Advances in Developmental Biochemistry, Volume 5

ADVANCES IN DEVELOPMENTAL B I OC HEMISTRY

Volume 5

1999

This Page Intentionally Left Blank

ADVANCES IN DEVELOPMENTAL BIOCHEMISTRY

Editor:

PAUL WASSARMAN Department of Cell Biology and Anatomy Mount Sinai School of Medicine New York, New York

VOLUME 5

1999

@ JAI PRESS INC. Stamford, Connecticut

Copyright 0 1999 /A/ PRESS INC. 100 Prospect Street Stamford, Connecticut 06907

All rights resewed. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any way, or by any means, electronic, mechanical, photocopying, recording, filming or otherwise without prior permission in writing from the publisher.

ISBN: 0-7623-0202-X Manufactured in the United States of America

CONTENTS

vi i

LIST OF CONTRIBUTORS PREFACE Paul M . Wassarman

IX

GENETIC CONTROL OF MESODERM PATTERNING AN D DIF F E RE NTIAT1ON DURING DROSOPHllA EMB RYOGENES IS Manfred Frasch and Hanh T. Nguyen

1

ACROSOMAL PROTEINS OF ABALONE SPERMATOZOA Victor D . Vacquier, Willie 1. Swanson, Edward C. Metz, and C. David Stout

49

CAPACITATION OF THE MAMMALIAN SPERMATOZOON Gregory S. Kopf, Pablo E. Visconti, and Hannah Galantino-Homer

83

OVARIAN NITRIC OXIDE: A LOCAL REGULATOR OF OVULATION, OOCYTE MATURATION, AND LUTEAL FUNCTION Lisa M . Olson

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THE REGULATION AND REPROGRAMING OF GENE EXPRESSION IN THE PREIMPLANTATION EMBRYO Richard M . Schultz

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ROLES OF METAL LOPROT EASE-DIS I NTEGRINS I N CELL-CELL INTERACTIONS AND IN THE CLEAVAGE OF TNFa AND NOTCH Carl P. Blobel

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V

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CONTENTS

A N I NTIMATE B IOC HEMISTRY: EGG-REGULATED ACROSOME REACTIONS OF MAMMALIAN SPERM Harvey M. Florrnan, Christophe Arnoult, lrnrana C. Kazam, Chungqing Li, and Christine M.B. O’Toole

199

INDEX

235

LIST OF CONTRIBUTORS

Chrisrophe Arnoult

Laboratoire de Biophysique Moleculaire et Cellulaire CNRS Grenoble, France

Carl P. Blobel

Cellular Biochemistry and Biophysics Program Memorial Sloan-Kettering Cancer Center New York, New York

Harvey M . Florrnan

Department of Anatomy and Cellular Biology Tufts University School of Medicine Boston, Massachusetts

Manfred Frasch

Brookdale Center for Developmental Molecular Biology Mount Sinai School of Medicine New York, New York

Hannah Callantino-Homer

Center for Research on Reproduction and Women’s Health University of Pennsylvania Medicz! Center Philadelphia, Pennsylvania

irnrana C. Kazarn

Department of Anatomy and Cellular Biology Tufts University School of Medicine Boston, Massachusetts

Gregory S. Kopt‘

Center for Research on Reproduction and Women’s Health University of Pennsylvania Medical Center Philadelphia, Pennsylvania vii

...

Vlll

LIST OF CONTRIBUTORS

Chunying L i

Department of Anatomy and Cellular Biology Tufts University School of Medicine Boston, Massachusetts

Edward C. Metz

Center for Marine Biotechnology and Biomedicine Scripps Institution of Oceanography University of California San Diego La Jolla, California

Hahn T. Nguyen

Division of Cardiology Albert Einstein College of Medicine Bronx, New York

Christine M.B. O’Toole

Department of Anatomy and Cellular Biology Tufts University School of Medicine Boston, Massachusetts

Lisa M . Olsen

Department of Obstetrics and Gynecology Washington University School of Medicine St. Louis, Missouri

Richard M. Schultz

Department of Biology University of Pennsylvania Philadelphia, Pennsylvania

C. David Stout

Department of Molecular Biology The Scripps Research Institute La J o b , California

Willie 1. Swanson

Center for Marine Biotechnology and Biomedicine Scripps Institution of Oceanography University of California San Diego La Jolla, California

Victor D. Vacquier

Center for Marine Biotechnology and Biomedicine Scripps Institution of Oceanography University of California San Diego La J o b , California

Pablo E. Visconti

Center for Research on Reproduction and Women’s Health University of Pennsylvania Medical Center Philadelphia, Pennsylvania

PREFACE Advances in Developmental Biochemistty was launched as a series by JAI Press in 1992 with the appearance of Volume 1. The series is inextricably linked to the companion series Advances in Developmental Biology, which was launched at the same time. As stated in the preface to Volume I-“Together the two series will provide annual reviews of research topics in developmental biology/biochemistry, written from the perspectives of leading investigators in these fields. It is intended that each review draw heavily from the author’s own research contributions and perspective. Thus, the presentations are not necessarily encyclopedic in coverage, nor do they necessarily reflect all opposing views of the subject.” Volume 5 of the series follows these same guidelines. Volume 5 of Advances in Developmental Biochemistry consist: of seven chapters that review specific aspects of development in several different organisms including mollusks, flies, and mice. Five of the seven chapters address aspects of fertilization, including capacitation of sperm (Chapter 3), the acrosome reaction (Chapter 7), gamete adhesion (Chapters 2 and 6), and oocyte maturation and ovulation (Chapter 4). In Chapter 1, Frasch and Nguyen discuss the genetic control of mesoderm patterning and differentiation during Drosophila embryogenesis. The authors present a comprehensive description of the origins, genetics, patterning, and specification of mesoderm, from gastrulation through organ development. Their insights into the genetic and molecular mechanisms that regulate interactions during mesoderm development provide a framework for understanding of mesoderm development not only in Drosophila, but also in other invertebrate and vertebrate species. IX

X

PREFACE

In Chapter 2, Vacquier and coauthors present a detailed account of their research on two acrosomal proteins of abalone sperm. This research has significantly advanced our understanding of how species-specificity evolves. Knowledge of the behavior and structure of these sperm proteins has suggested possible mechanisms of evolution of species-specific gamete interactions and has demonstrated that these proteins may play essential roles in establishing reproductive isolation between species. In Chapter 3, Kopf and coauthors summarize recent studies on capacitation of mammalian sperm. The chapter examines the role of media constituents, membrane events, and transmembrane and intracellular signaling during sperm capacitation. The authors provide detailed information about the biochemical and molecular events that form the basis of this critical event in mammalian fertilization. In Chapter 4, Olson discusses ovarian nitric oxide, a local regulator of ovulation, oocyte maturation, and luteal function in mammals. Although this is a relatively new area of investigation, there is compelling evidence that nitric oxide is a local regulator of all of these key ovarian processes in mammals. The author provides an experimental basis for this conclusion and for future studies. In Chapter 5 , Schultz reviews research on the regulation and reprogramming of gene expression in preimplantation embryos. The presentation focuses on the molecular basis of the maternal-to-zygotic transition (zygotic genome activation) in which maternal transcripts that direct early mammalian development are replaced by transcripts expressed from the embryonic genome. In a detailed analysis, the author summarizes the latest exciting findings in this area of research. In Chapter 6, Blobel discusses the roles of metalloprotease-disintegrins in cell-cell interactions and in the cleavage of T N F a and Notch. A brief summary of the known properties of snake venom disintegrins and metalloproteases is followed by a discussion of the role of membrane-anchored metalloprotease-disintegrins in cell-cell interactions and proteolysis of extracytoplasmic or extracellular protein domains. This timely and exciting area of research bears on aspects of mammalian fertilization and on cell fate decisions during development. In Chapter 7, Florman and coauthors review the mechanisms of egg-regulated acrosome reactions of mammalian sperm. Among the aspects discussed are the nature of primary signal transducers and targets stimulated by binding of acrosomeintact sperm to receptors in the egg zona pellucida. In a detailed presentation, the potential roles of several sperm components including G proteins, cation channels, tyrosine kinases, calcium, and cellular pH are considered. I am grateful to the authors for their excellent contributions, as well as for their cooperation and great patience during the preparation of this volume. I hope that the final product justifies the wait. Paul M. Wassarman Series Editor

GENETIC CONTROL OF MESODERM PATTERNING AND DIFFERENTIATION DURING DROSOPHlLA EMBRYOGENESIS

Manfred Frasch and Hanh T. Nguyen

I. Introduction

..........

A. Formation and Morphogenetic Movements of the Mesoderm . . . . . . . . . . . . . . 3 C. Genes Regulating Mesoderm Invagination: concertina, folded gastrulation, Rho, RhoGEF. . . . . . . . . . . . . . . . . . . . . . . . . . 4 D. heartless, A Gene Regulating the Dorsal Spreading of the Invaginated Mesoderm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 111. Embryonic Origin and Morphogenesis of Mesodermal Tissues. . . . . . . . . . . . . . . .5 A. Somatic Musculature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 B. Visceral Musculature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 C. Fat Body and Gonadal Mesoderm. . . . . . . . . . .9 D. Dorsal Vessel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 E. Hemocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 F. Dorsal Median (DM) Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Advances in Developmental Biochemistry Volume 5, pages 1-47. Copyright 0 1999 by JAI Press Inc. All right of reproduction in any form reserved. ISBN: 0-7623-0202-X

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MANFRED FRASCH and HANH T. NCUYEN

IV. Mesodermally-Expressed Genes Determining Broad Areas and Specific Tissues in the Mesoderm , . . , . . . . . . . . . . . . . 11 A. tinman, a Homeobox Gene Required for the Formation of Visceral Musculature, Dorsal Somatic Muscles, and the Heart . . B. hagpipe, a Homeobox Gene Required for Visceral Muscle Forma C. serpent, a GATA Gene Regulating Fat Body and Hemocyte Development . . . . . . . . . . . . . . . . . . . . . . . . .15 D. The Roles of clift, zJh-1, tin, and Abd-A in Gonadal Mesoderm Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 E. huttonless, A Homeobox Gene Required for DM Cell Formation . . . . . . . . . . 17 V. Early Patterning Mechanisms of the Mesoderm. . . . . . . . . . . . . . . . . . . . . . . . . . . 17 A. Dorsoventral Patterning. . . . . . . . . . . . . . . B. Anteroposterior Patterning. . . . . . . . . . . . . C. A Combinatorial Model of Mesoderm Patt D. Molecular Aspects of Early Mesoderm Patterning . . . . . . . . . . . . . . . . . . . . . . 26 VI. Patterning and Specification within the Developing 27 Heart and Visceral Mesoderm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... A. The Diversification of Heart Cells. . . . B. Anteroposterior Patterning of the Midgut Visceral Mesoderm. . . . . . . . . . . . . 28 VII. Patterning and Specification of Body Wall Muscles . A. A Role of twist in Muscle Development . . . . . . . . . . . . . . . . . . . . B. The Founder Cell Concept of Somatic Muscle De C. Genetic Mechanisms in Formation and Specification of Founder Cells. . , . , . 3 0 VIII. Genetic Control of Mesodermal Tissue Differentiation . . . . A. The Role ofmej2 in the Differentiation of Somatic Muscles,Visceral Mesoderm, and the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 B. Distinct Enhancer Modules of mej2 Linking Patterning and Differentiation Events . . . . . . . . . . . . . . . . . . . . . . . . . 36 XI. Concluding Remarks . . . . . . . . . . . . . . . . .40 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof .................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40

1.

INTRODUCTION

The mesodermal germ layer forms during gastrulation and gives rise to a variety of internal tissues and organs in all triploblastic animals. The composition, structure, and function of many of these tissues have clear similarities in different species, indicating that at least some of the mechanisms that regulate their development have been evolutionarily conserved. In recent years, the fruit fly Drnsophila has become one of the premier model systems to study the processes of gastrulation, patterning processes that subdivide the mesoderm, and regulatory events that control the differentiation of individual mesodermal tissues. Much has been learned about the genetic hierarchies that are responsible for the invagination and spreading of the mesoderm

Genetic Control of Mesoderm Patterning and Differentiation

3

into the interior of the embryo, progressive subdivisions generating the anlagen ofthe mesodermal derivatives, and differentiation of these anlagen into the mature tissues. It has become evident that many of these steps involve an interplay between mesoderm-intrinsic regulators and spatially-restricted inductive signals that are released from ectodermal cells. We summarize the insights into the genetic and molecular mechanisms of these regulatory interactions that have been obtained in recent years and that have provided a framework for the understanding of mesoderm development not only in Drosophilu, but also in other invertebrate and vertebrate species.

II. A.

MESODERM FORMATION, GASTRULATION, AND MESODERM MIGRATION Formation and Morphogenetic Movements of the Mesoderm

The Drosophila mesoderm is specified during the blastoderm stage in the ventral quadrant ofthe embryo, corresponding to about a20-cell-wide area that extends between about 5-95% egg length (0%=posterior pole) along the anterior/posterior axis. Gastrulation initiates at cellular blastoderm with the apical flattening and constriction of mesodermal cells, which subsequently elongate and invaginate to form a ventral furrow. Upon completion of the invagination, the hollow tube of mesodermal cells collapses and contacts the inside of the ectoderm. Thereafter, the mesodermal layer spreads dorsally and finally forms a monolayer of cells that extends from the ventral midline to the border between the dorsal ectoderm and amnioserosa (Poulson, 1950; Sonnenblick, 1950; Leptin and Grunewald, 1990; Sweeton et al., 1991). Until this stage (stage 10; 4.5 hrs. after egg laying), no clear morphological differences among individual mesodermal cells can be discerned and mesodermal cells are not yet committed to particular developmental fates (Beer et al., 1987). Genetic and molecular studies have defined a number of genes that have essential roles in the specification and morphogenetic movements of the early mesoderm. as summarized below. B.

Genes Determining the Mesodermal Anlage: Dorsal, twist, snail

The formation of the Drosophilu mesoderm is largely determined by autonomous regulatory mechanisms that act within the nuclei or cells that acquire mesodermal fates. Mesoderm formation is initiated by a nuclear gradient of the NKKB-related morphogen Dorsal, which is synthesized from maternally provided, ubiquitously distributed mRNA (reviewed in Rusch and Levine, 1996). Peak levels of nuclear Dorsal protein are present along the ventral midline of blastoderm embryos and are required to activate two zygotic genes, mist and snail, which are essential for mesoderm formation. Whereas the expression of twist extends to both poles of the embryo and is tapered toward the lateral sides of its expression domain, snailexpression is limited to a

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MANFRED FRASCH and HANH T. NGUYEN

ventral area between about 5-95% egg length with sharp lateral borders. It is precisely the area of cells coexpressing twist and snail that gives rise to the mesoderm. twist encodes a basic helix-loop-helix (bHLH) protein and snail a zinc-fingercontaining protein, indicating that the products of both genes act as transcription factors (Boulay et al., 1987; Thlsse et al., 1988). Mutations of either of these two genes cause a complete absence of invagination and differentiation of the mesoderm (Grau et al., 1984; Simpson, 1983). Genetic evidence also suggests that the two genes play different roles in mesoderm development. snail is thought to function largely, although not exclusively, to repress nonmesodermal genes in the prospective mesoderm and thus has a predominantly permissive role in mesoderm formation. By contrast, twist appears to have a key role in activating downstream genes that are required for the processes of invagination, patterning, and differentiation of the mesoderm (Kosman et al., 1991; Leptin, 1991). Recent studies have identified some twist target genes and have functionally defined the interaction of Twist protein with sequences In their enhancer elements (see below).

C. Genes Regulating Mesoderm Invagination: concertina, folded gastrulation, Rho, RhoCEF

The complete lack of mesoderm invagination in twist mutant embryos indicates that twist activates one or several target genes that mediate the cell shape changes required for gastrulation. Observations with particular alleles of snail that fail to repress neuroectodermal genes ventrally but still allow mesoderm invagination suggest that snail may also activate some genes that mediate gastrulation movements (Ip et al., 1994; Hemavathy et al., 1997). folded gastrulation (fog) is presently the only known gene that is expressed in a twist-dependent manner i n ventral areas of the blastoderm and that is required for regulating mesoderm invagination. In fog mutants, the concerted apical constriction of mesodermal cells does not occur, leading to their failure to invaginate, Sincefog encodes a secreted protein, it is thought to act as a signal that coordinates apical constrictions of mesodermal cells (Costa et al., 1994). While the receptor of Fog has not yet been identified, concertina (cta), the a-subunit of a trimeric G-protein, is likely to be involved in the intracellular transduction of theFog signal. This is indicated by the gastrulation phenotype of embryos lacking maternally derived Cta activity, which is very similar to the defects observed infog mutant embryos (Parks and Wieschaus, 1991). Other effectors of cell shape changes during gastrulation include the Rho GTPase and its exchange factor, RhoGEF.fog requires RhoGEF to elicit invagination events, and in embryos that are mutant for RhoGEF or express a dominant negative form of Rho, mesoderm invagination does not occur (Barrett et al., 1997; Hacker and Perrimon, 1998). Taken together, these observations suggest a signaling pathway in which twist and snail transcriptionally activate f o g and probably additional genes encoding signaling molecules that bind to transmembrane receptors on ventral cells of the blastoderm embryo (Barrett et al.,

Genetic Control of Mesoderm Patterning and Differentiation

5

1997; Morize et al., 1998). Activated receptor molecules may transmit these signals through a trimeric G-protein that includes the G a subunit Cta, which may directly or indirectly activate RhoGEF and, in turn, Rho. Rho has been shown to induce cell shape changes in cultured cells, at least partly by triggering a reorganization of the actin cytoskeleton (Ridley and Hall, 1992). However, the molecular targets of Rho that induce changes in mesodermal cell morphologies during gastrulation in Drosophila remain to be identified. D. heartlesr, A Gene Regulating the Dorsal Spreading of the lnvaginated Mesoderm

The collapse of the hollow mesodermal tube after invagination occurs during a wave of mitosis in the mesoderm and may therefore be a passive event. By contrast, the subsequent process of dorsal spreading of the mesoderm appears to be regulated by specific signaling mechanisms. It has been shown that the heartless gene is a critical component in regulating dorsal migration of the mesoderm (Beiman et al., 1996; Gisselbrecht et al., 1996; Shishido et al., 1997). In heartless mutant embryos, the mesoderm invaginates normally; however, it subsequently fails to spread towards the dorsal ectoderm and remains as a multilayered mass near the site of invagination. Interestingly, heartless encodes an FGF-receptor molecule that is specifically expressed in the mesoderm in a mist-dependent manner, indicating that the dorsal migration of the invaginated mesoderm is regulated by FGF-signaling. Because the ligand of Heartless has not yet been identified, it is unknown which cells produce the FGF signal molecules that are expected to trigger mesoderm migration. However, stainings of embryos with antibodies that recognize epitopes that are phosphorylated by activated receptor tyrosine kinase (RTK) molecules have revealed specific staining of cells in the lateral edges of the mesoderm, presumably corresponding to activated Heartless receptors (Gabay et al., 1997). This indicates that the FGF signals are released from lateral or dorsal cells of the ectoderm andor amnioserosa and trigger thc directed movement and spreading of the responding mesodermal cells toward the source of the signals. Because the ectoderm later releases important differentiation signals to the underlying mesodermal cells (see below), the internal spreading of the mesoderm below the ectoderm is a prerequisite for normal differentiation of the mesoderm at later stages of development.

ill.

EMBRYONIC ORIGIN AND MORPHOGENESIS OF MESODERMAL TISSUES

The determination of the anlagen of different mesodermal derivatives and their morphogenesis is initiated shortly after the dorsal migration of the invaginated mesoderm has been completed (see IV.). As a result of morphological studies and the availability of early molecular markers for these derivatives, we now have a

MANFRED FRASCH and HANH T. NCUYEN

6

relatively clear picture of the developmental origins and subsequent morphogenesis of the major mesoderm tissues. A.

Somatic Musculature

The mature larval somatic musculature (body wall musculature) is composed of syncytial striated muscle fibers. Individual fibers consist of single syncytia which include between approximately 5 and 15 nuclei, depending on the particular muscle type. Each larval segment includes a defined set of muscle fibers, and the abdominal segments A2 to A7 (and A1 with minor modifications) have an identical pattern of 30 different muscles on either side. Each of these muscles can be identified based on its specific shape, orientation, size, position, and epidermal attachment sites (Figure 1B). It is thought that most, if not all of the somatic muscles are derived from segmentally repeated areas of the early mesoderm that extend from the ventral midline to the dorsal border. These areas correspond to the stripes of high-twist expression that are observed at stage 10 ( 5 hours of development) in the mesoderm (see 1V.B. and V1.A.). The formation of muscles is preceded by the appearance of a special type of myoblasts called muscle founders (Bate, 1990). Muscle founders can be identified based upon their specific expression of molecular markers, including homeobox genes that mark subsets of founders and an enhancer trap insertion, rP298-lacZ, which appears to mark most, if not all, of these cells (Figure 1A) (Dohrmann et al., 1990; Nose et al., 1998). rP298-lacZ-expressing muscle founders arise from four distinct domains along the dorsoventral mesoderm axis that give rise to the dorsal, dorsolateral, lateral, ventrolateral, and ventral groups of muscle fibers, respectively. Most of the muscle founders develop into muscles near the positions where they are formed, but several of them first undergo cell migration to specific locations before developing into muscles. Beginning at stage 12, the muscle founders fuse with surrounding myoblasts, which are thought to be undetermined and are called fusion-competent cells. The resulting muscle precursors continue to recruit additional myoblasts until the syncytia reach their final size. At the same time, they form filopodial extensions, which form specific contacts with muscle attachment cells of the epidermis. The expression of myofiber-specific genes is also initiated during this period and the final differentiation and innervation of the muscle is completed by stage 17 (16 hours of development).

B.

Visceral Musculature

The visceral musculature, which surrounds the endoderm of the foregut, midgut and hindgut, is formed by mononucleate, highly elongated muscle cells. The midgut musculature consists of two layers, an inner layer of circular muscles and an outer layer of longitudinal muscles. By contrast, fore- and hindgut have only a single layer of circular muscles. As described below, each of these types of gut muscles

Genetic Control of Mesoderm Patterning and Differentiation

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has a diffcrent origin and undertakes a specific developmental pathway during its m orphogenesis. Circular Muscles of the Midgut

The circular muscles ofthe midgut are derived from eleven rectangular, segmentally distributed fields of mesodermal cells, which are located in the dorsal mesoderm in the region between parasegments 2 and 12 of the early embryo (stage 10;Figure 1C)(Azpiazu and Frasch, 1993;Dunin-Borkowski et al., 1995). At late stage 10,these cells ofthe inidgut visceral mesoderm primordia detach from the ectoderm and, at the same time, the surrounding cells of the somatic and fat body mesoderm spread into their former positions. These processes lead to the segregation of the midgut visceral mesoderm toward the interior, where it forms a second mesodermal layer dorsally while the outer mesodermal layer consists of somatic mesoderm. During this segregation process, the visceral mesoderm primordia elongate along the anterior-posterior axis withn each segment and ultimately merge with one another to form a continuous band of cells along the middle body region. The cells of this band elongate during late stage 12 in the dorsoventral direction and subsequent migration and further elongation around the midgut endoderm leads to the formation of the circular midgut musculature (Figure 1D) (Goldstein and Burdette, 1971; Sandborn et al., 1967). Longitudinal Muscles of the Midgut

The precursors of the longitudinal midgut muscles originate near the caudal end of the mesoderm (Campos-Ortega and Hartenstein, 1997). The earliest available marker, DHLH53E identifies the anlage of the longitudinal visceral mesoderm in the caudal mesoderm, which is already present during the late blastoderm stage, thus indicating that it is determined prior to gastrulation (Georgias et al., 1997). During stage 1 1, the cells of the caudal visceral mesoderm divide into two bilateral clusters from which they start migrating toward the anterior on either side ofthe embryo (Figure 1E). This migration occurs in close association with the midgut visceral mesoderm that will form the circular muscles and results in the scattering of the caudal visceral mcsoderm cells along the future midgut. During midgut formation, these cells elongate and align with one another to form longitudinal rows of fibers that are evenly distributed around the midgut. These migration events have been examined by PGal expression from certain tirrrnudhcZ and nief2/fucZ reporter constructs which is specifically detected in caudal visceral mesoderm and longitudinal gut muscles from stage 1 1 until late stages of embryogenesis (Figure 1F) (M.F., unpublished data; Nguyen and Xu, 1998). Foregut and Hindgut Muscles

The precursors of the foregut and hindgut muscles (Figure 1C) appear to be derived from the anterior- and posterior-most regions of the mesoderni, respectively.

heinocytes

DM cells

dorsal vessel fat bodyfgonadalins.

longit. gut musc.

circ. midgut musc.

somatic muscles

Genetic Control of Mesoderm Patterning and Differentiation

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Figure 7. Developmental origin and morphogenesis of individual mesodermal tissues in the Drosophila embryo. The left column shows the anlagen of different tissues and the right column the corresponding tissues after differentiation. Anterior is to the left and dorsal is up, unless noted otherwise. (A) Stage 11. Segmental repeats of founder cells of somatic muscles (arrows)asvisualized by pCal stainingwith enhancer trap line rP298. (B) Mature somatic muscle pattern as visualized by staining for myosin heavy chain protein (filet preparation courtesy of S. Knirr). (C)Stage 11. Clusters of the circular midgut muscle primordia between PS2 and PS 1 2 are stained for bap mRNA, as is the hindgut visceral mesoderm at the posterior of the embryo. (D)The differentiated circular midgut musculature is visualized with a bap//acZ reporter gene. (E) The caudal visceral mesoderm prior to its migration (stage 1 I), as visualized with a mef2//acZ reporter gene. (F) Differentiating longitudinal midgut muscles (arrows), stained as in E. (G)Segmental anlagen of the fat body (arrows)and gonadal mesoderm (in posterior segments) in a stage 11 embryo stained forsrp mRNA. ( H l ) Cross sectioned embryo (stage 13)stained forsrp mRNA and fasciclin Ill protein to visualize the differentiatingfat body and midgut visceral mesoderm, respectively. (H2) Stage 14 embryo (cross section) stained for Tinman protein to visualize the differentiating gonadal mesoderm and dorsal vessel. (I) Stage 11 embryo stained for Even-skipped protein to visualize the segmental heart anlagen (note that only pericardial progenitors are positive). 0) Dorsal view of dorsal vessel formation short before completion of dorsal closure. The internal rows of nuclei belong to cardioblasts and the external ones to pericardial cells (nuclear pGal staining of an enhancer trap line). (K) Stage 11 embryo stained for D M cell precursors in a bt///acZ enhancer trap insertion (Chiang et al., 1994). (L) Ventral view of late-stage embryo showing differentiated D M cells (stained as in K). (M)Blastoderm embryo (ventrolateral view) stained for srp mRNA expressionto show the mesoderm anlagen of the hemocytes (arrow). (N)Late stage embryo stained with antibodies against peroxidasin to visualize the differentiated migratory hemocytes. Abbreviations: cvm: caudal visceral mesoderm; en: endoderm; fb: fat body; gm: gonadal mesoderm; hg vm: hindgut visceral mesoderm; hp: heart progenitors; mg vm: midgut visceral mesoderm.

While the exact location of the foregut muscle anlagen has not yet been defined, it appears that the anlagen of the hindgut muscles are formed just posteriorly to those of the longitudinal midgut muscles (M.F., unpublished observations). During foregut and hindgut formation, these cells spread along the ectdermally-derived portions of the gut and form circular gut muscle fibers.

