are susceptible to the influences of sex steroids prior to birth.
4. FUTURE DIRECTIONS TO AUGMENT EXISTING KNOWLEDGE As snakes are a distinct suborder of vertebrates with nearly 3000 species, it is important that we obtain a thorough understanding of their reproductive endocrinology. It is imperative that we distinguish traits that are relatively conserved among snakes from those that may be unique to individual species or small groups of species. Thus, we are in great need of data from a larger diversity of snake species. Currently, the reproductive endocrinology of snakes is grossly biased (and possibly misrepresented) by data from a single species, T. s. parietalis. Studies of T. s. parietalis have led the way in our understanding of snake reproductive endocrinology, and such leadership in the field
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must continue. However, evaluating large taxonomic groups based on the study of a single species is dangerous. Applying findings based on studies of T. s. parietalis to snakes in general may be especially problematic given that T. s. parietalis has numerous traits that are atypical of most snakes, including its high latitude, cold climate distribution, and its mating system in which reproduction entails massive mating balls formed immediately at female emergence. Strong bias resulting from the evaluation of a single or few species to represent larger taxonomic groups is unfortunately not unique to snakes, as ‘frog’ physiology is largely based on data from the African clawed frog (Xenopus laevis) or American bullfrog (Lithobates (Rana) catesbeiana) and ‘turtle’ physiology on red-eared sliders (Trachemys scripta). Even avian and mammalian physiology are biased by studies on the domestic chicken (Gallus gallus) and on the laboratory mouse (Mus musculus) and rat (Rattus norvegicus), respectively. The bias regarding snake study species goes beyond T. s. parietalis in that data from other species come disproportionately from temperate species, particularly viperids. There is a great need for studies of the reproductive endocrinology of tropical snakes and snakes other than Viperidae and Colubridae (e.g., Scolecophidia, basal Alethinophidia, and the speciose Elapidae). Beyond being a relatively large taxonomic group, snakes show diverse reproductive strategies, and this diversity provides a unique opportunity for comparative studies to better understand reproductive endocrinology of vertebrates in general. Data collected thus far strongly indicate that snakes utilize a hypothalamicepituitarye gonadal hormonal axis similar to that of other vertebrates, at least on a broad scale. However, the diversity of reproductive tactics within the squamates (lizards and snakes) is unmatched by other major terrestrial taxonomic groups. While the group is ancestrally oviparous, viviparity has evolved over 100 times among squamates (Shine, 1985). Additionally, some squamates, including snakes, are parthenogenic, with the ability to produce offspring asexually. Parthenogenesis is best documented as a result of hybridization and polyploidy and thus an obligate process (reviewed in Dowling & Secor, 1997). Such forms of parthenogenesis have been well documented in lizards (e.g., particularly in lizards of the genus Aspidoscelis (Price, LaPointe, & Atmar, 1993)) as well as the blind snake Ramphotyphlops braminus (Ota, Hikida, Matsui, Mori, & Wynn, 1991). However, numerous squamates including snake species are capable of facultative parthenogenesis (Dubach, Sajewicz, & Pawley, 1997; Schuett et al., 1997b; Groot, Bruins, & Breeuwer, 2003). That is, species that are diploid and normally sexually reproductive can, at times, reproduce asexually, producing diploid offspring. Interestingly, there appear to be inter-species differences in the mechanism by which diploidy is
Hormones and Reproduction of Vertebrates
maintained despite meiosis, since in some species only males are produced while in others only females result (reviewed in Groot et al., 2003). While understanding the mechanisms for such unique reproduction among vertebrates may prove extremely valuable to identifying the regulators of snake reproduction, studies on facultative parthenogenesis are logistically difficult because it is not known what stimulates females to reproduce asexually rather than foregoing reproduction when a mate is not present. Similarly valuable, but less extreme physiologically, are the variations in the timing and frequency of reproduction among snakes. Specific environmental cues (e.g., increasing temperatures after a cool period) are thought to be critical in regulating snake reproductive cycles. However, some species can have multiple reproductive events in a given year and under different environmental conditions. The reproductive events may be limited to multiple mating periods (e.g., as described earlier for many rattlesnakes) or entail the production of multiple clutches. Multiple clutch production is typically limited to captive snakes (Ford & Seigel, 2006), although it has been documented in wild keelbacks, Tropidonophis mairii (Brown & Shine, 2002). This does not negate the value in using that potential to understand reproductive stimuli in snakes. To more fully examine the details of snake reproductive endocrinology, it is imperative that studies move away from the predominant use of descriptive studies and utilize manipulative experiments. Only in T. s. parietalis have a considerable number of manipulative studies been conducted to better understand the causal relationships between reproductive behavior, gonad function, and hormones. Potential manipulative approaches are numerous and range from hormone modulation (e.g., organ removal, hormone treatment, hormone antagonist treatment), to modification of organism state (e.g., energy balance, water balance, stress, function of senses), to environmental manipulation (e.g., temperature, photoperiod, humidity, rainfall, pheromonal cues, social structure). Conducting such experiments in both the laboratory where variables can be more tightly controlled and in the field where conditions are more natural will be essential. Given that data thus far collected suggest that the snake reproductive axis is generally similar to that of other vertebrates, it is not too risky to presume that stimuli for reproductive activity act through the brain. Unfortunately, literature on the reproductive neuroendocrinology of snakes is extremely limited. It is astounding that we do not yet know which, or even how many, GTHs exist in any snake, let alone among snakes. Our knowledge of snake hypothalamic hormones involved in reproduction is nearly non-existent. Although multiple variants of the GnRH decapeptide exist among vertebrates, only chicken-I GnRH has been identified, and
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only from a single snake species: not surprisingly, T. s. parietalis (Sherwood & Whittier, 1988; Smith et al., 1997). The failure to detect other GnRH variants from snakes is likely due to a lack of effort more than the variants not being present, since mammalian GnRH has yet to be identified in any reptile despite its presence in fishes, amphibians, and mammals (King & Millar, 1997). Similarly, gonadotropin-inhibiting hormone (GnIH) has yet to be found in any reptile. Since its recent discovery in quail (Tsutsui et al., 2000), GnIH has been identified in multiple species of fishes, birds, and mammals (reviewed in Ubuka, McGuire, Calisi, Perfito, & Bentley, 2008; Volume 4, Chapter 2). Despite its novelty, gonadotropin-inhibiting hormone is potentially an important regulator of reproduction, and thus a thorough understanding of the regulation of reproduction in snakes is likely impossible without the consideration of GnIH. Numerous other hormones are worthy of study to assess their roles in snake reproduction. Hormones such as prolactin (Ng, Lee, Cheng, & Wong, 1990), arginine vasotocin (Silveira, Schiripa, Carmona, & Picarelli, 1992), and growth hormone (Ng, Lee, Cheng, & Wong, 1993) have been identified in at least one snake species, yet their potential roles in reproduction have thus far been completely ignored. Hormones such as leptin and relaxin have yet to even be identified in snakes, yet their existence is likely considering their presence among diverse vertebrates. One final area of study that has received considerable attention in other taxa, especially birds, is that of maternal transfer of sex steroids to offspring via the yolk. Since initially described by Schwabl (1993), maternal steroid transfer has been shown to have dramatic effects on offspring phenotype (reviewed in Radder & Shine, 2007), although the mechanisms of transfer between the female and the yolk as well as the yolk and the embryo are poorly understood (Moore & Johnston, 2008). Despite this broad attention, the demonstrated biological importance of yolk transfer has yet to be explored in snakes. Clearly, the study of snake reproductive endocrinology is in its infancy. Regardless, contributions by numerous investigators have expanded our understanding over the last several decades. It is essential to build on this foundation, as doing so will not only provide comparative data, but provide knowledge that can be utilized to assist in efforts to ensure species persistence as well as enhance general knowledge of reproductive endocrinology across all vertebrates.
ACKNOWLEDGEMENTS We thank M. Rockwell Parker and Gordon W. Schuett for reviewing drafts of this manuscript.
ABBREVIATIONS APOA CORT DHT E2 FSH FSHR GnIH GnRH GTH LH NS P4 PGF2a POA SHBP T T3 T4 TSH VMH
Anterior preoptic area of the hypothalamus Corticosterone Dihydrotestosterone 17b-estradiol Follicle-stimulating hormone Follicle-stimulating hormone receptor Gonadotropin-inhibiting hormone Gonadotropin-releasing hormone Gonadotropin Luteinizing hormone Nucleus sphericus Progesterone Prostaglandin F2a Preoptic area Steroid hormone-binding protein Testosterone Triiodothyronine Thyroxine Thyroid-stimulating hormone Ventromedial hypothalamus
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Chapter | 13
Hormones and Reproductive Cycles in Snakes
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Chapter 14
Endocrine Disruption of Reproduction in Reptiles Ashley S.P. Boggs, Nicole L. Botteri, Heather J. Hamlin, and Louis J. Guillette, Jr. University of Florida, Gainesville, FL, USA
SUMMARY The vertebrate endocrine system regulates many aspects of development, growth, and reproduction. Endocrine-disrupting chemicals (EDCs) are exogenous chemicals that mimic hormones or alter hormonal-signaling processes. Endocrine-disrupting chemicals modify regulation by affecting hormone synthesis, transport, or clearance, or by altering receptor number or binding. Depending on concentration, timing, and duration of exposure, effects on physiological processes can be permanent or transient. Initially, EDC research on reptiles focused on internal concentrations of EDCs, but reptiles are now considered ideal species for the study of endocrine disruption because of their wide range of habitats, diet, parity mode, and modes of sex determination. The majority of research on reptiles focuses on species from aquatic habitats. There is a large and growing body of research on EDC actions in alligators and turtles. Little is known about the effects of EDCs on squamates. The value of reptiles as model species for endocrine disruption is broadening the knowledge of EDCs and how these chemicals affect wildlife and human public health.
1. INTRODUCTION The endocrine system controls nearly every aspect of vertebrate life and is instrumental in regulating physiological processes such as metabolism, development, reproduction, tissue function, and behavior. It is a highly integrated system requiring proper signals (hormones) of the right magnitude, reaching the intended receiver (receptor) at the appropriate time. Hormones affect cells through their interactions with receptors. Often these receptors are either transcription factors that directly govern expression of gene pathways, or they initiate signaling cascades that then direct cellular functions. The endocrine system is tightly regulated and relies heavily on appropriate signaling to drive physiological and developmental processes. An exogenous chemical that changes these signals can have considerable impacts on animal health and functioning. Hormones and Reproduction of Vertebrates, Volume 3dReptiles Copyright Ó 2011 Elsevier Inc. All rights reserved.
Although we have been aware for decades that exogenous chemicals are capable of disrupting the endocrine system (e.g., natural goitrogens and phytoestrogens), our appreciation of the wide array of man-made chemicals capable of altering endocrine function only became widely known in the early 1990s (Colborn & Clement, 1992). Such chemicals are now commonly called endocrine-disrupting chemicals (EDCs). Endocrine-disrupting chemicals act as hormone analogs or interfere with the synthesis, transport, storage, or clearance of hormones (Guillette, Edwards, & Moore, 2007; Milnes & Guillette, 2008) and include compounds such as pesticides, plasticizers, fungicides, heavy metals, and pharmaceuticals (Guillette & Iguchi, 2003; Guillette et al., 2007; Zoeller, 2007a; Davey et al., 2008). The physiological consequence of EDC exposure depends greatly on the timing of exposure. Organizational changes are irreversible and often occur when animals are exposed during development (Guillette, Crain, Rooney, & Pickford, 1995). These changes are usually related to disruption during organogenesis. Occasionally, effects due to embryonic exposure are not manifest until another major organizational event such as puberty occurs. For example, hypothyroid male children develop large but normal testes, yet after puberty fibrosis of the seminiferous tubules occurs, causing infertility (Jannini, Ulisse, & Darmiento, 1995). Therefore, although a neonate could appear physiologically normal, the second wave of major hormonal cues can trigger deleterious physiological changes, including the failure of the hormone target to respond appropriately. Organizational disruption often occurs during distinct periods of developmentdwindows of vulnerabilitydwhen tissues are responsive to hormonal cues (Guillette et al., 1995). These windows of vulnerability suggest that tissues can be responsive to hormones at certain times when that signal is necessary for development, whereas signals received outside this period potentially could have no 373
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effect. These critical windows can vary depending on the species examined and the contaminants involved. Alternatively, exposure to EDCs outside of critical windows of vulnerability can still result in considerable health consequences. Endocrine disruptions in adult animals, for example, can reduce fertility, alter metabolism, change sex-steroid hormone concentrations, and reduce sperm count (Guillette et al., 1995; Gray et al., 2006; Trokoudes, Skordis, & Picolos, 2006). These disruptions, which do not necessarily result in permanent changes to the structure and function of the organism, are termed activational disruptions. In juveniles, activational disruptions can lead to reduced growth, altered steroid profiles, and reduced hepatic function. Endocrine-disrupting chemicals can disrupt the endocrine system through several mechanisms including interfering with actions of occupied receptors at several levels, up- or downregulation of hormonal synthesis, altering plasma hormone carrier proteins, changing clearance or hepatic biotransformation of hormones, and altering the expression or number of hormonal receptors (Figure 14.1). One mechanism of EDC action is alteration of the synthesis
Hormones and Reproduction of Vertebrates
of hormones. Hormone production can be altered by modifying the enzymes and other molecules involved. One example involves the upregulation of aromatase (P450aro), the enzyme that converts testosterone (T) to estradiol (E2), by the pesticide atrazine (Hayes et al., 2002; Fan et al., 2007). Atrazine induces the steroidogenic factor 1 (SF-1) pathway, which increases the production of P450aro, leading to a decrease in androgens and an increase in estrogens (Fan et al., 2007). Contaminants also can disrupt hormone signaling by direct interference with the hormone receptor (Figure 14.2). Endocrine-disrupting chemicals can bind to the receptor, triggering the normal response of the natural ligand (agonist), or can bind and block the receptor from the natural ligand without activating it and thus preventing transcription (antagonist). A wide array of chemicals (e.g., pesticides and plasticizers) bind to the vertebrate estrogen receptor (ER) and can act as either an agonist or antagonist (Soto et al., 1995; Rooney & Guillette, 2000). Further, a growing list of chemicals (e.g., fungicides and phthalates) can alter androgen receptor (AR) functioning, with many acting as anti-androgens (Gray et al., 2006).
FIGURE 14.1 Schematic representation of the hypothalamus–pituitary–gonadal (HPG) axis. Steroid hormones interact with receptors in target tissues, altering gene expression profiles. Plasma steroid hormone concentrations are affected by plasma-binding proteins synthesized by the liver as well as by hepatic biotransformation and clearance. To date, a number of environmental contaminants have been identified that can alter hormonal synthesis, storage, clearance, and receptor binding (examples are given in the double-line boxes). Potentially, contaminants also could alter hypothalamic and pituitary regulation of gonadal steroids, as observed in mammals, but there are no data for reptiles.
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FIGURE 14.2 Endocrine disruption can occur at various points of hormone signaling but many contaminants have been shown to act as exogenous estrogens or anti-androgens. Hormones travel through the bloodstream attached to plasma proteins. Steroids, such as estradiol (E2) and testosterone (T), leave the bloodstream and cross the lipid bilayer to enter the cell. Once inside the nucleus, a hormone attaches to its receptor (R). The hormonereceptor complex then attaches to the hormone response element (RE) on the DNA to begin transcription of mRNA. Endocrine-disrupting chemicals (EDCs) can cause disruption by binding to plasma transport proteins or the receptors preventing binding of the hormone. They also can disrupt linkage of the receptors to the response element in the promotor. All these processes can alter or halt transcription.