The mature fat body is composed of a larger lateral lobe and a smaller dorsal lobe on either side of the embryo (Figure 1H1). The precursors of the dorsal lobe are largely derived from the dorsal mesoderm in parasegment 13 (which does not give rise to midgut visceral mesoderm; see II.B.l) (Riechmann et al., 1998). By contrast, the precursors of the lateral lobe arise from segmentally repeated clusters of cells that are located in parasegments 4 to 13 (Hoshizaki et al., 1994; Azpiazu et al., 1996; Riechmann et al., 1997). One field of fat body precursors is

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MANFRED FRASCH and HANH T. NLIJYEN

located just ventrally to the inidgut visceral inesoderm primordia in each of these parasegments (Figure IC). However. in parasegments 10 to 12. these fields take up only half their normal size and the missing (anterior) portions are occupied by the precursors ofthe gonadal mesoderm (Cumberledge et al., 1992; Brookman et al.. 1992; Riechmann et al.. 1998). A second. smallcr field offat body precursors is located more ventrally and posteriorly in each ofthe parasegments 4 to I ? . During fat body morphogenesis? these clusters of cells merge with one another and move between the somatic and visceral mesoderm to form the continous lateral lobes 0 1 the fat body. The three clusters of gonadal mesoderm primordia in parasegments 10 to 12 also coalesce and, together with the germ cells, form the gonads (Figure lH2). D.

Dorsal Vessel

The mature dorsal vessel, or heart, consists of a tubelike structure extending along the dorsal midline. Its lumen is formed by two longitudinal rows of cardioinyocytcs called cardioblasts or cardial cells (Figure 1J) (Rugendorff et al., 1994). There are about six cardioblasts on either side in each middle body scgment, which are surrounded by pericardial cells that are not myogenic (Mills and King, 1965). Like the inidgut visceral mesoderm and fat body, the anlagen of the heart are also formed as segmentally repeated clusters during stage 10 (Dunin-Borkowski et al., 1995; Azpiazu et al., 1996). The clusters of the heart anlagen are located at the dorsal margins of the mesoderm between the clusters of midgut visceral mesoderm precursors in each ofthe parasegments 2 to 12 (Figure 11). During the morphogenetic movements that lead to the multiple layers in the mesoderm, the heart progcnitors remain in contact with the ectoderm. As a result of cell rearrangements during stage 1 I , heart progenitors from adjacent parasegmental clusters come into contact to form a continuous longitudinal band of cells along the dorsal crest of the mesoderm on either side of the embryo. Following stage 12, the cardioblasts form a row at the dorsalmost positions of the mesoderm, and upon dorsal closure of the germ band. the cardioblast rows from either side ofthe embryo join at the dorsal midline t o form the heart tube.

E.

Hemocytes

Hemocytes function mainly as macrophages that remove debris from apoptotic cells in the developing embryo (Tcpass et al.. 1994). In addition, they appear to deposit extracellular matrix components of the basement membrane (KuscheGullberg et al.. 1992; Hijrtsch et al., 1998). Similar to the anlage of the caudal visceral mesoderm (H.B.),the blood cell anlage can already be detected at blastoderm stage, i.e.. prior to gastrulation. At this stage, the blood cell anlage occupies a small area of the head mesoderm that is locatcdjust anteriorly to the position of thc future ccplialic furrow (Figure 1 M ) (Rehorn et al., 1996).After gastrulation, the progeni-

11

C;enetic: Control of Me5oderr-n Patterning and Differentlatirjn

tors of hcmocytcs delaminate into the interior of the embryonic head and subsequently scatter throughout the embryonic body cavity (Figure 1 N). F.

Dorsal Median (DMj Cells

Pairs of dorsal median (DM) cells are located medially on the dorsal surface of' the ventral nerve cord and close to the segmental boundaries in each ofthe three thoracic and in the first seven abdominal segments (Figure 1 L). DM cells have glia-like properties since they develop laterally extended processes that pioneer and ensheath axons of motoneurons exiting from the dorsal midline of the nerve cord (Gorczyka et al.. 1994). The DM cell precursors form in small segmental clusters along the ventral midline ofthe mesoderm in stage 1 1 embryos in close contact with the mesectoderm (Figure 1K) (Chiang et al., 1994; Dunin Borkowski et al., 1995). During ncuroblast segregation and ganglion mother cell proliferation, the DM cell precursors. together with the rest of the ventral mesodermal cells, separate from the epidermis and assume their final locations on the dorsal surface of the CNS.

IV. MESODERMALLY-EXPRESSEDGENES DETERMINING BROAD AREAS AND SPECIFIC TISSUES IN THE MESODERM A.

tinman, a Homeobox G e n e Required for the Formation

of Visceral

Musculature, Dorsal Somatic Muscles, and the Heart Structure and Expression of tinman

tirirmm (tin)encodes a homeodomain protein ofthe NK family (Kim and Nirenberg, 1989). Its closest vertebrate homologs, Nkx2-3, Nkx-2-5,Nkx-2-6. Nkx2-7. and Nkx2-8, share between 63% and 68%)amino acid identity within the homeodomain (reviewed in Harvey, 1996). Binding site selection experiments have defined an optimal binding site with a consensus sequence 5'-TNNAGTG-3' for Nkx2-5. The most commonly found Tinman binding sites share this motif ;r,d contain the sequence YTCAAGTG-3' (Chcn and Schwartz, 1995; Gajewski et al., 1997; Xu et al., 1998). I n additicn t o the homeodomain. Tinman contains ahighly conserved dei'ppiiclc ~ t - y u c r i ~LCc :it11 unl\riovvii funLiiui1 iicili it> N-tzimiinua ~ d l l e dilie TN dumain. Initial ririrticiri expression occurs at cellular, blastoderm in the prospective mesoderm and is confined to the trunk mesoderm where it is initally modulated in a periodic pair-rule pattern (Bodmer et al.. 1990; A q i a z u and Frasch. 1993). During imxgination and dorsal migration of the mesoderm, the entire trunk mesoderm expresses tiiiriiciri mRNA i n a uniform pattern. However. shortly before the mesoderm reaches the dorsal border of the ectoderm. firirtictri expression decreases in the ventral poi-tions and its levels increase i n the dorsalmost cells 01' thc mcsodcrm. Be-

12

MANFRED FRASCH and 1 iANH I. NCIJYCK

tween stages 10 and I 1. tirirrrciri mRNA and protein expression is entirely restricted to the dorsal portions ofthe mesoderm. These domains occupy about one third of the distance between the dorsal margin and the ventral midline on either side. At early stage 12. the tirrrriar7 domains are further refined and become restricted to cells ;it the dorsal margins of the mesoderm that correspond to heart progenitors. riririirrrr remains expressed i n heart cells until late embryogenesis. This expression includes I'our out 01' six cardioblaxts and four pericardial cells per hemisegment, While rirr1?1(iti rnKNA is exclusively observed in the heart of late-stage embryos. tiririiuri protein perclures in segmentally repeated cells of the visceral mesoderm and in the gclnadal mcsotlerm. in addition to its continued expression in cardioblasts and pericat-dial cells (Figure 1 H2;Manfred Frasch, unpublished data).

Cenetic and Developmental Function of tinman The genetic function of tirirriari has been investipted with ethylmethanesulfonate~(EMS-) induced alleles, including one that carrics a small delet i o n in the coding region and another with a stop codon in the homeodomain. which arc presuniably null-alleles (AzpiaLu and Frasch, 1993; Bodmer. 1993).The ina.jor defects in IiomoLygous mutant embryos are observed in tissues derived from the clot-sal mesoderm. Notably, both the dorsal vessel and midgut visceral mesoderm ;ire entircly missing in the absence oftirirrim function (Figure 2A-D). The absence ol'an enhancer trap marker that is specific for all dorsal somatic muscles in tirlrriur? mutant embryos strongly indicates that tirrriiurz is also rcquired tor the formation 01' dot-sal body wall muscles (Figure 2E.F) (Yin and Frasch, 1998). This is consistent with the severe disruption ofthe muscle pattern in dorsal areas and the concomitant clongation of dot-solateral muscles into the areas normally occupied by dorsal types ol'niuscIes. Earl!. markers for the precursor cells ofeach of these tissues are also not expressed in firir)/(/r? mutants. Specifically. the homcobox gene l x q p i p o . which niai-ks the anlagcn of t h e midgut visceral mesoderm (see 1II.B.). is not activated. rr-.~kipp(~d and l~irh~hir-d (see V.A.) I'ail to be expressed and the liomeobox genes in early heart precursors (Azpiazu and Frasch. 1993; Bodmet-. 1993; Jagla CI d . . 1997).The expression oftlie homeobox gene r r i s h . which is normally cxprcss~ilin muscle founder cells. is also missing in this area in f i r r r i i m r mutimts d o t - a a l l ~locntccl . (Yin and Fi-asch. I998). Thus. i t appcars that rirrriiciri is required tor the specification 01a i i dorsai iiicsocicrmai tissues 'oeiween siages i O ;ind i i u i ' c l i i h i - ~ ~ i ~ i i i Thi:; z~is. function is consistent with its specific expression in dorsal portions of the mesode rni d 11r i n p this per i od t Iiu s s ug ge s t i ng t li at tin r r i m ful fi I1 s its major de \T Iop men t a I l'un c t i c) 11s during t h i s second . dorsal Iy rest I-ict ed phase of me s ode rni al expression . Apai-t I'rorii these m;i,jor defects i n dorsal mesodermal derivatives. r i r r r r r m m w tants also exhibit inore subtle defects in other areas ofthe mesodcrni. Fot-example.a specific subset ol' \,entral and lateral body w;ill m u s c l e s and thcir founder cells ;ire not forincd (1:igui-c 2G.H) ( A q i a l u and F3xcIi, 1993).hlorc o~ e t-. r i r l r r i r l r i rnutant embryos lack the DM c e l l s i i n d thcit-pi-ecursors (Figure 21.5) ( G o r c ~ y k ; t c;d.. c 1994:

bnman

wild type

The tunction of tinman i n the tormation o t mesodermal tissues. The left column shows wild-type embryos and the right column tinman mutant embryos of the iaine stage and stained with the same markers. (A, Bi Keither cardioblastj nor pericardial cells are formed in tin mutants (embryos stained as i n Figure 11). ( C , Di 'The midgut inusculatul-e (stained tor PCaI expression from ,In enhancer trap insertion) i s completely !??d +? lt 'l r 7 l t.ypr?w.i(:n +
figure 2.

.~'I.'

reporter insertion; Yin and Frasch, 14981 are n o t properly specitied i n t i ~ ~ r r i amutants. ii (G, HI Earlv stage 12. The muscle founders ot the S5Y cluster I (as dettxc-ted \vith an S59
14

hANFRED FRASCH and HANH T. NGUYEN

Yin and Frasch, 1998). Together with the zinc finger homeobox gene zjh-1, tinman is also required for normal formation of the gonadal mesoderm (Boyle et al., 1997; Broihier et al., 1998). It is likely that the functions of tinman in thesedevelopmental processes are required during its early phase of expression in the entire trunk mesoderm. Ectopic expression experiments have also been performed to study the functional potential of the tinman gene. Perhaps surprisingly, the consequences of prolonged ubiquitous tinman expression in the mesoderm or in the whole embryo were relatively mild. The main effect was a transient increase in the number of heart precursors during stage 1 1, but the formation of the heart, visceral muscles, and body wall muscles appeared not to be disturbed (Yin and Frasch, 1998). These results indicate that coregulators, whose expression or activity is spatially restricted, are obligatory for all known aspects of tinman function. B.

bagpipe, a Homeobox Gene Required for Visceral Muscle Formation

bagpipe (bap) is another member of the NK homeobox gene family and is located immediately downstream of tinman on the chromosome (Kim and Nirenberg, 1989; Azpiazu and Frasch, 1993). Closely related homologs, which aremembers of the Nkx3 series of homeobox genes that encode proteins with about 88% amino acid identity with Bagpipe within their homeodomains, have been recently identified in several vertebrate species (Newman et al., 1997;Tribioli et al., 1997).bagpipe is expressed during stages 10 and 11 in the anlagen of the circular gut musculatures (Azpiazu and Frasch, 1993). During early stage 10, bagpipe mRNA appears in the eleven rectangular patches of the midgut visceral mesoderm primordia, which are located in the dorsal mesoderm in each of parasegments 2 to 12 (Figure 1C). During stage 1 1, bugpipe expression is also initiated in the primordia of the foregut and hindgut visceral mesoderm. The activation of bagpipe in the midgut visceral mesoderm primordia, but not in the foregut and hindgut visceral mesoderm, requires the function of tinman. Based upon the functional dependency and closely overlapping temporal and spatial patterns of tinman and bagpipe expression, it appears that bagpipe may be a direct downstream target of tinman. While bagpipe expression in the midgut visceral mesoderm is transient and ceases upon visceral mesoderm segregation, its expression in the foregut and hindgut visceral mesoderm persists until late stages of development. Following stage 12, weak expression is also observed in a subset of heart precursors. The genetic function of bagpipe is consistent with its expression profile. Embryos homozygous for a hypomorphic EMS-allele, b ~ p ~show " ~ ,a strong reduction of the midgut visceral mesoderm (Azpiazu and Frasch, 1993). Moreover, homozygous deficiency embryos that lack the bagpipe completely, but retain tinman, do not form any visceral mesoderm of the midgut (Manfred Frasch, unpublished data). These observations indicate that bagpipe function is essential for specifying the formation of the circular midgut musculature from metamerically repeated cell

Genetic Control of Mesoderm Patterning and Differentiation

15

clusters in parasegments 2 to 12. By contrast, foregut and hindgut muscles are formed in bagpipe mutants, but it is not known whether they are differentiating in a normal fashion. The target genes that are activated by bagpipe and mediate its function in gut muscle development are largely unknown. One candidate gene, vimar, encodes an armadillo-repeat-containing protein and is activated in a bagpipe-dependent manner in the early midgut visceral mesoderm (Lo and Frasch, 1998).

C.

serpent, a GATA Gene Regulating Fat Body and Hemocyte

Development The serpent (srp)gene encodes a transcription factor of the GATA family and is required for the formation of two different mesodermal cell types: fat body and hemocytes. serpent is expressed in all fat body progenitors and represents the earliest known marker for the fat body anlagen (Figure 1G; see 1I.C). Its expression is maintained until the stages when the fat body is fully differentiated. The serpent gene was originally isolated based on the activity of its gene product to bind to regulatory sequences of the Adh (alcohol dehydrogenase) promoter (Abel et al., 1993). Adh is specifically expressed in the differentiated fat body and is likely to be a downstream target of serpent in this tissue. serpent mutants have a more severe phenotype and exhibit a total failure to form fat body, thus indicating that serpent also has a role during a very early stage of fat body development (Rehorn et al., 1996; Sam et a]., 1996). An analysis of srp embryos suggested that fat body progenitors are initially formed but fail to progress beyond the early stages of their development and subsequently appear to undergo apoptosis. This indicates that serpent is required at an early step of fat body differentiation to activate yet unknown target genes that are essential to propel the further development of this tissue. Two genes have been shown to act upstream of srp in fat body development: tinman and the zinc finger/homeobox-containing gene zjh-I (Lai et al., 1991; Broihier et al., 1998). Double mutant embryos lacking the activities of both tin and $1-1 fail to form fat body precursors, although in mutants for either of the two genes,the fat body is not as severely affected. It has been suggested that tin and zfh-1 have partially redundant roles in early fat body determination, and that the two genes fulfill this role during their early phase of expression in the entire mesoderm. serpent is also the earliest known marker for the hematopoietic mesoderm. Its expression in the blood cell anlagen is first seen at blastoderm, where it is restricted to a roughly square-shaped domain between about 85% and 15% egg length in the prospective mesoderm (Figure 1M) (Rehorn et al., 1996). srp expression is maintained during invagination of the cephalic mesoderm and the segregation of the prohemocytes into the interior of the embryo, but it is downregulated during their differentiation into mature hemocytes. srp mutant embryos are devoid of any mature hemocytes. Closer analysis has shown that early hemocyte progenitors are formed in the mutants but fail to proliferate and to migrate and subsequently die.

16

MANFRED FRASCH and HANH T. NCUYEN

Thus, serpent also has an essential role in early hematocyte development. One of its downstream targets in this pathway may be the gene glial cells missing (gem), which is expressed slightly later in the blood cell anlage and is required for normal differentiation of the hemocytes (Bernardoni et al., 1997). D. The Roles of clift, zfh-1, tin, and Abd-A in Gonadal Mesoderm Development clllft (cli, also called eyes absentleya) encodes a novel nuclear protein for which several vertebrate homologs exist. At stage 11, clift is specifically expressed in the three clusters of gonadal mesoderm precursors that are located in the lateral mesoderm of parasegments 10, 11, and 12 (Boyle et al., 1997). In clift mutants, the gonadal mesoderm precursors appear to be specified, but during stage 12, they gradually disappear and gonads are not formed. The phenotype suggests that clift is required for the maintenance of the fate and continued development of the gonadal mesoderm. Since, prior to stage 11, cliftis expressed more broadly in the mesoderm, it is not known whether its function is already required during this early period or only during its tissue-specific phase of expression. However, because clip is also required for normal fat body development, it has been suggested that it may affect the development of a common pool of lateral mesodermal cells that either develops into fat body (in parasegments 4-9) or into gonadal mesoderm (in parasegments 10-12) (Moore et al., 1998). Similar to the fat body, the gonadal mesoderm requires tin and zfh-1 for its normal development. In either, tin or ~$4-1mutants, the number of gonadal mesoderm precursors (as detected by cli expression) is significantly reduced, while ectopic expression of ~$5-1results in increased numbers of precursors (Boyle et al., 1997; Moore et al., 1998). tin zfh-1 double mutants lack gonadal mesoderm completely, indicating that the two genes cooperate in early gonadal mesoderm development (Broihier et al., 1998). Both genes have an early phase of expression in the entire mesoderm and, during stages 11 to 12, their products are specifically detected in the gonadal mesoderm (While $4-1 mRNA appears to be actively expressed in the gonadal mesoderm (Moore et al., 1998), the Tin protein seems to perdure preferentially in this tissue and can be detected in the gonads until stage 15; Figure 1H2). Therefore it is not clear at what stage(s) tin andzfh-1 are required for gonadal mesoderm development. However, the mutant phenotypes indicate that, at the latest, they are required during the early stages of differentiation of this tissue. What are the mechanisms that differentiate between fat body and gonadal mesoderm formation in the lateral mesoderm? srp has the capacity to block gonadal mesoderm development; it exerts this ability in parasegments 4 to 9 thereby allowing fat body development in these areas (Moore et al., 1998; Riechmann et al., 1998). However, in parasegments 10 to 12, this activity of srp is masked, even though it is expressed there. It appears that the homeotic gene Abd-A (via its expression from the iab-4 enhancer) is responsible for functionally inactivating srp in

Genetic Control of Mesoderm Patterning and Differentiation

17

parasegments 10 to 12 and, thus, the srp-expressing clusters of lateral mesoderm cells in these parasegments are able to form gonadal mesoderm (Cumberledge et al., 1992; Brookman et al., 1992; Warrior, 1994; Greig and Akam, 1995; Moore et al., 1998; Riechmannet al., 1998).The ground state (which requires the activities of tin, ~$4-1, and cli) of the lateral mesoderm in the P domains therefore is to develop into gonadal mesoderm, but it is only able to enter this developmental pathway if the function of srp is inhibited by AbdA. E.

buttonless, A Homeobox Gene Required for DM Cell Formation

buttonless (btn) is a homeobox gene most closely related to the vertebrate mox and gax genes (78% amino acid identities within the homeodomain) (Chiang et al., 1994). btn is expressed in a highly restricted pattern during embryogenesis and is first observed in segmentally arranged groups of two to four mesodermal cells each that are located along the ventral midline of stage- 1 1 embryos (Figure 1K). During the subsequent neuroblast segregation, the mesoderm splits along the ventral midline but maintains segmentally arranged connections bridging the left and right halves. The btn-expressing cells are part of these mesodermal crossbridges that cover the ventral nervous system and are later the only remaining mesodermal cells (being reduced to two neighboring cells per segment) along the dorsal midline of the ventral nerve cord. These correspond to the dorsal median (DM) cells that extend lateral processes during later stages of embryogenesis (Figure 1L; see 1I.F.). In btn mutants, DM cells are completely missing (Chiang et al., 1994). Although segmental clusters of mesodermal cells transiently express btn-lacZ reporter genes, these cells fail to develop into DM cells. As a consequence, the pathfinding of transverse nerves is disrupted in btn mutant embryos. These observations show that btn IS required for the specification or early differentiation of DM cells. tinman is also required for DM cell specification (Gorczyka et al., 1994). It was found that tinman acts upstream of btn, since there is no transcriptional activation of btn in tin mutant embryos (Figure 2J) (Yin and Frasch, 1998). Because of the ventral location of the btn-expressing cells, tin must fulfill this function during its early phase of expression in the whole trunk mesoderm.

V.

EARLY PATTERNING MECHANISMS OF THE MESODERM

As described in chapters 2 and 3, the primordia of the different mesodermal tissues arise at precisely defined positions within the mesodermal layer. In large part, this is helieved to be a result of the spatially restricted activation (or repression) of control genes that determine (or suppress) the formation of particular tissues in a cellautonomous fashion. Each primordium or field of precursors of a particular tissue is defined by its position along the dorsoventral axis and anteroposterior axis. Classi-

MANFRED FRASCH and HANH T. NCUYEN

18

cal embryological experiments involving localized ablation of mesodermal or ectodermal portions, which were performed in lacewing flies in the 1930s, had shown that inductive influences from the ectoderm have important roles in the specification of mesodermal tissues at defined locations (Seidel et al., 1940). Specifically, Seidel and colleagues concluded: “In the lateral portions of the ectoderm, there are factors that are necessary for the formation of cardioblasts and gut muscle cells, whereas the medial portions of the ectoderm are lacking these factors”. In the recent years, much has been learned about the molecular and genetic processes underlying these patterning mechanisms that determine dorsoventral and anteroposterior coordinates of gene expression within the mesoderm and thus generate the diversification of mesodermal tissues. A.

Dorsoventral Patterning

The Roles of Dpp and Its Downstream Effectors in Dorsal Mesoderm Induction

The homeobox gene tinman appears to be a central component in the dorsoventral patterning of the mesoderm because its activity is required to generate all dorsal mesodermal derivatives, midgut visceral mesoderm, heart components, and dorsal somatic muscles. This correlates well with the spatial pattern of tin expression which, upon internal spreading of the mesoderm, becomes confined to broad domains in the dorsal portions of the mesodermal layer. It appears that high levels of tin expression in the dorsal mesoderm are necessary for the activation of target genes that specify visceral mesoderm (e.g., bagpipe), dorsal muscle precursors (e.g., msh; Yin and Frasch, 1998), and heart progenitors. A number of genetic studies have demonstrated that influences from the dorsal ectoderm are critical in triggering dorsal mesodermal expression of tin and the formation of dorsal mesodermal derivatives. For example, in a genetic background engineered to produce less mesoderm (with snaif under the control of a weakened twist promoter, which generates a narrower ventral snail domain in a snail mutant background), the mesoderm is apparently unable to spread as far as in wild-type embryos and few mesodermal cells reach the dorsal ectoderm (Maggert et al., 1995). Indeed, in these embryos, tin is expressed only in a few dorsal cells of the mesoderm during stage 10, and the formation of heart and midgut visceral mesoderm is severely reduced. Similar observations in heartless mutant embryos were made in which mesoderm migration toward the dorsal ectoderm is also disrupted (Beiman et al., 1996; Gisselbrecht et al., 1996; Shishido et al., 1997; see I.D.) and in embryos treated with drugs that prevent or reduce gastrulation movements (Baker and Schubiger, 1995).These observations are consistent with the conclusion of Seidel and colleagues that the dorsal ectoderm must produce a factor (or, in contemporary terms, signaling molecule) that triggers the determination of dorsal mesodermal derivatives in the underlying mesoderm.

Genetic Control of Mesoderm Patterning and Differentiation

19

The major signaling molecule involved in this process has been identified as the product of the decapentaptegic gene (dpp).Dpp is a TGF-P family member and belongs to the subfamily of the bone morphogenetic proteins (BMPs). Dpp is expressed along the dorsal circumference of blastoderm embryos and, during stage 9, its expression becomes confined to a broad band of dorsal ectodermal cells on either side of the germ band (St. Johnston and Gelbart, 1987). It is during this period when the mesoderm spreads dorsally and reaches the dorsal ectodermal cells expressing Dpp. Ultimately, the domains of maintained tin expression in the dorsal mesoderm are located precisely beneath the ectodermal Dpp domains. Indeed, dpp activity is required for this dorsal mesodermal phase of tinman expression. In dpp mutants, tinman expression ceases without being restricted to the dorsal mesoderm, and dorsal mesodermal derivatives are not specified (Frasch, 1995). Conversely, upon ectopic expression of Dpp in the ventral ectoderm, tinman expression is ectopically induced in the ventral mesoderm, and at least somedorsal derivatives (including the midgut visceral mesoderm) are expanded towards the ventral midline. Together, these data suggest that Dpp has a key role in dorsoventral patterning of the mesoderm and is specifically functioning to induce the formation of dorsal mesodermal derivatives including the heart, visceral muscles, and dorsal body wall muscles (Frasch, 1995; Staehling-Hampton et al., 1994). An early and essential response to Dpp signaling appears to be the activation of tin expression in the dorsal mesoderm, which in turn is necessary to activate downstream genes such as bagpipe that specify dorsal mesodermal derivatives. However, it appears that tinman is not sufficient to mediate the Dpp responses since ectopic expression of tin does not result in ectopic bagpipe expression and ventral expansion of the visceral mesoderm or other dorsal mesodermal derivatives (Manfred Frasch, unpublished data). Either Dpp induces additional genes in the dorsal mesoderm that are required in combination with tinman to activate target genes, or Tinman requires components of the intracelM a r signal transduction cascade of Dpp as coactivators to turn on its targets in the dorsal mesoderm (see 1V.D.). Some of the components of the Dpp signal transduction cascade have been identified and shown to mediate the function of Dpp in mesodermal tinman induction. Specifically, the type-I Dpp receptor Thickveins (Tkv), a transmembrane receptor kinase related to type-I BMP-receptors, is required for tin induction by Dpp, and pan-mesodermal expression of a constitutively active version of Tkv leads to ventrally expanded expression of tin (Yin and Frasch, 1998). Medea, a Drosophila member of the Smad family of intracellular TGF-P effector proteins, is also essential for dorsal mesodermal tin induction (Xu et al., 1998). Injection of mRNAs encoding constitutively active Tkv does not rescue the mesodermal defects in Medea null mutants, as would be expected if Medea acts downstream of the Dpp receptor. Interestingly, tinman activity itself is required for the Dpp-dependent induction of its own gene, thus indicating that tinman and the Dpp signals act synergistically during this process. Some of the molecular details underlying these interactions have been clarified and are described in 1V.D.

MANFRED FRASCH and HANH T. NGUYEN

20

The Roles of ECF Signaling in Ventral Mesoderm Patterning

The fates of mesodermal cells near the ventral midline appear to be also determined largely by inductive processes. These processes have been studied in most detail for the development of the DM cells. Dye injection experiments have suggested that DM cell precursors are formed from cells that are located at the lateral borders of the mesoderm during gastrulation and that abut the rows of mesectoderma1 cells flanking the mesoderm (Luer et al., 1997). Experiments involving genetic or mechanical ablation of mesectodermal cells, which normally develop into midline neurons, have demonstrated that these cells are essential for DM cell formation. Furthermore, transplantation of ectopic mesectodermal cells into the ventrolateral ectoderm yielded supernumerary DM cells, indicating that mesectodermal cells can induce DM cell formation in the adjacent mesoderm. The mesectodermal cells are the sites of processing of the EGF-like mo!ecule Spitz and thus constitute a ventral source of EGF signals in the early embryo. Genetic studies have shown that components of the EGF signaling pathway, including Spitz, the modulatory proteins Rhomboid and Star, and the EGF receptor Faint little ball (Flb), are indeed necessary for the formation of a normal number of DM cells (Luer et al., 1997; Zhou et al., 1997). These experiments suggested that EGF signaling has an early role in DM cell induction as well as a later role in controlling cell divisions that generate two DM cells from one progenitor in each segment. However, since in EGF receptor mutants, DM cell formation is strongly reduced but not completely abolished, it appears that additional, yet unknown signals from the mesectoderm are involved in DM cell induction. The additional requirement of mesodermally expressed tinman for DM cell formation suggests that these signals act either by modifying the Tinman protein or through synergistic interactions between Tinman and intracellular signaling components. These activities result in the activation of buttonless expression, which in turn is required to specify the differentiation of the DM cells. In addition to the DM cells, signals from the mesectoderm are also required for the specification of certain ventral body wall muscles that are formed near the ventral midline (Lewis and Crews, 1994; Luer et al., 1997).

B.

Anteroposterior Patterning

Anteroposterior patterning of the mesoderm occurs in broad areas along the longitudinal axis of the embryo and results in the formation of cephalic, trunk, and caudal mesodermal tissues. In addition, the early trunk mesoderm is further patterned in a metameric fashion such that each parasegment acquires an anteroposterior polarity. Consequently, the anterior parasegmental portions give rise to derivatives that are different from those in their posterior portions. As described below, segmentation genes including the gap genes, pair-rule genes, and segment polarity genes, have critical roles in determining these mesodermal patterns along the anteroposterior embryo axis.

Genetic Control of Mesoderm Patterning and Differentiation

21

The Subdivision into Head versus Trunk Mesoderm

The broad subdivision of the mesoderm along the anteroposterior embryo axis leads to the formation of pharyngeal muscles and hemocytes from the cephalic mesoderm, longitudinal gut muscles from the caudal mesoderm, and a variety of mesodermal tissues from the trunk mesoderm. Although the mechanisms regulating these subdivisions have not yet been extensively studied, some insights into the distinction between head and trunk mesoderm have been obtained. During late blastoderm, serpent is expressed in a cephalic mesodermal domain that marks the hemocyte primordium, whereas tinman is expressed posteriorly adjacent in the prospective trunk mesoderm. One important regulator for this differential pattern of expression has been identified as the head gap gene buttonhead (brd), which encodes a SP1-related zinc finger transcription factor (Yin et al., 1997). brd is expressed as a transverse stripe that includes the mesoderm in the prospective head of blastoderm embryos and overlaps with the domain of srp expression (Wimmer et al., 1991). Importantly, in brd mutants, srp expression in the head mesoderm is almost completely abolished, while the expression of tinman is expanded into the same area. As a consequence, the number of hemocytes is significantly reduced in btd mutants and the residual hemocytes appear to be defective in their differentiation (Yin et al., 1997). This defines brd as an important patterning gene for the anteroposterior subdivision of the mesoderm and the most upstream gene in hemocyte determination known to date. However, there is a small amount of residual srp expression and the trunk mesodermal derivatives do not expand into the head region in brd mutants; this suggests that additional head gap genes are involved in the regulation of this subdivision. The distinction between trunk mesoderm and caudal visceral mesoderm probably requires analogous regulators in the posterior region of the embryo. One of them appears to be the terminal gap gene tailless (tll),since rll mutants fail to form caudal visceral mesoderm (Manfred Frasch, unpublished observations). This function of tll could be mediated by a yet unknown tll target gene(s) that is expressed in a narrow transverse stripe defining the domain of bHLH53F and perhaps other genes (see 1I.B.) in the caudal mesoderm. Segmental Patterning and Cell Fate Determination in the Mesoderm

Histological and genetic studies have revealed that the early trunk mesoderm, like the early ectoderm, is composed of metamerically repeated units (DuninBorkowski et al., 1995; Azpiazu et al., 1996; Riechmann et al., 1997). These units are in exact register with the ectodermal parasegments, the borders of which are marked by the expression of the engrailed gene (parasegments are shifted by half a unit with respect to the future segments). The trunk mesoderm extends from parasegment (PS) 2 to PS 13, and the basic pattern within each mesodermal parasegment between PS 2 and PS 12 appears to be identically repeated. In essence,

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MANFRED FRASCH and HANH T. NGUYEN

each parasegment is divided anteroposteriorly into two portions, which give rise to different mesodermal derivatives. The first portion, which is positioned largely underneath an ectodermal P compartment and shares its anterior border, has accordingly been named the “P domain”. The P domains are characterized by their expression of bagpipe (in dorsal areas) and serpent (in lateral areas) and thus contain the anlagen for the circular midgut musculature and the fat body. The second portion of each mesodermal parasegment underlies largely the A compartment of the ectoderm, shares its posterior border, and has been named the “A domain”. The A domains are characterized by their expression of markers for heart progenitors (near the dorsal margins), the perdurance of high twist levels (see VI.A.), and, following stage 1 1, the expression of markers for somatic muscle precursors. Thus, the A domains contain the anlagen for the dorsal vessel and the majority of the body wall muscles. While the concept of mesodermal A and P domains is generally accepted (although some authors have used different terminologies, see below), it remains debatable whether these domains represent true compartments or not. In the ectoderm, compartments have been defined by clonal analysis, which demonstrated, for example, that all daughter cells derived from a progenitor in the P compartment remain in areas contiguous with the P compartment. This is thought to be due to some unknown mechanisms of differential cell adhesion or repulsion between cells of the A and P compartments. Because of the important role of compartment borders as organizing centers for ectodermal pattern formation and growth (Lawrence and Struhl, 1996),it is crucial that mixing of cells between the A and P compartments is prevented. In the mesoderm, clonal analysis has not been performed as extensively. Lineage analysis has concluded that the somatic musculature is not composed of A and P compartments (Lawrence, 1982), which can be explained by the recent findings that at least the majority of somatic muscles are derived exclusively from the mesodermal A domains. Moreover, when clones were generated at blastoderm stage or during early gastrulation, they could give rise to mixed lineages consisting of different mesodermal derivatives, such as somatic muscles and fat body, showing that during these early stages, compartment borders have not yet been laid down in the mesoderm (Beer et al., 1987; Klapper et al., 1998). It is conceivable that mesodermal A and P domains do not function as compartments in their entirety, but rather as discrete units of gene expression. Perhaps compartmental units are only formed as individual mesodermal tissues start to segregate within each domain and precursors of different tissues become distinct from each other. In this case, each A and P domain would be composed of several different compartments along its dorsoventral extent during stage 11. Many of the segmentation genes that determine the periodic subdivisions of the ectoderm are also involved in generating the mesodermal A and P domains, as well as in the resulting diversification of tissues in the trunk mesoderm. For example, mutations in pair-rule genes that reduce the number of ectodermal parasegments in half also produce half the number of mesodermal parasegments. Notably, null mutations

Genetic Control of Mesoderm Patterning and Differentiation

23

in the pair-rule gene even-skipped, which cause a loss of segmental subdivisions in the ectoderm also produce a virtually unsegmented mesoderm. In this mutant situation, the P domains appear to be missing, as indicated by the lack of hap and srp expression and the absence of midgut visceral mesoderm and most of the fat body at later stages (Azpiazu et al., 1996; Riechmann et al., 1997) (Based upon the function of eve in allowing the formation of the P domains, they are also termed eve domains). Experiments with genetic mosaics have further shown that the activity of eve to allow formation of P domains is required in the mesoderm itself, and that eve activity in the ectoderm alone is not sufficient (Azpiazu et al., 1996). However, there are several indications that the homeodomain-containing product of eve does not directly activate genes like bup or srp in the P domains; first, eve is expressed in a seven-striped pattern in alternating parasegments while hap and srp are activated in P domains of every segment; second, eve expression ceases prior to the onset of hap and srp expression; and third, when eve is removed along with certain other pair-rule genes (e.g., sloppy paired; Riechmann et al., 1997), gene expression in P domains comes back, although only half the number of parasegments are formed. Based upon these considerations, it is likely that eve, together with other pair-rule genes, participates in the regulation of downstream genes within the mesoderm that are expressed in stripes either in the A domains or in the P domains of every parasegment. These pair-rule targets are in turn required to activate or repress the expression of mesodermal control genes such as hap and srp within their striped domains. What are the candidates for such pair-rule targets in the mesoderm? The homeobox gene engruiled, which is a prominent target of pair-rule genes in the ectoderm, does not appear to be a significant mediator of pair-rule gene function in the mesoderm (Lawrence and Johnston, 1984; Azpiazu et al., 1996). However, a good candidate is the segmentation gene sloppy paired (slp), which is expressed in a pair-rule gene-dependent manner in the ectodermal A compartments and the mesodermal A domains (Manfred Frasch, unpublished observations) of every parasegment. Interestingly, in slp mutants, the A domains are missing and the P domains appear expanded (Riechmann et al., 1997). (Therefore, the A domains have also been named slp domains). This is manifested in a complete loss of heart progenitors and a nearly complete loss of somatic muscles in slp mutants, whereas bup and srp display uniform expression along the anteroposterior axis in the dorsal or lateral mesoderm, respectively. The simplest explanation for these observations would be that slp, which encodes a forkhead domain protein (Grossniklaus et al., 1992), is required downstream of the pair-rule genes to activate mesodermal control genes in the A domains that specify heart and somatic muscle development, and is simultaneously required to repress P domain-specific control genes such as hap and srp in these areas. However, since ectopic expression of slp in the P domains does not have any noticable effects (Riechmann et al., 1997), additional pair-rule gene targets must be active. These target genes could either be required together with slp in the A domains, or they could be expressed in a complementary pattern in the P domains and activate P domain-specific control genes such as bup and srp in those areas.

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MANFRED FRASCH and HANH T. NGUYEN

Apart from these mesoderm-autonomous mechanisms, inductive influences from the ectoderm also have important roles in mesoderm segmentation. wingless ( w g ) ,which encodes a secreted molecule of the Wnt family and is expressed in portions of the A compartments, has a major influence in the development of the mesodermal A domains. Thus, in wg mutants, heart progenitors are completely missing, and the number of somatic muscle precursors is severely reduced (Wu et al., 1995; Baylies et al., 1995; Ranganayakulu et al., 1996) (The residual muscle precursors may be derived from the P domains). Conversely, the expression of genes from the P domains, such as bap and srp, is significantly broadened in the absence of wg function. The same phenotypes are observed when the function of components of the wg signaling pathway, including dishevelled (dsh)and armadillo (arm),is absent (Park et al., 1996; Azpiazu et al., 1996). After gastrulation, wingless is only expressed in stripes in the ectoderm, but at the beginning of mesoderm invagination, the wg stripes surround the entire circumference of the embryo and therefore include mesodermal cells. In genetic mosaics for wg, it was determined that wingless can induce heart formation either from within the mesoderm or by being secreted from the ectoderm (Lawrence et al., 1995). In the normal situation, both ectodermal and mesodermal sources of Wg are likely to act additively although the perdurance of wg expression in the ectoderm seems to make the route through induction across germ layers more significant. hedgehog (hh), a secreted factor related to vertebrate Sonic hedgehog, is expressed in ectodermal P compartments and functions in the normal development of the mesodermal P domains. Since the expression domains of bap and srp are reduced, but not abolished, in hh mutants, it appears that hh has a supporting and not an obligatory function in the development of mesodermal P domains (Azpiazu et al., 1996). hh and wg appear to have opposing roles in mesoderm segmentation, which is most obvious from the results of ectopic expression experiments. In an otherwise wild-type background, ectopic expression of either of the two genes does not have any major effects. By contrast, when wg is ubiquitously expressed in an hh mutant embryo, P domains appear to be completely lost and visceral mesoderm and fat body formation are abolished. The opposite effect is observed upon ubiquitous hh expression in wg mutant embryos, which show a loss of the A domains and a concomitant expansion of the P domains (Azpiazu et al., 1996). This results in strong expansions of visceral mesoderm and fat body, similar to what is seen in slp mutants. These results further indicate that in the normal situation, where the Wg and Hh proteins may diffuse into each other’s domain of expression, the “resident” signal could prevent the “intruding” signal from functioning thus contributing to the separation of A and P domains. It is presently not known at which level of the signaling cascades or responses this interference occurs. In addition, it will be important to clarify how mesoderm-intrinsic regulation and inductive regulation by wg and hh are interconnected. In this context, it has been suggested that wg and hh are required for the maintenance of the striped expression of genes that are initially activated by pair-rule genes in the A and P domains of the mesoderm (Figure 3) (Azpiazu et al., 1996).

Genetic Control of Mesoderm Patterning and Differentiation

P

A

P

P

A

P

P

25

A

P

D

E 0 0

E

BP P

A

P

early gastrulation

post-gastrulation

post-migration

Figure 3. Model of cornbinatorial inputs of segmental and dorsoventral cues that determine the formation of individual anlagen in the early mesoderm (see text). Abbreviations: A, P: A and P domains/compartments; cm: cardiac mesoderm; fb: fat body anlagen; gm: gonadal mesoderm anlagen; dsm, Ism, vsm: dorsal, lateral, and ventral somatic mesoderm, respectively; vm: midgut visceral mesoderm anlagen.

C. A Combinatorial Model of Mesoderm Patterning As discussed in the previous paragraphs, the subdivision of the mesoderm patterning involves patterning events along both the dorsoventral and the anteroposterior axis. Taken together, these observations indicate that the specification of individual mesodermal tissues involves combinatorial mechanisms and that specific anlagen are determined i n quadrants at the intersection ofdefined dorsoventral and anteroposterior cues (Figure 3) (Azpiazu et al., 1996). For example, cells at the intersection of the dorsal domains of Dpp and Tin with the transverse stripes of Hh (and presumably mesodermal stripes of an unknown molecule "M") would activate bup and develop into midgut visceral mesoderm. Similarly, cells at the intersection ofthe Dpp/Tin domains with the stripes of Wg/Slp or "N" would d v a t e genes that are required for heart and dorsal somatic muscle development in the A domains. Analogous intersections could also determine the formation of particular tissues in more ventral areas. This could include the activation of btn and formation of DM cells at the intersections of ventral EGF signals with striped regulators (Zhou et al., 1997). However, there are still many open questions with regard to this proposed combinatorial model of mesoderm patterning. For example, are there dorsoventral signals in addition to Dpp and EGF that are important for the distinction between heart and dorsal muscle precursors, for the specification of fat body anlagen in the dorsolateral mesoderm, and for the formation of ventral and lateral body wall muscles in the ventrolateral mesoderm? Do the Dpp and EGF signals elicit mesodermal responses in a graded fashion and thus act as morphogens? Perhaps, high activities

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MANFRED FRASCH and HANH T. NCUYEN

of Dpp might be required to activate tin and bup in dorsal portions of the dorsal mesoderm, whereas lower activities might be sufficient to activate srp (or an upstream regulator of it) in dorsolateral areas. Likewise, it is conceivable that Wg and Hh have morphogenlike functions within the A and P domains. Although these and other questions remain to be resolved, the combinatorial model of mesoderm patterning provides an important framework for future studies and offers the possibility of testing some of its predictions with molecular approaches. It is interesting to note that many of the signaling events require the activity of tinman in the mesoderm, suggesting that tinman provides competence to the mesoderm-initially to the whole trunk mesoderm and subsequently to the dorsal mesoderm-to respond to these signals in an exquisite fashion. D. Molecular Aspects of Early Mesoderm Patterning

Because much has been learned about the genetic inputs controlling the tinman pattern, there have been significant efforts to determine how these inputs act on the molecular level to regulate tin expression. Reporter gene analysis with genomic sequences from the introns and flanking regions of the tin gene have identified distinct enhancers whose activities mimic individual aspects of the endogenous tin expression (Yin et al., 1997). These tin enhancers act in a modular fashion and each of them appears to receive separate genetic inputs. One 180-bp enhancer that is located in the first intron drives reporter gene expression in the early trunk mesoderm and was therefore expected to receive inputs from twist. Indeed, this enhancer contains three E-box sequences (CATGTG or CATATG) that bind Twist in vitro and that are essential for driving reporter gene expression. These and additional findings suggest that Twist acts through these binding sites to activate tin in the early mesoderm. Another portion of this enhancer acts to prevent tin activation by Twist in the cephalic mesoderm and appears to be negatively influenced by the btn gene (see 1V.B.). A second enhancer of about 300 bp, which is located about 700bp downstream of tin, is specifically active in cardioblasts, thus resembling the late phase of tin expression. The molecular inputs to this enhancer have not yet been defined. By contrast, much has been learned about the molecular function of a third enhancer that is located about 2 kb downstream of tin and is specifically active in the dorsal mesoderm of stage 10 and 11 embryos. Since this reflects the Dpp-dependent expression of tin, this enhancer was a strong candidate for a “Dpp-response element”. In addition to Dpp, the activity of this enhancer also depends on the activity of tin itself, indicating that the Dpp signaling cascade needs to synergize with mesodermal Tin proteins for the localized induction of tin during stage 9 to 10 (Xu et al., 1998). Indeed, this Dpp-response element contains two in vitro Tin binding sites that are required for normal levels of reporter gene induction in vivo. A second, more unexpected result from mutating these Tin binding sites (TCAAGTGG) is ectopic reporter gene expression specifically in the dorsal ectoderm, that is, in the cells that

Genetic Control of Mesoderm Patterning and Differentiation

27

produce the Dpp signals. This indicates that, in the normal situation, there might be a ubiquitous repressor that binds to a sequence overlapping with the Tin binding sites while, in the mesoderm, Tin can compete for binding and thus allow induction by Dpp. Two candidate sequences for receiving inputs from the Dpp signaling cascade have been identified within this enhancer, and the deletion of either of them abolishes enhancer activity. A yeast screen to identify cDNAs encoding factors that bind to these sequences identified Medea, the Drosophila Smad4 homolog that is required to transmit Dpp signals to the tin promoter (see 1V.A.).Subsequent experiments showed that the Dpp response element of tin contains at least eight in vitro binding sites for Medea and Mad, the Drosophila homolog of SmadlI5, four of which are contained within the two essential sequences (Xu et al., 1998). In addition to binding of Medea, Mad, and Tin, binding sites for an unknown factor that are closely linked to the Medea/Mad binding sites are also required. These results suggest that Dpp signaling induces the formation and binding of a large protein complex to the Dpp response element of tin consisting of Tin itself, heteromers of Medea and Mad, and additional factor(s), which in turn activates tin transcription in the dorsal mesoderm. By contrast, in the ectoderm, a repressor appears to bind at or near the Tin binding sites and to prevent activation of tin transcription even though active MededMad heteromers are present in this tissue. Such a mechanism would ensure that tin induction is targeted exclusively to the mesoderm and not to other tissues that receive Dpp signals. Dpp-dependent tin activation may serve as a paradigm for the induction of other mesoderm-specific genes by Dpp and other signal molecules that are released by ectodermal cells.

VI. PATTERNING AND SPECIFICATION WITHIN THE DEVELOPING HEART AND VISCERAL MESODERM A.

The Diversification of Heart Cells

There is clear evidence that the segmental character of the dorsal vessel is maintained even after the segmentally repeated heart primordia have merged to form a contiguous structure along the anteroposterior axis. For example, tinrnan is expressed in only four of the six cardioblasts per hemisegment, and several enhancer trap lines also express lacZ in defined subsets of cardioblasts in each segment (Hartenstein and Jan, 1992). The expression of the ladybird homeobox genes (lbe and 161) represents another interesting example for the segmental organization of the dorsal vessel. lb expression is observed in two of the four tinman-expressing cardioblasts in each hemisegment of late stage embryos, and two pericardial cells (Jagla et al., 1997). These pericardial cells are distinct from even-skipped-expressing pericardial cells (e-PCs) and have been termed I-PCs. Results from ectopic lb expression and homozygous lb deficiency embryos strongly indicate that lb has a role in specifying distinct cell fates within the cardiac mesoderm, although specific

MANFRED FRASCH and HANH T. NCUYEN

28

lb mutations would be required to obtain insights into the physiological relevance of the diversification into different types of pericardial and cardial cells within each segment (Jagla et al., 1997). Comparisons of eve and lb expression during earlier stages has indicated that the cardioblasts and pericardial cells are originally (at stage 1 1) specified by anteroposterior patterning processes and subsequently realigned by morphogenetic movements into dorsal cardial and ventrally adjacent pericardial cells. Experiments with temperature-sensitive alleles of wingless and hedgehog indicated that these two segment polarity genes are important for this diversification process. The results suggested that wingless is required for the specification of lb-expressing cells, whereas hedgehog is needed to prevent the more posterior cells in each parasegment from becoming “lb cells”. These activities of wg and hh appear to occur after their initial function in mesoderm segmentation and precardiac mesoderm specification (see 1V.B).

B.

Anteroposterior Patterning of the Midgut Visceral Mesoderm

A number of studies have shown that the cells of the midgut visceral mesoderm acquire different identities along the anteroposterior extent of this tissue. This becomes apparent when midgut morphogenesis takes place during later stages of embryogenesis. At this stage, visceral mesoderm cells at defined anteroposterior positions are instrumental in generating defined midgut structures such as gastric caecae in the anterior and three separate constrictions in the main portion of the midgut. It has been shown that these differential properties are largely determined by homeotic genes that are expressed in spatially restricted domains along the visceral mesoderm, with Scr being expressed anteriorly, followed by Antp, Ubx, and Abd-A towards the posterior (Tremml and Bienz, 1989; Reuter and Scott, 1990). Mutations in these homeotic genes aEect midgut morphogenesis specifically in the areas of their expression. For example, gastric caecae formation is disrupted in Scr mutants, the first constriction is absent in Antp-, formation of the second constriction is disrupted in U b r and AbdA-, and the third constriction requires only AbdA. The regulatory hierarchies that specify visceral mesoderm cells to form the second constriction have been analyzed most extensively. Ubx is expressed in parasegment (PS) 7 of the visceral mesoderm where it activates dpp expression. AbdA is expressed posteriorly adjacent to PS 7 and, together with Dpp emanating from PS 7, activates wg expression in this area. Wg (perhaps in cooperation with Dpp) is in turn required for the activation of the expression of the zinc-finger-ncoding gene teashirt (tsh)in a slightly larger domain, and teashirt is necessary for the formation of the second midgut constriction in this area (Immergliick et al., 1990; Reuter et al., 1990; Hursh et al., 1993; Capovilla et al., 1994; Mathies et al., 1994). Although the crossregulatory interactions during this process are relatively complex and the target genes of tsh have not been identified, this represents one of the few examples where the role of homeotic genes in a morphogenetic process has been clarified in some detail.

Genetic Control of Mesoderm Patterning and Differentiation

VII.

29

PATTERNING AND SPECIFICATION OF BODY WALL MUSCLES A.

A Role of twist in Muscle Development

During stage 10, the pattern of twist changes from uniform expression to a striped pattern. The domains of high twist levels correspond to the mesodermal A domains from which the majority of somatic muscles are derived, whereas the domains of low twist levels coincide with the P domains and therefore include the domains of bup expression. Genetic analysis has shown that this modulation of twist levels is functionally significant (Baylies and Bate, 1996). Ectopic expression of twist in a uniform pattern at this stage interferes with the development of midgut visceral mesoderm, suggesting that twist has to be absent in order for bup to function in specifying this tissue. Conversely, removal of twist function during this period leads to a severe reduction of somatic muscle formation, indicating that high levels of twist are necessary for myogenesis in the A domains. This requirement for twist must be transient because, during stage 12, twist expression disappears from all mesodermal cells in the embryonic trunk region with the exception of the adult muscle precursors. Thus, twist appears to regulate an early step of muscle development at the transition between mesoderm segmentation and myogenesis. B.

The Founder Cell Concept of Somatic Muscle Development

During stages 12 to 14, groups of somatic myoblasts fuse with one another to form multinucleate muscle fibers. An important finding was that apparently not all partners in this fusion event are equivalent, but rather they fall into two distinct groups, founder myoblasts and fusion-competent cells. In general, each muscle fiber is generated by the fusion of one or two founder cells with a larger number of fusion-competent myoblasts (Bate, 1990; Dohrmann et al., 1990). There are a number of observations that suggest that only the founder myoblasts are specified with respect to their developmental identity, whereas the fusion-ccmpetent cells remain uncommitted to a particular fate until they fuse with a founder cell. For example, many founder cells have been identified based upon their specific expression of certain markers such as the luczreporter gene in some enhancer trap lines or the homeobox genes S59, upterous (up),and the zinc-fingerxncoding gene Kruppel ( K r ) (Figure 1A) (Dohrmann et al., 1990; Bourgouin et a]., 1992; Ruiz-Gomez et al., 1997; Nose et al., 1998). As described below (VI.C.), some of these genes, which encode transcription factors, have indeed important roles in cell fate specification of these founders. By contrast, specific markers for subsets of fusion-competent cells have not been identified. It was observed that upon fusion of a S59-expressing founder cell with neighboring fusion-competent cells, S59 protein initially spreads into the nuclei derived from the fusion-competent cells and subsequently these nu-

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MANFRED FRASCH and HANH T. NCUYEN

clei also appear to express this marker (Dohrmann et al., 1990). This event probably reflects the process of “entraining” the newly acquired uncommitted nuclei into the specific genetic program of the founder-cell nucleus. The second major evidence for the founder-cell concept came from studies on myogenesis in mutants that are defective in myohlast fusion. In the absence of fusion, the founder cells form at their normal locations and appear to proceed with-theirnormal developmental program, albeit as small, mononucleate muscle cells. For example, they express appropriate founder cell-markers, elongate into fibers that attach to the epidermis, and become innervated and contractile. By contrast, the fusion-competent myoblasts fail to develop any of these features and ultimately degenerate. Because of this pivotal role of muscle founder cells in myogenesis and muscle specification, it is of major interest to determine how these cells are generated and how they acquire their specific identity. C.