Carrier or binding proteins, found in the circulatory system, retain hormones in the circulation. Unbound lipophilic hormones such as steroids do not travel well in hydrophilic blood, and are rapidly removed from circulation by the liver and excreted. Hydrophilic carrier proteins attached to hormones allow for more efficient transport. Therefore, altering the affinity of a carrier protein for its hormone ligand can change its availability and clearance (Cheek, Kow, Chen, & McLachlan, 1999). The upregulation of metabolic enzymes and increased excretion following EDC exposure is part of the body’s natural defense. Within the liver, steroids and other compounds are chemically modified prior to excretion. Xenobiotics are detected by a promiscuous receptor that upregulates metabolic pathways to remove the invading chemical (Blumberg et al., 1998). Because these receptors are not highly specific, they can also increase clearance of endogenous steroids, resulting in an overall reduction of circulating steroid concentrations. Indeed, EDCs also are known to alter the concentrations of metabolic enzymes,
thereby altering retention or clearance of both endogenous steroids and EDCs alike (Li, Dehal, & Kupfer, 1995; Guillette & Gunderson, 2001; Gunderson, Oberdorster, & Guillette, 2004). The complexities surrounding EDC modes of action are further compounded by mixtures. Environmental contamination is rarely due to a single EDC, but rather to a milieu of EDCs of varying concentrations and potency. Whereas the traditional toxicological approach considered the additive effects of mixtures, the study of endocrine disruption has challenged this approach by demonstrating the synergistic effects of some EDCs. Polychlorinated biphenyl (PCB) congeners studied in combination can display synergistic effects on estrogenic activity through increased potency above an additive effect (Bergeron, Crews, & McLachlan, 1994). A possible mechanism for this response could be due to different pathways of disruption such as combination of an estrogen mimic and an androgen antagonist to produce a larger feminizing effect than an estrogen mimic alone. Other experiments
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have demonstrated a decreased effect of mixtures, possibly due to competition for a similar pathway among the compounds in the mixture (Carpenter et al., 1998). Thus, when designing experiments and assessing data, it is necessary to realize the importance of confounding factors introduced by mixtures.
2. REPTILES AS MODELS OF EDC EXPOSURE Over the past twenty years, reptiles have received increasing attention as biomonitors of contaminantinduced endocrine disruption. The natural history of reptiles, including their persistence in a wide variety of habitats and wide geographic distribution, makes them suitable candidates for studies of endocrine disruption within a broad range of species. Reptiles can be found in widely different ecosystems including arboreal, terrestrial, and aquatic habitats. This diversity allows for the existence of a variety of physiological responses to contaminant exposure. Additionally, many reptiles have high site fidelity (McNease & Joanen, 1975), permitting a unique opportunity to follow individuals through different life stages in their local environment. Reptiles occupy varying dietary niches, allowing research on the effects of trophic level and accumulation of toxicants. Many reptiles are long-lived scavengers or top-tier predators and are excellent models for the study of bioaccumulation and biomagnification of contaminants (Selcer, 2006). Common reptilian diets include fish, which have the potential to be highly exposed to aquatic EDCs, or insects, which are the targets of many pesticides. Additionally, reptiles are poikilotherms, a characteristic that affects their ability to metabolize and clear contaminants (Selcer, 2006). This is evidenced by the bioaccumulation and biomagnification of contaminants in reptiles to levels equal to or greater than those demonstrated in mammals and birds (Hall & Henry, 1992). Similarly to mammals, some groups of reptiles undergo genotypic sex determination (GSD), in which sex is genetically programmed. In other groups of reptiles, sex is determined by the incubation temperature of the egg. Crocodilians and some turtles, reptiles that commonly demonstrate temperature-dependent sex determination (TSD), appear to be more susceptible to endocrine disruption because of a suggested increased role of estrogens and androgens during development of the gonad compared to GSD (Crews, Bergeron, & McLachlan, 1995; Crews, 2003). In addition, reptiles display several parity modes (oviparity or viviparity; see Chapter 9, this volume). The variety in both sex determination and parity modes in reptiles allows for diverse comparisons of physiological responses to EDC exposure. These comparisons among oviparous and viviparous reptilian
Hormones and Reproduction of Vertebrates
species can help clarify the complexity surrounding the maternal transfer of contaminants. Oviparous animals liberate their fat stores to generate yolk nutrients; thus, maternal contaminants that bioaccumulate in the mother can be transferred to the yolk (Rauschenberger, Sepulveda, Wiebe, Szabo, & Gross, 2004; Rauschenberger et al., 2004b; Rauschenberger, Wiebe, Sepulveda, Scarborough, & Gross, 2007). Contaminants present in the egg at oviposition are absorbed throughout embryonic development and affect development in combination with other biotic and abiotic factors (Figure 14.3). Lipoproteins in the eggs of birds, turtles, and other oviparous vertebrates have been shown to contain contaminants such as 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) (a metabolite of dichlorodiphenyltrichloroethane (DDT)), PCBs, dioxins, and dibenzofurans at concentrations higher than in the ambient environment due to maternal concentration of these contaminants (Fox, 1992). Because reptiles have thick and poorly permeable skin, topical exposure of EDCs to adult reptiles is presumed to be limited. However, the composition of reptilian eggshells permits effective topical application of EDCs to the embryo, making embryonic exposure scenarios possible (Kern & Ferguson, 1997). In some viviparous reptiles, embryonic exposure to contaminants could be a continuous and dynamic process facilitated through placental transfer. This range in reproductive strategies provides numerous experimental strategies in studying endocrine disruption of embryonic development. Although the diversity of life-history traits, ecological niches, and parity modes of reptiles assist researchers in identifying a range of responses to EDCs, this diversity of information is often difficult to synthesize in order to determine common mechanisms of disruption. Much of today’s research uses a multi-system approach, evaluating a variety of effects ranging from gene expression to population level effects. Responses of individuals, and even populations, to EDCs could be attributed to changes in cellular regulation. If cells produce incorrect signals, often in the form of mRNA or proteins, tissues and organs manifest these changes through altered tissue morphology and function. Though organisms are not the sum of their parts, by implementing a multi-system approach, researchers have begun to identify mechanisms of disruption common to reptiles and other vertebrates alike (see Volume 5, Chapter 14).
3. ENDOCRINE-DISRUPTING CHEMICALS (EDCs) AND REPTILIAN REPRODUCTION Our knowledge concerning the impact of EDCs on reptilian reproduction has grown substantially over the last decade and a half. Contaminant-induced endocrine disruption has
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FIGURE 14.3 The developing embryo is subject to both environmental and maternal influences during development in oviparous reptilian species. Endocrine-disrupting chemicals from the environment and the mother (via her diet and deposition in the yolk) can alter embryonic development.
been linked to a suite of endocrine effects including altered fertility, reduced viability of offspring, impaired hormonal activity, modified reproductive anatomy, and, more recently, altered steroidogenic enzyme and steroid hormone receptor gene expression. For many years, alligators and turtles have been the predominant reptilian models for EDC research. Recent advances have expanded our knowledge of reptilian exposure to EDCs to include other crocodilians, snakes, and lizards. Advances also have been made in understanding the organizational effects of embryonic EDC exposure and establishing the permanence of effects of EDCs throughout ontogeny. Recent studies have described developmental abnormalities resulting from in ovo EDC exposure via maternal transfer to the embryo. Such studies provide evidence that endocrine disruption can result in permanent organizational changes.
3.1. Tissue Concentrations of Endocrinedisrupting Chemicals (EDCs) in Reptiles The American alligator has been a useful model for endocrine disruption and many of the disruptive effects identified in alligators have paralleled effects later seen in humans and other mammals. Since the early 1990s, researchers have been investigating the adverse reproductive effects on alligator populations inhabiting Lake Apopka, FL. Lake Apopka, subject to nearly forty years of agricultural and pesticide runoff from adjacent agricultural operations, suffered a large pesticide spill in 1980 that consisted primarily of dicofol and sulfuric acid. Dicofol contains several DDT analogs including DDE and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD). In the years following the spill, the hatchling and juvenile alligator population plummeted due to a decline in egg
378
Hormones and Reproduction of Vertebrates
FIGURE 14.4 Average concentrations (ng/ml) of serum contaminants in male and female juvenile alligators from Lake Apopka and reference Lake Woodruff. Data from Guillette, Brock, Rooney, and Woodward (1999a).
viability rates and increased juvenile mortality (Guillette et al., 1994b). Yolk samples from Lake Apopka alligator eggs revealed contamination from several organochlorine pesticides and their metabolites, including DDT, DDE, DDD, toxaphene, dieldrin, trans- and cis- chlordane, and trans-nonachlor, providing evidence for direct contaminant transfer to offspring (Heinz, Percival, & Jennings, 1991). Further, studies of Lake Apopka alligators have examined contaminant levels in muscle, blood, and liver, and have noted elevated levels of many organochlorine contaminants as well as PCBs in these tissues as compared to alligators from a relatively pristine and ecologically similar site in FloridadLake Woodruff National Wildlife Refugedwhich has been used as a reference site for more than 20 years (Delany, Bell, & Sundlof, 1988; Guillette, Brock, Rooney, & Woodward, 1999a; Garrison, Guillette, Wiese, & Avants, 2009) (Figure 14.4; Table 14.1). Organochlorine (OC) contaminants have been detected in several crocodilian species including the American alligator, the American crocodile (Crocodylus acutus), Morelet’s crocodile (Crocodylus moreletii), the Australian freshwater crocodile (Crocodylus johnstoni), and the Nile crocodile (Crocodylus niloticus) (Campbell, 2003; Pepper et al., 2004; Rauschenberger et al., 2004a; Sepulveda et al., 2004; Yoshikane et al., 2006). Conversely, squamates have received relatively little attention in the study of endocrine disruption. Early studies in squamates describe contaminant concentrations in tissues or compare contaminant loads in animals from
different locations. Recent studies link sublethal contaminant effects with exposure concentrations (Lambert, 1997; Lambert, 1999; Sanchez-Hernandez & Walker, 2000). Cadmium (Cd) is the most studied heavy metal in squamates. Cadmium biomagnifies (Croteau, Luoma, & Stewart, 2005) and, because snakes and lizards are middleto top-tier predators, they could be especially vulnerable to Cd exposure. In reptiles, Cd preferentially accumulates in the liver upon short-term exposure. However, chronic Cd exposure will shift accumulation to other tissues, particularly the kidney (Linder & Grillitsch, 2000). Studies of European lacertid lizards report that Cd accumulates preferentially in the gut, followed by the liver, kidney, and finally in the carcass (Mann, Sanchez-Hernandez, Serra, & Soares, 2007). Transfer to other internal organs, such as the reproductive system, is generally low.
3.2. Sex Steroid Hormone Alterations Many reptiles, such as the crocodilians, are protected species. Therefore, analyses such as blood hormone concentrations provide a non-lethal and minimally invasive method for evaluating the effects of EDC exposure. Abnormal plasma sex-steroid concentrations are important reproductive endpoints that have been studied extensively in alligator populations. T and E2 are essential for normal reproductive activity in both male and female vertebrates and play integral roles during sex determination in reptiles (see Chapter 1, this volume). Alterations in
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Lake Apopka Alligator Eggs 1984
Lake Apopka Alligator Livers
1985
2001
1985
Pesticides
Detections n¼3
Average Conc. ppb
Detections n¼23
Average Conc. ppb
o,p0 -DDE
-
-
4
7
0
3
5800
23
3500
0
o,p -DDD*
-
-
-
-
p,p0 -DDD
3
820
22
370
o,p0 -DDT*
-
-
-
-
p,p0 -DDT
-
-
6
20
trans-Nonachlor
3
110
23
cis-Nonachlor
-
-
Oxychlordane*
-
trans-chlordane* cis-chlordane*
p,p -DDE
Lake Apopka Alligator Muscle
Detections n¼1 0
Average Conc. ppb ND
Detections n¼12
Average Conc. ppb
1
1.9
12
114
2
1.6
12
2
1
2.3
2
11.3
1
30
1
160
150
-
-
12
6.9
-
-
-
-
11
3.2
-
22
30
-
-
3
10
-
-
2
6
-
-
2
16
1
70
22
60
-
-
1
1.7
Endocrine Disruption of Reproduction in Reptiles
TABLE 14.1 Concentrations of contaminants in alligator samples from Lake Apopka, FL, from different years. Muscle analysis reports a combined value for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), and dichlorodiphenyltrichloroethane (DDT).
())
chiral pesticides (-) not tested ND nondetect Data from Delany, Bell, and Sundlof (1988); Guillette, Brock, Rooney, and Woodward (1999); Garrison, Guillette, Wiese, and Avants (2009) See text for explanation of the contaminants. Chiral pesticides include both enantiomers
379
380
their concentrations during critical developmental windows can cause sex reversal and altered sex ratios (Lance & Bogart, 1994; Wibbels & Crews, 1995). Hatchling alligators reared in the laboratory from eggs collected at sites contaminated with known estrogenic compounds can exhibit abnormal reproductive effects that manifest very early post-hatching. For example, sixmonth-old male alligators (Alligator mississippiensis) from Lake Apopka show depressed plasma T concentrations, threefold lower than Lake Woodruff males but comparable to Lake Woodruff females. Lake Apopka females have elevated plasma concentrations of E2 as compared to hatchling alligators from Lake Woodruff raised under similar conditions (Guillette et al., 1994b). In similarly designed experiments, female Lake Apopka hatchling alligators raised to nine months of age display a twofold depression in plasma T concentrations and no difference in E2 concentrations compared to the females from Lake Woodruff, despite reduced ovarian aromatase activity in females from Lake Apopka (Crain, Guillette, Rooney, & Pickford, 1997). Another study in which male and female alligators were raised in captivity for 13 months under similar conditions showed no difference in plasma T for males and no difference in plasma E2 concentrations in females compared to the Lake Woodruff alligators. However, a loss of sexually dimorphic gene expression (discussed below) was found in the animals from Lake Apopka (Milnes et al., 2008). Taken together, these studies suggest complex alterations in steroid synthesis and/or clearance in neonatal and juvenile alligators that change with age. Studies analyzing hormone concentrations in wildcaught juveniles display a different pattern to those of juveniles examined in captivity where animals were raised under similar conditions. Juvenile male alligators from contaminated sites such as Lake Apopka show depressed concentrations of plasma T and elevated concentrations of plasma E2 compared to animals of the same age and size from Lake Woodruff. Similarly, juvenile female alligators from Lake Apopka show elevated plasma E2 concentrations when compared to reference populations (Guillette, Crain, Rooney, & Woodward, 1997; Guillette et al., 1999b; Milnes, Woodward, Rooney, & Guillette, 2002). Subsequent studies of Florida alligator populations from Lakes Griffin and Okeechobee, also contaminated with a complex mixture of agricultural pesticides and excess nutrients, have shown altered plasma sex steroid hormone concentrations (Guillette et al., 1999b; Gunderson et al., 2004a). Thus, without a depuration period such as that found in captivity, normal plasma T and E2 concentrations in juvenile alligators from contaminated sites continue to be altered and show a consistent pattern. Recent studies have examined sex steroid concentrations of adult Australian freshwater crocodiles living in the
Hormones and Reproduction of Vertebrates
Ord River of Western Australia, an area of p,p’-DDE and toxaphene contamination (Yoshikane et al., 2006). Unlike previous results reported in juvenile alligators from Lake Apopka, adult freshwater crocodiles living in these contaminated areas did not show significant modifications in plasma T and E2 concentrations. Thus, altered plasma sex steroid concentrations are not always indicative of environmental contamination. A common effect of EDCs in wildlife populations is the loss of sexual dimorphism in those animals from contaminated environments. All juvenile alligators from Lake Apopka frequently have low plasma T concentrations when compared to a marked sexual dimorphism in reference populations. A disruption in the sexually dimorphic expression of genes regulating steroidogenesis and steroidmetabolizing enzymes could explain the altered sex steroid concentrations seen in animals from Lake Apopka (see Gunderson, LeBlanc, & Guillette, 2001; Milnes et al., 2008). Altered gene expression patterns occur in hepatic degradation enzymes in alligators inhabiting waters contaminated with DDT, DDE, and DDD, thereby demonstrating a plausible mechanism that could explain the abnormal sex steroid concentrations (Gunderson et al., 2001). Normal sexually dimorphic gene expression of two enzymes involved in the biotransformation of T, testosterone hydroxylase, and oxido-reductase, is altered in juvenile alligators from contaminated lakes throughout Florida. Female alligators from these lakes display a male pattern of androgen metabolism (Gunderson et al., 2001). In comparison, juvenile alligators from Lake Woodruff display a sexually dimorphic pattern, with females having higher expression of these two enzymes than males (Gunderson et al., 2001). Hepatic CYP3A, another important regulator of the biotransformation of T, could provide the mechanism for the depressed plasma T concentrations observed in male juvenile alligators from contaminated sites. Organochlorine compounds upregulate CYP3A in other vertebrates, which would increase urinary clearance of T and possibly skew sex steroid hormone concentrations. Toxaphene, a compound found in the yolks of Lake Apopka alligator eggs and a known CYP3A inducer, was shown to significantly induce CYP3A gene expression in juvenile alligators 24 hours after exposure (Gunderson, Kohno, Blumberg, Iguchi, & Guillette, 2006). However, plasma T concentrations have not been shown to change significantly following toxaphene treatment (Gunderson, Kohno, Blumberg, Iguchi, & Guillette, 2006). Contrary to the results seen in testosterone hydroxylase and oxido-reductase gene expression, CYP3A does not demonstrate sexually dimorphic gene expression in juveniles inhabiting reference lakes. The lack of variation seen in CYP3A gene expression among juvenile alligators collected from contaminated and uncontaminated lakes could be
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FIGURE 14.5 Comparison of human (ERa) full-length proteins with other vertebrate estrogen receptors (data from GenBank). The functional A/B to F domains are schematically represented with percent similarities of sequence presented. Note the high sequence similarity (values at or close to 100%) between human and most of the reptilian species for the DNA-binding (DBD) and ligand-binding (LBD) domains.