Genetic Mechanisms in Formation and Specification of Founder Cells

Segregation and Specification of Muscle Founders

As described in sections 1V.B. and VLA., the majority of the somatic muscles is derived from the mesodermal A domains, which transiently maintain high levels of mist expression. Genetic and histological studies have revealed that each muscle founder cell is derived from a defined cluster of mesodermal cells in these areas. In most, if not all cases, the formation of founder cells is preceded by the segregation of a distinct cell, called muscle progenitor cell, from these “preclusters”. The muscle progenitor then divides and usually gives rise to two muscle founders that can either go on to form two different muscles or in some cases form a single muscle. A number of observations suggest that initially, all cells of aprecluster have the potential to become muscle progenitors, but processes of mutual inhibition ensure that only one of them will do so. These processes are analogous to those of neuroblast segregation from proneural clusters and appear to utilize some of the same genetic mechanisms. The major components that mediate the mutual inhibition are the products of neurogenic genes, including the signaling molecules Notch, Delta, and the bHLH transcription factors of the E(sp1) group. In the absence of the activity of any of these molecules, there is a strong hypertrophy of muscle founder cells apparently as a result of the formation of supernumerary muscle progenitors from the preclusters (Corbin et al., 1991; Bate et al., 1993; Baker and Schubiger, 1996). In the neurogenic preclusters of the ectoderm, signaling by the neurogenic gene products leads to the downregulation of genes of the Achaete-Scute Complex (AS-C), which encode bHLH transcription factors and are necessary for neuroblast formation. Thus, only the cells that escape this inhibitory process maintain AS-C gene expression and develop into neuroblasts. The downstream targets of the neurogenic genes in the mesoderm are not yet known, although at least one of the AS-C genes, lethal of scute (l’sc)seems to play an analogous role. l’sc is initially expressed in de-

Genetic Control of Mesoderm Patterning and Differentiation

31

fined mesodermal clusters, which presumably correspond to the postulated mesodermal preclusters. Due to the action of neurogenic genes, expression in most clusters is gradually restricted to a single cell, which appears to become the muscle progenitor. In l’sc mutants, particular muscles are occasionally missing, which would be expected if l’sc has a function in the specification of muscle progenitors. However, since the majority of the muscle founder cells and muscles are normal, there must be additional genes that, analogously to the proneural genes during neurogenesis, determine a single cell from a mesodermal precluster to become a muscle progenitor. A major function of l’sc and analogous “promuscular” genes is presumably to activate the expression of specific transcription factors in the muscle progenitors and founders that, in turn, have important roles in specifying the development and identity of individual body wall muscles. Several examples of genes encoding such factors have been identified and, interestingly, each of them is expressed in a defined subset of muscle progenitors and/or founders. The prototype for a putative “muscle identity gene” of this type is the homeobox gene S59, which is expressed in the progenitors of six larval muscles, although expression is only maintained in three of the multinucleate fibers (Dohrmann et al., 1990). Recent results have demonstrated that in S59 mutants, several of these muscles are missing and are apparently transformed into different types of muscles that are then present in duplicate. Ectopic expression of S59 in all myoblasts also causes transformation of muscle identities (S. Knirr and Manfred Frasch, unpublished data). Similar observations have been made with respect to the expression and function of the homeobox gene msh, the zinc finger-encoding gene Kriippel (Kr), and the Drosophilu myoD homolog nautilus (nau).Each of these genes is expressed in a specific subset of muscle progenitors and founders, with nuu showing the broadest distribution among the three. In embryos that are mutant for any of these genes, some of the muscles that would normally develop from the respective founders are missing or abnormally’ shaped. Once again, ectopic expression of msh, Kr, or nau results in cell fate switches and, as aconsequence, in disruptions of the mature muscle pattern (Lord et al., 1995; Keller et al., 1997; Ruiz-Gomez et al., 1997; Nose et al., 1998). The LIM/homeodomain encoding gene upterous (up) is yet another .candidate for a muscle identity gene although, in contrast to the previously described mutants, the muscle phenotype in u p mutants only has weakpenetrance (Bourgouin et al., 1992). Taken together, these observations have led to the idea that the expression of certain muscle identity genes is activated in distinct, but overlapping, subsets of muscle progenitors and founders, where they act combinatorially to determine the developmental fate and identitiy of the muscles that are derived from them. It is obvious that the “promuscular” genes, which are presumably expressed in all muscle progenitors, must act in combination with spatially restricted regulators to activate the expression of muscle identity genes in such intricate patterns. Although some of these regulators could include extrinsic signals such as Dpp, EGF, or Wg, the exact mechanisms of this patterning process remain unknown. However, it has been dem-

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MANFRED FRASCH and HANH T. NCUYEN

onstrated that positional information in the somatic mesoderm is in part provided by mesoderm-intrinsic mechanisms that are due to the spatially restricted expression of homeotic genes (Hooper, 1986;Greig and Akam, 1993;Michelson, 1994). These inputs have important roles in creating unique muscle patterns in different body parts, which for example leads to a modulation of the muscle pattern in the thoracic versus the abdominal segments of the larva. The Roles of Lineage Genes in Asymmetric Cell Divisions of Muscle Progenitors

Recent studies have identified an important mechanism that contributes to the diversification of muscle founder cells and involves asymmetric cell division of muscle progenitors. This appears to be the result of asymmetric distribution and segregation of certain cytoplasmic determinants prior to and during the mitotic division of the progenitors. Two such determinants are encoded by the lineage genes numb and inscutable (insc, also called not enough ntuscleslnem; Burchard et al., 1995;reviewed in Knoblich, 1997).The role of numb and insc in assymmetric muscle progenitor divisions has been examined particularly well for the development of the ventral muscles 26 (VA1)and 27 (VA2)(Ruiz Gomez and Bate, 1997).These two muscles are derived from a common progenitor that expresses both Kr and S59, which are nuclearly localized. Numb and Insc are asymetrically distributed in the cytoplasm of this progenitor, and each of them is localized within a crescent but at opposite poles of the cell. It was shown that insc activity is required for the asymmetric distribution of Numb, but it is not known how the asymmetric distribution of Insc itself is determined. As a result of its asymmetric distribution, Numb segregates only into one daughter cell, which becomes the founder cell of muscle 27 but is excluded from the other, which then becomes the founder of muscle 26.In turn, the asymmetric segregation of Numb determines the differential specification of these two muscle founders. Thus, in mutants of numb, both develop into muscle 26, whereas upon ectopic expression of numb in all myoblasts, both of these founders develop into muscle 27. How does numb determine the identities of these founder cells? Apparently, it does so, at least in part, by regulating the maintenance of Kr and S59 expression in the founder cell of muscle 27 that inherits Numb protein. By contrast, its sibling lacks Numb protein, which results in the downregulation of Kr and S59, and consequently this cell assumes the identity as a founder cell of muscle 26.Kr seems to act upstream of S59 in this maintenance process. Thus, in Krmutants, S59 is not maintained in either of the two founder cells and both develop into muscle 26,while upon ectopic Kr expression in all myoblasts, both maintain S59 expression and develop into muscle 27 (Ruiz Gomez and Bate, 1997). It is not known how numb maintains Kr expression in the muscle 27 founder. In neuronal specification, numb has been shown to act through the inhibition of Notch signaling, and this seems also to be the case for some other muscle founders that have been examined (Ruiz Gomez and Bate, 1997;Carmena et al., 1998).Thus, it

Genetic Control of Mesoderm Patterning and Differentiation

33

p26/27

Notch

Not

....I

m26

m27

!

lnsc Numb Kr

s5 9 M27

Figure 4. The role of asymmetric cell divisions in the diversification of muscle identities. Through the function of lnscutable (Insc), Numb protein is asymmetrically localized in the dividing progenitor of muscles 26 and 27 (p26/27). Therefore, Numb segregates into only one of the daughter cells, which will become the founder of muscle 27. Notch may function in the siblings analogously as has been shown for other founder cells. In this specific situation, it may downregulate Kruppel (Kr) expression in one cell, which will then become the founder of muscle 26 (m28), but is prevented by Numb from downregulating Kr in the other, which will become a muscle 2 7 founder (m27). Kr maintenance in m27 i s required for maintaining S59 expression, and the expression of both genes appears to function in m27 specification. Upon fusion with uncommitted myoblasts, m27 forms a multinucleated muscle 27 (M27) that continues559 expression, while its sibiing, which has lost Kr and S59, fuses to form muscle 26 (M26).

appears that Notch signaling has at least two roles in muscle development-an early one in lateral inhibition processes leading to the segregation of muscle progenitors and a later one during the asymmetric specification of the two daughter cells that are derived from the progenitor. If this mechanism also operates in the founders of muscles 26 and 27, Notch signaling would be expected to downregulate

MANFRED FRASCH and HANH T. NCUYEN

34

Kr in the m 26 founder, whereas the presence of Numb in the m 27 founder blocks this activity, thereby allowing Kr expression to be maintained (Figure 4). A major question for the future will be how the asymmetry in the muscle progenitor is established initially, an attribute that ultimately determines the differential development of the sibling founder cells and the muscles derived from them.

VIII. A.

GENETIC CONTROL OF MESODERMAL TISSUE DIFF E RENT1A l l ON

The Role of mef2 in the Differentiation of Somatic Muscles, Visceral Mesoderm, and the Heart

Drosophila mej2 is amember of the MEF3 (myocyte-specific enhancer factor 2) family of transcription factors, which have in common an evolutionarily conserved MADS box type of DNA binding domain and an adjacent MEF2-specific domain (reviewed by Olson et al., 1995; Shore and Sharrocks, 1995; Taylor, 1995). As with its vertebrate counterparts, the Drosophila mej2 gene product can also recognize the consensus sequence PyT(A/T)(A/)AAATAPu, which has been found in the regulatory region of various vertebrate and Drosophila muscle-specific and myogenic genes (Olson et al., 1995; Lin et al., 1996). Both mef2 RNA and protein are present in all muscle progenitors and the corresponding differentiated musculatures (Lilly et al., 1994; Nguyen et al., 1994; Bour et al., 1995; Taylor et al., 1995), suggesting a functional role in all three muscle types. Genetic analysis of me$? mutations has indicated that it is essential for the terminal differentiation of all three major musculatures in the Drosophila embryo (Bow et al., 1995; Lilly et al., 1995;Ranganayakulu et al., 1995). Loss of me$? function does not affect the early muscle patterning events. In mej2 mutant embryos, the general population of P3-Tubulin expressing myoblasts is present and somatic muscle progenitors are specified, as evidenced by the existence of nautilus-, S59-, and apterous-expressing founder cells (Bour et al., 1995; Lilly et al., 1995). Similarly, tinman and bagpipe gene expression is not affected, leading to normal formation of heart progenitors and visceral mesoderm. By contrast, late aspects of differentiation events are defective. In the absence of MEF2 activity, the specified somatic myoblasts do not undergo terminal differentiation to form multinucleate, MHCexpressing muscle fibers. Midgut morphogenesis is disrupted as a consequence of abnormal differentiation of the visceral mesoderm and the heart is not fullydifferentiated since muscle-specific transcription is not observed in mej2 mutant embryos. A growing list of presumptive targets of mej2 activity is emerging. It is clear that in the mef2 mutants, myosin heavy chain (MHC) expression is not activated in any of the musculatures (Bour et al., 1995; Lilly et al., 1995). It is currently not known whether the regulation of MHC occurs at the transcriptional level. Troponin T and

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apS2 integrin expression is also affected in mej2 mutant embryos (Nguyen, unpublished). Interestingly, tropomyosin I (Tml) gene transcription is specifically affected in the somatic musculature and not in the visceral muscle or the heart of mef2 mutant embryos. In vivo analysis indicated that mutation of MEF2 binding sites, which are present in both the proximal and distal enhancers of the Tmf gene, results in a significant reduction in reporter gene expression in embryonic, larval, and adult somatic muscles (Lin and Storti, 1997). This analysis has also shown that the MEF2 site is required, although not sufficient, for full activity of the proximal enhancer. Indeed, it appears that the MEF2 site interacts cooperatively with a second cis-regulatory muscle activating (MA) region within the Tmf proximal enhancer, which is recognized by yet uncharacterized factor(s). Notably, a recent study described the cloning of PDPl (PAR domain protein- 1 ), a factor that binds to the MA region (Lin et al., 1997). PDPl is a member of the PAR subfamily of bZIP family of transcription factors and appears to function as a transcriptional activator in trans-activation assays. PDPI is expressed in the body wall and pharyngeal muscles, as well as several nonmuscle tissues, including the epidermis. Mutational analysis has shown that PDPl sites are functionally important for the cooperative interactions between the MA region and MEF2 sites. However, the combination of PDPl and MEF2 is still not sufficient to drive somatic muscle expression, implying the existence of yet additional regulatory factor(s) that interact with MEF2 andor PDPl . A second target of mej2 activity in the visceral musculature has been uncovered in addition to MHC. Because of the abnormal gut morphology in me@ mutants, which is similar to the phenotype of the myospheroid and inflated mutations (Brabant and Brower, 1993; Brown, 1994), defects in adhesion properties of the visceral mesoderm to the underlying endoderm were suspected. Indeed, in mej2 mutant embryos, neither protein nor RNA expression of the aps2 integrin gene is observed whereas there is normal pps gene expression (Ranganayakulu et al., 1995; Nguyen, unpublished). The absence of aps2 gene expression in the mutant embryos has been further correlated with the inability of an aps2 integrin enhancer, which can bind MEF2 in vitro, to drive reporter gene expression in the visceral musculature (Ranganayakulu et al., 1995). Besides the absence of activation of muscle-specific structural genes, another hallmark of the mej2 mutant phenotype is the nearly total lack of myoblast fusion. The fusion process can be characterized by a sequence of events that includes differentiation, cell-cell recognition, adhesion, alignment, and membrane fusion (Knudsen and Horwitz, 1978; Wakelam, 1985). An increasing number of gene products has been identified that are needed for myoblast fusion. To date, analysis of loss-of-function mutations of myoblast city, rolling stone, blownfuse, or altered forms of Drac I has suggested that each of these genes acts at adifferent point in the myoblast fusion process (Luo et al., 1994; Rushton et al., 1995; Doberstein et al., 1997; Erickson et al., 1997; Paululat et al., 1997) Whether any of these genes or yet to be identified ones are under mej2 regulation as well as the mechanisms through which they mediate their function remains to determined.

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mej2 appears to also play a role in the processes that mediate organization of the contractile apparatus and muscle attachments to the epidermis. A recent study showed that muscleblind (mlb) is an important mediator of these processes and a target of mej2 function (Artero et al., 1998). In mej2 mutants, muscleblind expression is decreased and ectopic MEF2 expression in the epidermis can lead to ectopic Musclebind expression. Mlb is a Cys,His-type, zinc finger-containing protein that was previously shown to be critical for terminal differentiation of photoreceptors (Begemann et al., 1997). rnbl mutant embryos have normal direct types of muscle attachments; however, the assembly of indirect types of muscle attachments is defective. The formation of these indirect muscle attachments involves the linking of a great number of muscle tips to a limiting epidermal surface via a tendon matrix (TM). It was observed that in rnbl mutants, the TM, which is composed of musclederived extracellular matrix, is greatly reduced. The analysis further showed that, in addition to its potential role in TM formation, mbl function appears to be needed for the correct assembly of Z-bands of the sarcomere. Yet another aspect of muscle differentiation in which mej2 is involved is the formation of functional neuromuscular junctions (NMJ). In mej2 mutant embryos, target recognition by motor axons occurs normally, based upon the observations that Connectin- and Vestigial-expressing founder cells do become innervated by motorneurons (Prokop et al., 1996). However, the further differentiation of target muscles that is necessary to generate functional NMJ requires mej2 function. Formation of synapses and localization of presynaptic active zones at the sites of synapses is not observed in the mej2 mutants. Further studies will be needed to identify additional molecules within the postsynaptic muscle cell, some of which are likely to be mej2 targets, that are important for the differentiation of the presynaptic terminal. In summary, it has become evident from the various studies that mej2 function is critically needed for several distinct differentiation programs that culminate in the generation of terminally-differentiated functional musculatures.

B.

Distinct Enhancer Modules of mef2 Linking Patterning and Differentiation Events

It appears that appropriate spatial distribution and level of activity are important for normal mej2 function. It has been observed that .ectopic MEF2 expression or overexpression of MEF2 in the mesoderm results in embryonic lethality and defective muscle development (Lin et al., 1997). This strict requirement for regulated nzej2 expression suggested that it might be under stringent regulatory control during embryonic development. It has been documented that mej2 is expressed in a dynamic fashion during embryogenesis (Lilly et al., 1994; Nguyen et al., 1994; Taylor et al., 1995; Bour et al., 1995). Initial mej2 expression is observed in the ventral mesodermal primordia and is ubiquitous in the mesoderm after gastrulation. Subsequent to the internal migration of the mesodernial layer, mej2 undergoes a transient dorsal mesoderm-

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restricted phase of expression, as observed with tinman expression. In subsequent phases of expression, mej2 is activated in all muscle progenitors and then maintained in the differentiated musculatures. Efforts toward defining the regulatory regions have established that all the necessary regulatory sequences for reproducing the endogenous mej2 expression pattern during embryogenesis are localized within approximate 12Kb of 5' flanking sequences of the mej2 gene. Dissection of these genomic regions has indicated that regulated mej2 in the various muscle lineages is controlled by a set of separable enhancers that are responsive to different genetic cues (Gajewski et al., 1997; Cripps et al., 1998; Nguyen and Xu, 1998). Reporter gene analysis has shown that the expression in the early mesoderm is under the control of a twist-responsive enhancer. This enhancer can bind Twist in vitro, and ectopic activation of this enhancer is obtained in response to ectopic Twist activity (Cripps et al., 1998;Nguyen et al., 1998). It is also active and dependent upon twist function in adult muscle precursors (Cripps et al., 1998). While it has been observed that a reduction of Twist activity during larval stages results in reduced MEF2 activity, which in turn leads to patterning defects in the adult musculature (Cripps et al., 1998), the functional significance of the early mej2 expression is not obvious. However, it is possible that early MEF2 activity is needed for the segregation a n d o r maintenance of twist-expressing adult precursors, and hence, this particular function would not be manifested during embryogenesis. Regulation of dorsal mesoderm-restricted niej2 expression is also mediated through adpp-responsive enhancer, as documented for tinman (Frasch, 1995; Xu et al., 1998; Nguyen and Xu, 1998). A sequence comparison of the Dpp-responsive targets in the mej2 and tinman genes has uncovered several conserved elements, suggesting that there might be common features between the corresponding mechanisms that mediate this inductive process. One notable possibility is that SMAD family members transduce the Dpp signal to the mej2 regulatory region. However, unlike the tinman scenario, the activation of the Dpp-dependent mej2 enhancer does not appear to require Tinman binding activity (Nguyen, unpublished). The function of mef2 activity in the dorsal mesoderm might be to induce differentiation, rather than specification, of the dorsal mesoderm derivatives such as the heart and dorsal somatic muscles. In support of this proposed function is the fact that the heart and dorsal somatic muscle progenitors are formed normally in mej2 mutant embryos; however, they do not undergo terminal differentiation. TWOdistinct enhancers have been identified that are responsible for mej2 expression in tinman-expressing cardioblasts. One heart element contains two potential Tinman binding sites, both ofwhich have been shown to bind Tinman in vitro and to be of functional importance in vivo (Gajewski et al., 1997). In contrast to the first heart enhancer, which is activated from stage 12 on, the second one is active at a later stage (stage 14 and later), suggesting the existence of different sets of coregulators that are necessary for conferring temporal specificity to these two heart enhancers (Nguyen and Xu, 1998). The combined activity of the two heart enhancers would ensure that robust levels of me$? activity is achieved in the differentiated

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heart musculature. In addition, since mej2 is expressed in all six cardioblasts per hemisegment, there must exist additional factor(s) for regulating mej2 expression in the remaining two Tinman-negative cardioblasts. Distinct regulatory elements were identified that can direct mej2 expression in the two types of visceral mesoderm (Nguyen and Xu, 1998). Two enhancer elements were shown to be capable of driving reporter gene expression in the caudal visceral mesoderm and its derivative, the longitudinal gut musculature (Figure lE,F). Expression from a different enhancer is observed in the trunk visceral mesoderm and circular gut muscles. Candidate regulators include bagpipe (Azpiazu et al., 1993) and the recently-described bHLH54F protein (Georgias et al., 1997) for activation in the trunk and caudal visceral mesoderm, respectively. However, there must exist additional regulatory factors for maintaining me$? activity in the trunk visceral mesoderm because bagpipe is only transiently expressed until early stage 12. mej2 appears to be expressed in both musc!e founder cells and fusion-competent cells based upon three lines of evidence: (1) MEF2 expression is observed in a large number of mesodermal cells (Bour et al., 1995); (2) expression from an enhancer trap line, which marks founder cells, coincides with MEF2-positive mesodermal cells (Nguyen and Xu, 1998); and (3) somatic founder cells that express Ladybird protein are also MEFZpositive (Nguyen, unpublished). The more recent cis-regulatory analysis has indeed uncovered multiple elements that can drive reporter gene expression differentially in the somatic mesodermal cells and fully differentiated musculature. Two distinct “early somatic” enhancers are capable of activating expression in subsets of muscle founders. Among the three additional “late somatic” enhancers, one mediates transient expression in presumed fusion-competent cells and two are involved in maintaining expression in the fully differentiated somatic musculature. The spatial and temporal specificity of these enhancers suggests that common regulators(s) may function in concert with different co-factors to control their differential activation. Consequently, regulated mej2 expression in the forming mesoderm and muscle lineages is achieved through the action of various genetic inputs upon regulatory modules that are located in the flanking region of the mej2 gene. It is likely that sets of spatially and temporally specific activators act upon these multiple regulatory elements, which appear to function in an additive manner, to ensure that mej2 is expressed appropriately throughout embryogenesis. Thus far, the mesoderm-intrinsic regulators include twist and tinman, and extrinsic signals are derived from wingless and dpp function. The majority of regulatory inputs (intrinsic and extrinsic), especially those converging on the later acting elements, remain to be identified in future studies.

IX.

CONCLUDING REMARKS

Significant progress has been made in unraveling the genetic and molecular processes that control the development of the mesoderm in Drosophila. Three mesodermally expressed genes, twist, tinman, and rnej2, have been found to play key

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roles in mesoderm formation, patterning, and differentiation, respectively. twist functions in the activation of a large set of target genes in the mesoderm primordium, including folded gastrulation, heartless, tinrnan and rnej2, which are required for invagination, migration, subdivision, and differentiation events during later periods of development. Thus, twist sets in motion aregulatory cascade that is subsequently elaborated to determine all aspects of mesoderm development. The subdivision of the mesoderm is achieved by combinatorial cues that intersect at defined anteroposterior and dorsoventral positions, which define the formation of the anlagen of specific mesodermal derivatives at these sites. Many of these cues are mediated by inductive signals from the ectoderm, which is patterned prior to the mesoderm. The homeodomain protein Tinman appears to have a major function in mediating mesodermal responses to a number of these signals and integrating them to induce specific target genes. This has been best studied with respect to the dorsal mesoderm, where tinman is required to mediate apparently all inductive responses to Dpp and integrate them with segmental cues. From the analysis of the induction of tinman expression itself by Dpp in the dorsal mesoderm, we are beginning to understand how the integration of mesoderm-intrinsic and -extrinsic cues functions at the molecular level. Related mechanisms, perhaps with additional levels of complexity, may also be involved in the regulation of tinman target genes in combination with various additional cues. Examples include the activation of segmental bagpipe expression or cardiogenic genes in the dorsal mesoderm, activation of specific regulators for muscle progenitor formation in other areas, and the activation of buttonless expression by a combination of tinman, EGF signaling, and segmental regulators in the ventral mesoderm. The determination of the anlagen of individual mesodermal derivatives is followed by unique differentiation programs in each of them. Significant progress has been made in elucidating the central role of mej2 in activating differentiation genes, in the myogenic lineages. A future direction would be to determine how me$?acts in combination with additional regulators to activate specific target genes in different myogenic lineages. From the genetic and molecular analysis of the regulation of the mej2 gene, we are obtaining important information on the links between early patterning events and the differentiation of myogenic tissues. The results of this analysis indicate that a complex array of regulatory regions of the mej2 gene integrates this patterning information to allow defined levels of mej2 expression in myogenic tissues, but not in nonmyogenic derivatives of the mesoderm. It will be important to determine whether (yet unknown) differentiation control genes in mesodermallyderived, but nonmyogenic, tissues are regulated by analogous mechanisms. In the past few years, comparisons between invertebrate and vertebrate systems have led to the conclusion that an astonishing number of regulatory mechanisms in mesoderm development have been evolutionarily conserved. In particular, genes that are homologous to tinman and bagpipe appear to have related roles in heart and visceral muscle development, respectively, in vertebrate embryos (Lyons et al., 1995; Tribioli et al., 1997). Furthermore, the Dpp-related bone morphogenetic proteins

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(BMPs) have a central function in the dorsoventral axis determination of the vertebrate mesoderm, although the axis appears inverted between vertebrates and invertebrates. Analogous to the Drosophila system, BMPs activate the nested activation of certain homeobox genes, including Xventl & 2 and the tinman-related Nkr2.5, in ventral and ventrolateral areas of the vertebrate mesoderm, and induce formation of the heart, lateral plate (visceral) mesoderm, and striated muscles (reviewed in Ferguson, 1996; Schultheiss et al., 1997; Dosch et al., 1997). These signaling events involve Smad proteins, which are related to Drosophila Mad and Medea (reviewed in Baker and Harland, 1997), and future studies will show whether these proteins form similar complexes on enhancers of Nkr2.S or other targets, as has been shown for the Dpp response enhancer of Drosophila tinman. The conservation of the structure and function of rnep genes between invertebrates and vertebrates indicates that not only patterning processes, but also regulation of differentiation, have been highly conserved between species (reviewed in Olson et al., 1995). Nevertheless, there are also some apparent differences. An example is the myoD homolog nautilus, which seems to have a much more specific role in Drosophila myogenesis as compared to the broad requirement of myoD in vertebrate myogenesis (Abmayr and Keller, 1998). In conclusion, comparative studies of invertebrate and vertebrate systems have produced important insights into mesoderm development. For the near future, we can expect that studies of both similarities and variations among different biological systems will give us a more detailed understanding of the regulatory hierarchies that control the formation and differentiation of mesodermal tissues.

ACKNOWEDCMENTS Manfred Frasch was supported by grants from the NIH, Pew Foundation, and American Heart Association and Hanh T. Nguyen by a grant from the American Heart Association (AHA).

NOTE ADDED IN PROOF Since its submission, anumber of additional papers have appeared on topics that are discussed in the present article. The most relevant of these are as follows: Bilder et al., 1998; Buffet al., 1998; Carmena et al., 199%; Farrell and Keshishian, 1998; Fuerstenberg and Giniger, 1998; Gajewski et a]., 1998;Lu et al., 1998;Michelson et al., 1998a and 1998b; Park et al., 1998a and 1998b; Vincent et al., 1998.