a consequence of stress. Prior to necropsy, wild animals were held in cloth bags for two to seven hours, and Guillette et al. (1997) and Gunderson et al. (2003b) have demonstrated that b-corticosterone concentrations rise after two hours of capture (Guillette et al., 1997; Gunderson, Kools, Milnes, & Guillette, 2003). In Gunderson et al. (2006), a synthetic glucocorticoid, dexamethasone, induced CYP3A mRNA expression. Taken together, these data provide a plausible scenario where elevated b-corticosterone concentrations could induce CYP3A expression, thus masking potential differences between the lakes (Gunderson et al., 2006). Whereas altered hepatic clearance provides one possible mechanism for modified sex steroid concentrations, recent work has investigated changes in gonadal gene expression for various steroidogenic enzymes and steroid hormone receptors as another mechanism causing altered plasma steroid concentrations. Gene expression profiles of the two estrogen receptors, ERa (ESR1) and ERb (ESR2), in the testes and ovaries of juvenile American alligators indicate that both testes and ovaries from the American alligator express more ERa than ERb (Kohno, Bermudez, Katsu, Iguchi, & Guillette, 2008). A more refined pattern of expression exists for ERa and ERb among the Lake Woodruff animals. Females from Lake Woodruff express significantly more ovarian ERb than that seen in the testes of males. Interestingly, 60% of male animals from Lake Apopka demonstrated the female patterning of ERa/ ERb expression (Kohno et al., 2008). These results indicate that males from Lake Apopka have
altered ERa and ERb expression. Estrogenic EDCs bind and activate ERb more effectively as compared to ERa, despite ERb demonstrating a lower affinity for E2 than ERa (Kuiper et al., 1997; 1998). These findings, combined with our previous understanding of the binding affinity of the alligator ERs for many of the estrogenic contaminants found in the EDC milieu in Lake Apopka (Vonier, Crain, McLachlan, Guillette, & Arnold, 1996; Guillette, Vonier, & McLachlan, 2002) suggest a greater responsiveness in Apopka males, as compared to Lake Woodruff males, for environmental estrogen contamination. It is also worth noting that the ERs of reptiles and mammals have highly conserved gene and protein sequence similarity in the DNA- and ligand-binding regions when compared to birds, mammals such as mice and humans, and amphibians such as Xenopus laevis (see Katsu et al., 2004; 2006; 2008) (Figure 14.5). These data suggest that although some variation in estrogenic response among species would be expected, it is likely that the responses seen in one reptile would predict estrogenic effects in other reptilian, avian, and mammalian species. Changes seen in gene expression could identify mechanisms for the anomalies in hormone profiles observed in juvenile females from contaminated lakes in Florida. The gene DAX1 is regulated by P450aro, and gonadal expression of P450aro and DAX1 mRNA is depressed in Lake Apopka females compared to Lake Woodruff females (Wang et al., 2001; Kohno et al., 2008). It is possible that, through
382
a negative feedback loop, gonadal mRNA expression of P450aro and DAX1 could decline due to increased levels of E2, which is documented in Apopka females. Because of their role as transcription factors, activated sex steroid receptors are responsible for the production of other proteins such as vitellogenin (Vtg), a phospholipoglycoprotein used to form yolk proteins in the oocytes. Vitellogenin previously has been used as a biomarker of endocrine disruption in wildlife, since it can be induced upon exposure to exogenous natural estrogens and environmental xenoestrogens (Sumpter & Jobling, 1995; Palmer, Huth, Pieto, & Selcer, 1998; Hutchinson & Pickford, 2002). Initial studies of Morelet’s crocodiles reported multiple organochlorine contaminants in eggs and tail scutes (Wu, Rainwater, Platt, McMurry, & Anderson, 2000; Pepper et al., 2004; Wu et al., 2006); thus, it was predicted that C. moreletii juveniles of both sexes, as well as adult males, would exhibit plasma Vtg production due to OC exposure. However, Vtg was only detected in adult females that were collected during the breeding season (Rainwater et al., 2008). This finding is consistent with other reptilian studies in which Vtg is not produced despite EDC exposure (Matter et al., 1998a; Gunderson et al., 2003a). Vitellogenin induction in juvenile alligators exposed in ovo to multiple OCs could be due to the lack of properly functioning biochemical machinery in an immature animal (Matter, McMurry, Anthony, & Dickerson, 1998b). Further, low exposure might not elicit a vitellogenic effect, or a mixture of EDCs with varying mechanisms of action could inhibit an estrogenic effect, which could otherwise occur (Gunderson et al., 2003a). Expanding urbanization and industrialization have led to an increase in environmental Cd, which is a potent nonsteroidal estrogen that can form a high-affinity binding complex with the hormone-binding domain of the ER (Nesatyy, Rutishauser, Eggen, & Suter, 2005). Cadmium can inhibit Vtg gene expression and subsequently disrupt vitellogenesis through direct binding to the rainbow trout ER (Flouriot, Pakdel, Ducouret, Ledrean, & Valotaire, 1997; Le Guevel et al., 2000). Additionally, Cd depresses ER mRNA in the liver, which could indicate lower levels of the ER protein (Flouriot et al., 1997). Vitellogenin production is dependent upon circulating E2, and E2 action is mediated by the ERs. Cadmium exposure could therefore impact Vtg production via this mechanism (Vetillard & Bailhache, 2005). Whether Cd exposure presents the same issues in reptiles has yet to be explored. Field studies on adult painted turtle (Chrysemys picta) populations living near the Massachusetts Military Reservation (MMR), a Superfund site in Cape Cod, Massachusetts, report several reproductive modifications that could be related to exposure to heavy metals, including Cd, found in the surface water and sediment. Female turtles from this site displayed decreased E2 and Vtg levels compared to
Hormones and Reproduction of Vertebrates
turtles from the reference site, Washburn pond. Massachusetts Military Reservation turtles also displayed reduced gonadal size and females had a reduced number of large follicles, suggesting that Cd contamination could cause a decline in reproduction in these animals (Rie, Kitana, Lendas, Won, & Callard, 2005).
3.3. Reproductive Organ Abnormalities Laboratory-reared female alligators from Lake Apopka demonstrate an ovarian pathology that is characterized by multi-oocytic (polyovular) follicles (MOFs) and multinucleated oocytes (Guillette et al., 1994b) (Figure 14.6). Similarly, broad-snouted caimans exposed to both low and high dosages of E2, as well as male-to-female sex-reversed animals, have an increase in the frequency of MOFs (Stoker et al., 2008). These gonadal abnormalities are very similar to those observed in mice exposed to the estrogenic compound diethylstilbestrol (DES) (Iguchi, 1992) and suggest that the reproductive anomalies observed in Lake Apopka alligators and E2-exposed caiman are organizational in origin. These modifications in ovarian morphology could provide a potential explanation for the reduced clutch viability seen in Apopka animals. Studies in mice demonstrate that fertilized ova from MOFs are significantly less likely to develop to implantation-stage embryos than uniovular follicles (Iguchi, Kamiya, Uesugi, Sayama, & Takasugi, 1991). A disruption in the GTH–estrogen–inhibin–activin signaling pathway via estrogenic contaminants could be responsible for the MOF occurrence seen in Apopka females (Guillette & Moore, 2006). Although the etiology of MOF formation is still unclear, one hypothesis is that they arise from a failure of primary follicular cells, i.e., granulosa cells, to differentiate and surround primordial oocytes during early development (Guillette & Moore, 2006). This activity is partially regulated by the presence of activin A, one isoform of activin. Activin action is regulated in two ways: through the actions of inhibin, which competitively binds the activin type II receptor (ActRII), and through the actions of follistatin, an activin-binding protein that sequesters activins for degradation (Martens et al., 1997; Pangas & Woodruff, 2000). Follicle-stimulating hormone (FSH) can stimulate production of inhibin a and follistatin (Tuuri, Eramaa, van Schaik, & Ritvos, 1996). Perhaps an exogenous estrogenic signal, potentially acting at multiple points, is capable of prematurely initiating this delicate pathway, resulting in the formation of MOFs. If follistatin and inhibin a are upregulated due to a premature FSH signal or an inappropriate estrogen signal, activin A activity will decline. This will result in altered follicular formation due to lack of granulosa cell proliferation. Ovaries from hatchling alligators do not possess follicles, because their assembly occurs slowly over several
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FIGURE 14.6 Photomicrographs of ovarian follicles from juvenile American alligators. Normal and multi-nucleated oocytes (a) and multi-oocyte follicles can be seen (b). Multi-oocytic (polyovular) follicles have been reported from females alligators from Lake Apopka, FL, as well as female caimans treated with various estrogenic contaminants. o, oocyte; MOF, multi-oocytic follicle; mno, multi-nuclear oocyte.
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months post-hatching (Moore, Uribe-Aranzabal, Boggs, & Guillette, 2008; Moore et al., 2010b). During germ cell nest breakdown and subsequent follicle assembly, an activindominated signaling milieu appears to be critical in alligators as it does in mammals (Moore et al., 2010b). Activins participate in signaling crosstalk with steroid hormones. Our data reveal sexually dimorphic mRNA expressions of transforming growth factor b (TGFb) signaling factors in gonads of neonatal and 13-month-old American alligators similar to patterns established in other vertebrates with genetic sex determination (Moore et al., 2010b). Varying incubation temperature did not have an overt effect on gene expression levels of sex steroid hormone receptors within the gonad of Lake Woodruff animals (Moore et al., 2010d). However, differing exposure to EDCs (e.g., the lake of origindLake Apopka vs. Lake Woodruff) altered certain mRNA expressions that are crucial for reproductive function, namely inhibin bB subunit (INHBB), follistatin (FST), and growth differentiation factor 9 (GDF9) (Moore, Hamlin, Botteri, & Guillette, 2010a). The loss or reduction of sexually dimorphic mRNA expression for various factors involved in TGFb and steroid signaling in alligators from Lake Apopka (contaminant-exposed animals) could be associated with the previously reported losses in sexual dimorphism in Lake Apopka animals, such as phallus length, gonadal steroidogenic factor mRNA expression, and circulating steroid levels (see Milnes et al., 2008; Moore et al., 2010a; 2010d). Further, exogenous FSH can alter expression of inhibin and activin genes and upregulate follistatin gene expression in neonatal one-week-old American alligators (Moore et al., 2010c). Taken together, these studies are providing evidence for EDC-induced MOFs and support of the hypothesis that altered activin, inhibin, and steroid hormone signaling are likely involved in the development of this pathology (Moore et al., 2010a; 2010c; 2010d). Abnormal sex steroid concentrations experienced during neonatal and juvenile development can lead to modifications in anatomical features reliant upon these hormones for maturation and differentiation. For example, male neonates from Lake Apopka exhibit higher plasma T concentrations (Milnes, Bryan, Medina, Gunderson, & Guillette, 2005). Yet, paradoxically, phallus tip length and cuff diameter, two morphological endpoints reliant upon androgen signaling for development, are reduced in these Apopka males. Further, juvenile male alligators in Lake Apopka have smaller penises and lower plasma T concentrations, which taken together have been hypothesized to indicate a lifetime abnormality in plasma T concentrations and or androgen signaling (Guillette, Pickford, Crain, Rooney, & Percival, 1996). Altered reproductive organ formation following EDC exposure has been observed in other reptiles as well.
Hormones and Reproduction of Vertebrates
Caimans exposed to low doses of E2 or bisphenol A (BPA) show disrupted seminiferous tubules, similarly seen in neonatal rats treated with DES and juvenile male alligators living in Lake Apopka (Guillette et al., 1994a; Atanassova et al., 1999). These subtle changes experienced early in development could alter spermatogenesis and reproductive function later in life. Whereas changes in circulating sex steroids (elevated E2/depressed T) also have been documented in neonatal caiman exposed in ovo to BPA and E2, these hormonal changes are accompanied by conflicting observations in ovarian morphology that are dependent upon dose. Although high dose male-to-female sexreversed caimans demonstrate delayed ovarian maturation, animals exposed to low doses show advanced ovarian maturation (Stoker et al., 2003). However, histology of adult C. johnstoni from an area of p,p’-DDE and toxaphene contamination displayed no obvious morphological changes in the gonad (Yoshikane et al., 2006). Adult painted turtle populations living near the MMR display altered ovarian follicular dynamics including reduced numbers of large follicles, possibly due to Cd contamination (Rie et al., 2005). Neonates hatched from contaminant-exposed wild females located near MMR have not shown changes in gonadal structure nor the number of proliferating germ cells (Kitana & Callard, 2008). In contrast, laboratory-reared red-eared slider hatchlings exposed in ovo to Cd demonstrate an increase in oocyte apoptosis, which continues at least three months posthatch. A decrease in the total number of germ cells also has been reported in red-eared slider turtles exposed to Cd during embryonic development (Kitana & Callard, 2008).