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Ferguson, E. (1996). Conservation of dorsal-ventralpatterning in arthropods and chordates. Cum. Opin. Genet. Dev. 6:424-431. Frasch, M. (1995). Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophilu embryo. Nature 374:464-467. Fuerstenberg, S. and Giniger, E. (1998). Multiple roles for Notch in Drosophilu myogenesis. Dev. Biol. 201~66-77. Gabay, L., Seger, R., and Shilo, B.-Z. (1997). MAP kinase in situ activation atlas during Drosophilu embryogenesis. Development 124:3535-3541. Gajewski, K., Kim, Y., Choi, C.Y.,and Schulz, R.A. (1998). Comhinatorial control of Drosophilu mef2 gene expression in cardiac and somatic muscle cell lineages. Dev. Genes Evol. 208:382-392. Gajewski, K., Kim, Y., Lee, Y., Olson, E., and Schulz, R. (1997). D-me@ is a target for Tinman activation during Drosophilu heart development. EMBO J. 16:515-522. Georgia, C., Wasser, M., and Hinz, U. (1997). A basic-helix-loop-helix protein expressed in precursors of Drosophilu longitudinal visceral muscles. Mech. Dev. 69: 115-124. Gisselbrecht, S., Skeath, J., Doe, C., and Michelson, A. (1996). heurtless encodes a fibroblast growth factor receptor (DFRI/DFGF-R2) involved in the directional migration of early mesodermal cells in the Drosophilu embryo. Genes Dev 10:3003-3017. Goldstein, M. A,, and Burdette, W. J. (1971). Striated visceral muscle of Drosophilu melunoguster. J. Morphol. 134:315-334. Gorczyka, M. G., Phyllis, R. W., and Budnik, V. (1994). The role of tinrnun, a mesodermal cell fate gene, in axon pathfinding during the development of the transverse nerve in Drosophilu. Development 120:2143-2152. Grau, Y., Carteret, C., and Simpson, P. (1984). Mutations and chromosomal rearrangements affecting the expression of snuil, a gene involved in embryonic patterning in Drosophilu mehnoguster. Genetics 108:347-360. Greig, S., and Akam, M. (1993). Homeotic genes autonomously specify one aspect of pattern in the Drosophih mesoderm. Nature 362:630-632. Greig, S.. and Akam, M. (1995). The role of homeotic genes in the specification of the Drosophilu gonad. Curr. Biol. 5:1057-1062. Grossniklaus, U., Pearson, R., and Gehring, W. (1992). The Drosophilu sloppy puired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors. Genes Dev. 6:1030-1051. Hacker, U. and Perrimon, N. (1998). DRhoCEF2 encodes a member of the Dbl family of oncogenes and controls cell shape changes during gastrulation in Drosophilu. Genes Dev 12:274-284. Hartenstein, V . and Jan, Y. N. (1992). Studying Dr(J.Fophih embryogenesis with P-fuczenhancer trap lines. Roux’s Arch. Dev. Biol. 201:194-220. Harvey, R. (1996). NK-2 homeobox genes and heart development. Dev. Biol. 178.203-216; Hemavathy, K., Meng, X., and Ip, Y. (1997). Differential regulation of gastrulatio.n.andneuroectodemal gene expression by snuil in the Drowphilu embryo. Development 1243683-3691. Hooper, J . E. (1986). Homeotic gene function in the muscles of Dr(J.F/JphilU larvae. EMBO J. 512321-2329. Hortsch, M., Olson, A., Fishman, S., Soneral, S., Marikar, Y., Dong, R., and Jacobs, J . (1998). The expression of MDP- I , a component Of Dr(J.wphi/U embryonic basement membranes, is modulated by apoptotic cell death. Int. J. Dev. Biol. 42:33-42. Hoshizaki, D., Blackburn, T., Price, C., Ghosh, M., Miles, K., Ragucci, M., and Sweis, R. (1994). Embryonic fat-cell lineage in Drosophilu melanoguster. Development 120:2489-2499. Hursh, D., Padgett, R., and Gelbart, W. (1993). Cross regulation of decugenruplegic and U[trubirhorux transcription in the embryonic visceral mesoderm of Drosophilo. Development 117:1211-1222. lmmergliick, K., Lawrence, P., and Bienz, M. (1990). Induction across germ layers in Drosophilrr mediated by a genetic cascade. Cell 62:261-268.

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ACROSOMAL PROTEINS OF ABALONE SPERMATOZOA

Victor D. Vacquier, Willie J. Swanson, Edward C. Metz, and C. David Stout

I. Introduction. . . . .................. 50 11. Species-Specifici . . . . . . . . . . . . . . . . . . . . . . . . . . . .50 111. The Abalone as an Experimental Organism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 IV. The Egg Vitelline Envelope ......... 52 V.TheAbaloneSpermatozoon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 VI. Sperm-Egg Interaction in Abalones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55 VII. The Species-Specificity of Lysin . . . . . . . . . . . . . . . . . . . . . . . . . . . .58 VII1. Function of the 18K Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 9 IX. Obtaining Sequences of Lysin and 18K Proteins . . . . . . . . . . . . . . . . . . A. Lysin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ;.._ B. The 18K Protein. . . . . . . . . .............. X. Analysis of Lysin and 18K Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61 A. Lysin Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61 B. 18K Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 XI. Crystal Structure o rom the Red Abalone ... 64

Advances in Developmental Biochemistry Volume 5, pages 49-81. Copyright 0 1999 by JAI Press Inc. All right of reproduction in any form reserved. ISBN: 0-7623-0202-X

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XII. Possible Mechanism of Lysin’s Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Variable Features of Lysins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Conserved Features of Lysins . . . . . . . . . . . . . . . . . . . . . . . . . XIII. Lysin and the 18K Protein Arose by Gene Duplication . . . . . . . . . . . . . . . . XIV. Lysin and 18K Evolve by Positive Darwinian Selection . . . . . . . . XV. Identification of Lysin’s Receptor in the Egg Vitelline Envelope XVI. Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments ......... .......... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

.69

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.77 79 79

INTRODUCTION

Our understanding of living systems is deeply rooted in the knowledge of how biological molecules interact to create form, phybiology, and behavior. We understand much about gene activity; soon we will understand how differential gene expression directs embryonic development. We have a detailed knowledge of the molecular and cellular interactions of the immune system. Regarding the reproductive biology of metazoans, we know how egg and sperm cells differentiate in a variety of animal phyla. After the sperm and egg pronuclei fuse to reconstitute the diploid genome and activate the egg, we have considerable knowledge of the control of the cell division cycle. However, in comparison to these examples where detailed knowledge exists of how molecular interactions underlie cellular behavior, we know little about the process of sperm-egg interaction leading to gamete fusion and zygote formation. Fertilization occurs only once in the life history of sexually reproducing animals. As fascinating as it is to those of us who study it, sperm-egg interaction preceding gamete fusion remains one of the least studied, least understood, fundamental biological processes.

II.

SPECIES-SPECIFICITY OF SPERM-EGG INTERACTION

In most animal species, whether internally or externally fertilizing, sperm-egg interaction leading to gamete fusion usually exhibits some degree of speciesspecificity (Giudice 1973; O’Rand 1988; Yanagimachi 1988a, 1988b). This means that sperm and eggs of the same species are almost always more efficient at forming zygotes than are combinations of sperm and eggs from different species. There is a great range in species-specificity, with some interspecies cross mixtures of gametes forming zygotes as efficiently as homospecific combinations and other interspecies combinations yielding no zygotes. For example, among sea urchin species, Stongylocentrotus purpuratus spermatozoa are very efficient at fertilizing Allocentrotus fragilis eggs; however, the reciprocal cross yields no interspecies zygotes (Fedecka-Bruner et al., 197 1; Vacquier, unpublished). The species-specificity of sperm-egg interaction suggests the process is mediated by gamete recognition

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molecules acting in ligand-receptor combinations. The steps in the sperm-egg interaction cascade that could involve species-specific ligand-receptor binding are as follows: chemotaxis of sperm to egg, the binding of the sperm cell to the extracellular coat surrounding the egg, the induction of the sperm acrosome reaction, the interaction of the contents of the acrosome vesicle with the egg coat(s), the penetration of the egg coat by the sperm, the binding between the plasma membranes of egg and sperm, and finally, the fusion of the two plasma membranes leading to the incorporation of the sperm into the egg cytoplasm (Epel, 1997). For cell biology, the species-specificity of gamete interaction during fertilization is a valuable model for studying the molecular events of intercellular recognition and adhesion that underlie such basic processes as embryonic development, wound healing, metastasis, and invasion of host cells by parasites. For evolutionary biology, fertilization proteins also provide unique opportunities. In most animals, the species-specificity of reproduction involves elaborations of morphology and behavior that have a complex genetic basis. Among free-spawning organisms, however, the specificity of reproduction is determined by relatively few loci encoding gamete recognition proteins on the surfaces of sperm and egg. The isolation and cloning of these fertilization proteins and the analysis of their sequences will yield clues as to how gamete specificity evolves.

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THE ABALONE AS A N EXPERIMENTAL ORGANISM

The species-specificity of fertilization represents an interesting phenomenon to study by the isolation of interacting cell surface macromolecules. Advantageous model organisms must be chosen to explore the process biochemically. The three highest vertebrate classes (Reptilia, Aves, Mammalia) exhibit internal fertilization in which the cell surface interactions and gamete fusion occur in the upper portions of the female reproductive tract. Much has been learned about mammalian fertilization in a variety of species in which sperm-egg interaction can be studied in vitro. Small mammals such as mice, rats, guinea pigs, and hamsters have been invaluable in the identification of sperm and egg surface macromolecules mediating gamete binding. The mouse is a most important model because of the ability to perform gene knockout experiments to delete gamete recognition proteins (Baba et al., 1994; Lu and Shur, 1997), or to modify them in transgenic animals (Litscher and Wassarman, 1996; Liu et al., 1995). The appeal of studying fertilization in mammalian species is that such work may lead to the discovery of homologous molecules that mediate sperm-egg interaction in humans. However, due to the rapid evolution of fertilization proteins, the discovery of homologous human proteins may be difficult. For example, the mouse sperm protein sp56, which binds to the egg’s ZP3, cannot be detected outside of rodents (Bookbinder et al., 1995). The disadvantages of studying the biochemistry of mammalian fertilization are the small amounts of gametes (especially eggs) that can be obtained and the substantial monetary expense.

V.D. VACQUIER, W.J.SWANSON, E.C. METZ, and C.D. STOUT

52

Marine invertebrates such as sea urchins, starfish, clams, mussels, snails, and worms have been used for about 120 years in studies of gamete interaction during fertilization. The advantages of most invertebrates for fertilization studies are adults spawn gametes into seawater where sperm-egg interaction and fertilization occur, large quantities of gametes can be obtained at low cost, and the speciesspecificity of gamete interaction can be readily observed and quantitative assays devised to study the biochemical basis for the specificity. The vast quantities of gametes available permit the preparative isolation of the gamete recognition molecules. The biochemistry of the activation of sea urchin eggs at fertilization has contributed enormously to our general knowledge of cellular activation (Epel, 1997). Abalone (genus, Halioris) are large, single-shelled, marine archeogastropod mollusks of separate sexes that yield vast amounts of sperm and eggs permitting the isolation of the egg VE and the sperm acrosomal vesicle proteins. Approximately 60 to 70 abalone species exist in the world, most as clusters of species that share habitat and breeding season. The habitats of seven abalone species overlap along the coast of California (Hahn, 1989; Lindberg, 1992). The eggs and sperm of abalone can be obtained by the artificial induction of spawning by addition of hydrogen peroxide to seawater, or by dissection of the single gonad and differential sedimentation to obtain the isolated gametes. The major advantage of abalone gametes is that hundreds of milligrams of sperm acrosomal proteins and egg vitelline envelopes can be isolated with ease at low expense. This article reviews the work of our laboratory on the two proteins released from the acrosomal vesicle of abalone spermatozoa onto the surface of the egg vitelline envelope. The objectives of our studies are to discover the mechanism of interaction of these sperm proteins with their specific egg surface receptors and to understand how species-specificity evolves. The two abalone sperm proteins have illuminated the possible mechanisms of evolution of species-specific gamete interaction and have also shown that these proteins may play essential roles in establishing reproductive isolaticn between species.

IV.

THE EGG VlTELLlNE ENVELOPE

As viewed with a light microscope, abalone eggs are surrounded by an elevated vitelline envelope (VE) outside of which is a hydrated jelly coat. The jelly coat can be removed by sieving the eggs through nytex netting or by exposure for two minutes to pH 5.5 seawater followed by readjustment to pH 7.8. The egg VE remains intact, but the amount of egg jelly remaining bound to the VE is unknown (Figure 1). It is not known if the egg jelly is the inducer of the sperm acrosome reaction (AR) as it is in sea urchins (Vacquier and Moy, 1997). No studies have been done on the composition of abalone egg jelly. The “dejellied” abalone eggs fertilize efficiently. The VEs can be isolated in quantity by homogenization of eggs and differential sedimentation (Figure 2). VEs are 60% carbohydrate and 40% protein (Lewis et al., 1982). Transmission electron microscopy of thin sections of VEs (Lewis et al.,

Acrosornal Proteins of Abalone Spermatozoa

53

Figure 7. An unfertilized egg of the red abalone Hahotis rufescens. The dark egg is 1 75 pm in diameter. The vitelline envelope is 0.6 pm in thickness and 245 p m in diameter (from Lewis et al., 1982).

Figore2. Dark field microscopy of isolated H. rufescens vitelline envelopes (from Lewis et al., 1982).

1982; Usui and Haino-Fukushima, 1991) and freeze fracture deep-etch replicas of VEs (Mozingo et al., 1995) show VEs have a complex, fibrous ultrastructure. The VE is a tough, elastic envelope 0.6 pm in thickness that remains intact following homogenization of eggs (Lewis et al., 1982). After isolation, the VEs are stable for several years if stored at 4°C in EDTA, Tris and azide at pH 8 (Lewis et a]., 1982).

V.

THE ABALONE SPERMATOZOON

Almost all animal sperm, when near to, or when contacting the outermost egg investment, undergo the exocytosis of an acrosome vesicle (AV), which releases pro-

54

V.D. VACQUIER, W.J. SWANSON, E.C. METZ, and C.D. STOUT

teins involved in the species-specific steps of the fertilization cascade (Darszon et al., 1996; Allen and Green, 1997). The isolation of AVs from sea urchin sperm allowed the identification and characterization of bindin, the first sperm-egg adhesive protein to be isolated (Vacquier and Moy, 1977; Vacquier et al., 1995). From anterior to posterior, abalone spermatozoa are composed of an acrosome vesicle (AV), a nucleus with approximately two picograms of DNA (Hinegardner, 1974) a midpiece cluster of five mitochondria, and a 45-pm long flagellum (Lewis et al., 1980; Figure 3). The “head” of the sperm, composed of AV, nucleus and midpiece, is approximately 7 pm in length and 1 pm in diameter. The abalone AV is the largest AV known among marine invertebrates (in H . rufescens it is 2.5 x 1.1 pm). The AV is so large that it could never maintain its shape or integrity if only bounded by the plasma membrane and the outer AV membrane. The stability of the walls of the AV is provided by the construction of an acrosomal shell (or hull) under the outer AV membrane, which is a paracrystalline array of tightly packed, spiral, 13-nm diameter fibers (Lewis et al., 1982; Usui, 1987). This is a very unusual type of construction for a secretory vesicle. The AV shell is constructed under the composite of the plasma membrane-outer AV membrane of the differentiating spermatid and is then filled with two major proteins that comprise the major components of the AV. Spermatogenesis in abalone has never been studied; thus no further information exists about the AV shell. The AV is fixed to the anterior tip of the sperm nucleus by apreformed, 2-pm-long rod of actin filaments (Figure 3 ) ; the rod extends an additional

Figure 3. (Upper Panel). Scanning electron micrograph of an H. rufescens spermatozoon. The sperm head, from mitochondrion (M)to tip of the acrosome vesicle (granule; AV) is 7 pm. The width of the nucleus (N) is 1 pm. (Lower Panel).Transmission electron micrograph of the acrosomal vesicle showing it attached to the nucleus (NF) by the rod of actin filaments (AF). The darker material labelled 1 shows the location of the 18K protein and 2 shows the location of lysin (from Lewis et al., 1980).

55

Acrosomal Proteins of Abalone Spermatozoa

5 pm during the acrosome reaction (Lewis et al., 1980;Shiroya and Sakai, 1993). The two major proteins released from the AV during the acrosome reaction (AR), 16K lysin (hereafter termed “lysin”), and the 18K protein (hereafter termed “ 1SK’) are differentially distributed within the AV (Lewis et al., 1980; Figure 3 ) . Immunogold electron microscopic localization shows that lysin is located proximal to the nucleus and the 18K is distal with a clear separation between the two proteins (Figure 3 ; Haino-Fukushima and Usui, 1986).When the acrosomal actin rod extends from the anterior tip of the sperm, it becomes coated with the 18Kprotein (Swanson and Vacquier, 19951).

VI.

SPERM-EGG INTERACTION IN ABALONES

The steps in abalone sperm-egg interaction were deduced from light and electron microscopic studies (Figure 4). First, the sperm plasma membrane covering the tip

A

PERIVITELLINE SPACE

i b!$

2 ACROSOME REACTION

ACROSOMAL PROCESS

3 LYSlS OF V.E.

4. PASS THROUGH YE.

i ’

w*

Figure 4. Scheme showing sperm-egg interaction in the abalone. 1. The sperm binds to the egg VE by the plasma membrane at the tip of the AV (AC), (F, flagellum; M, mitochondrion; N, nucleus). 2. The sperm acrosome reacts releasing lysin and the 18K protein from its anterior tip. 3. Lysin disrupts the fibers of the VE and the 18K coats the extending acrosome process as it extends. 4. The sperm passes through the hole in the VE and the membrane covering the tip of the acrosomal process fuses with the egg (from Vacquier and Lee, 1993).

56

V.D. VACQUIER, W.J. SWANSON, E.C. METZ, and C.D. STOUT

of the AV adheres to the egg VE. Hundreds of acrosome-intact sperm can bind to the VE. Very few of the bound sperm undergo the acrosome reaction. Nothing is known regarding the induction of the abalone sperm AR by egg surface molecules. Second, exocytosis of the sperm AV occurs only at its anterior tip. Lysin and 18K are released and the actin rod (the acrosomal process) extends to a total length of 7 pm (Lewis et al., 1980;Shiroya and Sakai, 1993).The empty AV hull is visible by phase contrast microscopy (Lewis et al., 1982). Third, a 3 pm diameter hole forms in the VE , and fourth, the sperm passes through the hole as the acrosomal process continues to lengthen by actin polymerization. Fifth, the membrane covering the tip of the acrosomal process fuses with the egg plasma membrane activating the egg’s cytoskeletal mechanism that draws the sperm into the egg cytoplasm. Almost every sperm that undergoes the AR penetrates the egg VE. For this reason, abalone eggs have evolved a potent, long duration electrical block to polyspemic fusion (Stephano, 1992). When the edge of the hole created by the sperm is viewed by electron microscopy (Figure 5), it appears as if the fibrous structure of the VE has been unravelled by the sperm proteins; the 13-nm fibers comprising the VE appear to be splayed apart forming the hole. The electron-dense, osmiophilic, oval structures in the VE appear as if they are composed of more densely packed fibrous material than is the VE matrix surrounding the dense ovoids. No visible changes occur to the egg VE after fertilization. There is no evidence of a cortical granule reaction in abalone eggs, nor are there visible changes to the

Figure 5. Thin section transmission electron micrograph of the hole in the VE made by lysin. A longitudinal grazing section through the AV hull shows its paracrystalline repetitive structure with 1 3 nm diameter spacing. The semi-intact VE is at bottom right. The fibers of the VE appear to be unraveled and splayed apart by lysin. (bar = 1 pm; from Lewis et al., 1982).

Acrosomal Proteins of Abalone Spermatozoa

57

egg VE. At the two- and four- cell stage, the VE surrounding the embryo will still bind fresh sperm, and the sperm will acrosome react and penetrate the VE (Vacquier, unpublished). Thus, the abalone egg VE appears quite different from other animal egg investments that become refractory to sperm binding and penetration after the activation of the egg. The abalone sperm AR can be artificially induced by raising the calcium ion concentration of seawater from the normal 1OmM to 50mM in seawater buffered with 10 mM Tris at pH 8.2. Unlike other species used for fertilization studies, the abalone AR does not lead to the rapid death of the sperm. In abalone sperm, the acrosomal compartment is sealed off from the respiratory compartment; acrosome-reacted, sperm will continue to swim for days if stored in the cold room at 4°C. The acrosomal exudate of these sperm is composed predominantly of soluble lysin and 18K protein. Reducing and denaturing polyacrylamide gel electrophoresis of whole sperm, AV exudate, and purified lysin shows that abalone spermatocytes make a substantial investment in the synthesis of these two acrosomal proteins (Figure 6).

Figure 6. Polyacrylamide gel electrophoresis of abalone sperm and AV contents. Lanes A and E are standard proteins of known molecular mass. Lane B, whole sperm dissolved in SDS. Lane C, the acrosome vesicle content released to seawater when exocytosis of the sperm is induced by high calcium ion concentration. Lane D, purified 76-kDa lysin. (from Lewis et al., 1982).

58

V.D. VACQUIER, W.J. SWANSON, E.C. METZ, and C.D. STOUT

VII. THE SPECIES-SPECIFICITYOF LYSlN Lysin and 18K proteins can be purified in hundreds of milligrams by CM-cellulose chromatography (Vacquier and Lee, 1993; Swanson and Vacquier, 1995a; 1995b). Both isolated proteins are stable for extended periods if stored in seawater at 4°C or frozen at -70°C. Experiments show that lysin is the protein responsible for creating the hole in the VE by a nonenzymatic mechanism. Purified 18K does not enhance lysin's ability to dissolve VEs (Swanson and Vacquier, 1995b).The nonenzymatic nature of lysin's action on VEs was shown by the tight binding of lysin to the VE, the facts that no new N-termini are created and that no VE proteins are degraded to smaller sizes, and the kinetics of the VE dissolution reaction with purified lysin and isolated egg VEs (Haino-Fukushuna, 1974;Lewis et al., 1982;Vacquier et al., 1990; Vacquier and Lee, 1993; Swanson and Vacquier, 1997). Two simple assays were developed to quantitate the species-specificityof VE dissolution by lysin. In one assay, therelease to the supernatant of '251-labelledVE molecules from isolated VEs as a function of lysin concentration was measured (Vacquier et al., 1990).In a second assay, the decrease in turbidity of a suspensionof isolated VEs is measured as a function of lysin concentration (Figure 7; Vacquier and Lee, 1993; Swanson and Vacquier, 1997). Both assays show that lysin from the red abalone (Haliotis rufescens) is poor at dissolving VEs of the pink species (H. corrugata). However, at high enough concentrations, both pink and black lysins will dissolve red VEs (Figure 7). At 12 pg red lysin, red VEs are completely dissolved, whereas 27 pg of pink lysin, or 42 pg of black abalone lysin, are needed to completely dissolvered VEs. Black abaloneVEs are dissolved equally well by black and pink species lysins, but red lysin is poor at dissolving black VEs. Pink abalone egg VEs are dissolved almost equally well with pink and black lysins, but almost no dissolution occurs by red lysin. However, at 70 pg, red lysin will completely dissolve pink VEs (Swanson

Figure 7. Species-specific dissolution of isolated VEs by purified lysins as determined by the light scattering assay. Vertical axis, percent VE dissolved; horizontal axis, pg lysin ,, VEs from the red abalone, ti.rufescens. B ,, VEs from the black abalone, H. added. ,R cracherodii. ,P ,, VEsfrorn the pinkabalone, H. corrugata. ( 0 )red lysin; (A) pink lysin; and (m) black lysin (from Vacquier and Lee, 1993).

Acrosomal Proteins of Abalone Spermatozoa

59

and Vacquier, 1997).These data show that the molecular recognition between purified lysin and isolated egg VE occurs in vitro and can be quantitatively assayed.

VIII.

FUNCTION OF THE 18K PROTEIN

The function in fertilization of the 18K protein remains uncertain. Although it appears to play no role in VE dissolution in our experiments with the California species, similar studies with Japanese species suggest that it enhances the activity of lysin to dissolve VEs (Usui and Haino-Fukushima, 1991). Our experiments suggest that one role for 18K may be as a mediator of gamete membrane fusion. Before the AR, 60%of the AV protein content is lysin and 40% is 18K. In acrosome-reacted sperm, the amounts of the two proteins remaining associated with the sperm are 38% lysin and 62% 18K.%s apparent enrichment for the 18K protein relative to lysin after the AR indicates that relatively more 18K remains associated with the reacted sperm than does lysin (Swanson and Vacquier, 1995a). Indirect immunofluorescence shows that the 18K protein coats the surface of the membrane covering the acrosomal process of the acrosome-reacted sperm. The position of the 18Krelative to lysin in the intact AV, would favor the coating of the acrosomal process as it lengthened by actin polymerization (Figures 3 and 4). Purified 18K protein will aggregate liposomes composed of PS:PC:PE (1: l:l), but liposomes made of PC are not aggregated. The 18K protein also induces the release of dye from liposomes. Fluorescence-based assays showed that both lysin and 18K are capable of fusing negatively charged liposomes; however, the 18K is a much more potent fusagen than is lysin (Figure 8). Analysis for secondary structure 2.5

10

SLQ

5

MIN

5

w

5

Figure 8. The fusion of artificial phospholipid vesicles induced by 18K protein (a) and lysin (b) at the three concentrations indicated above (c, buffer alone). The upper panels are with sperm proteins from the red abalone (Hr; H. refescens); the lower panels are with sperm proteins from the green abalone (Hf; H. fulgens). The 18K proteins are more potent fusagens than lysin. Although the two 18K proteins are only 33.8% identical in primary structure, their five predicted amphipathic helices have similar hydrophobic moments (from Swanson and Vacquier, 1995a).

60

V.D. VACQUIER, W.J. SWANSON, E.C. METZ, and C.D. STOUT

of the 18Kprimary structure suggested it contains five strongly amphipathic a-helices with hydrophobic moments equal to, and even greater than, mellitin, one of the most potent fusagenic peptides known (Swanson and Vacquier, 1995a). The 18K coats the sperm membrane that fuses with the egg membrane. This location and its high liposome-fusing potential are indirect evidence that 18K may be involved in membrane fusion between the gametes. Other sperm proteins such as sea urchin sperm bindin (Glabe et al., 1990)and guinea pig fertilin-p (Wolfsberg and White, 1996) are also fusagenic. The lack of an in vitro assay for species-specific membrane fusion precluded testing the species-specificity of 18K.

IX.

OBTAINING SEQUENCES OF LYSIN AND 18K PROTEINS A.

Lysin

As a first step in attempting to determine the mechanism of the speciesspecificity of lysin’s ability to dissolve VEs, we cloned the cDNA for lysin from the pink and red species. The N-terminal sequences of the two mature lysin proteins had been previously determined (Fridberger et al., 1985). Being highly represented as protein in mature sperm (Figure 6), it was not surprising to find the lysin (and 18K) mRNAs to be abundant in testis polyA+ RNA. The lysin open reading frames are 465 bp for the pink and 462 bp for the red and show a 13% difference. The 3’untranslated regions are 170 bp for the pink and 165 bp for the red and show 7% difference. The 18-amino-acid signal sequences differ by two amino acids. Mature pink lysin is 137 amino acids and red lysin is 136; the two proteins are 78% identical indicating considerable divergence has occurred since the speciation event. The most variable region between the two lysins is in the N-terminal segment of positions 2 through 15 in which 11 residues vary between species. The surprising findings of this first study were that the variation at the nucleotide level in the open reading frame is twice that of the 3’ untranslated region, the two proteins are 22% different in amino acid sequence, and the N-terminal is hypervariable, which could account for the observed species-specificity in dissolving VEs (Vacquier et a]., 1990). This first study encouraged us to continue to obtain additional lysin sequences. We found that ethanol fixation (50%-90%) of fragments of abalone testis preserved the lysin mRNA, making possible procurement of testis samples from approximately 30 species world wide. Following the synthesis of “universal” lysin primers in the 3‘and 5’ untranslated regions of lysin cDNA, it was a simple process to obtain lysin sequences from an additional 25 species using RT PCR (Lee et al., 1995; Lee and Vacquier, 1995).

B.

The 18K Protein

We purified the 18K protein by CM cellulose ion exchange chromatography and obtained the sequence of the first 41 residues by gas phase protein sequencing.

61

Acrosomal Proteins of Abalone Spermatozoa LYSIN

-1 tl

-18

*.*

tt

ffff..

10

I

t

f

H.rufescens MKLLVLCIFMlMATLWS H.sOrenseni H.kamtSchatkana F........ H.corrugat.3 L V. W V... H.fuIqens

.................. .- .... ... ...... .-.. .. ....... ........ ...... ...... .-

...*

70

60

H. rufeicenr

80

I * .*** I *

.Iff*

.*

t

..

20

*

30

.... ....