3.4. Sex Determination The abnormal reproductive effects observed to date in juvenile alligator populations led our group to question whether these effects were organizational or activational in origin. In addition, questions arose as to how the EDCs found in Apopka alligator eggs impacted sex determination in an animal that exhibited TSD. A number of previous in ovo experiments in reptiles showed that environmental estrogens could override the influence of temperature during this critical period. Temperature, dosage, and the synergistic or additive activities of EDCs provide a complicated matrix of effects that makes comparisons across species difficult to predict. Ecologically relevant concentrations of the synthetic estrogen, ethinylestradiol (EE2), DDT, p,p’-DDD, o,p’-DDE, p,p’DDE, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), indole-3-carbinol, trans-nonachlor, and the anti-estrogen tamoxifen cause male-to-female sex reversal in American alligator embryos incubated at a male-producing temperature (see Matter et al., 1998b). Competitive binding assays have demonstrated partial binding of o,p’-DDE, p,p’-DDE,
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trans-nonachlor, p,p’-DDD and various PCBs with the alligator uterine ER (Vonier et al., 1996). p,p’-DDE is unique in that it apparently has the ability to produce multiple endocrine effectsdestrogenic, anti-estrogenic, and no estrogenic activityddepending on the endpoints, species, and dosages studied (Guillette & Milnes, 2001). p,p’-DDE can cause sex reversal from male to female at high concentrations, can synergize with its isoform, o’pDDE, to produce 100% sex reversal, and can act as a partial anti-estrogen when combined with EE2 (Matter et al., 1998a; 1998b). Further, the effect of p,p’-DDE can be directly influenced by the temperature at which the embryo is incubated. Milnes et al. (2005) reported that no sex reversal could be demonstrated in response to p,p’-DDE treatment when eggs were incubated at 33 C (an all-male temperature) but almost complete sex reversal to females occurred at the intermediate temperature of 32 C. In contrast to the alligator, p,p’-DDE does not sex reverse embryos of the sea turtle Chelonia mydas (Podreka, Georges, Maher, & Limpus, 1998) but does sex reverse at least one species of freshwater turtle, the red-eared slider (Willingham & Crews, 1999), further complicating our understanding and highlighting a complex species diversity in response to p,p’-DDE exposure. This also highlights our relatively poor understanding of not only the role of estrogens in TSD but also the interaction of EDCs with steroid receptors. As we presented previously (see Figure 14.5), the DNA- and ligand-binding domains of ERa from various species are highly conserved in sequence, yet we can obtain diverse responses given changing environmental conditions such as incubation temperature. The herbicides 2-4-dichlorophenoxyacetic acid (2,4-D) and atrazine and the fungicide vinclozolin do not induce male-to-female sex reversal in alligators incubated at a male-producing temperature. However, atrazine can induce increased P450aro expression in testicular tissue in male alligators, whereas 2,4-D has not been shown to increase P450aro expression (Crain et al., 1997). These studies indicate that EDCs can produce a complicated array of effects and that multiple endpoints need to be evaluated in order to fully understand these effects. Studies in caimans have investigated the estrogen-like effects of BPA, a controversial chemical in plastics, on gonadal development (Stoker et al., 2003; 2008). Like American alligators, sex determination in caiman is temperature-dependent (Lang & Andrews, 1994). Caiman embryos are sensitive to BPA when exposed prior to the period of sex determination. Bisphenol A exposure mimics E2 effects related to sex reversal and gonadal disruption. Because BPA is a weak estrogen, concentrations 2000- to 100 000-fold higher than E2 are required in order to produce the same effect (Krishnan, Stathis, Permuth, Tokes, & Feldman, 1993; Olea et al., 1996). The strength of BPA is contested by another study in caimans reporting that
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the BPA dose required to cause male-to-female sex reversal (140 ppm) is 100 times higher than that of E2 (1.4 ppm). While much lower in concentration than previously reported, BPA can still be considered a weak estrogen compared to E2. In addition to the crocodilians, turtles have been the only other reptiles extensively studied for EDC-induced sex reversal. Many turtles, like crocodilians, rely upon TSD. The red-eared slider turtle (Trachemys scripta elegans) has shown male-to-female sex reversal upon exposure to exogenous estrogens and estrogenic EDCs including E2, p,p’-DDE, trans-nonachlor, cis-nonachlor, chlordane, aroclor 1242, 2’,4’,6’-trichloro-4-biphenylol, and 2’,3’,4’,5’tetrachloro-4-biphenylol (Bergeron et al., 1994; Crews, 1996; Willingham & Crews, 1999). This has led researchers to question how these compounds exert their effects. Aromatase inhibition causes male red-eared slider turtles to be produced at a normally female-producing temperature, whereas applying an estrogen to eggs incubated at a normally male-producing temperature will produce females (Crews, 1996). Endocrine-disrupting chemicals also have considerable effects on steroidogenic pathways. Steroidogenic factor 1, a gene that regulates aromatase expression, has sexually dimorphic expression in the red-eared slider turtle and therefore could be a suitable target for endocrine disruption (Fleming, Wibbels, Skipper, & Crews, 1999). Aromatase activity in the brain of turtle embryos is higher in embryos incubated at female temperatures (Willingham, Baldwin, Skipper, & Crews, 2000a). Arochlor 1242 caused a significant increase in aromatase activity in the brain during the temperature-dependent window of development and in the adrenal–kidney–gonad complex before hatching in turtles, suggesting one possible mechanism for femalebiased sex reversal (Willingham et al., 2000a; Willingham, Rhen, Sakata, & Crews, 2000). The biological actions of sex steroid hormones also can be regulated by the presence or absence of their receptors. Experimentally induced sex reversal changed the normal expression patterns of ERa, ERb and AR in red-eared slider turtles (Ramsey & Crews, 2007), demonstrating that changes in steroid receptor gene expression can negatively impact hormonal signaling during sex determination, providing a framework for sex reversal. In reference to mixture experiments, the freshwater turtle T. s. scripta has been the most thoroughly studied reptile. A mixture of PCBs causes sex reversal in red-eared slider embryos (Bergeron et al., 1994) and a study of eight commonly identified compounds from Lake Apopka demonstrated that trans-nonachlor, cis-nonachlor, aroclor 1242, p,p’-DDE, and chlordane, when applied singly or as a mixture in ovo, result in sex reversal (Willingham & Crews, 1999). Aroclor 1242, chlordane, and trans-nonachlor alter T and progesterone (P4) concentrations in hatchling turtles (Willingham et al., 2000b). Conversely,
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studies in juvenile snapping turtles (Chelydra serpentina) have not demonstrated altered sex steroid profiles in sexreversed turtles (Rhen, Elf, Fivizzani, & Lang, 1996). Individuals sex reversed by in-ovo E2 exposure exhibited normal plasma sex steroid hormone profiles for T and E2 (Rhen et al., 1996).
4. ENDOCRINE-DISRUPTING CHEMICALS (EDCs) AND REPTILIAN THYROID FUNCTION Although steroid hormone effects have been dominant in EDC research, current research trends are beginning to extend beyond the estrogen- and androgen-driven reproductive processes (see Guillette, 2006). Chemically induced alterations in thyroid function are gaining recognition as a consequence of EDC exposure. Thyroiddisrupting contaminants can behave much in the same way as steroid-disrupting contaminants. Polychlorinated biphenyls (PCBs) can disrupt thyroid hormones (THs) via the TH receptors (TRs) (Zoeller, 2007b). Other studies suggest that some PCB congeners can alter the binding ability of the TRs to the thyroid response element (TRE) in the promoter of various genes (Miyazaki, Iwasaki, Takeshita, Kuroda, & Koibuchi, 2004). Without proper binding of the THs–TRs–TRE complex, gene transcription is inhibited. Perchlorate blocks the sodium iodide symporter of the thyroid gland. Therefore, iodide, the limiting element in the production of THs, cannot enter the gland, leading to hypothyroidism and goiter (Hooth et al., 2001). Thyroid gland development essentially is similar across extant reptiles (Lynn, 1960). The thyroid gland is composed primarily of TH-producing follicular cells. Changes in thyroid gland histology such as follicular cell size and colloid space are tightly correlated with pituitary thyrotropin (TSH) disruption (Hood, Liu, & Klaassen, 1999). Increased follicular cell height, follicular cell hyperplasia, and decreased colloid space are reliable markers of hypothyroidism. The hypothalamus–pituitary–thyroid (HPT) axis has changed little throughout vertebrate evolution and THs consisting of triiodothyronine (T3) and thyroxine (T4) are identical among vertebrate taxa (Escriva, Delaunay, & Laudet, 2000; Zoeller & Tan, 2007). A recent study used ovine TSH to successfully stimulate production of THs in the thyroid cells of the Chinese soft shell turtle and the Chinese bullfrog, exemplifying the conservation of the thyroid system across vertebrates (Huang et al., 2009). The critical organizational role of THs in vertebrates is becoming well-understood. Thyroid hormones regulate the growth and metabolism necessary for embryos to develop and grow. Thyroid hormones also are necessary
Hormones and Reproduction of Vertebrates
for proper growth and gonadal development (Figure 14.7). Hypothyroid mice display delayed ossification in bone development (Bassett et al., 2008). Developing hypothyroid human males display delayed and reduced Sertoli cell differentiation and fibrotic seminiferous tubules, causing sterility (Jannini et al., 1995). Although we have some understanding about the biology of reptilian thyroid function, little information exists on the effects of EDCs on proper thyroid functioning. Previous studies have been limited in scope and have examined TH disruption by evaluating hatching rates and mortality. However, advances in molecular techniques now allow for the analysis of small quantities of tissue, such as those acquired from embryos. These advances have broadened the scope and diversity of scientific inquiry and increased our understanding of endocrine disruption at the embryonic level. The effects of THs on reproduction of reptiles include actions of THs on TRs found in reproductive tissues, TH regulation of gonads during mating seasons, development of embryonic tissues, and time of hatching, thereby affecting hatchling survival. Further, THs can act indirectly by altering metabolism and growth, possibly affecting aspects of reproduction such as lifetime fitness and age of maturation. We will discuss THs in virtually all stages of life through its roles in development, metabolism, growth, and reproduction.
4.1. Hatching and Neonatal Growth The developing embryo receives some THs from egg yolk stores. However, approximately one third of the way through embryonic development, TH levels in the embryos of the viviparous lizard Xantusia vigilis and two oviparous crocodilians, the American alligator and saltwater crocodile, begin to increase, implying that the increase in THs is of embryonic origin (Miller, 1963; Medler & Lance, 1998; Shepherdley et al., 2002a). In the case of saltwater crocodiles, the pre-hatching spike in circulating concentrations of T3 and T4 are 22 times and 10 times higher, respectively, than those concentrations found in juvenile saltwater crocodiles (Shepherdley et al., 2002b). There are several possible mechanisms to account for the increasing concentrations of circulating T4 and T3 from embryonic day 50 to hatching. Inner ring deiodination (IRD) decreases at this time, possibly to reduce conversion of T4 to reverse T3 (rT3) or T3 to T2 and reduce TH clearance. Additionally, the TH plasma carrier protein transthyretin (TTR) is only present in the late stages of development to hatching, possibly keeping more THs in circulation (Richardson et al., 2005). Thyroid hormone concentrations remain high until three weeks post-hatching (Shepherdley et al., 2002b), suggesting a critical role of THs during late development, hatching, and neonatal life.
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FIGURE 14.7 Schematic representation of the hypothalamus–pituitary–thyroid (HPT) axis. In mammals, thyrotropin-releasing hormone (TRH) from the hypothalamus travels to the pituitary, which triggers the release of thyrotropin-stimulating hormone (TSH). This signal then travels to the thyroid gland, which uptakes iodide to produce thyroid hormones (THs). Release of THs from the follicular cells is triggered by TSH. There are two functional forms of TH: thyroxine (T4) and triiodothyronine (T3). Although T4 is the major hormone in circulation, T3 is believed to be the active form within the cell. Thyroxine is converted to T3 or reverse T3 (rT3) via outer ring deiodinase (ORD). An inner ring deiodinase (IRD) converts T4 into rT3, which is one of the excreted metabolites of T4. The HPT axis is set up on a negative feedback loop so that, if circulating levels of THs are low, the pituitary is triggered to upregulate release of TSH. In this way, circulating levels of THs are tightly regulated by the HPT axis. Various EDCs (double-lined boxes) can act upon the HPT axis at different points of regulation.
Several studies in the avian literature have highlighted the critical role THs play during hatching and early growth. Normally, a spike in THs coincides with pipping, an initial break in the eggshell by the embryo. Studies have shown that chicks that are hypothyroid and subsequently do not generate a TH spike will either be delayed in hatching or fail to hatch entirely (McNabb, 2006). Thyroid hormonedependent hatching is also evident in reptiles. Cadmium, a thyroid-disrupting heavy metal, reduces survival rates of embryonic fence lizards (Sceloporus undulatus) exposed via contaminated substrate. Thyroid hormone levels are reduced in lizards that survive, but there is no effect on body size (Brasfield et al., 2004). In contrast, exposure to thiourea, a known anti-thyroidal drug, in snapping turtle embryos causes a delay in hatching as well as a decreased growth rate and poor yolk absorption, all of which are known to be thyroid-dependent processes (Dimond, 1954). A similar reduction in growth rate is seen in red-eared slider turtles (Willingham, 2001). Hatchlings exposed to low doses of trans-nonachlor and p,p’-DDE lost weight before
feeding and turtles exposed to higher doses of trans-nonachlor grew faster than controls once ad libitum feeding began, possibly by a compensatory mechanism. Because of the pivotal role of THs in embryonic development, hatching, and neonatal growth, EDC-induced hypothyroidism could have major impacts on reptilian populations. Concentrations of THs can decrease hatching rates and increase the time to hatching (Dimond, 1954). Decreased hatching rates could lead to population declines. Further, decreased hatchling weights due to decreased embryonic growth could reduce hatchling survivability (Brown & Shine, 2009). Therefore, proper TH production and regulation are critical for survival and reproduction in the early life stages of reptiles.
4.2. Growth, Metabolism, and Effects on Reproduction Thyroid hormones can alter reproduction by altering growth and metabolism. In many reptiles, sexual
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maturation is closely related to size (Shine & Charnov, 1992). Reduced growth rates due to reduced food availability can delay sexual maturation in American alligators (Wilkinson & Rhodes, 1997). Reduced growth rates due to an imbalance of THs could delay sexual maturity in the same manner and likewise delay sexual maturation. Body condition and fat stores are regulated by THs, which can affect clutch size and egg size. Reduced egg size can lead to reduced hatchling size and survivability (Brown & Shine, 2009). Therefore, EDCs that affect thyroid hormones can affect reproduction by altering growth and metabolism. During juvenile life stages, a large fraction of the energy budget is allocated to growth. Therefore, growth and metabolism can be excellent endpoints for evaluating endocrine disruption of TH. Thyroidectomized juvenile Eastern fence lizards (S. undulatus) had nondetectable levels of thyroid hormone and a three-fold reduction in growth (Gerwien & John-Alder, 1992). It was clear from this study of at least one reptilian species that THs during the juvenile stage are necessary for optimal growth. Do EDCs that impair TH production have the same effect, however? Could EDCs that cause hypothyroidism lead to decreased growth in reptiles? Studies of growth, circulating T4 concentrations, and thyroid gland morphology in alligators from lakes of varying degrees of contamination in Florida have addressed these questions. Juvenile alligators studied from three central Florida lakes show pronounced seasonal cycles in circulating T4 concentrations that did not vary between males and females (Bermudez et al., 2005). Male alligators examined in the spring from contaminated Lake Okeechobee exhibited elevated plasma TH concentrations when compared to alligators from other Florida lakes (Crain, Guillette, Pickford, Percival, & Woodward, 1998; Hewitt, Crain, Gunderson, & Guillette, 2002; Bermudez et al., 2005). A more complex question to be addressed involves changes in seasonal patterns among juvenile alligators living in contaminated and reference lakes. Seasonal plasma TH profiles were significantly different between juvenile populations when contaminated Lake Apopka was compared to the reference, Lake Woodruff, but the pattern was complex (Bermudez et al., 2005). Furthering possible explanations for this complexity, an additional study found that circulating TH concentrations measured in alligators from contaminated waters in Florida showed no significant differences in plasma TH concentrations among the populations. Rather, increased variation in plasma concentrations appeared to be a better indicator of disruption (Gunderson et al., 2002). Circulating TH concentrations can vary widely from individual to individual due to various factors such as temperature, season, nutritional state, and contaminant exposure, making the detection of a significant and consistent difference in circulating TH concentrations difficult. Often, complex
Hormones and Reproduction of Vertebrates
patterns of disruption of circulating hormone concentrations can be seen more clearly in thyroid morphology. Alligators from the most contaminated areas of Lake Okeechobee (Belle Glade) displayed significantly larger thyroid follicular cell height and significantly smaller colloid space than animals from other less contaminated Florida sites, suggesting altered thyroid performance (Hewitt et al., 2002). Growth is the result of complex physiological processes that can be disrupted at many levels of biological organization. Studies evaluating growth rates of wild juvenile alligators in Florida have not found altered growth in animals from contaminated vs. reference lakes (Milnes et al., 2002). However, growth rate is only one of the physiological responses that THs mediate. Further, Lake Apopka and Lake Okeechobee contain EDCs that affect a number of hormones, not just THs. Physiological processes such as growth might not be affected due to the complexity of an open ecological system. Evaluations of environments contaminated by EDC mixtures can lead to complicated results, and highlight the need to assess multiple endpoints. Although there is extensive literature on heavy metal accumulation in reptiles that describe multiple techniques used for assessment, few studies correlate body burden with the physiological effects of endocrine disruption. Coal ash contains various trace elements including selenium, Cd, arsenic (As), strontium, and vanadium. Low doses of As given during tadpole tail absorption (a TH-dependent process) assays cause an increase in absorption whereas high doses inhibit tail absorption (Davey et al., 2008). This biphasic response, by acting on TRs and preventing THmediated processes, was also demonstrated in cell culture. Growth hormone receptor, mineralocorticoid receptor, progesterone receptor, AR, ER, and retinoic acid receptor displayed the same biphasic response after arsenic exposure, suggesting a similar mechanism of disruption (Davey et al., 2008). Despite this biphasic response, many of these heavy metals readily bioaccumulate and, thus, increase the likelihood of overexposure and hypothyroidism. A population of juvenile banded water snakes (Nerodia fasciata) inhabiting areas contaminated with coal ash displayed an increased metabolic rate but decreased growth rates compared to snakes from environments without coal ash (Hopkins, Rowe, & Congdon, 1999). Hopkins et al. (1999) suggest that the increased metabolism was necessary to clear the heavy metals and less energy was allocated to growth. However, captive-raised southern water snakes (N. f. fasciata) fed a diet of contaminated fish did not display similar increased metabolism (Hopkins et al., 2002). Southern water snakes from pristine locations fed a diet of contaminated fish displayed fibrosis of the liver in one third of the animals but metabolic rate and growth were not affected (Ganser et al., 2003). Although it appears that
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heavy metals alter endocrine function, the results vary in physiological response. Certainly, other environmental factors apart from the degree of exposure are likely playing a role in altered growth and metabolism. During the juvenile-to-adult transition, metabolic processes that were devoted to growth in juveniles are allocated, in part, to other activities such as maturation of the reproductive system and then mating behaviors and reproductive activity in adults (Kohel, MacKenzie, Rostal, Grumbles, & Lance, 2001; Cox, Skelly, & John-Alder, 2005). In alligators, size generally correlates with age and THs are negatively correlated with size (Crain et al., 1998). Therefore, the role of thyroid hormones will vary depending on the life stage of the organism.
4.3. Thyroidal–Gonadal Axes and Effects on Reproduction Thyroid hormones can alter reproduction through many mechanisms, for example by acting directly on the gonads via the TRs present in reproductive tissues. However, much of the literature on adult reptilian TH concentrations is focused on seasonal data. Because reptiles depend on external sources of heat for metabolism, one would expect that TH profiles, thyroid gland synthesis, and plasma concentrations of reptiles depend greatly upon temperature. Temperature and THs have a strongly positive correlation in reptiles (Kohel et al., 2001). This, in combination with low food availability during colder months, could also factor into the seasonal profiles of THs in adult reptiles. Despite these confounding factors, TH levels appear to be highly intertwined with reproduction in seasonal mating and reproductive processes. In the American alligator, juvenile species appear to go through a pubertal phase. Plasma T4 concentrations in peripubertal alligators fluctuate in a similar manner to E2 and T but have a low correlation (r2 < 0.07 – 0.20) with body temperature (Bermudez et al., 2005), illustrating the complex pattern between THs and reproduction in the presence of seasonal variation in both ambient and body temperatures. Many seasonally reproducing adult reptiles experience gonadal regression during the non-mating season. In many male reptiles, seasonal profiles display a spike in TH concentrations during the time of testicular recrudescence and spermatogenesis. This seasonal peak is seen in snakes such as the Chinese cobra (Naja naja) and the adder (Vipera berus), lizards such as the Western fence lizard (Sceloporus occidentalis), and turtles such as the desert tortoise (Gopherus agassizii) (Bonagallo, Licht, Mackenzie, & Lofts, 1980; Nilson, 1982; Cardone, Angelini, Esposito, Comitato, & Varriale, 2000; Kohel et al., 2001; Brasfield, Talent, & Janz, 2008). In a study by Cardone et al. (2000),
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a specific response in the testes due to T3 injections given to Italian wall lizards (Podarcis sicula) during the recrudescence period caused an increase in AR gene transcription independent of T. This suggested that T3 could prime the testes for increased sperm production. In female reptiles of the same species (P. sicula) as well as the snakes V. berus, Vipera aspis, and Natrix maura, peaks in plasma TH concentrations often occur during vitellogenesis and ovulation (Nilson, 1982). When West African lizards are thyroidectomized or given T4 injections, there is a high incidence of atresia, especially of large yolky ovarian follicles (Eyeson, 1970). While some have considered the T4 injection portion of this study to contradict the observation of high plasma TH concentrations in other female reptiles during normal ovulation (Bonagallo et al., 1980), it is generally accepted today that perturbations both below and above normal endogenous concentrations could have the same affect; i.e., the induction of ovarian follicular atresia. Understanding TH roles in spermatogenesis and ovulation serves to highlight the sensitivity of reptilian reproduction to disruption of the thyroidal–gonadal axis of reptiles. Thyroid hormones are required for high-energy activities such as territorial defense and mating, and have been linked to changes in body condition and fat storage. Activation of peroxisome proliferator-activated receptor (PPAR) induces peroxisome proliferation. Peroxisomes are involved in many pathways of metabolism and catabolism including lipid metabolism, a major action required for yolking of oocytes. Italian wall lizards (P. sicula) exposed to methyl thiophanate, a fungicide, displayed induced peroxisomal proliferation at sublethal concentrations. Thyroid morphology was altered and animals showed decreased follicular cell height, elongated follicular cell nuclei, and retracted colloid. Further, plasma THs and TSH concentrations were decreased (Sciarrillo et al., 2008). The increase in peroxisomes could also liberate fat stores at inappropriate times, as they are required for reproduction. Therefore, these animals are likely at risk of induced hypothyroidism, reduced body condition, or altered reproduction due to methyl thiophanate exposure. In the desert tortoise, peaks in plasma TH concentrations are seen during territorial defense and fighting in males that occur outside of spermatogenesis (Kohel et al., 2001). Disruption of adult TH concentrations that affect body condition and energetic needs likely affect mating behaviors or reproduction. Indeed, this was observed in a study linking body condition index (BCI) (percent fat O total body mass) to mate choice in V. aspis. In all combinations of mating scenarios between low- and high-BCI males and females, only the high-BCI males and females copulated. Low-BCI females and low-BCI males had no successful mating attempts despite the BCI of the partner (Aubret, Bonnet, Shine, & Lourdais, 2002).
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Increased TH activity and increased metabolism, due to EDC exposure, could prevent mating due to mate choice behaviors relating to body condition.
5. CONCLUSIONS Despite major advances in the study of endocrine disruption in reptiles, there still remains a considerable gap in our knowledge, which current research continues to address. Although a great deal of research has focused on the deleterious effects of EDCs on reptilian reproduction, advances in our understanding of the effects of EDCs are hampered by our limited knowledge of basic biological processes in most species. In order to recognize perturbations in a system, it is necessary to understand normal functioning of that system under normal environmental conditions. Although much has been learned about the pathways involved in sex differentiation and determination and reproductive activity in reptiles, for example, studies continue to refine our understanding of the stability of sex and the complexities of the gene and hormonal cascades involved in these processes. The detection of changes to thyroid morphology is an effective method to determine thyroid disruption in reptiles as many of the assays used to detect altered thyroid hormone signaling in mammals, for example, have not been developed for this group. However, changes in morphology are detected only after the contaminant has adversely affected the animal. Unfortunately, an effective early biomarker of thyroid disruption has not been developed in reptiles but changes in gene expression of thyroid-related proteins such as the sodium iodide symporter (NIS), TRs, or PPAR could prove to be important future indicators of thyroid disruption. By detecting changes in the expression of the genes involved in metabolism or other aspects of thyroid function, thyroid disruption could be detected before morphological changes to the thyroid gland occur. Our understanding of sexually dimorphic gene expression and the role of THs in reproduction is increasing our awareness of the molecular mechanisms involved in regulating changes in circulating hormone concentrations, hormone receptors, and other reproductive endpoints. While this review is not exhaustive, it highlights recent advances in the field of endocrine disruption of reptiles. The more we understand about reptilian physiology, the more we understand not only about endocrine disruption of reptiles, but about the health of the environment. To highlight the importance of reptiles as sentinels of endocrine disruption, it would be appropriate to end this chapter with a case that is currently unfolding. During the time of writing this chapter, it has been reported that gharials from the river shores of India have begun dying in great numbers (Gharial Conservation Alliance, 2008). The cause is
Hormones and Reproduction of Vertebrates
speculated to be the bioaccumulation of toxins due to the specialized fish diet of gharials. Autopsies have reported that the deaths are most likely due to kidney disease and goutda buildup of uric acid caused by the kidney’s inability to clear the metabolic toxin. Surprisingly, only the middle size class is affected and only during the winter months. Gharials are poikilotherms and therefore rely upon solar energy to warm themselves in the winter for proper metabolic function. It is hypothesized that the larger animals are expending energy in territorial defense rather than growth and therefore are not consuming as many fish. On the other hand, while both the small- and medium-size classes of gharials are eating relatively large quantities of contaminated fish to keep pace with their accelerated growth, the smaller animals are able to warm themselves faster and metabolize the toxins more rapidly, whereas the medium-sized individuals are not able to warm as quickly. What does the future hold for the surviving gharials? Will we uncover organizational defects in the smaller age classes that survive and will the adult gharials continue to reproduce successfully? This interesting case involving the mass deaths of an endangered reptilian species highlights the necessity of understanding reptilian physiology and toxicology so that we may continue to learn from these sentinels of environmental health.
ABBREVIATIONS 2,4-D ActRII AR As BCI BPA Cd DBD DDD DDE DDT DES DI E2 E3 EDC EE2 ER ESR1 ESR2 FSH FST GDF9 GSD GTH HPG HPT INHBB
2,4-Dichlorophenoxyacetic acid Activin type II receptor Androgen receptor Arsenic Body condition index Bisphenol A Cadmium DNA-binding-domain Dichlorodiphenyldichloroethane Dichlorodiphenyldichloroethylene Dichlorodiphenyltrichloroethane Diethylstilbestrol Deiodinase Estradiol Estriol Endocrine-disrupting chemical Ethinylestradiol Estrogen receptor see ERa see ERb Follicle-stimulating hormone Follistatin Growth differentiation factor 9 Genotypic sex determination Gonadotropin Hypothalamus–pituitary–gonadal Hypothalamus–pituitary–thyroid Inhibin bB subunit
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IRD LBD MMR MOF OC ORD P4 P450aro PCB PPAR SF-1 T T3 T4 TCDD TGFb TH TR TRE TRH TSD TSH TTR Vtg
Endocrine Disruption of Reproduction in Reptiles
Inner ring deiodinase Ligand-binding Massachusetts Military Reservation Multi-oocytic follicle Organochlorine Outer ring deiodinase Progesterone Aromatase Polychlorinated biphenyl Peroxisome proliferator-activated receptor Steroidogenic factor 1 Testosterone Triiodothyronine Thyroxine 2,3,7,8-Tetrachlorodibenzo-p-dioxin Transforming growth factor b Thyroid hormone Thyroid hormone receptor Thyroid response element Thyrotropin-releasing hormone Temperature-dependent sex determination Thyrotropin Transthyretin Vitellogenin
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Endocrine Disruption of Reproduction in Reptiles
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Species Index Index Acanthodactylus erythrurus, 149 Acanthophis praelongus, 249 Acris crepitans, 232 Acrochordus granulatus, 357, 359 Agama agama, 78, 79, 226 Agama atra, 254 Agkistrodon contortrix, 184, 228, 231, 358, 364 Agkistrodon piscivorus, 126, 187, 358 Alligator lizard, see Gerrhonotus coeruleus principis Alligator mississippiensis, 6, 21, 71, 90, 99, 129, 177, 179, 187, 189, 196, 231, 263, 306, 307, 308, 312, 313, 314, 315 Alligator snapping turtle, see Macrochelys temminkii Amazonian river turtle, see Podocnemis expansa Amblyrynchus cristatus, 43, 188, 190, 196, 198, 199, 225, 226, 228, 335 American alligator, see Alligator mississippiensis American crocdile, see Crocodylus acutus Amphibolurus caudicinctus, 231 Amphibolurus maculosus, 226 Amphibolurus muricatus, 9, 19, 20, 22, 24, 25, 26, 202, 338 Amphibolurus nuchali, 231 Ampibolurus ornatus, 126 Amyda japonica, 123 Andean ground lizard, see Liolaemis bellii Anguid slow worm, see Anguis fragilis Anguis fragilis, 151 Aniella pulchra, 337 Anolis acutus, 327 Anolis carolinensis, 19, 39, 40, 41, 46, 47, 48, 76, 100, 101, 105, 108, 125, 127, 132, 173, 174, 175, 179, 180, 181, 182, 183, 188, 190, 191, 192, 226, 230, 232, 255, 326, 329, 331, 332, 333 Anolis equestris, 46 Anolis limifrons, 327 Anolis opalinus, 327 Anolis pulchellus, 108, 181 Anolis sagrei, 78, 82, 177, 182, 183, 190, 230, 231, 329, 331, 332 Anops kingii, 335 Apalone mutica, 18 Argus monitor lizard, see Varanus panoptes Ascincid lizard, see Eulampsus heatwoli Aspic viper, see Vipera aspis Aspidoscelis spp, 366 Aspidoscelis uniparens, 19, 227 Augrabies flat lizard, see Platusaurus broadleyi
Australian flatback turtle, see Natator depressus Australian freshwater crocodile, see Crocodylus johnstoni Australian jacky dragon, see Amphibolurus muricatus Australian skink, see Bassiana duperreyi Australian snapping turtle, see Elysa dentata Australian tree skink, see Egernia striolata Barisia imbricata imbricata, 254, 337 Bassiana duperreyi, 20, 29, 200, 202 Batagur baska, 285 Batagur trivittata, 285 Bearded dragon, see Pogonus barbata Black swamp snake, see Seminatrix pygaea Black-lined polated lizard, see Gerrhosaurus nigrolineatus Black-tailed rattlesnake, see Crotalus molossus Blanus cinereus, 145, 146, 335 Blind snake, see Ramphotyphlops braminus Blood python, see Python curtus Blotched blue-tongued lizard, see Tiliqua nigrolutea Blue-tongued skink, see Tiliqua nigrolutea; Tiliqua scincoides Boiga irregularis, 157, 171, 179, 180, 363, 365 Bothrops spp., 252 Bothrops jaracara, 92, 232, 361 Bradypodion pumilis, 252, 252, 338 Brazilian skink, see Mabuya heathi Broad-headed skink, see Eumeces laticeps Brookesia spp, 338 Brown anole, see Anolis sagrei Brown tree snake, see Bioga irregularis Brown tree snake, see Boiga irregularis Caiman, see Caiman crocodilus Caiman crocodilus, 96, 119, 120, 145, 306, 309 Caimen latirostris, 306, 307 Caimen yacare, 306, 312 California legless lizard, see Aniella pulchra Calotes versicolor, 75, 78, 89, 92, 101, 103, 104, 122, 329, 330, 338 Cape girdled lizard, see Cordylus cordylus Caretta caretta, 103, 105, 171, 189, 195, 231, 279, 280, 282, 283, 284, 288, 290, 296 Carettochelys inscupta, 282 Cerberus rhynchops, 357, 359 Chalcides spp, 102, 249 Chalcides chalcides, 94, 251, 261, 262 Chalcides ocellatus, 79, 89, 251, 252