40

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50

.I

.*

*

R-SWRYVEPKnNIULPEVALI[VOIIAGIDRGLVI(WtRVBGRTLS~QK~Y~~ R P................ T T..G...H.............. T O.............E E. .VRH R.A.....N.GP..R....... .HRFRFIPA.YIR.E E. T.........GR................. R.TF.R~YI...Y..~.I S....Q.TA R.T"...T.F

...... .. ......

................

90

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100 (It

.I

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....... .......

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110

120

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130

* .*

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~ ~ ~ N I [ X I D A L G R T P W G D Y T R L W L E I G R R I D U A Y ~ ~ ~ K ~ I 136 P K ~ P ~ E I ~ ~ ~ ~ G K li.rorensenl T..... ~.kdmtschdtkand T ..... H.CDrriiqata a ......VR .T H. fulqens .Q.. .VKR.. X.

.............. ..............

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18kDa

..

.....

...

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-1 +1 10 20 30 40 50 . I I * I I I I I f MRSLVLLCVLI---UAI~ ~ X - - - - - - K S ~ S K E N ~ ~ ~ I ~ F L D S R ~ ~ R - I E K I G X P ~ T P ~ - ~ ~

-17

.

H rufescens ~.sorenseni

H.assimiIis H.corruqata ~.fuiyens

H. r u feIcenS H.s~re"seni H.assirnllis H.corruqata ~.f~igens

f f

...........----......

..F.L .....MGAVSQ. V. -.......... I----AVG.V. 60

70

*

* I

...........I.......A.AGI(....-V........ TS .....i.... ...n .nxlr

.. ........

T

W Q... X EI-...........n........... IB R.RPMrWF.IV.KEK.K....IGR(BY..AXL(m..RBWLV~K~EIIR.T.AII(..IK FD------ DW ROEQSYYORG .vH... E ~ L V . -FRDPIRWNLGPGWVPL.IUV....~.R .

..

80

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90

*' I

100

I

110

I

120

I

130

*.

W C H N Y Y ~ V S K K I I L L G G W L N K ~ F ~ G R I I G H K H Q W I L K ~ Q ~ ~ ~ - - - -132 ~~~~AI~IV~~LP~ 132 T F...... I T V......E.--ST F.RS FLAL A L..S..AVRQ...G.----...S..ATST....R............ 132 V..G.TIW.ORt.H.KTRP..E.Y.KKV..YLh.R.-~IVF.fl.IG YL-- ~~K.SEImPHP.LLDT.DE PVRXIEG 1 4 6 Y.nn .~Y.... ~a.xnLPv TLTK...RI. YR.-YGVI.ELYADVFI(D.QGF.GP.MT.~.YSS~~PGTB..I[IIB~ 1 4 1

... ...

.......... ............ .............. ......... ...

........ ................. ...

Figure 9. The alignments of lysin and the 18K proteins from five abalone species. Dots denote identity to the top sequence and dashes are inserted for alignment. Asterisks denote positions of perfect identity. In lysin, -1 8 to -1 is the signal sequence; in 18K it is -1 7 to -1. The lengths of the mature proteins are given at the C-terminal ends. H. assimilis (threaded abalone) is closely related to H. kamtschatkana (pinto abalone). H. sorenseni is the white abalone (from Vacquier et al., 1997).

Primers were made to obtain the full length sequences of five species by PCR (Swanson and Vacquier, 1995b). Instead of presenting an alignment of all 27 lysin sequences and the five 18K sequences, we will present the sequences of both proteins from five species of California abalone (Figure 9; Vacquier el a]., 1997). The sequences of both acrosomal proteins are known for four species. Haliotis kamtschatkana (known for lysin) and H. assimilis (known for 18K) are considered to be comparable, closely related species.

X.

ANALYSIS OF LYSIN A N D 18K SEQUENCES A.

Lysin Sequences

The signal sequences of 18 residues (- 18 to - 1) are highly conserved among lysins and are typical of eukaryotes (Figure 9). The N-terminal Arg (position + I ) is known from gas phase sequencing of isolated lysins from red and pink sequences (Fridberger et al., 1985; Lee and Vacquier, 1992). The five mature lysins are 136 to 137 residues in length, the pink species having the insertion of a His residue between positions 1 and 2 (among 27 species, the length variation is 126-138 residues; Lee and

62

V.D. VACQUIER, W.J. SWANSON, E.C. METZ, and C.D. STOUT

Vacquier 1995;Lee et al., 1995). Of the 136 aligned positions, 64 (47%) are variable and 72 (53%) fixed. The two longest regions of identical sequence are runs of 9 and 11 contiguous residues in the central regions of the sequences. The most variable region of lysin is the N-terminal domain composed of residues 2 to 12 in which all 27 species have unique sequences. Although different in sequence, the amino acid compositions of all abalone lysins, and hence their molecular weights (16K), are quite similar. All lysins have a high net positive charge with PI values of 10 to 11.There are no Cys residues in any of the 27 mature lysin sequences and no evidence of glycosylation. The most surprising aspect of the lysin sequence alignment is the high diversification of primary structure for this single copy gene (Lee, 1994). For example, in the 10 pairwise comparisons of these five mature lysins (Table l), black and green species are 65% identical, meaning that 48 amino acid positions out of 136 are replaced between these two species. The majority of amino acid replacements are nonconservative for class of residue, but there is a tendency to preserve the size of the residue (Vacquier et al., 1997). The evolution of lysins is thus constrained in length, amino acid composition, charge, and size of residue, but unconstrained in placement of many residues. Table 7. Pairwise Comparisons of the Percent Identity of the Mature Acrosomal Proteins, Number of Synonymous (Ds), and Nonsynonymous (Dn) Substitutions Per 100 Sites Species % Comparison LYSlN Hr - Hs Hr-Hk Hr - Hc Hr - Hf HS- Hk HS- Hf H s - HC H k - Hc Hk - Hf Hc - Hf 18 kDa Hr - Hs Hr - Ha Hr - Hc Hr - Hf Hs - Ha Hs - Hf HS- HC Ha - Hc Ha - Hf Hc- Hf Notes:

Ds

Identity

90 a2 78 65

65

1.6t 2.8 t 10.6 t 21.3 t 3.3 2 21.1 t 9.9 t 9.8 t 22.7 t 16.0 t

1.3 1 .a 3.5 5.3 1.9 5.3 3.4 3.4 5.5 4.4

87 75 31 34 77 35 33 31 35 27

1.8 t 4.9 t 87.0 t 78.2 t 3.1 t 83.5 t 78.0 t 92.2 t 84.0 t 174.2 t

1.7 2.5 17.5 15.4 1.9 16.8 15.5 I 8.6 16.6 23.8

85 64 a0 77 65

Significant at the p

i'0.025,

Dn : Ds

Dn

**0.005 level.

5.8 t 10.7 t 14.6 t 24.9 t 8.4 t 25.4 rt 13.7 rt 18.1 2 24.5 2 24.0 5

8.1 t 18.5 t 82.6 t 81.1 t 14.5 t 76.9 175.6 t 82.3 t 85.5 t 86.9 t

1.4 1.9 2.3 3.2 1.7 3.3

2.2 2.6 3.2 3.2

1.7 2.7 8.6 8.5 2.3 8.0 7.8 8.8 9.0 8.9

3.63* 3.82'* 1.38 1 ,I 7 2.55* 1.20 I .3a 1.85*

I .oa 1.50

4.50** 3.78 0.95 1.04 4.67** 0.92 0.97 0.89 1.02 0.76

Acrosornal Proteins of Abalone Spermatozoa

63

B. 18K Sequences The sequences of 18K proteins from five species of California abalone were deduced from cDNA sequencing (Figure 9). The N-terminal sequences of the mature 18K proteins from the red and pink species were obtained by gas phase sequencing of isolated protein. The alignment shows that the signal sequence (- 17 to - 1) is identical in the first three species listed but is different in the next two species. The mature 18K protein of the first three species listed is 132 residues in length, the fourth is 146, and the fifth 141. There are two conserved Cys residues at positions 60 and 131. Aromatic residues are highly conserved in position among the five species. The isoelectric points of the five 18K sequences vary from 10.3 to 10.7. Considerable evolutionary divergence has occurred among the five mature sequences as shown by the percent identities of the 10 pairwise comparisons, which range from 27% to 87% (Table 1). Regardless of their extreme divergence, the five 18K sequences are homologs in that all pairwise alignments have Z scores of at least 18 standard deviation units above randomized alignments (Doolittle, 1987). Although they are only 38.8% identical in primary structure, the a-helical wheel diagrams of red and green species 18K proteins are essentially identical (Swanson and Vacquier, 1995a). There are no repetitive elements in either lysins or 18K proteins. In contrast to the high divergence among the mature 18K sequences, both the 3' and 5' untranslated regions are highly conserved (Swanson and Vacquier, 1995b). The 18K protein might be one of the most divergent proteins yet discovered in metazoa. Neighbor-joining trees of the two acrosomal proteins from the five species show the same topology; they also show that 18K is two to three times more divergent than lysin (Figure 10). H. rufescens (Red)

LYSIN

1

91

H. sorenseni (White)

H. kamtschtkana (Pinto)

I

H. conugata (Pink)

H. fuupns (Green)

I

0.1

18 kDa

I

99

H. rufescens (Red) H. sorenseni (White) H. assirnilis (Threaded)

H. corrugata (Pink) H. fuigens (Green)

0.1

-1

Figure 10. Neighbor-joining trees of lysin and 18K proteins from the five species. The scale bar shows amino acid p-distances. The topology of both trees is the same, however 18K i s two to three times more divergent than lysin (from Vacquier et al., 1997).

V.D. VACQUIER, W.J. SWANSON, E.C. METZ, and C.D. STOUT

64

XI.

CRYSTAL STRUCTURE OF LYSlN FROM T H E RED ABALONE

Diffraction quality crystals of lysin from the red abalone (H. rufescens) were grown under several conditions (Baginsky et al., 1990; Diller et al., 1994). The structures of both monomeric and dimeric lysins were solved. Monomeric red abalone lysin is 65% a-helical, has no j3 sheet and is a novel fold. There are five a-helices, with helices 1 through 4 forming the core of the molecule and having extensive interactions along their internal surfaces (Figure 1 1). The monomeric crystal structure reveals lysin’s three major structural features (Shaw et al., 1993; 1994). First, the species unique N-terminal segment of positions 1 to 12 projects out away from the helical bundle where it is free to interact with other molecules. The moderately variable Cterminal domain and the hypervariable N-terminal domains lie on the same side of the monomer. Second, one side of the monomer has two roughly parallel tracks of basic residues extending the length of the molecule. Red abalone lysin has 12 Arg and 13 Lys residues (Arg’ and Lys’” are not resolved in the crystal structure). One N

Q Figure 7 7.

The crystal structure of the red abalone lysin monomer. The a-carbon trace shows the five a-helices numbered a-1 to a-5 and the two basic tracks of Arg and Lys residues. The left basic track contains nine residues and the right track 14 residues (Arg’ and L ~ s are ’ ~ not ~ visible in the crystal structure). The two termini are labeled N and C. The N-terminal segment of residues 1 to 1 2 extends away from the helical bundle and the hypervariable N-and C- termini are in proximity (from Shaw et al., 1993). In the Arg and Lys side chains, carbon atoms are white and nitrogen atoms dark gray.

Acrosornal Proteins of Abalone Spermatozoa

65

basic track contains nine residues (Arg7*,Lys7' Lys7*,ArgR7,Arg", Arg", Arg", Lys", and ArgTh)and the other track 14 residues (Lys', Lys", LYS''~,Lys'", Arg12', Arg", Arg", Argl", Lys4*,Lys"', Lys"@,Lyslo*L Y S ~and ~ ,Arg4'). The basic tracks are each 45 long and, when viewed from the side, make this surface of lysin essentially flat. Third, on the side opposite the basic tracks, there is a patch of 1 1 solventexposed hydrophobic residues comprising 10%of the surface area of the monomer (Figure 12; positions Tyr", Tyrbs,Leuh7,Trpb8,IleY2,He", Me?, Ty?, Phel'l', Phel', and Met""). The finding of the exposed hydrophobic patch explained the earlier observation that lysin bound tenaciously to paraffin (Lewis et al., 1982). In crystals of the monomer, the hydrophobic patch is occluded by crystal-packing contacts. Being highly positively charged on one side and hydrophobic on the other, lysin is a highly amphipathic protein. The amphipathic character of the a-helices of lysin (and also 18K) is the structural reason for the ability of these proteins to fuse negatively charged liposomes (Figure 8). Lysin appears to be a surface-active protein, lacking a binding cleft or pocket characteristic of enzymes and lectins (Shaw et al., 1993; 1994).

A

Figure 72. Placement of the 11 residuesof the hydrophobic patch on the surface of the lysin monomer. The patch represents 10% of lysin's surface area (Shaw et al., 1993). The single letter code for amino acids is used. in the hydrophobic patch side chains, carbon atoms are black and nitrogen, oxygen,and sulfur atoms are dark gray, gray, and light gray, respectively.

66

V.D. VACQUIER, W.J. SWANSON, E.C. METZ, and C.D. STOUT

Conservation of the placement of aromatic residues in lysins suggests that all lysins have a similar fold. The thermodynamically unfavorable presence of the solvent-exposed hydrophobic patch suggested that lysin might exist as a dimer in seawater. A chemical crosslinker was used to show this is indeed the case; above 8 pM, essentially all lysin is present as dimers (Shaw et al., 1995).The dimers can be dissociated by Triton X- 100 and other detergents. Fluorescence energy transfer experiments showed the K, for dimer formation is approximately 1 pM.This low affinity constant is not surprising considering that the strong electrostatic repulsive forces of the four basic tracks of a lysin dimer would tend to dissociate dimers, while the strong hydrophobic affinity of the two hydrophobic patches would try to associate them. The half time of monomer exchange between dimers is approximately eight minutes and follows a single exponential curve. The crystal structure of the lysin dimer resembles an extended S shape when viewed along its local twofold axis, which relates the helical bundles of the two monomers (Figure 13A; Shaw et al., 1995).When viewed from the side, the dimer is essentially flat on one side and slightly convex on the other (Figure 13B). A polar view of the dimer emphasizes the asymmetric clustering of the hydrophobic patches of the two monomers (Figure 13C).The four termini of the two monomers

A

Figure 13 continued

B

C

Figure 13. Three views of the crystal structure of the lysin dimer. The hydrophobic patch and basic track residues are depicted as in Figures 11 and 12. Arg', Lys', and Lys13" are not visible in the structure of the dimer. (A) View along the local twofold axis relating monomers of the dimer. Residues of the hydrophobic patch interdigitate at the dimer interface while residues of the basic tracks are on opposite surfaces of the dimer. (B) The dimer viewed from the side, showing that one side of the dimer is essentially flat and the opposite side slightly convex. (C) The polar view of the dimer showing the hydrophobic patch residues are clustered to one side of the dimer and all four termini in proximity on the same face of the dimer are shown in the central region at the bottom of the structure (from Shaw et al., 1995).

67

68

V.D. VACQUIER, W.J. SWANSON, E.C. METZ, and C.D. STOUT

lie on the same face of the dimer. The most important feature of the interaction of the two hydrophobic patches is the interdigitated stack of aromatic residues of His", Tyr57,Phe'"', Phe""', Phe104,Pheiol',T Y ~ and ~ ~ Hish1' ' , (where prime indicates the other monomer). There are only two hydrogen bonds in the dimer, one between ArgShand IleiIi' and the other between Aspg7and L ~ S ' ~Of ' ~ 20 ' . contacts at the dimer interface, 14 are hydrophobic or stacking interactions, two are hydrogen bonds, and four are nonspecific polar side-chain interactions. Approximately 60% of the hydrophobic patch is occluded in the dimer leaving 40% still exposed to solvent. The four basic tracks of the two monomers project their side chains onto opposite surfaces of the dimer (Figure 13). This arrangement gives each dimer a net positive charge at pH 7 of +26 and suggests that the recognition of a VE ligand by lysin involves the basic track residues. Addition of isolated egg VEs to a cuvette containing fluorescent lysin dimers resulted in the immediate dissociation of the dimer into monomers (Figure 14). Although this experiment suggests that the monomer is the active species, the experiment could not distinguish whether dissociation of the dimer is merely a result of its interaction with its VE ligand or whether dissociation is a mandatory requirement for lysin's action to dissolve VEs. Thus, the reason for the existence of lysin dimers is not immediately apparent. The dimer could be important in lysin's proper recognition of the VE or merely needed for the correct packaging of the protein at high concentration within the acrosome vesicle (Shaw et al., 1995).

0

50

100

TIME

150

200

(secl

Figure 14. Kinetics of lysin dimer dissociation when isolated VEsare added to a cuvette with fluorescently labeled lysin. Vertical axis, relative FlTC fluorescence; horizontal axis, seconds after VE addition. The increased fluorescence emission (release of quenching) indicates dissociation of the dimer. The immediate increase in signal could not be captured. Jaggedtrace is the actual measurements and the smooth trace the fit of the data to a single exponential equation showing at,,> of approximately 30 seconds (from Shaw et al., 1995).

Acrosornal Proteins of Abalone Spermatozoa

XII.

69

POSSIBLE MECHANISM OF LYSIN’S ACTION

The fact that lysin is a nonenzymatic, surface-active protein, and knowing its sequence variability among abalone species and also the crystal structures of both monomeric and dimeric lysin of one species, allows us to propose a two-step model to explain lysin’s action at dissolving the VE. The first step would be the speciesspecific molecular recognition between lysin and the VE that would bring lysin close to its VE ligand creating the “proper fit”. This step would be mediated by the surface-exposed, species-variable residues located throughout the molecule. This species-specificity appears to be most important for the initiation of the dissolution reaction and less important after VE dissolution is underway (Figure 7). The species-specificity can be bypassed by the addition of large quantities of heterospecific lysin. After proper molecular recognition is made by lysin’s species-variable domains, highly conserved domains would mediate the second step, which is the general mechanism by which all lysins cause the fibers of the VE to nonenzymatically lose cohesion to each other and splay apart (Figure 5). A.

Variable Features of Lysins

The N-terminal domain of residues 2 through 12 is always species-unique and hypervariable between species. The C-terminal is moderately species-specific. Other regions in lysin, for example between positions 20 to 45 and 100 to 110,could also be involved in mediating species-specific recognition of the VE (Figure 9). These variable residues are distributed over the entire surface of the molecule (Shaw et al., 1994). The VE receptor (ligand) for lysin and all the other VE proteins is heavily glycosylated. It is possible that recognition of glycosidic chains could underlie lysin’s specificity. If so, one would predict that glycopeptide digests of egg VEs might inhibit lysin’s action. However, it is also possible that glycosylation of lysin’s cognate VE receptor is involved in dictating the proper folding of the ligand and that the glycoside chains themselves are not involved per se in the molecular recognition between lysin and the VE. Because lysin is relatively small, unglycosylated and contains no disulfide bonds, it is an excellent protein on which to perform site-directed mutagenesis to attempt to identify the residues critical for speciesspecific recognition. B.

Conserved Features of Lysins

The positions of the basic track residues are highly conserved in the sequences of lysins from 27 species of abalone (Figure 1 I ; Lee and Vacquier, 1995).In the seven species of California abalones (Lee and Vacquier, 1992; Vacquier and Lee, 1993), among the common 18 positions comprising the basic tracks, four positions are exclusively held by Lys and eight by only Arg. The conservation and surface exposure of these basic track residues (Figures 1 1 and 13) suggests they could be involved in

V.D. VACQUIER, W.J. SWANSON, E.C. METZ, and C.D. STOUT

70

displacement of hydrogen bonds among VE fibers. Evidence that VE integrity is maintained by hydrogen bonding among the fibers comes from several observations. Isolated VEs are not dissolved by high concentrations of nonionic detergents, small micelle detergents such as deoxycholate, dithiothreitol, EDTA, or high salt. Isolated VEs are dissolved between pH 5.0 and 4.5 with 50% dissolution at pH 4.7. The only amino acids having pKa values in this range are Glu and Asp. Urea dissolves VEs with 50% dissolution at 2.7 M. Heating dissolves VEs with 50% dissolution at 52°C. Inclusion of 0.4% Triton X-100 in any of these experiments does not shift the dissolution curve. Also, 30% isopropanol in seawater rapidly dissolves VEs; this could occur by competitive dissociation of VE hydrogen bonds by the small alcohol molecules (isolated VEs are devoid of lipid). The strong amphipathic nature of lysin monomer created by the basic tracks on one side and the hydrophobic patch on the other side could be key to lysin’s mechanism of action. The hydrophobic patch of 11 residues is highly conserved in lysin sequences (Figures 9 and 12). Monomeric lysin binds tenaciously to paraffin and can only be displaced by strong ionic detergents such as hot SDS. Large micelle nonionic detergents such as Triton X-100 decrease, but do not block, lysin’s activity to dissolve VEs. However, detergents forming small micelles, such as Zwittergent 3-10, octylglucoside, and sodium cholate totally block lysin’s activity to dissolve VEs. In these experiments, it is not known if the inhibiting detergent is acting on the lysin or on its VE ligand. The hydrophobic patch of lysin is the only part of the monomer that would bind detergent. One hypothesis to explain these results is that large micelle detergents such as Triton X-100 cannot block the hydrophobic patch from its proper interaction of the VE, whereas because of their small micelle size, detergents such as octylglucoside can bind the hydrophobic patch more effectively and block its interaction with its cognate recognition motif on lysin’s VE ligand. Lysin may use the two basic tracks to displace hydrogen bonds between the 13 nm diameter fibers comprising the VE bonds and may use the hydrophobic patch to severe the VE fibers. To summarize, variable structural features of lysins suggest ways to attack the elucidation of the molecular mechanism of species-specific sperm-egg recognition in abalones. The invariant structural features of lysins suggest ways to explore the molecular mechanism lysin uses to destroy nonenzymatically the integrity of the VE to allow the sperm to pass through this protective envelope and contact the egg cell membrane.

XIII.

LYSIN A N D THE 18K PROTEIN AROSE BY GENE DUPLICATION

The study of the evolution of fertilization proteins is just beginning. This is an important field of inquiry because the evolution of gamete recognition proteins must underlie development of prezygotic barriers to reproduction and hence be involved

Acrosornal Proteins of Abalone Spermatozoa

71

in the formation of new species. The study of the evolution of abalone sperm acrosoma1 proteins has illuminated the importance of this approach. Sequences of seven lysins of California abalones were the first to be deposited in GenBank; they matched no other proteins in the data base (Lee and Vacquier, 1992). When sequences of the five 18K proteins from the same species were used to search GenBank, no significant matches were found. A “profile” of the five 18K sequences was made and used to search GenBank (Gribskov et al., 1990); the only significant matches found were to the seven abalone lysins, suggesting that lysin and the 18K were related proteins. Next, prediction of lysin’s secondary structure was made and the positions of the predicted a-helices compared to the actual helices known from the crystal structure of the monomer; the match between predicted and actual was excellent. The prediction of secondary structure was then done for the red abalone 18K protein; the placement of the a-helices was in excellent agreement with that of lysin. The red 18K sequence was then threaded onto the crystal structure of lysin , showing that 18K could have the same fold as lysin (Swanson and Vacquier, 199%). Finally, genomic sequences of lysin and 18K from the red abalone were obtained. The sequences showed that both genes are composed of five exons and four introns. Most importantly, the positions of introns 1,2, and 4 are in identical positions in both genes and have the same phase within the interrupted codon (Figure 15). Repeated attempts to determine the position of the third intron of lysin in red, pink, and green abalone species failed. A third intron may be present because it

i

Exon 1 Exon 2 Lysin -RSWHYVEPKFLNKAPEVALKVQIIAGFDRGLVKW---LRVHGRTLSTVQ 18K DK------ KSTVSKENAAAMKVAMIKFLDSRTDRFKKRIEKIGYPITPPQ -

4

Exon 3

1

A2

?

KlCAZlYFVNRRYMQTHWANYMLWINKXIDALGRTPVVGDYTRLGAEI PTTLLYYNRERLMDWCHNYVVSKKII~GGNICLNKKNFARMGRII

4

Exon 5

Exon 4

GRRIDMAYFYDFL--KDKNMIPKYLPYMZEINRMRPADVPVKYMGK GWKNQWILKRRQYKASAIAKKIVAMKVADLPCN

4 1

Figure 75. Alignment of lysin and 18K proteins from red abalone. Both proteins have five exons (numbered) and four introns (positions shown by arrows). The position of intron three in lysin could not be determined and is shown by a question mark corresponding to the third 18K intron. For both proteins, the known introns are in the exact same positions and have the same phase in the interrupted codon (phase numbered above arrow head). These data indicate the two proteins arose by gene duplication.

72

V.D. VACQUIER, W.J. SWANSON, E.C. METZ, and C.D. STOUT

proved impossible to amplify from lysin exon 3 into exon 4 in any species using a variety of primers. The most likely explanation is that the third intron of lysin is too large to be amplified by standard PCR. The hypothetical position of the third intron of lysin is therefore labeled with a question mark corresponding to the position of the third 18Kintron. To summarize, the above dataindicate that lysin and 18K arose by gene duplication and subsequently diversified in primary structure while retaining many common structural features at the genomic and protein secondary structural levels. Assuming that the third lysin intron is at the position shown by the question mark (Figure 15),the exons of lysin correspond to domains revealed by the crystal structure of the monomer (Figure 16). Exon 1 encodes the N-terminal hypervariable domain of \

Exonl

\

Exon2

Figure 76. Mapping the lysin exons onto the crystal structure of the monomer shows that the separate exons code for the structural domains of the protein and supports the idea that lysin arose by exon shuffling. Exon 1 codes for the hypervariable N-terminus, exon 2 codes the first a-helix, exon 3 codes for the second a-helix, exon 4 codes for a-helices 3,4, and 5, and exon 5 codes for the moderately variable C-terminal segment (from Metz et al., unpublished).

73

Acrosomal Proteins of Abalone Spermatozoa

positions 1 to 15; exon 2 encodes the first a-helix of positions 16 to 43; exon 3 encodes the second a-helix of positions 44 through 76; exon 4 encodes the third, fourth, and fifth a-helices of positions 77 to 124; and exon 5 encodes the C-terminal moderately variable domain of positions 125 through 136. The length of exon 4 suggests that it has lost at least one intron. The introns between these structural units are large, which may enhance recombination. The encoding of separate a-helices by separate exons is a well known phenomenon of the relationship of gene structure to protein structure (De Souza et al., 1996) and supports the hypothesis that lysin (and 18K) arose by exon shuffling (Long et al., 1995). We hypothesize that before the gene duplication event, one acrosomal protein possessed the two activities of making a hole in the VE and mediating the fusion of the gametes. In extant abalone, the 18K no longer acts on the VE but has differentiated to be a potent fusagen of gamete plasma membranes. Lysin creates the hole in the VE but still retains the ability to fuse membranes, although it does so with less efficiency than 18K (Figure 8; Swanson and Vacquier, 1995a; 1995b).

XIV.

LYSIN AND 18K EVOLVE BY POSITIVE DARWINIAN SELECTION

When the lysin sequences of the first seven species were obtained, we were impressed at how much divergence had occurred between their primary structures (Figure 9; Table 1). We also discovered that amino acid replacement was mainly nonconservative regarding the class of residue replaced. Next, we made pairwise comparisons of the aligned cDNA sequences and scored the numbers of amino acid altering (nonsynonymous) and silent (synonymous) nucleotide changes in the 2 1 pairwise comparisons of the seven sequences. The data (Table 2) showed that the vast majority of codon differences between any two lysins are amino acid altering. For example, in the comparison of mature red and pinto abalone lysins of 136 codons, 25 of the codon differences are nonsynonymous and only one is silent (Lee and Vacquier, 1992). Computer programs were used to calculate the percentage of nonsynonymous nucleotide substitutions per nonsynonymous site and the percentage of Table 2.

Ratio of Amino Acid Altering to Silent Codon Differences in Pairwise Comparisons of Mature Lysins (136 Codons)

Species

Red

White Flat Pinto Pink Black Green

13:l 23:l 25:l 29:G 40:4 48:G

White

Flat

16:2 21 :2 26:5 38:6 49:9

33:3 30: 7 38:6 47:ll

Pinto

Pink

Black

27:s 46:8

48:11

37:l 47:3

51:9

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V.D. VACQUIER, W.J.SWANSON, E.C. METZ, and C.D. STOUT

synonymous substitutions per synonymous site in pairwise comparisons of the lysin sequences (Nei and Gojobori, 1986; Kumar et al., 1993). Usually, because most proteins are subjected to purifying selection (most mutations are deleterious), the synonymous rate is much greater than the nonsynonymous rate; for mammalian proteins, the ratio of nonsynonymous to synonymous substitution is about 0.2 (Li et al., 1985; Li and Graur, 1991). If selection is neutral, the expected ratio of nonsynonymous to synonymous is 1. However, there are at least 20 examples in GenBank in which the ratio of nonsynonymous (Dn) to synonymous (Ds) nucleotide substitution in pairwise comparisons of homologous proteins is much greater than 1. In these cases, there is adaptive value to the organism to diversify a protein sequence. This phenomenon is termed “positive Darwinian selection” and is usually found in proteins involved in cell surface recognition phenomena involved in disease. Many of the examples of positive selection involve interspecies interactions in which the two species are pitted against each other for survival. For example, selection on the porB gene of N . gonorrhoeae (Smith et al., 1995) favors diversity in the porB protein sequence so that the bacterium can escape the human immune response. The same survival strategy is applied in positive selection acting on surface antigens of Plasmodium. (Hughes and Hughes, 1995). Viral surface antigens also show positive selection, allowing mutant viruses to escape complexing with antibody (Fitch, 1996). There is recent evidence that transcription factors that function in reproduction also exhibit positive selection (Sutton and Wilkinson, 1997). Lysin and 18K are both subjected to strong positive selection (Table 1). For the 10 pairwise comparisons of the five lysin sequences, Dn:Ds ratios as high as 3.82 are observed (Lee and Vacquier, 1992; Vacquier and Lee, 1993). For pairwise comparisons of other abalone lysins, Dn:Ds ratios as high as 4.10 have been found (Lee et al., 1995). For the 18Kprotein,the highest Dn:Ds ratio is 4.67, probably the highest yet found in the analysis of a full-length mature protein (Table 1; Swanson and Vacquier, 1995b; Vacquier et al., 1997). These remarkable high ratios of Dn:Ds do not result from nucleotide nor codon usage bias (Lee et al., 1995; Swanson and Vacquier, 199%). The observation of such high Dn:Ds ratios in full length sequences of lysin and 18K means there is strong selective pressure driving the diversification of these two acrosomal proteins. We have no data on the nature of the strong selective force but believe it may be adaptive change to maintain proper interaction with the egg surface receptors for these two proteins. This is because both lysin and 18K function in fertilization in a time span of seconds and act only on egg surface molecules. For example, if rapid, continuous evolutionary change occurred in lysin’s receptor in the egg VE, lysin would have to change equally rapidly to remain compatible with its receptor. Thus, what we observe as positive selection in lysin and 18K is probably adaptive change to allow both proteins to interact with their cognate receptors on the egg surface, both of which play crucial roles in the fertilization cascade. We hypothesize that the egg surface receptors (ligands) for lysin and 18K are changing rapidly for an as yet unknown reason. The sperm proteins

Acrosomal Proteins of Abalone Spermatozoa

75

thus show adaptive change to match their egg surface receptors. In other words, the surface recognition molecules of the egg change first, and mutant sperm proteins are selected for which are a better “match” with the egg surface receptors. Thus, changes in the female gamete select for adaptive changes in the male gamete. As species diverge, species-specific steps in the process of gamete interaction may develop (the female component changes first and the male component follows). Lysin and 18K are two of the most statistically robust examples of positive selection yet discovered. Their adaptive evolution by positive selection may be to enhance the development of prezygotic barriers to fertilization between species.

XV.

IDENTIFICATION OF LYSIN’S RECEPTOR IN THE EGG VlTELLlNE ENVELOPE

The VE of pink and red abalone species could be isolated in large quantity (Figure 2). When dissolved by ever increasing amounts of lysin, no changes occur to the VE bands. The VE glycoproteins resolve as a collection of bands from 30 to 50 kDa and large material remaining at the top of the stacking gel. Our goal was to determine which component(s) was the VE receptor for lysin. Isolated VEs were dissolved in pH 4.0 seawater, readjusted to pH 7.8, and clarified by low speed centrifugation, and the supernatant passed over a column of immobilized lysin. After washing in seawater, bound material was eluted at pH 2.8 and immediately neutralized. The only material binding the lysin column (and not two control columns) was the large material that remains on top of the stacking gel (Swanson and Vacquier, 1997). Isolated VEs were dissolved, mixed with a small amount of ‘251-labelledlysin, and sedimented in a sucrose gradient in the ultracentrifuge; all labelled lysin migrated with the large molecular mass VE components remaining in the stacking gel. This large molecular mass VE material was a potent inhibitor of lysin-mediated VE dissolution. A gradient gel system was developed that allowed this material to enter the separating gel; it migrated between muscle titin (2,800K) and nebulin (770K) with a relative molecular mass of approximately 2,000 kDa (Figure 17). Different preparations ran as a family of 1 to 5 bands, one-dimensional peptide maps of the different bands produced the same pattern, showing they were different size forms of the same material. We named this material VERL for vitelline envelope receptor for lysin (Swanson and Vacquier, 1997). Quantitative densitometry of VEs showed that VERL made up about 30% of the protein staining material of VEs. Analysis showed VERL was 50% protein and 50% carbohydrate. Quantitative sugar analysis yielded 52% glucose, 20% mannose, 16%fucose, 7% galactosamine, and 5% glucosamine. Negatively stained images of VERL showed it to be a rigid, unbranched, 13-nm-diameter rod of varying lengths (630 k 170 nm). VERL‘s length and rigidity would account for the elastic nature of isolated VEs (Figure 17). Molecular sieving on Sephacryl-500 yielded VERL in the void volume, indicating a molecular mass of over 1,000kDa. Amino

V.D. VACQUIER, W.J.SWANSON, E.C. METZ, and C.D. STOUT

76

2,800 770

205

45

Figure 77. The vitelline envelope receptor for lysin (VERL)is a giant glycoprotein. (left panel), electrophoresis of VERL on 2.5% acrylamide gels (silver staining) shows it resolves as two sharp bands between titin (2,800K) and nebulin (770K). Lane 1, rabbit muscle extract myosin (205); lanes 2-6, different loads of pink abalone VERL resolved into two components. (Right panel), electron micrography of VERL molecules negatively stained with uranyl acetate. TheVERL fibersare 13 nm in diameter (from Swanson and Vacquier, 1997).

acid analysis shows isolated VEs are 29% Thr, however VERL is only 9.5% Thr, showing it to be distinct from the mass of the other VE components. VERL is rich in Glu and Asp, indirectly supporting the idea that these residues may interact with the basic tracts of lysin during VE dissolution. Fluorescence polarization was used to study lysin binding to isolated VERL in solution. Similar binding kinetics were obtained using isolated VERL or total solubilized VEs, supporting the idea that VERL is the only lysin-binding molecule in VEs. Sigmoidal lysin-VERL binding curves were obtained indicating positive cooperativity in lysin binding with a Hill coefficient of approximately 3. The fractional occupancy of lysin binding sites on VERL increased from 10% to 90% over 1.1 log units of VERL concentration. Species-specificity was also shown for soluble VERL binding to lysin, although specificity was not of the magnitude seen using intact VEs. For homospecific combinations, the effective concentration for half maximal binding (EC,,,)ranged from 9.54 to 9.96 nM. In this original work, we estimated there are an average of 134 lysins bound per VERL molecule. However, based on the size of the mRNA of 13.5 kB, we now believe this to be an overestimate by at least a factor of two (approximately 60 lysins bind each VERL). Regardless of the true maximum number of lysins that bind each VERL, the data suggest that VERL has a repetitive motif with high affinity for lysin. Several hypotheses could explain the large apparent size of VERL. Like muscle titin, VERL could be encoded by a single giant mRNA. VERL subunits could be linked together after their synthesis by a transglutaminase reaction. VERL's size

Acrosomal Proteins of Abalone Spermatozoa

77

might derive from the covalent linkage of several gene products. The importance of VERL is that we can now isolate both the sperm and egg components mediating species-specific fertilization in one animal species. The rapid, adaptive evolution of lysin by positive selection must be in response to changes occurring in VERL. Molecular cloning of VERL will be of importance to the study of evolution and structural basis of gamete interaction (Swanson and Vacquier, 1997).

WI. FUTURE DIRECTIONS We know the crystal structure of lysin from one species of abalone, H. rufescens. The evolutionary homology of lysin, its identical function among species, conservation in length, amino acid composition, and placement of aromatic residues lead to the prediction that all lysins have a similar fold. Lysin from the red and green abalone differ in the placement of 48 out of 136 residues. Being able to compare crystal structures of these two lysins would be of paramount importance in understanding the species-specificity of lysin’s action. One species, H. tuberculata,has a lysin that lacks the N-terminal segment of residues 1 through 12 (Lee et al., 1995). This species is one of the few examples of a singly occurring abalone species. Is this species singly occurring because the lack of the N-terminal segment precludes the evolution of species-specific fertilization? Or, has the N-terminal segment been lost because there is no selective pressure to maintain species-specific identity to prevent interspecies hybridization? It would be important to also know the crystallographic structure of this truncated lysin. If the crystal structures of the most variable lysins were known, it would be possible to map the other lysin sequences onto the composite crystal structure. Clearly, protein crystallography is an important future direction for this research. Lysin is ideal for site-directedmutagenesis since it is not glycosylated and has no Cys residues. There are two major questions to address. First, what is the lysin primary structural basis for species-specific recognition between lysin and the VE? Second, what is the structural basis for the general mechanism all lysins use to dissolve VEs? Would exchanging the N-termini between red and pink lysins yield a red lysin capable of dissolving pink VEs with the same kinetics as pink lysin (Figure 7)? In addition to the N-terminal segment of residues 1 through 12, there are 19 replacements between pink and red lysins (Figure 9). Nine of these differences involve a change in charge and could be as important to species recognition as is the N-terminus. Conversely, the N-terminus could play no role in species recognition or in the VE dissolution process. Selection on it may be totally relaxed, allowing it to express ail mutations. Making a mutant lysin missing the N-terminus might answer this question. We believe that lysin uses the Arg and Lys residues of the basic tracks to displace hydrogen bonds between VERL molecules in the VE. Mutating the basic tracks would be important to test this idea. Some of the basic track positions are held exclusively by either Arg or Lys. For example, among the California

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abalone (Lee andvacquier, 1992), positions 1,29,36,55,56,78,94,95,and 125 are exclusively held by Arg, and positions 14,20,48, 72, and 136 are exclusively held by Lys. What would happen to the crystal structure and activity if one or more of these residues was mutated to Ala or if Lys and Arg residues were switched? Above 8 pM lysin is a dimer, dimerization being mediated by residues of the hydrophobic patch. The dimer dissociates into monomers upon binding to the VE. Is monomerization crucial for lysin’s activity? Engineering a Cys residue into the hydrophobic patch might produce a disulfide bond between monomers, and this would prevent their monomerization. One advantage this study has is that the functional biological assay for lysin’s activity is precise, simple, and quick and uses small amounts of recombinant protein. Also, by mapping specific mutant lysins onto the known crystal structure, the role of novel structural features can be determined. VERL, the vitelline envelope receptor for lysin, has been isolated from pink and red abalone species and shown to bind approximately 60 lysins. Cloning and sequencing of VERL from different species will reveal its species-specific differences. Does VERL also evolve by positive selection? Is the glycosylation of VERL involved in the lysin recognition? Does lysin sever VERL filaments as actin filaments are severed by specific actin-binding proteins such as gelsolin (McLaughlin and Weeds, 1995). VERL is functionally constrained in that the oocyte must construct a protective, tough vitelline envelope around it using VERL molecules as a major component; yet the VE must also remain capable of being penetrated by sperm. It is important to determine the crystal structure of the 18Kprotein. Because 18K and lysin arose by gene duplication, the two proteins may have a similar crystal structure. The 18K has two conserved Cys residues (positions 60 and 131) not found in lysin. When mapped onto the lysin crystal structure, the two Cys residues are at an appropriate distance to form a disulfide bond. The 18K may have another function other than that of gamete membrane fusion. Its remarkable sequence divergence by positive selection suggests that, as is true with lysin, 18K is showing adaptive change to achanging cell surface receptor. It is possible that VERL and the 18K receptor also evolved by gene duplication. Freeze fracture deep etch replicas of the abalone egg surface show it to be covered by a coat of fibers to which the sperm bind (Mozingo et al., 1995). Did these fibers and the VERL fibers in the VE arise from a gene duplication as did lysin and the 18K? Duplication of a gene for an egg surface receptor may have provided the selective force underlying the duplication of the gene giving rise to lysin and 18K. Thus, we have two abundant gamete recognition proteins evolving by positive selection, which may be involved in the speciation process. Further studies of abalone fertilization protein genes in a phylogenetic context and in comparison with other abalone loci may clarify our understanding of the patterns, rates, and causes of the evolution of fertilization specificity. The significance of these studies to cell and developmental biology is that sperm lysin dissolving a hole in the egg VE is an example where one cell, nonenzymatically and specifically, destroys the extracellular matrix of another cell. Could such a

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process be more widespread in various cells and tissues, or does it only occur in acheogastropod fertilization? There are many examples in embryogenesis, regeneration, wound healing, and metastasis where single cells move through fields of other cells by disruption of the extracellular matrix and intercellular junctions. A nonenzymatic, amphipathic protein-based mechanism for loosening extracellular matrices to allow cells to pass into other compartments may be found in future studies of nongamete cell types.

ACKNOWLEDGMENTS This research was supported by NIH grant HD12986 to V.D.V., NSF grant MCB951342 to C.D.S. and a United States-MexicoFoundation for Science grant to V.D.V.

REFERENCES Allen, C.A. and Green, D.P. (1997). The mammalian acrosome reaction: gateway to sperm fusion with the oocyte. Bioessays 1 9 :241-247. Baba, T., Azuma, S . , Kashiwabara, S., and Toyoda, Y. (1994). Sperm from mice carrying a targeted mutation of the acrosin gene can penetrate the oocyte zona pellucida and effect fertilization. J. Biol. Chem. 269:31845-31849. Baginsky, M.L., Stout, C.D., and Vacquier, V.D. (1990). Diffraction quality crystals of lysin from spermatozoa of the red abalone (Hulioti.7 rufe!fescens).J. Biol. Chem. 265:4958-4962. Bookbinder, L.H., Cheng, A., and Bleil, J.D. (1995). Tissue and species-specific expression of sp56, a mouse sperm fertilization protein. Science 269:86-89. Darszon, A., Lievano, A,, and Beltran, C. (1996). Ion channels: Key elements in gamete signaling. Cum. Top. Dev. Biol. 34:117-167. De Souza, S.J., Long, M., Schoenbach, L., Roy, S.W. and Gilbert, W. (1996). Intron positions correlate with modular boundaries in ancient proteins, Proc. Natl. Acad. Sci. U.S.A. 93: 14632-14636. Diller, T.C., Shaw, A., Stura, E.A., Vacquier, V.D., and Stout, C.D. (1994). Acid pH crystallization of the basic protein lysin from the spermatozoa of red abalone (Huliotis rufescen.7). Acta Crystallog., Sect. D 92620.627. Doolittle, R.F. (1987). Of URFS and ORFS: A Primer on How to Analyse Derived Amino Acid Sequences. University Science Books, Mill Valley, CA. Epel, D. (1997). Activation of Sperm and Egg during Fertilization.In: Handbook of Physiology,Section 14: Cell Physiology. (Hoffman, J.F. and Jamieson, J.J., Eds.), pp. 859-884. Oxford Press, New York. Fedecka-Bruner, B., Anderson, M., and Epel, D. (1971). Control ofenzyme synthesis in early sea urchin development: Aryl sulfatase activity in normal and hybrid embryos. Dev. Biol. 25:655-671. Fitch, W.M. (1996). The variety of human virus evolution. Mol. Phylogen. Evol. 5:247-258. Fridberger, A,, Sundelin, J., Vacquier, V. D., and Peterson, P.A. (1985). Amino acid sequence of an egg-lysin protein from abalone spermatozoa that solubilizes the vitelline layer. J. Biol. Chem. 260:9092-9099. Giudice, G. (1973). Developmental Biology of the Sea Urchin. pp. 162-174. Academic Press, San Diego, CA. Glabe, C.G., Hong, K., and Vacquier, V.D. (1990). Fusion of the sperm and egg membrane during fertilization. In: Cellular Membrane Fusion. (Wilschut, J. and Hoekstra, D., Eds.), pp. 627-246. Marcel, Dekker Inc., New York.

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Gribskov, M., Luthy, R., and Eisenberg, D. (1990). Profile Analysis. Meth. Enzymol. 183146-149. Hahn, K. 0.(1989). Handbook of Culture of Abalone and Other Marine Gastropods. CRC Press, Boca Raton, FL. Haino-Fukushima, K. (1974). Studies on the egg membrane lysin of Tegulu pfe@rzr The reaction mechanism of the egg membrane lysin. Biochim. Biophys. Acta. 352:179-191. Haino-Fukushima, K. and Usui, N. (1986). Purification and immunocytochemical localization of the vitelline coat lysin of abalone spermatozoa. Dev. Biol. 115:27-34. Hinegardner, R. (1974). Cellular DNA content of the mollusca. Comp. Biochem. Physiol. 4 7:A447-A460. Hughes, M. K. and Hughes, A. L. (1995). Natural selection on Plasmodium surface proteins. Mol. Biochem. Parasitol. 71:93-113. Kumar, S., Tamura, K., and Nei, M. (1993). MEGA: Molecular evolutionary genetics analysis, version 1.01. The Pennsylvania State University, University Park, PA. Lee, Y.-H. (1994). Abalone Sperm Lysin. Ph.D. Thesis, University of California, San Diego, CA. Lee, Y.-H. and Vacquier, V.D. (1992). The divergence of species-specific abalone sperm lysins is promoted by positive Darwinian selection. Biol. Bull. 182:97-105. Lee, Y.-H. and Vacquier, V.D. (1995). Evolution and systematics in Haliotidae (Mollusca, Gastropoda): Inferences from DNA sequences of sperm lysin. Marine Biology 124267.279. Lee, Y.-H., Ota,T., and Vacquier, V.D. (1995). Positive selection is a general phenomenon in the evolution of abalone sperm lysin. Molecular Biology and Evolution 12:231-239. Lewis, C.A., Leighton, D.L., and Vacquier, V.D. (1980). Morphology of abalone spermatozoa before and after the acrosome reaction. J. Ultrastruct. Res. 72:39-47. Lewis, C.A., Talbot, C. F., and Vacquier, V.D. (1982). A protein from abalone spermdissolves theegg vitelline layer by a nonenzymatic mechanism. Dev. Biol. Y2:227-240. Li, W.-H., Wu, C.-I., and Luo, C.-C. (1985). A new method for estimating synonymous and nonsynonymous rates of nucleotide substitution considering the relative likelihood of nucleotide and codon changes. Mol. Biol. Evol. 2:150-174. Li, W.-H. and Graur, D. (1991). Fundamentals of Molecular Evolution. Sinauer Associates, Inc. Sunderland, Massachusetts. Lindberg, D.R. (1991). Evolution, distribution, and systematics of Haliotidae. In: Abalone ofthe World: Biology, Fisheries, and Culture. (Shepherd, S.A., Tegner, M.J., and Guzman Del Proo, S.A., Eds.), Chapter 1. Blackwells Scientific Publishers, London. Litscher, E.S. and Wassarman, P.M. (1996). Recombinant hamster sperm receptors that exhibit species-specific binding to sperm. Zygote 4:229-236. Liu, C., Litscher, E. S., and Wassarman, P.M. (1995). Transgenic mice with reduced numbers of functional sperm receptors on their eggs reproduce normally. Mol. Biol. Cell. 6:577-585. Long, M., Rosenberg, C., and Gilbert, W. (1995). Intron phase correlations and the evolution of the introdexon structures of genes. Proc. Natl. Acad. Sci. USA 92:12495-12499. Lu, Q. and Shur, B.D. (1997). Sperm from ~l,4-galactosyltransferase-null mice are refractory to ZP3 induced acrosomereactions and penetrate the zonapellucidapoorly.Development 124:4121-4131. McLaughlin, P.J. and Weed, A.G. (1995). Actin binding protein complexes at atomic resolution. Ann, Rev. Biophys. and Biomol. Struct. 24:643-675. Mozingo, N.M., Vacquier, V.D., and Chandler, D.E. (1995). Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization. Mol. Reprod. Dev. 41:493-503.

Nei, M. and Gojobori, T. (1986). Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3:418-426. O’Rand, M.G. (1988). Sperm-egg recognition and barriers to interspecies fertilization. Gamete Res. 1Y:315-328.

Shaw, A., McRee, D.E., Vacquier, V.D., and Stout, C.D. (1993). The crystal structure of lysin a fertilization protein. Science 262: 1864-1867.

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Shaw, A., Lee,Y.-H., Stout, C.D., and Vacquier, V.D. (1994). The species-specificity and structure of abalone sperm lysin. Sem. Dev. Biol. 5:209-216. Shaw, A., Fortes, P.G.A., Stout, C.D., and Vacquier, V.D. (1995). Crystal structure and subunit dynamics of the abalone sperm lysin dimer: Egg envelopes dissociate dimers. J. Cell Biol. 130: 11 17-1 126. Shiroya, Y. and Sakai, Y.T. (1993). Organization of actin filaments in the axial rod of abalone sperm revealed by quick freeze technique. Dev. Growth and Differ. 35:323-329. Smith, N.H., Maynard-Smith, J., and Spratt, B.G. (1995). Sequence evolution of the porB gene of Neissericr gonorrhoeae and N. rnerningifidis:Evidence of positive Darwinian selection. Mol. Biol. Evol. 12:363-370. Stephano, J. L. (1992). A study of polyspermy in abalone. In: Abalone ofthe World: Biology, Fisheries, and Culture. (Shepherd, S.A., Tegner, M.J., and Guzman Del Proo, S.A., Eds.) Chapter 39. Blackwells Scientific Publishers, London. Sutton, K. A. and Wilkinson, M.F. (1997). Rapid evolution of a homeodomain: Evidence for positive selection. J. Mol. Evol. 45:579-588. Swanson, W.J. and Vacquier, V.D. (1995a). Liposome fusion by a Mr 18,000protein localized to the acrosomal region of acrosome-reacted abalone spermatozoa. Biochemistry 34: 14202-14209. Swanson, W.J. and Vacquier, V.D. ( 1995b).Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa. Proc. Natl. Acad. Sci. USA. 92:4957-4962. Swanson, W.J. and Vacquier, V.D. (1997). The abalone egg vitelline envelope receptor for sperm lysin is a giant, multivalent molecule. Proc. Natl. Acad. Sci. USA. 94:6724-6730. Usui, N. (1987). Formation of the cylindrical structure during the acrosome reaction of abalone spermatozoa. Gamete Res. 16:37-45. Usui, N. and Haino-Fukushima, K. (1991). Two major acrosomal proteins act on different parts of the oocyte vitelline coat in the abalone Hufiotis discus. Mol. Reprod. and Dev. 28:189-198. Vacquier, V.D. and Lee, Y.-H. (1993). Abalone sperm lysin: Unusual mode of evolution of a gamete recognition protein. Zygote 1:181-196. Vacquier, V.D. and Moy, G.W. (1977). Isolation of bindin: The protein responsible for adhesion of sperm to sea urchin eggs. Proc. Natl. Acad. Sci. USA 74:2456-2460. Vacquier, V.D. and Moy, G.W. (1997). The fucose sulfate polymer of eggjelly binds to sperm REJ and is the inducer of the sea urchin sperm acrosome reaction. Dev. Biol. 192:125-135. Vacquier, V.D., Camer, K.R., and Stout, C.D. (1990). Species-specificsequences of abalone lysin, the sperm protein that creates a hole in the egg envelope. Proc. Natl. Acad. Sci. USA 87:5792-5796. Vacquier, V.D., Swanson, W.J., and Hellberg, M.E. (1995). What have we learned about sea urchin sperm bindin? Develop. Growth Differ. 37 1-10, Vacquier, V.D., Swanson, W.J., and Lee, Y.-H. (1997). Positive Darwinian selection on two homologous fertilization proteins: What is the selective pressure driving their divergence? J . Mol. Evol. 44 (Suppl. 1): S15-S22. Wolfsberg, T.G. and White, J.M. (1996). ADAMs in fertilization and development. Dev. Biol. lRO:389-40 1, Yanagimachi, R. (1988a). Mammalian Fertilization. In: The Physiology of Reproduction. (Knobil, E. and Neill, J., Eds.), pp. 135-185. Raven Press, New York. Yanagimachi, R. (1988b). Sperm-egg fusion. CUE. Top. Membr. Transport. 32:3-43.

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CAPACITATION OF THE MAMMALIAN SPERMATOZOON

Gregory S. Kopf, Pablo E. Visconti, and Hannah Galantino-Homer

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Definition and Assays of Capacitation . . . . . .

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A. In Vivo and In Vitro Capacitation. ................................. .85 B. Relationship of Capacitation to the Acrosome Reaction and Motility . 87 C. Assays for the Measurement of Capacitation . . . . . . . . . . . . . . . . . .88 . . . . . . . . . . . . . . . . . . 90 111. Molecular Basis of Capacitation . . . . . . . . . . . . . A. Role of the Media Constituents, Albumin, Calcium, and Bicarbonate . . . . . . . 90 B. Membrane Events . . . . . . . . . ................... C. Transmembrane and Intracellu ng . . . . . . . . . . . . . . . . . IV. Conclusions and Future Directions. .......................... Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References .......... ............................ 103

Advances in Developmental Biochemistry Volume 5, pages 83-107. Copyright 0 1999 by JAI Press Inc. All right of reproduction in any form reserved. ISBN: 0-7623-0202-X

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1.