Chamaeleo calyptratus, 338 Chamaeleon chamaeleon, 232 Chelodina oblonga, 285, 293, 294 Chelodina steindachneri, 293, 294 Chelonia mydas, 45, 75, 77, 103, 144, 171, 174, 187, 189, 193, 194, 195, 196, 223, 281, 282, 286, 288, 290, 296, 297, 385 Chelydra serpentina, 9, 25, 75, 108, 122, 129, 134, 177, 178, 181, 182, 285, 287, 386 Chersine angulata, 28, 76, 293 Chicken, see Gallys gallus Chinese cobra, see Naja naja Chinese green tree viper, see Viridovipera stejnegeri (Trimeresurus stejnegeri) Chrysemis dorbigni, 288 Chrysemys picta, 9, 18, 21, 24, 75, 96, 103, 104, 119, 120, 122, 125, 128, 129, 131, 133, 134, 143, 177, 218, 231, 285, 287, 288, 382 Cnemidophorus burti, 336 Cnemidophorus inornatus, 45, 49, 51, 54, 82, 230, 231, 232, 329, 336 Cnemidophorus lemniscatus, 45, 173 Cnemidophorus nativo, 327 Cnemidophorus sexlineatus, 78, 189, 196 Cnemidophorus tigris, 78, 330 Cnemidophorus uniparens, 45, 49, 51, 54, 82, 96, 189, 232, 251, 254, 336 Coleonyx variegatus, 78, 149 Collared lizard, see Crotophytus collaris Common chameleon, see Chamaeleon chamaeleon Common gecko, see Hoplodactylus maculatus Copperhead snake, see Agkistrodon contortrix Cordylis nigra, 76 Cordylus cordylus, 148, 151 Cordylus giganteus, 148, 340 Cordylus polyzonus, 340 Corucia zebrata, 339 Cottonmouth snake, see Agkistrodon piscivorus Crocdylus siamensis, 309 Crocodile lizard, see Shinisaurus crocodilurus Crocodylus acutus, 311, 378 Crocodylus johnstoni, 196, 307, 309, 378 Crocodylus moreletii, 306, 309, 312, 378, 382 Crocodylus niloticus, 76, 306, 312, 378 Crocodylus palustris, 306, 309, 311 Crocodylus porosus, 306, 307, 309, 311 Crotalus atrox, 190, 191, 251, 357, 358, 360 Crotalus durissus, 121 Crotalus durissus terrificus
397
398
Crotalus durissus terrificus, 358 Crotalus molossus, 358 Crotalus scutulatus, 358 Crotaphytus collaris, 175, 184, 228, 253, 254, 334, 335 Ctenophorus pictus, 226 Ctenosaura pectinata, 98 Cyrtodactylus malyanus, 327 Death adder, see Acanthophis praelongus Dermochelys coriacea, 277, 285, 288 Desert iguana, see Dipsosaurus dorsalis Desert tortoise, see Gopherus agassizii Diamondback turtle, see Malaclemys terrapin centrata Dipsochelys dussumieri, 282 Dipsosaurus dorsalis, 148, 150, 151, 188 Draco melanopogon, 327 Dwarf chameleon, see Bradypodion pumilum Earless monitor lizard, see Lanthanotus borneensis Eastern fence lizard, see Sceloporus undulatus Eastern hermann’s totoise, see Testudo hermanni boettgeri Eastern three-lined skink, see Bassiana duperreyi Egernia spp, 225, 237 Egernia stokesii, 149, 152 Egernia striolata, 149, 152 Egernia whitii, 178, 194, 195, 196, 197, 203, 237 Elaphe climacophora, 360 Elysa dentata, 285 Emydura krefftii, 294 Emys orbicularis, 19, 21, 24, 143 Eretmochelys imbricata, 171, 177, 193, 195, 196, 282, 288, 290, 297 Erymnochelys madagascariensis, 282, 295 Eublepharus macularis, 16, 41, 52, 77, 96, 104, 146, 150, 151, 174, 217, 218, 230, 329, 341 Eulamprus heatwoli, 196 Eulamprus tympanum, 15 Eumeces spp, 339 Eumeces fasciatus, 79 Eumeces laticeps, 149, 151, 152 Eumeces obsoletus, 254, 340 European adder, see Vipera berus European common lizard, see Lacerta vivipara European pond turtle, see Emys orbicularis European wall lizard, see Podacris muralis Fan-throated lizard, see Sitana ponticeriana Flying lizard, see Draco melanopogon Furcifer labordi, 322, 338 Furcifer verrucosus, 338 Galapagos marine iguana, see Amblyrynchus cristatus Galapagos tortoise, see Geochelone nigra Gallus gallus, 263, 366
Species Index
Gavialis gangeticus, 309 Gecko gecko, 232 Gekko japonicus, 341 Geochelone carbonaria, 143 Geochelone denticulata, 143 Geochelone nigra, 75, 105, 189, 285, 293, 297 Geoclemys reevesii, 77 Gerrhonorus coeruleus, 99 Gerrhonotus coeruleus principis, 337 Gerrhosaurus nigrolineatus, 149, 340 Giant arabian tortoise, see Dipsochelys dussumieri Gila monster, see Heloderma suspectum Girdle-tailed lizard, see Cordylis nigra Glass snake, see Ophiodes fragilis Gopher tortoise, see Gopherus polyphemus Gopherus spp, 143 Gopherus agassizii, 134, 143, 171, 189, 285, 288, 290, 296, 297, 389 Gopherus berlandieri, 143 Gopherus gopherus, 291 Gopherus polyphemus, 189, 231, 285, 285, 291, 297 Graptemys flavimaculata, 288 Graptemys ouachitensis, 21 Great plains skink, see Eumeces obsoletus Green anole, see Anolis carolinensis Green iguana, see Iguana iguana Green sea turtle, see Chelonia mydas Gymnophthalamus underwoodi, 336 Hardwick’s spiny-tailed lizard, see Uromastix hardwickii Heloderma horridum, 337 Heloderma suspectum, 337 Hemidactylus brooki, 76 Hemidactylus flaviviridis, 66, 68, 70, 76, 81, 83, 123, 148, 341 Hemidactylus turcicus, 258 Hermann’s tortoise, see Testudo hermanni Holbrookia propinqua, 334, 335 Homonota darwini, 327 Homopus signatus signatus, 293 Hoplodactylus maculatus, 132, 178, 190, 194, 195, 196, 199, 200, 201, 255, 327, 328 Iberian rock lizard, see Iberolactera spp.; Lacerta monticola Iberian wall lizard, see Podacris hispanica Iberian worm lizard, see Blanus cinereus Iberolacerta cyreni, 149, 150, 151, 152, 153, 154, 155 Iberolacerta monticola, 156 Iguana iguana, 126, 146, 148, 151, 157 Imbricate alligator lizard, see Barisia imbricata imbricata Indian chequered water snake, see Natrix piscator Indian keeled lizard, see Mabuya carinata Indian wall lizard, indian house lizard, see Hemidactylus flaviventris Italian wall lizard, see Podacris sicula; Podacris sicula sicula
Japalura mitsukurii formosensis, 338 Japanese grass lizard, see Takydromus tachydromoides Karoo girdled lizard, see Cordylus polyzonus Keelback, see Tropidonophis mairii Keeled earless lizard, see Holbrookia propinqua Kemp’s ridley turtle, see Lepidochelys kempii Kinosternum odoratum, 143 Kinosternum spp, 143 Komodo dragon, see Varanus komodoensis Lacerta agilis, 153 Lacerta lepida, 149 Lacerta monticola, 42 Lacerta muralis, 82 Lacerta schreiberi, 149 Lacerta sicula, 94 Lacerta viridis, 149 Lacerta vivipara, 89, 94, 97, 103, 120, 122, 123, 124, 148, 152, 188, 189, 199, 200, 201, 217, 220, 225, 226, 233, 235, 237, 252, 254, 256, 257, 259, 262, 266, 329, 332, 335 Lampropholis guichenoti, 130 Lanthanotus borneensis, 337 Laticauda colubrina Laticauda colubrina, 357, 359 Leatherback turtle, see Demochelys coriacea Leiolopisma laterale, 101 Leopard gecko, see Eublepharus macularis Lepidochelys kempii, 171, 282, 288, 290, 296 Lepidochelys olivacea, 77, 171, 194, 195, 196, 197, 285, 288, 297 Lerista bouganvillii, 256, 257, 266 Liford’s wall lizard, see Podacris lilifordi Liolaemis bellii, 326 Liolaemis fabiani, 148 Liolaemus monticola, 42 Liolaemus pictus, 328 Liolaemus spp, 101, 147, 148, 151 Liolaemus tenuis, 150 Lissemys punctata punctata, 120, 122, 133, 134, 288, 297 Lithobates catesbeiana, 366 Little striped whiptail lizard, see Cnemidophorus inornatus Lizard, see Agama stellio Llama, see Lamia glama Loggerhead turtle, see Caretta caretta Lophognathus temporalis, 14 Mabuya spp, 102, 249, 261 Mabuya capensis, 76, 253 Mabuya carinata, 78, 100, 121, 174, 178, 179, 181, 182, 252 Mabuya heathi, 102, 339 Macrochelys temminkii, 9 Malaclemys terrapin, 288 Malaclemys terrapin centrata, 19, 285 Malacochersus tornieri, 277 Mauremys leprosa, 143 Mauremys rivulata, 285
399
Species Index
McCann’s skink, see Oligosoma maccanni Mediterranean gecko, see Hemidactylus turcicus Menetia spp, 339 Mexican beaded lizard, see Heloderma horridum Mexican gecko, see Phyllodactylus lanei Microlophus albemarlensis, 226 Microlophus delanonis, 226 Mojave rattlesnake, see Crotalus scutulatus Monopeltis anchietae, 335 Morelet’s crocodile, see Crocodylus moreletii Mountain lizard, see Japalura mitsukurii formosensis Mountain spiny lizard, see Sceloporus jarrovi Mud turtle, see Kinosternum odoratum; Stenotherus odoratus Mus musculus, 366 Musk turtle, see Kinosternum odoratus; Stenotherus odoratus Naja naja, 78, 94, 254, 357, 358, 360, 362, 389 Natator depressus, 288 Natrix maura, 173, 389 Natrix piscator, 362 Natrix sipedon pictiventris, 107 Nerodia spp, 96, 97, 131, 132, 252, 361 Nerodia fasciata, 388 Nerodia fasciata fasciata, 388 Nerodia rhombifera, 362 Nerodia sipedon, 223, 252, 254, 358, 360 Nerodia taxispilota, 254 Nile crocodile, see Crocodylus niloticus Nile monitor, see Varanus niloticus Niveoscincus decresiensis, 329 Niveoscincus metallicus, 101, 253 Niveoscincus metallicus, 106, 254, 329, 339 Niveoscincus microlepidotus, 107, 152, 251, 253, 254, 255, 261, 339 Niveoscincus microlepidus, 152 Niveoscincus ocellatus, 15, 255, 339 Northern fence lizard, see Sceloporous undulatus hyacinthinus Oligosoma spp, 339 Oligosoma maccanni, 340 Olive ridley turtle, see Lepidochelys olivacea Opheodrys aestivus, 358 Ophiodes fragilis, 337 Oriental garden lizard, see Calotes versicolor Painted turtle, see Chrysemys picta; Trachemys (Chrysemys) picta Pelomedusa subrafa, 295, 296 Peltocephalus spp., 295 Pelusios castanoides, 295 Phrynosoma cornutum blainvillei, 334 Phrynosonoma cornutum, 171, 190, 191, 195, 332, 334, 339 Phyllodactylus lanei, 327, 341 Phymaturus antofagastensis, 327 Phymaturus patagonicus, 327 Phymaturus punae, 327
Physignathus lesueurii, 29 Platysaurus broadleyi, 226, 340, 343 Podarcis spp, 148, 335 Podarcis hispanica, 42, 149, 150, 151, 154, 155 Podarcis lilifordi, 150 Podarcis melisellensis, 43, 225, 226 Podarcis muralis, 71, 225, 226 Podarcis sicula, 76, 78, 79, 96, 100, 102, 105, 131, 132, 191, 232, 329, 332, 335, 339, 389 Podarcis sicula compestris, 78, 330 Podarcis sicula sicula, 68, 75, 77, 79, 107, 108, 175, 177, 182, 189 Podocnemis expansa, 25, 282, 284, 285 Pogona barbata, 91, 103, 175, 190, 195, 338 Pogona vitticeps, 29 Psammobates tentorius tentorius, 292 Psammodromus algirus, 149, 155, 226, 335, 336 Pseudemoia entrecasteauxii, 261 Pseudemydura umbrina, 282, 285, 294, 295 Pseudemys nelsoni Pseudomoia spenceri, 259 Pygmy monitor lizard, see Varanus brevicaudus Python curtus, 364 Python reguis, 232 Rainbow whiptail lizard, see Cnemidophorus lemniscatus Ramphotyphlops braminus, 366 Rat snake, see Elaphe climacophora Rattus norvegicus, 366 Red-eared slider, see Pseudemys scripta elegans; Trachemys scripta; Trachemys (Psuedemys, Chrysemys) scripta elegans Red-sided garter snake, see Thamnophis sirtalis parietalis Red-spotted garter snake, see Thamnophis sirtalis concinnus Reeve’s turtle, see Geoclemys reevesii Rough greensnake, see Opheodrys aestivus Ruin lizard, see Podacris sicula compestris Sagebrush lizard, see Sceloperus graciosus Saiphos equali, 266 San diego coast horned lizard, see Phrynosoma cornutum blainvillei Sand lizard, see Lacerta agilis Sceloperus jarrovi, 132, 233, 251, 253, 262, 329, 332, 334 Sceloporus aeneus arenus, 257 Sceloporus aeneus bicanthalis, 257 Sceloporus cyanogenys, 105, 251, 252, 254 Sceloporus formosus, 328 Sceloporus graciosus, 42 Sceloporus grammicus, 328 Sceloporus jarrovi, 104, 230, 253, 254, 255, 326, 329 Sceloporus malachiticus, 328 Sceloporus mucronatus, 328 Sceloporus occidentalis, 79, 182, 189, 195, 389
Sceloporus poinsettia, 8 Sceloporus pyrocephalus, 225, 333 Sceloporus scalaris, 256 Sceloporus torquatus torquatus, 98 Sceloporus undulatus, 89, 102, 174, 190, 251, 253, 254, 329, 332, 333, 388 Sceloporus undulatus erythrocheilus, 226 Sceloporus undulatus hyacinthinus, 333 Sceloporus virgatus, 108, 333, 339 Sea snakes, see Laticauda spp Seminatrix pygaea, 121 Sharp-mouthed lizard, see Anolis pulchellus Shingleback skink, see Tiliqua (Tachydosaurus) rugosa Shinisaurus crocodilurus, 337 Side-blotched lizard, see Uta stansburiana Sitana ponticeriana, 119, 120, 121, 122, 123 Six-lined race runner, see Cnemidophorus sexlineatus Snapping turtle, see Chelydra serpentina Snow skink, see Niveoscincus microlepidus Soft-shelled turtle, see Lissemys punctata punctata Solomon island skink, see Corucia zebrata South African giant girdled lizard or sungazer, see Cordylus giganteus South American red-footed tortoise, see Geochelone carbonaria South American yellow-footed tortoise, see Geochelone denticulata Southern snow lizard, see Niveoscinus macirolepidotus Spanish terrapin, see Mauremys leprosa Sphenodon punctatus, 63, 103, 119, 144, 171, 175, 177, 189, 191, 196, 231 Sphenomorphus fragilis, 256 Sphenomorphus indicus, 15 Spiny-footed lizard, see Acanthodactylus erythrurus Spotted snow skink, see Niveoscinus ocellatus Staurotypus salvinii, 18 Staurotypus triporcatus, 18 Stenotherus odoratus, 75, 77, 122, 123, 177, 285, 286, 287, 290 Steppe’s tortoise, see Testudo horsfieldii Stoke’s skink, see Egernia stokesii Storeroa dekayi, 252 Striped plateau lizard, see Sceloporus virgatus Takydromus tachydromoides, 78 Tasmanian skink, see Niveoscincus metallicus Tasmanian skink, see Niveoscincus ocellatus Testudo hermanni, 120, 143, 285 Testudo hermanni boettgeri, 291 Testudo hermanni hermanni, 291 Testudo horsfieldii, 285, 292 Texas horned lizard, see Phrysonoma cornutum Thamnophis elegans Thamnophis elegans, 131, 202, 251, 252, 366 Thamnophis sirtalis, 122, 125, 225, 231, 252, 363, 365, 366
400
Thamnophis sirtalis concinnus, 178, 190, 193, 197, 198 Thamnophis sirtalis parietalis, 41, 45, 82, 91, 157, 158, 131, 159, 175, 178, 179, 184, 186, 187, 190, 193, 194, 195, 197, 198, 226, 355, 356, 359, 360, 361, 362, 363, 364, 365, 366, 367, 385 Tiliqua nigrolutea, 91, 94, 98, 99, 102, 231, 251, 254, 328, 339 Tiliqua rugosa, 76, 98, 132, 152, 175, 177, 231, 339 Tiliqua scincoides, 150 Trachemys scripta, 18, 19, 69, 71, 75, 76, 77, 79, 123, 125, 127, 131, 144, 174, 196, 226, 232, 366 Trachemys (Psuedemys, Chrysemys) scripta elegans, 52, 76, 144, 173, 385 Trachydosaurus rugosus, 252 Tree lizard, see Urosaurus ornatus Trimeresurus stenegeri, see Viridovipera stejnegeri Trionyx sinensis, 133 Tropidonophis mairii, 366 Tuatara, see Sphenodon punctatus Tungara frog, see Physalaemus pustulosus Turtle-headed sea snake, see Emydocephalus annulatus
Species Index
Uromastix hardwickii, 79, 107, 148, 254 Urosaurus ornatus, 42, 44, 50, 51, 54, 55, 104, 174, 175, 177, 184, 194, 195, 196, 197, 198, 200, 216, 217, 225, 226, 227, 228, 232, 234, 235, 236, 329, 334 Uta palmeri, 225 Uta stanisburiana, 42, 78, 101, 174, 177, 183, 184, 189, 198, 199, 200, 216, 217, 218, 219, 223, 225, 226, 228, 230, 233, 235, 236, 237, 238, 334 Varanus albigularis, 231, 337 Varanus brevicaudus, 337 Varanus komodoensis, 337 Varanus niloticus, 322 Veiled chameleon, see Chamaeleo calyptratus Vipera aspis, 102, 231, 254, 358, 359, 364, 389 Vipera berus, 25, 125, 225, 226, 280, 358, 362, 364, 389 Vipera spp, 102 Viridovipera stejnegeri (Trimeresurus stejnegeri), 358 Wall lizards, see Podacris spp. Water dragon, see Physignathus lesueurii Water snake, see Natrix maura maura
Water snake, see Nerodia fasciata Water snake, see Nerodia fasciata fasciata; Nerodia sipedon Westerm swamp tortoise, see Pseudemydura umbrina Western banded gecko, see Coeonyx variegatus Western diamond-backed rattlesnake, see Crotalus atrox Western fence lizard, see Sceloporus occidentalis Western hermann’s tortoise, see Testudo hermanni hermanni Western whiptail, see Cnemidophorus tigris Whiptail lizard, see Cnemidophorus nativo White-throated savannah monitor lizard, see Varanus albigularis Xantusia vigilis, 76, 252 Xenopus laevis, 366 Yellow-bellied house gecko, see Hemidactylus flaviviridis Zonosaurus boettgeri, 340 Zonosaurus maximus, 340 Zygaspis quadrifrons, 335
Subject Index Index Accessory sex organs excurrent duct system, 81, 118–120 femoral glands, 81, 126 infundibulum, 127–128 isthmus, 130 oviduct, 131–132 renal sexual segment, 81, 125–126 reproductive cycle and variation, 122 uterine tube, 128–130 uterus, 130 vagina, 130–131 ACTH, see Corticotropin Activin, endocrine-disrupting chemical effects, 384 AGBGM, see Androgen-glucocorticoidbinding globulin Aggression Hawk–Dove interactions, 235 social groups, monogamy, filiative behaviors, and aggression suppression, 236–237 Allostasis, definition, 170 AmbX, see Nucleus ambiguus g-Aminobutyric acid (GABA), stress circuits, 172 Amygdala, reproductive behavior regulation, 49–51, 53–54, 361 Androgen receptor (AR) female function, 96 muscle expression and reproductive behavior, 47 Androgen-glucocorticoid-binding globulin (AGBG), corticosterone binding, 174, 197, 236 Androstenedione, steroidogenesis, 74–75, 93–94 Anterior preoptic area of the hypothalamus, see Preoptic area Anti-Mu¨llerian hormone, see Mu¨llerianinhibiting substance AR, see Androgen receptor Arginine vasotocin (AVT) corticotropin-releasing hormone colocalization, 173 crocodile oviposition role, 314 egg retention role, 253, 255 fitness correlation in males, 230, 232–233 oviposition control, 132 reproductive behavior regulation, 82, 227 turtle reproductive cycles, 290–291 Aromatase sex determination, 17, 19 steroidogenesis, 74–75
temperature-dependent sex determination role, 91 Atresia, follicular, 101 AVT, see Arginine vasotocin BCI, see Body condition index Behavior, reproductive developmental effects of hormones on behavior, 216–220 ecological context of displays anoles, 40–41 sex differences, 41–43 endocrine network theory natural, sexual, and social selection, 223, 225–227 overview, 222–223 prolactin as master regulator, 237–240 fitness correlations arginine vasotocin, 230, 232–233 neurotransmitter systems, 233 overview, 227–229 parental care by crocodiles, 233 testosterone aggression and territoriality, 228, 230 colors and badges, 228 courtship and copulation, 230–231 territory size, 230 hormonal control activational events, 221–222 alternative reproductive strategies, 44 anoles, 43–44 leopard geckos, 45 male behaviors, 227 red-sided garter snakes, 45 snakes, 82 turtles, 45 viviparous species, 220–221 whiptail lizards, 45 neural control anoles, 46–49 leopard geckos, 50 red-sided garter snakes, 50 whiptail lizards, 49–50 overview, 39–40, 215–216 season and hormonal manipulation anoles, 53–54 leopard geckos, 54 red-sided garter snakes, 54 side-blotched lizards, 54 tree lizards, 54–55 whiptail lizards, 54 social groups, monogamy, filiative behaviors, and aggression suppression, 236–237
social networks and endocrine networks, 233–236 stress effects, 183–187 trait development anoles and whiptail lizards, 50–51 reptiles with alternative reproductive morphs, 51–52 reptiles with temperature-dependent sex determination, 52–53 Bisphenol A (BPA), endocrine disruption, 385 Body condition index (BCI), 389 Body length, see Snout-to-vent length BPA, see Bisphenol A Bradykinin ovarian synthesis, 92 vitellogenesis role, 103–104 Bully genotype, 234–235 Cadmium, endocrine-disruption, 382, 387 Calcium-ATPase, eggshell thickness role, 259 CAM, see Chorioallantoic membrane CBG, see Corticotropin-binding globulin Charnov–Bull model, sex differentiation and adaptation, 26–28 Cholesta-5,7-dien-3-ol, 153, 155 Chorioallantoic membrane (CAM) formation, 260–261 maternal recognition of pregnancy, 263–264 placenta emergence, 262 CL, see Corpus luteum Cloacal gland, pheromone secretion lizards, 149 snakes, 157 Clutch number, variability in turtles, 285 Clutch size, determinants, 100–101, 219 Constant temperature equivalent (CTE), models, 24–25, 30 Cooperation Hawk–Dove interactions, 235 social groups, monogamy, filiative behaviors, and aggression suppression, 236–237 Corpus luteum (CL) egg retention role, 250–253, 255 hormone production relaxin, 107–108 steroids, 107 hormone secretion, 250–251 life span, 106, 251 morphological changes, 106–107 prostaglandin F2a and luteolysis, 108 CORT, see Corticosterone
401
402
Corticosterone (CORT), see also Stress androgen level correlations, 175–179, 182 behavior effects, 183–187, 219 binding proteins, 174 crocodile reproduction and seasonality, 311 endocrine network theory, 223 fitness effects of stress during reproduction adults, 198–199 mothers, 199–200 offspring, 200–202 lizard reproduction regulation, 341–342 pulsatile secretion, 173 reproductive behavior regulation, 44–45, 54–55 seasonal changes baseline levels, 188–190 pre-breeding, breeding, and postbreeding periods, 188, 191–192 stress levels, 188–190 snake reproduction effects in females, 357, 365 stress response modulation during reproduction, 193–198 testicular function, 78 turtle reproductive cycles, 297 yolk, 104 Corticotropin (ACTH) hypothalamic-pituitary-adrenal axis, 173–174 stress response modulation during reproduction, 197 Corticotropin-binding globulin (CBG), 97 Corticotropin-releasing hormone (CRH), hypothalamic-pituitary-adrenal axis, 173 CRH, see Corticotropin-releasing hormone Crocodiles development of reproductive system gonadal development, 307–308 juvenile growth and peripubertal seasonality, 309 key events, 306 secondary sex characteristics, 308–309 molecular endocrinology and prospects for study, 315–316 overview, 305–306 parental care, 233 pheromones, 145 reproductive cycle females ovulation, gravidity, and oviposition, 312–314 vitellogenesis and oocyte maturation, 312 males, 311 seasonality, 309–311 CTE, see Constant temperature equivalent CYP3A, endocrine-disrupting chemical effects, 380–381 Cyproterone acetate, male reproductive behavior impact, 43 2,4-D, see 2, 4-Dichlorophenoxyacetic acid DAX1
Subject Index
endocrine-disrupting chemical effects, 385 sex determination, 17–18 DDD, see 1,1-Dichloro-2,3-bis (p-chlorophenyl)ethane DDE, see 1,1-Dichloro-2,3-bis (p-chlorophenyl)ethylene DDT, see Dichlorodiphenyltrichloroethane Deception, Hawk–Dove interactions, 235 Dehydroepiandrosterone (DHEA), steroidogenesis, 74–75, 93–94 Dewlap structure color, 219, 225 polymorphism in tree lizard, 47, 225 reproductive behavior regulation, 46–47 DHEA, see Dehydroepiandrosterone DHT, see Dihydrotestosterone 1,1-Dichloro-2,3-bis(p-chlorophenyl)ethane (DDD), endocrine disruption, 377 1,1-Dichloro-2,3-bis(p-chlorophenyl)ethylene (DDE), endocrine disruption, 376 2,4-Dichlorophenoxyacetic acid (2,4-D), endocrine disruption, 385 Dichlorodiphenyltrichloroethane (DDT), endocrine disruption, 376, 378, 390 Dihydrotestosterone (DHT) corticosterone level correlations, 175 lizard reproduction regulation, 331–333, 335–336, 341 ovarian synthesis, 93–94 reproductive behavior regulation, 43–45, 50–51 sex determination, 17, 19 snake reproduction females, 357–358 males, 358 testicular function, 78, 80–81 Dmrt1, sex determination, 16–17, 63 Dopamine aggression studies, 233 stress circuits, 172–173, 175 Dove genotype, 234–235 Ductus deferens, anatomy and histology, 121, 122 EDCs, see Endocrine-disrupting chemicals EGF, see Epidermal growth factor Eggshell formation, 130, 256–257 thickness reduction mechanisms, 255–259 ELISA, see Enzyme-linked immunosorbent assay Endocrine-disrupting chemicals (EDCs), see also specific chemicals growth and metabolism effects, 387–389 hatching and neonatal growth effects, 386–387 mechanisms of disruption, 388 mixtures and additive effects, 375–376 overview, 373–376 reproductive organ alterations, 382–384 reptiles as exposure models, 376–377
sex determination effects, 384–386 steroid hormone alterations, 378, 380–382 thyroid function effects, 386, 390 tissue concentrations, 377–378 windows of vulnerability, 373–374 Enzyme-linked immunosorbent assay (ELISA), hormone assays, 280 Epidermal growth factor (EGF), eggshell thickness role, 258 Epididymus anatomy, 119 anatomy, 120 development, 81 histology, 120, 121 secretory granule formation and regulation, 122–124 sperm storage, 125 Epinephrine, turtle reproductive cycles, 297 ER, see Estrogen receptor Ergosterol, 153 Estradiol corpus luteum production, 107 crocodile reproduction ovulation, 312–313 seasonality, 310 vitellogenesis, 312 eggshell thickness role, 257–258 endocrine-disrupting chemical effects, 376, 385 estrogen forms, 91 lizard reproduction regulation, 332–336, 338–340 maternal recognition of pregnancy, 262–263 organizational role in behavior, 216–218 reproductive behavior regulation, 44–45 reproductive cycles and secretion, 131 sex determination, 17, 19 snake reproduction role in females, 357, 366 steroidogenesis, 74, 76, 95 stress response in females, 178 testicular function, 79 turtle reproductive cycles, 286, 288–290, 294 vitellogenesis role, 102–103 Estrogen receptor (ER) endocrine-disrupting chemical effects, 381 forms, 96 oviduct, 131–132 shell glands, 259 Ethinylestradiol, endocrine disruption, 384 Female-male pattern, temperature-dependent sex determination, 9, 14 Female-male-female pattern, temperature-dependent sex determination, 9, 14 Female mimicry, snakes, 159–160, 364 Femoral gland behavioral assays, 126 pheromone secretion, 146–147 regulation, 81, 126
403
Subject Index
Flutamide excurrent duct system effects, 81 male reproductive behavior impact, 43 Follicle-stimulating hormone (FSH) clutch size determination, 101 crocodile juvenile growth and peripubertal seasonality, 309 endocrine-disrupting chemical effects, 382, 384 functional overview, 174–175 lizard reproduction regulation, 330, 332 ovarian function, 92, 100 ovulation control, 105 receptors in snakes, 360–361 reproductive behavior regulation, 44 snake reproduction, 360–361 testicular function, 77–78, 80–81 turtle reproductive cycles, 286–287 Follicles, see also Corpus luteum; Ovaries; Ovulation atresia, 101 clutch size determinants, 100–101 development, 98–100 endocrine-disrupting chemical effects, 382–383 hormonal regulation, 100 recruitment, 98 Follistatin, endocrine-disrupting chemical effects, 384 FSH, see Follicle-stimulating hormone GABA, see g-Aminobutyric acid GDF9, endocrine-disrupting chemical effects, 384 Genotypic sex determination (GSD) hybrid mechanisms, 10–12, 29 patterns, 2, 8 phylogenetic distribution of patterns, 8, 12–15 GH, see Growth hormone Glucocorticoids, testicular function, 78 GnIH, see Gonadotropin-inhibiting hormone GnRH, see Gonadotropin-releasing hormone Gonadotropin-inhibiting hormone (GnIH), 330, 338 Gonadotropin-releasing hormone (GnRH) endocrine network theory, 222–223 hypothalamic-pituitary-gonadal axis, 174–175 lizard reproduction regulation, 330–331, 333, 338 ovarian function, 92 ovulation control, 105 snake reproduction, 360–361 testicular function, 76–77 types, 174, 330 vitellogenesis role, 103–104 Gonadotropins, see Follicle-stimulating hormone; Luteinizing hormone Growth hormone (GH), vitellogenesis role, 103 GSD, see Genotypic sex determination
Hawk–Dove system, 234–235 Histamine, testicular function, 80 Homeostasis, definition, 170 HSD, see Hydroxysteroid dehydrogenase Hydroxysteroid dehydrogenase (HSD) chorioallantoic membrane expression, 264 corpus luteum expression, 107 steroidogenesis, 74–75 IGF-1, see Insulin-like growth factor-1 IL-1, see Interleukin-1 Infundibulum anatomy and histology, 127–128 sperm storage, 133–134 Insulin-like growth factor-1 (IGF-1) eggshell thickness role, 258 crocodile reproduction, 313 Interferons classification, 262 maternal recognition of pregnancy, 262 Interleukin-1 (IL-1), placental synthesis, 262 Isthmus, anatomy and histology, 130 Kin recognition, pheromones, 152 K-strategist, 233–235 Leydig cell secreted factors in testicular function, 80 structure and function, 68–69, 71 LH, see Luteinizing hormone Lizards classification, 324–325 pheromones chemosensory recognition kin recognition, 152 scent trails, 152 sex and individual recognition, 150–152 feces, 149–150 iguanas, 148 male social dominance and evolution, 155–157 mate choice and evolution, 152–155 Scleroglossa, 148–149 secretory glands cloacal gland, 149 femoral gland, 146–147 preanal gland, 146–147 precloacal gland, 146–147 skin, 146 urodeal gland, 149 rationale for study environmental and conservation considerations, 325–326 evolutionary considerations, 322–323, 325 practical considerations, 321–322 reproductive cycles hormonal control Agamidae, 338 chameleons, 338 Cordylidae, 340
Dibamidae, 341 geckos, 340–341 Gerrhosauridae, 340 glass lizards, 336–337 Gymnophthalmidae, 336 Helodermatidae, 337 hypthalamic-pituitary-adrenal axis, 341–342 iguanas, 331–335 Lacertidae, 335–336 Lanthanotidae, 337 overview, 330–331 rhythms, 342–343 Shinisauridae, 337–338 skinks, 339–340 Varanidae, 337 whiptail lizards, 336 worm lizards, 335 Xantusiidae, 340 Xenosauridae, 337 prospects for study, 343–344 seasonality, 326–328 types, 328–330 Luteinizing hormone (LH) functional overview, 174–175 lizard reproduction regulation, 330 ovarian function, 92, 100 ovulation control, 105 reproductive behavior regulation, 44 snake reproduction, 360–361 testicular function, 77–78 turtle reproductive cycles, 286, 290 Macrophage, testicular secreted factors in testicular function, 80 ultrastructure, 68, 72 Male-female pattern, temperature-dependent sex determination, 9 Melatonin lizard reproduction regulation, 342–344 snake reproduction role, 362 testicular effects of photoperiod, 67 turtle reproductive cycles, 297–298 N-Methyl-D-aspartate (NMDA), brain receptors, 172 Methyl thiophanate, endocrine disruption, 389 MIS, see Mu¨llerian-inhibiting substance MOFs, see Multi-oocytic follicles Monogamy, hormonal control, 236–237 Mu¨llerian-inhibiting substance (MIS) crocodile secondary sex characteristics, 308 sex determination, 16–17, 63–64 Multi-oocytic follicles (MOFs), endocrinedisrupting chemical effects, 382–383 Nasal gland, pheromone secretion, 157–158 Nesting, cyclicity in turtles, 282, 284–285 NMDA, see N-Methyl-D-aspartate Norepinephrine ovarian function, 174 stress circuits, 172, 175 turtle reproductive cycles, 297
404
Nucleus ambiguus (AmbX), reproductive behavior regulation, 46–47 Nucleus sphericus, see Amygdala Ovaries, see also Corpus luteum; Follicles; Ovulation development, 89–91 endocrinology and conservation, 108 regulation by pituitary hormones, 92 steroids overview, 91–92 peripheral metabolism, 97–98 plasma binding proteins, 96–97 receptors, 95–96 steroidogenesis, 93–95 stress effects on function, 179–182 structure, 89 Oviduct hormonal regulation, 131–132 sperm storage, 133–134 Oviparity, see also Viviparity definition, 247 overview, 117, 118 Oviposition crocodile, 314 hormonal control, 132 Ovoviviparity, definition, 248 Ovulation crocodiles, 312–314 hormonal control, 105 mechanisms, 104–105 oocyte maturation, 105 P4, see Progesterone PCBs, see Polychlorinated biphenyl compounds Peroxisome proliferator-activated receptor (PPAR), thyroid hormone effects, 389 Pheromones Amphisbaenians, 145–146 behavioral assays, 142 crocodiles, 145 lizards chemosensory recognition kin recognition, 152 scent trails, 152 sex and individual recognition, 150–152 feces, 149–150 iguanas, 148 male social dominance and evolution, 155–157 mate choice and evolution, 152–155 Scleroglossa, 148–149 secretory glands cloacal gland, 149 femoral gland, 146–147 preanal gland, 146–147 precloacal gland, 146–147 skin, 146 urodeal gland, 149 overview, 141 prospects for study, 159–160
Subject Index
Rhynchocephalia, 144–145 snakes chemosensory recognition female mimicry, 159–160 mate assessment, 158–159 sex discrimination and trailing, 158 secretory glands cloacal gland, 157 nasal gland, 157–158 skin, 157 turtles Cheloniidae, 144 Emydidae, 143–144 functional overview, 143 sources of pheromones, 142–143 Testudinae, 143 vomeronasal system, 141–142 Pineal gland, see Melatonin Placenta emergence, 259–262 progesterone production, 261 types, 261 POA, see Preoptic area Polychlorinated biphenyl compounds (PCBs), endocrine disruption, 375–376, 385–386 PPAR, see Peroxisome proliferator-activated receptor PR, see Progesterone receptor Preanal gland, pheromone secretion, 146–147 Precloacal gland, pheromone secretion, 146–147 Pregnancy, see Viviparity Pregnenolone, steroidogenesis, 74, 93–94 Preoptic area (POA), reproductive behavior regulation, 46, 49–51, 53–54, 336, 361 PRL, see Prolactin Progesterone (P4) corpus luteum egg retention role, 250–253, 255 production, 107 production, 250–251 crocodile ovulation role, 312–313 immunomodulation, 262 levels in squamate reproductive cycle, 254 lizard reproduction regulation, 331–332, 334–340 metabolism, 98 oocyte maturation role, 105 organizational role in behavior, 217–218 oviposition control, 132 placental production, 261 reproductive behavior regulation, 44–45 reproductive cycles and secretion, 131 snake reproduction role, 357 steroidogenesis, 74, 93–94 stress response females, 179 males, 178 turtle reproductive cycles, 287, 289, 292 vitellogenesis role, 103 yolk, 104 Progesterone receptor (PR)
chorioallantoic membrane expression, 264 overview, 96 oviduct, 131–132 Prolactin (PRL), behavior regulation, 237–240 Prostaglandins crocodile oviposition role, 314 maternal recognition of pregnancy, 263 ovarian synthesis, 92 ovulation control, 105 prostaglandin F2a luteolysis role, 108 oviposition control, 132 reproductive behavior regulation, 44 snake reproduction role, 363 snake reproduction role, 359–364 turtle reproductive cycles, 290–291 Relaxin, corpus luteum production, 107–108 Renal sexual segment (RSS) regulation, 81 secretory granules, 125–126 Retractor penis magnus (RPM) muscle, reproductive behavior, 47–48 RPM, see Retractor penis magnus RSS, see Renal sexual segment R-strategist, 233–234 Scent trails lizards, 152 snakes, 158 SCN, see Suprachiasmatic nucleus SDM, see Sex determining mechanisms Seminiferous tubules development, 66 number, 119 Septum, reproductive behavior regulation, 361 Serotonin aggression studies, 233 stress circuits, 172–173, 175 Sertoli cell histology, 66–67, 69–70 secreted factors in testicular function, 79–80 Sex determining mechanisms (SDMs) dichotomies versus continuums, 10–12 diversity adaptive significance, 25–28 coexistence of genotypic sex determination and temperaturedependent sex determination, 29–30 temperature-dependent sex determination ecological relevance, 24–25 evolutionary potential, 28–29 endocrine-disrupting chemical effects, 376 evolutionary transitions, 15 family comparisons, 4–7 genotypic sex determination patterns, 2, 8 overview, 1–2 phylogenetic distribution of patterns, 8, 12–15
405
Subject Index
prospects for study, 30–31 proximate mechanisms, 16–20 temperature-dependent sex determination patterns, 8–10 thermosensitive period experimental determination, 20–22 variation and ecological implications, 21, 23 timing overview, 20 Sex hormone-binding globulin (SHBG), 97 Sf1, sex determination, 17–18, 63 SHBG, see Sex hormone-binding globulin SHBP, see Steroid hormone-binding protein She-male, see Female mimicry Snakes female mimicry, 159–160, 364 gonadotropins in reproduction, 360–361 neuroendocrinology of reproduction, 361 overview, 355 pheromones chemosensory recognition female mimicry, 159–160 mate assessment, 158–159 sex discrimination and trailing, 158 secretory glands cloacal gland, 157 nasal gland, 157–158 skin, 157 prospects for study, 365–367 reproductive cycles embryonic influences, 365 environmental influences, 362 overview, 355–356 physiological influences, 364–365 social influences, 362–364 steroids and reproduction females, 357–358 males, 358–359 red-sided garter snakes females, 359 males, 359–360 Snout-to-vent length (SVL) crocodile juvenile growth, 309 maternal stress effects on offspring, 201–202 Sox9, sex determination, 16–17 Spermatogenesis patterns and androgen levels, 73, 75 postnuptual cycle, 118 prenuptual cycle, 118 species comparison, 69, 73–74 Sperm storage epididymus, 125 female reproductive tract, 133–134 Sry, sex determination, 16–17 Steroid hormone-binding protein (SHBP), snakes, 361 Steroidogenesis, crocodile molecular endocrinology, 315 Stress corticosterone seasonal changes baseline levels, 188–190
pre-breeding, breeding, and postbreeding periods, 188, 191–192 stress levels, 188–190 definitions, 169–170 fitness effects during reproduction adults, 198–199 mothers, 199–200 offspring, 200–202, 365–367 prospects for study, 202–204 reproductive function relationship behavior, 183–187 neurotransmitters, 175 ovarian function, 179–182 overview, 171–172 reproductive hormones, 175–179 testicular function, 182–183 reptile study importance, 171 response mediation brain, 172–173 hypothalamic-pituitary-adrenal axis, 173–174 hypothalamic-pituitary-gonadal axis, 174–175 response modulation during reproduction evidence, 192–197 mechanisms, 197–198 Suprachiasmatic nucleus (SCN), circadian rhythm regulation, 342 SVL, see Snout-to-vent length TCDD, see 2,3,7,8-Tetrachlorodibenzop-dioxin Temperature-dependent sex determination (TSD) aromatase role, 91 ecological relevance, 24–25 evolutionary potential, 28–29 hybrid mechanisms, 10–12, 29 patterns, 8–10 phylogenetic distribution of patterns, 8, 12–15 proximate mechanisms, 18–20 reproductive trait development, 52–53 thermosensitive period experimental determination, 20–22 variation and ecological implications, 21, 23 Testes development, 63–65 functional regulation androgens, 78–79 environmental factors, 76 estrogens, 79 histamine, 80 hypothalamic hormones, 76–77 immune function, 80–81 Leydig cell-secreted factors, 80 model, 83 pituitary hormones, 77–78 Sertoli cell-secreted factors, 79–80 stress hormones, 78 testicular macrophage-secreted factor, 80 thyroid hormone, 78
spermatogenesis, 69–74 steroidogenesis, 74–76 stress effects on function, 182–183 structure interstitial compartment, 67–69 tubular compartment, 66–67 Testosterone corpus luteum production, 107 corticosterone level correlations, 175–179, 182 crocodile reproduction gonadal development, 307 juvenile growth and peripubertal seasonality, 309 male cycles, 312 seasonality, 310 secondary sex characteristics, 309 vitellogenesis, 312 endocrine-disrupting chemical effects, 378, 380 fitness correlation in males aggression and territoriality, 228, 230 colors and badges, 228 courtship and copulation, 230–231 territory size, 230 lizard reproduction regulation, 331–341 metabolism, 97, 380 organizational role in behavior, 216–217 ovarian synthesis, 91–92, 93–94 reproductive behavior regulation, 43–45, 50–51, 53–55, 227 reproductive cycles and secretion, 131 sex determination, 17, 19 snake reproduction females, 357–359 males, 358–360 spermatogenesis patterns and androgen levels, 73, 75 steroidogenesis, 74 turtle reproductive cycles, 286–289, 292–294, 296 yolk, 104 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), endocrine disruption, 384 TGF-b, see Transforming growth factor-b Thermosensitive period (TSP) experimental determination, 20–22 variation and ecological implications, 21, 23 Thyroid hormone endocrine-disrupting chemical effects, 386–389 forms, 296 snake reproduction role, 362 turtle reproductive cycles, 296 Tongue flick, pheromone detection assay, 141–142 TPN muscle, see Transversus penis muscle Transcortin, see Corticotropin-binding globulin Transforming growth factor-b (TGF-b), endocrine-disrupting chemical effects, 384
406
Transversus penis (TPN) muscle, reproductive behavior, 46–48 TSD, see Temperature-dependent sex determination TSP, see Thermosensitive period Turtles behavior hormonal control, 45 cyclicity of reproduction clutch number variability, 285 nesting, 282, 284–285 prenuptual and postnuptual cycles, 280–281 sexual maturation, 281–282 timing of reproduction, 285 gonadal cycle assessment, 278–279 hormonal and gonadal reproductive cycles catecholamines, 297 corticosterone, 297 Cryptodira freshwater turtles, 285–288 sea turtles, 288–290 tortoises, 290–293 pineal hormones and annual cycles, 297–298 Pleurodires Chelidae, 293–295 Pelomedusidae, 295–296 Podocnemididae, 295 thyroid hormone, 296
Subject Index
hormone assays, 280 overview, 277–278 pheromones Cheloniidae, 144 Emydidae, 143–144 functional overview, 143 sources of pheromones, 142–143 Testudinae, 143 Urodeal gland, pheromone secretion, 149 Uterine tube, anatomy and histology, 128–130 Uterus eggshell formation, 130, 256–257 epithelium, 130 shell glands, 256–257, 259 Vagina, anatomy, 130–131 Ventromedial hypothalamus (VMH), reproductive behavior regulation, 46, 49–51, 53–54, 336, 361 Vitamin D, provitamins as pheromones in lizard mate choice, 153–154 Vitellogenesis crocodiles, 312 hormonal regulation, 102–104 mechanisms, 102 overview, 101–102
steroids in yolk Vitellogenin cadmium effects on expression, 382 function, 102 sequestration, 102 Viviparity behavior hormonal control, 220–221 definition, 247–248 egg retention and gestation length, 250–255 eggshell thickness reduction mechanisms, 255–259 evolution selective forces, 248–250 squamates, 248 maternal recognition of pregnancy, 262–263 placenta emergence, 259–262 prospects for study, 265–268 reproductive morphology and physiology, 250 VMH, see Ventromedial hypothalamus VNO, see Vomeronasal organ Vomeronasal organ (VNO), functional overview, 141–142 Wt1, sex determination, 17–18, 63 Yolk, see Vitellogenesis
Color Plates
FIGURE 2.1 Male Anolis carolinesis, Louisiana, USA. Photo by M. Johnson.
FIGURE 3.2 Temperature-dependent cellular reorganization in the bipotential gonad of a turtle, Trachemys scripta. In the bipotential gonad, at stage 17 primordial germ cells (PGCs) (red) are associated with the coelomic epithelium. By stage 23, temperature-dependent sex differentiation occurs. At 26 C temperature, PGCs become enclosed within the testis cord, while at 31 C sex cords degenerate (DSC) and PGCs remains in the periphery. The primordial germ cells are highlighted by an anti-VASA antibody (red) and sex cords (SC) and testis cords (TC) are outlined by an anti-laminin antibody (green). Modified from Yao and Capel (2004).
FIGURE 3.11 The testicular macrophage (M) of the Indian wall lizard, showing phagocytosis of a heat-killed yeast cell (arrow). Numerous phagocytic processes can be seen; X 480.
FIGURE 3.13 The biosynthetic pathway of sex steroids. The conversion of cholesterol to androgen is carried out by three primary enzyme systems, namely C27 side-chain cleavage P450, C21 side-chain cleavage P450, and D5-3b-hydroxysteroid dehydrogenase and D5-4-isomerase. The C27 side-chain cleavage P450 enzymes are located in the mitochondria, whereas the other two enzyme systems are in the smooth endoplasmic reticulum.
FIGURE 3.14 Schematic representation of the regulation of testicular immune responses in the Indian wall lizard Hemidactylus flaviviridis. Red lines denote inhibition whereas green arrows indicate stimulation of a particular response. ROS, reactive oxygen species.
FIGURE 3.16 Model proposed for endocrine and paracrine regulation of testicular functions in squamates. Red lines denote inhibition/negative feedback whereas the green arrows represent stimulation of a particular response. LPS, lipopolysaccharide; ROS, reactive oxygen species.
FIGURE 10.1 Leatherback sea turtle (Dermochelys coriacea) covering her nest. Note the white spot on the skull (arrow), indicating the location of the well-developed pineal complex.
FIGURE 10.2 Laparoscopy on an adult male loggerhead sea turtle (Caretta caretta) following proper surgical procedures.
FIGURE 10.3 Ultrasound examination of the gonads in an adult male loggerhead sea turtle (Caretta caretta).
FIGURE 10.5 Laparoscopic (1, testis; 2, epididymis) and histological images (3) of reproductively active (a) and inactive (b) adult male loggerhead sea turtles (Caretta caretta) collected in Cape Canaveral, Florida. E, epididymis; T, testis; ST, seminiferous tubules. Reproduced from Blanvillain et al. (2008), with permission.
FIGURE 10.6 Soft plastron in a reproductively active male Caretta caretta with contour lines from the PLASTron software, as described in Blanvillain et al. (2008). Reproduced from Blanvillain et al. (2008), with permission.