INTRODUCTION

Unlike the sperm of many lower species, testicular mammalian sperm that have undergone spermatogenesis and spermiogenesis appear mature from a morphological standpoint but clearly are immature from a functional standpoint, that is, they have acquired neither progressive motility nor the ability to fertilize a metaphase IIarrested egg. The acquisition of progressive motility and fertilization competence are acquired in many mammals during transit through the epididymis, but complete fertilization capacity in vivo is conferred during residence in the female reproductive tract. The molecular and physiological events comprising this extratesticular maturational process that lead to the fertilization-competent state are referred to collectively as sperm capacitation. This term arose from independent experimental observations made by Austin (1951; 1952) and Chang (1951; 1955) that freshly ejaculated sperm do not have the ability to fertilize eggs but require a limited time of residence in the female tract in order for sperm to gain the “capacity” for fertilization. Subsequent to these seminal observations, capacitation in vitro has been demonstrated in a variety of mammalian species utilizing defined media with compositions that approximate the environment of the female reproductive tract. The ability to capacitate sperm in vitro has been of great importance to both scientists and clinicians, who have been able to capitalize on this property to study the basic biology of fertilization and to develop assisted reproductive technologies in a variety of species, including the human. Although capacitation was discovered nearly one-half century ago, relatively little is known about the molecular basis of this phenomenon. A complete understanding of the biochemical and biological underpinnings of capacitation will not only aid in our understanding of sperm biochemistry and fertilization, but will also provide a framework with which to develop new approaches to regulating fertility. This review is not meant to be exhaustive, as there are many excellent reviews dealing with this subject (Florman and Babcock, 1991; Storey and Kopf, 199 1; Yanagimachi, 1994;Cohen-Dayag and Eisenbach, 1994; Harrison, 1996; Cross, 1998; Florman et al., 1998). Rather, it is meant to focus on some of the recent developments in the field and to provide a discussion of future experimental approaches to understand this important sperm maturational event.

II. DEFINITION AND ASSAYS OF CAPACITATION From a historical perspective, capacitation has been defined as the time interval between sperm deposition in the female reproductive tract during natural mating and the time during which fertilization occurs. This time interval would therefore encompass all of the interactions of sperm with the female tract and vice versa (Smith, 1998; Suarez, 1998; Verhage et al., 1998).This definition was established following the observation that sperm taken from the female tract immediately fol-

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lowing mating did not have the ability to fertilize eggs and that residency of the sperm in the female tract in some way conferred fertilization capacity. Due, in part, to an increased understanding of sperm biology and to the establishment of more sophisticated assays of sperm function, the definition of capacitation has been narrowed and modified over the years to reflect many investigators’ biases regarding the physiological importance of this event. Although fertilization still represents the absolute confirmation that sperm have undergone capacitation, the ability of sperm to undergo a regulated acrosome reaction (e.g., in response to the zonu pellucidu [ZP]) can be taken as an earlier, upstream endpoint of this extratesticular maturational event. At this juncture, it must also be stressed that changes in sperm motility patterns are correlated with capacitation in a number of species; such motility changes are referred to collectively as sperm hyperactivation (Yanagimachi, 1994; Suarez, 1996). Although there are reports demonstrating the dissociation of capacitation and hyperactivation (Neil1 and Olds-Clarke, 1987), the complete independence of these two events has not been conclusively demonstrated (Suarez, 1996). With this in mind, experimental approaches to understand the molecular basis of capacitation must consider events that occur both in the head (i.e., acrosome reaction) and the tail (i.e., motility changes) of the sperm. A.

In Vivo and In Vitro Capacitation

As mentioned above, although capacitation in vivo must occur to insure SUCcessful fertilization, it can also be accomplished in vitro in defined media, in various biological fluids (e.g., heat-treated serum; oviductal fluid; follicular fluid; vitreous humor), and in a non species-specific manner (Yanagimachi, 1994). These observations suggest that specific aspects of the capacitation process can be initiated and controlled intrinsically by the sperm itself, and that certain minimal environmental requirements must be met; this will be discussed below. The intrinsic nature of the capacitation process is of great interest from a cell regulation standpoint and must be considered when designing and interpreting experiments directed at a molecular understanding of this maturational process. This by no means rules out the presence and physiologically relevant role of positive and/or negative modulators of capacitation present in the male and female reproductive tracts of various species. It is possible that the regulation of capacitation lies less in the stimulation of this process and more in the derepression of inhibitory modulators of capacitation through the removal of decapacitating factors (Hunter and Nornes, 1969; Yanagimachi, 1994). Such modulators might be very important, as they may function to extend the fertilizability of the sperm population in the ejaculate by exerting additional modulation over the aforementioned intrinsic regulatory aspects of capacitation. In fact, numerous studies have focused on the role of factors in the male reproductive tract that can prevent (Davis, 1978;Cross, 1996; Cross, 1998 and references therein) or promote (Therien et al.,

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1995,1997)capacitation. The oviduct or uterus represents the physiological sites of capacitation in vivo in many species (Yanagimachi, 1994). Consistent with this idea are the observations that factors in the female reproductive tract have been demonstrated to exert a positive effect on sperm capacitation, viability, and motility (Parrishet al., 1989; Ehrenwaldetal., 1990; Chian and Sirard, 1995; Verhage et al., 1998). It is postulated that these factors bind to the surface of the sperm and exert their modulatory functions on sperm in a manner that is, to date, poorly understood. A better understanding of their role in controlling sperm capacitation will undoubtedly be achieved once many of these factors are identified. Interactions of sperm with these factors in conjunction with interactions of the sperm with the cells lining different regions of the reproductive tract (Suarez, 1998; Smith, 1998) may constitute a concerted regulation of capacitation in vivo. Regulation of sperm function at this level may be extremely important in the selection of optimal subpopulations of sperm for the task of fertilization. In many species, the sperm composition of the ejaculate may be quite heterogeneous with respect to cellular age, morphology, motility characteristics, and ability to undergo capacitation (Bedford, 1983). Extrinsic regulation of such a heterogeneous sperm population may serve to select those subpopulations of sperm that ultimately could participate in the fertilization process, as well as to extend the fertilizable lifespan of the ejaculate by widening the window of capacitation in these subpopulations (Cohen-Dayag and Eisenbach, 1994), given the apparent stochastic nature of capacitation when assessed by in vitro assays. Such extrinsic modulation provides a higher order of regulation over the intrinsic properties of capacitation. Given the complex nature of interactions between the sperm and the reproductive tracts to regulate capacitation, it is of interest that capacitation can be accomplished in vitro in numerous species by incubating cauda and/or ejaculated sperm under a variety of conditions in defined media that mimic the electrolyte composition of the oviductal fluid. If one accepts the postulate of extrinsic, as well as intrinsic, control of capacitation, capacitation in vitro would result in a population of sperm fully competent to fertilize eggs but induced to undergo this maturational event in a different temporal fashion. Although it is easier to study capacitation in vitro and though the sperm capacitated under these conditions could be considered normal, one must always be careful in extending conclusions based on studies carried out in vitro to the in vivo situation. However, a molecular understanding of this poorly understood maturational event will have to come initially from studies of capacitation in vitro. As stated above, capacitation in vitro can be accomplished in media of defined composition. The composition of such media includes energy substrates such as pyruvate, lactate, and glucose (depending on the species), aprotein source (usually serum albumin), NaHCO,, and Ca2+.Recent experiments are starting to unravel the putative mechanism of action by which these media components promote capacitation at the molecular level, and this will be discussed below.

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Relationship of Capacitation to the Acrosome Reaction and Motility

Although the truest endpoint of capacitation is the ability of sperm to fertilize an egg and give rise to offspring, the number of variables that must be met to give rise to the “capacitated’state based on this rather broad definition is enormous. This difficulty must also be considered when trying to develop assays for the measurement of capacitation (see below). It has been necessary, therefore, to look for more narrow biological endpoints that can be taken as indices of capacitation. The identification of these endpoints could then be used as a starting point for the elucidation of the molecular events underlying this maturational process as well as for the development of assays to measure it. The first biological endpoint in the sequence leading to fertilization that was discerned as being part of the capacitation process was identified by Pavlok and McLaren (1972) as penetration of the zona pellucida (ZP). They concluded that capacitation was required for sperm penetration of the ZP but not for penetration of the egg itself. Subsequent to this report, Inoue and Wolf (1975; Wolf and Inoue, 1976) provided evidence that capacitation conferred on sperm the ability to establish binding to the ZP. Once it became apparent that the acrosome reaction was required for penetration of the ZP and that the ZP could indeed induce the acrosome reaction, two possible endpoints of capacitation could then be considered (i.e., the ability for sperm to bind to the ZP and/or the ability of the bound sperm to undergo an acrosome reaction in response to the ZP). Experiments by Storey and coworkers (Saling et al., 1978; Heffner and Storey, 1982) suggested that sperm binding to the ZP was not a component of capacitation. A consensus of opinion from experiments carried out in the mouse and in other species now supports the view that one endpoint of capacitation is the ability of the sperm to undergo an acrosome reaction in response to a physiologically relevant ligand such as the ZP. Recently, several groups have extended this definition by including acrosome reactions induced by progesterone, but for the most part this definition has been limited to studies with human sperm. Several investigators have noted that sperm, incubated under conditions supporting capacitation, exhibit a change in the flagellar beat pattern from cne of low amplitude favoring progressive motility to a high amplitude favoring a frenzied activity with little progressive motility (Yanagimachi, 1994). This change in motility has been referred to as sperm hyperactivation. This has now been observed in quite a few mammalian species, although the degree of these motility changes are different in the different species. To date, there are conflicting opinions as to whether hyperactivation of motility is an event that is a component of the capacitation process. It is clear that it is closely associated with capacitation but has been experimentally dissociated from this maturational event in a few instances, leading to the suggestion that it is closely associated but distinct from capacitation. For example, Olds-Clarke (1989) demonstrated that hyperactivation of mouse sperm carrying the t-haplotype occurs prior to capacitation in vitro. Other investigators have been able to dissociate these two events

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by modulating the ionic composition of the media, but conclusions based on this approach must be carefully considered given the fact that a variety of sperm functions are exquisitely sensitive to the ionic composition of the media. At this point, it is difficult to conclude whether any changes in motility are completely independent of the capacitation process. Since little is known about the regulation of sperm motility, and recent studies suggest that components of the flagellum may be undergoing posttranslational modifications during capacitation (see below), it is too early to say that changes in motility are completely independent of capacitation. C.

Assays for the Measurement of Capacitation

As stated above, since capacitation in its truest sense represents the ability of the sperm to fertilize an egg and fertilization is a multistep process, development of simple quantitative assays to measure this zxtratesticular maturational event have been difficult and must be carefully tested when introduced for use in a new experimental model. In fact, there are no direct assays of capacitation and all of the assays to evaluate this process are based on different definitions. These difficulties, no doubt, are due to our ignorance of the molecular underpinnings of this event, and knowledge gained in this area will be instrumental in the development of new and specific assays. A few representative assay approaches are listed below to provide an overview of the advantages and disadvantages of the more common assays presently in use. Chlortetracycline Fluorescence

The antibiotic chlortetracycline (CTC) yields different patterns of distribution on the sperm surface that can be visualized as distinct fluorescence patterns depending on the capacitation and acrosomal status of the sperm. These different patterns of CTC binding and their correlation with the capacitation status of the sperm were first described in the mouse (Saling and Storey, 1979; Ward and Storey, 1984). This probe has now been used to assess capacitation and the acrosome reaction in several other species including the human (Lee et al., 1987). It should be noted, however, that the distribution pattern in the sperm of other species is clearly different from those in the mouse (Lee et al., 1987)and, therefore, this assay must be carefully calibrated for each species. The advantage of this method is that it monitors the capacitation status of the sperm independently of the acrosome reaction. The disadvantage of this method is that the mechanism by which CTC yields the different patterns is not clearly understood and, therefore, the physiologicaVmo1ecular events comprising capacitation that give rise to these patterns are completely unknown. It is believed that changes in the distribution of Ca2+-CTC complexes bound to phospholipids in the plasma membrane are responsible for the different patterns observed, and this property of the sperm membrane during capacitation may turn out to be very important. As a consequence, any compound that changes

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the fluorescent absorption spectrum of the CTC or of Ca2+-phospholipid complexes, or quenches the fluorescence intensity of these complexes could potentially be interpreted as changing the capacitated state of the sperm. Interpretation of results using this assay would, therefore, be problematic if such changes were to occur nonspecifically. Induction of the Acrosome Reaction

As discussed above, the acrosome reaction induced by a physiologically relevant agent is considered to be an endpoint for the completion of capacitation (Florman and Babcock, 1991; Yanagimachi, 1994). The underlying assumption of this operational definition is that sperm that undergo the acrosome reaction have already become capacitated. Experiments from several laboratories have concluded that either cauda epididymal or ejaculated sperm do not immediately possess the ability to undergo an acrosome reaction in response to biological agents such as the ZP or progesterone (Ward and Storey, 1984; Yanagimachi, 1994; Shi and Roldan, 1995; Visconti et al., 1995a). It is well accepted that these compounds induce the acrosome reaction only in sperm that are already capacitated (Florman and Babcock, 1991). The advantage of this definition of capacitation (and the subsequent use of assays to assess this event) is that acrosomal exocytosis is closer timewise to capacitation than is fertilization, and acrosome reaction assays are easier to perform. A concern with the use of these assays is that compounds that are able to stimulate or inhibit the acrosome reaction cannot be assumed to do so by stimulating or inhibiting capacitation (e.g., A23 187). Specifically, acrosome reactions might be able to occur in uncapacitated sperm when the cells are challenged with compounds that bypass capacitation. Fertilization In Vitro

There are both advantages and disadvantages to the use of in vitro fertilization assays to assess capacitation. An advantage is that the endpoint measured follows the classical definition of capacitation (i.e., sperm that are able to fertilize eggs have undergone capacitation). There are several disadvantages, however. Although this technique is an excellent way to demonstrate that a particular incubation mediudcondition is capable of capacitating sperm, it has obvious limitations for analysis if one wishes to examine whether a particular compoundincubation condition affects capacitation. For example, it cannot be assumed that, if a specific compoundincubation condition inhibits in vitro fertilization, its mode of action is by inhibiting capacitation, since fertilization is a multistep process that involves various aspects of sperm function (e.g., motility; acrosome reaction) and interaction between gametes (e.g., ZP binding; plasma membrane binding, and/or fusion). In addition, the concentration of sperm used in the in vitro fertilization assays may dramatically impact interpretation of results since these assays normally use a

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much higher sperm to egg ratio than is normally encountered in vivo. Lastly, it is time-consuming and expensive to perform.

111.

MOLECULAR BASIS OF CAPACITATION

Although capacitation is still poorly understood, recent in vitro studies by several laboratories are starting to lead to a unified hypothesis of how this event may be controlled at a molecular level. As stated above, one must clearly recognize the limitations in interpretation using in vitro approaches, but at the same time, studies of capacitation in vitro represent an excellent approach to ultimately understanding this process in vivo. As previously stated, capacitation in many species occurs in vitro spontaneously in defined media without the addition of biological fluids, suggesting that the intrinsic cellular regulation of capacitation involves preprogrammed membrane, transmembrane, and/or intracellular signaling events that, once initiated, lead to the capacitated state. These events will be considered both individually and together below. It is also clear that different media support capacitation in sperm from different species and that certain components of these media (i.e., serum albumin, Ca”, and HCO,-) play critical regulatory roles in promoting capacitation in all species studied thus far. The mechanism by which these media components function and are coupled to membrane, transmembrane, and intracellular signaling events regulating capacitation will also be considered. The following discussion of the molecular aspects of capacitation will be considered from two perspectives. First, a discussion of the regulatory systems that appear to be common among different species will be considered thereby forming a unifying hypothesis of capacitation. Those regulatory processes that may be unique to one or more species will then be discussed and, where appropriate, will be integrated into this unifying hypothesis. The reader is referred to Figure 1 throughout this discussion as this figure incorporates many of the issues discussed into a working model of membrane, transmembrane, and intracellular signal transduction events that are proposed to regulate capacitation. A.

Role of the Media Constituents, Albumin, Calcium, and Bicarbonate

The mechanism by which albumin (in many cases bovine serum albumin; BSA) promotes capacitation in mammalian sperm is intriguing as it is believed to function during capacitation in vitro as a sink for the removal of cholesterol from the sperm plasma membrane (Go and Wolf, 1985; Langlais and Roberts, 1998; Cross, 1998). The association between cholesterol removal from the sperm plasma membrane, albumin, and capacitation was first proposed by Davis and colleagues (1980). Removal of this sterol likely accounts for the membrane fluidity changes observed during capacitation (Wolf et al., 1986). The consequence of the removal of this sterol is that the cholesterol to phospholipid ratio in the membrane decreases, and such changes in

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@ f

Ca”

HCO,

Figure 7. Working model depicting the transmembrane and intracellular signaling pathways hypothesized to play a role in regulating sperm capacitation. This model is based on the work from a number of different laboratories cited in this review. In this model, cholesterol efflux to an appropriate acceptor initiates changes in membrane architecture due, in part, to a decrease in the orientation order of membrane lipids and phospholipids leading to an increase in bulk membrane fluidity. This change in membrane dynamics results in the activation of signaling pathways leading to capacitation. (-1 indicates negative regulation; (+) indicates positive regulation; solid arrows indicate established pathways; dashed arrows indicate hypothesized pathways that are to be experimentally tested. Abbreviations used in this figure: AC, adenylyl cyclase; Chol. Acc., cholesterol acceptor; PTK, protein tyrosine kinase; PTP, phosphotyrosine phosphatase; PDE; cyclic nucleotide phosphodiesterase; PK-A, protein kinase A; pY-proteins, phosphotyrosine-containing proteins.

membrane composition could certainly influence cellular function (see below). Such changes may underlie the changes in cell surface antigen distribution during capacitation that have been described by several investigators (Moore, 1995; Harrison and Gadella, 1995; Rochwerger and Cuasnicu, 1992). It is not known whether cholesterol removal represents the sole function of BSA during capacitation, and little is known about the consequences of cholesterol removal on sperm membrane dynamics as it

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relates to capacitation. Experiments demonstrating that other cholesterol-binding proteins such as HDL can stimulate sperm cholesterol efflux (Langlais et al., 1988) and replace albumin in in vitro fertilization assays (Therien et al., 1997) suggest that the primary action of BSA may be in mediating cholesterol movement. Cross and co-workers (Cross, 1998 and references therein) recently demonstrated that human semen contains cholesterol and that this sterol'can account for the inhibitory effects of seminal plasma on human sperm capacitation, presumably by preventing cholesterol efflux from the sperm plasma membrane. Moreover, the mechanism by which cholesterol movement is initiated and the mechanism by which albumin captures cholesterol is not known (see below). Calcium has been demonstrated to be a key component of media for the regulation of a variety of sperm functions. However, the involvement of Ca2+in initiating and/or regulating capacitation is presently controversial. In mouse sperm, there is evidence that capacitation requires the presence of extracellular CaZ+(Dasgupta et al., 1993; Visconti et al., 1995a), although these investigators did not measure Ca2+ fluxes or intracellular Ca2+concentrations in these studies. Some investigators have demonstrated an increase in intracellular sperm Ca2+during capacitation, while others have shown that no changes occur during this maturational event (Yanagimachi, 1994 and references therein). The ambiguity between these studies could be due, in part, to the well demonstrated action of Ca2+on the acrosome reaction and the inherent difficulties in differentiating both of these events. As discussed below, however, the action of Ca2+at the level of effector enzymes involved in sperm signal transduction (e.g., adenylyl cyclase, cyclic nucleotide phosphodiesterase, phosphatases) suggests that this divalent cation is likely to play an important role in capacitation. To date, the requirement for HC0,- in capacitation has been established in the mouse (Lee and Storey, 1986;Neil1 and Olds-Clarke, 1987;Visconti et al., 1995a; Shi and Roldan, 1995) and in the hamster (Boatman and Robbins, 1991), although it remains to be demonstrated in other mammalian species. Although little is known about the mechanisms of HC0,- transport in sperm, the ability of DIDS and SITS, well known inhibitors of anion transporters, to block the actions of HC0,- on various sperm functions suggests that sperm contain anion transporters (Okamuraet al., 1988; Visconti et al., 1990;Spira and Breitbart, 1992; Parkkila et al., 1993).Sperm contain a protein that is immunoreactive with an antibody to the AE1 class of anion transporters (Parkkila et al., 1993), but little is known about the identification and function of this protein in these cells. Although it has yet to be shown unequivocally, the transmembrane movement of HC0,- anions could be responsible for the known increase in intracellular pH that is observed during capacitation (Uguz et al., 1994;Zeng et al., 1996; Cross, 1998). The role of this anion in the regulation of sperm function is intriguing as it has been demonstrated to modulate CAMPmetabolism in sperm through its unique effect to markedly stimulate the adenylyl cyclase of these cells by an as yet unknown mechanism (Okamura et al., 1985; Garty and Salomon, 1987; Visconti et al., 1990,1995b).It is of interest from a physiological point of view that HC0,- con-

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centrations are low in the epididymis and high in the seminal plasma and in the oviduct (Harrison, 1996). Since HC0,- present in the extracellular milieu has also been positively correlated with the motility of pig sperm (Okamura et al., 1985), the HC0,concentrations present in the male and female reproductive tracts, in conjunction with the other reproductive tract modulators discussed above, could impact on the development of the capacitated state. Specifically, low levels of HC0,- in the epididymis would be conducive to maintaining sperm in an environment that does not support capacitation, whereas the higher concentrations of this anion in the female tract might contribute to capacitation. B.

Membrane Events

Although capacitation is still poorly understood, numerous investigators have provided ample evidence that this maturational event is clearly associated with major changes in membrane architecture and composition, the consequence of which is likely to lead to transmembrane signaling and intracellular effector activation. Such changes include a reduction in membrane cholesterol with resultant decreases in cholesterollphospholipidratios as assessed by a variety of criteria (Davis, 198 1; Bearer and Friend, 1982; Tesarik and Flechon, 1986; Ehrenwald et al., 1988; Suzulu and Yanagimachi, 1989; Hoshi et al., 1990; Zarintash and Cross, 1996). Such changes likely account for the observed alterations in sperm membrane fluidity (Wolf et al., 1986), aggregation of intramembraneous particles and formation of particle-free patches (Koehler and Gaddam-Rose, 1975), and the documented membrane protein redistributions reported with lectins (Cross and Overstreet, 1987) and antibodies (Shalgi et al., 1990; Rochwerger and Cuasnicu, 1992). From a cell signaling standpoint, this change in membrane dynamics may have profound effects on transmembrane signaling and may represent some of the “intrinsic” control of capacitation described above. Transmembrane signaling may be initiated by changes in ion channel activity andlor the activity of membrane-associated enzymatic and nonenzymatic proteins (see below). In addition, this dramatic change in plasma membrane lipid architecture could also be functionally important, as it may ultimately prime the membrane for fusion with the outer acrosoma-!membrane during the acrosome reaction. Since cholesterol efflux appears to be the driving force behind these changes in membrane dynamics during capacitation, a clear understanding of the mechanism by which this sterol moves within the plasma membrane and out of the plasma membrane in response to an appropriate acceptor (e.g., serum albumin) is critical to a molecular understanding of this maturational event. Cholesterol Efflux

Based on the results of several investigators studying cholesterol efflux in sperm during capacitation, one could postulate that such an efflux mechanism might bear some similarities to reverse cholesterol transport observed in somatic cells, where

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the first step is the efflux of cellular cholesterol to an appropriate extracellular acceptor-usually HDL. Work from several labs support the idea that during reverse cholesterol transport, cholesterol efflux occurs via a mechanism involving aqueous diffusion in which this sterol de-adsorbs from the plasma membrane into the aqueous phase, diffuses, and is then solubilized by an acceptor molecule. However, other mechanisms have been proposed that include receptor-mediated mechanisms as well as collision-based mechanisms. The molecular basis of how this occurs is unclear and is the subject of intensive investigation by many labs. As stated above, many investigators studying capacitation have demonstrated that this maturational event is associated with the ioss of cholesterol from the plasma membrane of these cells. This sterol loss during capacitation in vitro appears to be mediated by proteins that can function as cholesterol acceptors (e.g., serum albumin; HDL). There is also evidence for the role of Lipid Transfer Protein-I (LTP-I) in this process (Ravnik et al., 1992,1995). If one accepts the notion that capacitation in vivo is also associated with cholesterol loss, some important questions must be considered. Firstly, what component of the female reproductive tract might serve as a cholesterol acceptor in vivo? It is clear that the composition of the fluids of the female tract arise in part as a transudate of the serum so that serum-derived sterol acceptorshinding proteins can function in vivo. The identity of such acceptors remains to be clarified. Secondly, if cholesterol loss plays a key role incapacitation both in vitro and in vivo, is there a mechanism by which efflux from the membrane is regulated by cholesterol binding or sensing proteins associated with the sperm andor the reproductive tracts? If one considers the in vitro capacitation model, is the pool of cholesterol associated with the plasma membrane immediately available for efflux and transport to serum albumin or is there some mechanism by which the availability of membrane cholesterol for efflux is regulated? Recent work in somatic cells suggests that there are different pools of cholesterol in the membrane that display different kinetics of release (Yancey et al., 1996). The mechanism by which these pools interconvert is not clear, and it is distinctly possible that there is some regulatory process that could control this event. On the other hand, is cholesterol loss initiated immediately upon incubation of sperm in medium supporting capacitation so that the sperm membrane starts to undergo changes in fluidity spontaneously? If this alternative is correct, it is distinctly possible that the aforementioned modulatory factors in the male and female reproductive tracts may represent a mechanism to extrinsically regulate the release of cholesterol from the sperm membrane duringcapacitation in vivo in order to extend the fertilizable lifespan of the ejaculate. How might changes in cholesterol content of the membrane regulate transmembrane signaling events in the sperm leading to capacitation? It is clear from a variety of studies that cholesterol alters the bulk biophysical properties of biological membranes. For example, this sterol can increase the orientation order of the membrane lipid hydrocarbon chains and can consequently reduce the ability of membrane proteins to undergo conformational changes that may control their functions due to the fact that the membrane is less fluid (Figure 2A, A). Therefore, h g h concentrations of cholesterol in the

A

B

a Chol.

+

+

Signal

Signal

B'

A'

v

\f

+ Signal

Signal

figure 2. Working models depicting the mechanisms by which cholesterol efflux and changes in membrane cholesterol concentrations may regulate transmembrane signaling. In the first model (A, A'), a cholesterol acceptor (Chol.Acc.) binds cholesterol through a poorly understood mechanism. The resultant loss of cholesterol from the membrane causes a decrease in the orientation order of membrane lipids and phospholipids leadingto an increase in bulk membranefluidity. Asa consequence ofthis change in lipid architecture, nonenzymatic or enzymatic proteins are now removed from constraint within the membrane and assume an active conformation (going from the inactive state in A to the active state in A).This would represent an indirect mode of cholesterol regulation of protein function. In the second model (B, B'), the removal of cholesterol from the membrane removes this sterol from the nonenzymatic or enzymatic proteins to which it is bound. This loss of binding to target proteins removes inhibitory modulation and the proteins assume an active conformation (going from the inactive state in B to the active state in 6'). This model represents a direct mode of cholesterol regulation of protein function. It should be noted that these two models are not mutually exclusive and that regulation of membrane protein function and signal transduction by cholesterol efflux could represent a combination of these two models. 9<

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H.GALANTINO-HOMER

membrane might inhibit membrane protein function. This “indirect” effect of cholesterol on membrane protein function might stabilize those membrane and transmembrane events that are part of the “intrinsic” regulatory nature of capacitation. Cholesterol has also been demonstrated to have “direct” effects by binding to and regulating membrane protein function; such binding may serve to exert a positive or negative modulatory effect on the protein in question (Figure 2B, B’). In fact, studies of several membrane-associated ion transporters (e.g., Na+, K+-ATPase, GABA transporter) by cholesterol supports the idea that both direct and indirect effects of this sterol on the regulation of enzyme activity could be invoked (Vemuri and Philipson, 1989; Shouffani and Kanner, 1990). With respect to capacitation, it is interesting to note that the loss of cholesterol from the human sperm plasma membrane under conditions conducive to capacitation has been postulated to be coupled to the increase in intracellular pH that accompanies this maturational event (Cross and RazyFaulkner, 1997; see below). This suggests that the cholesterol itself andor its concentration in the membrane could modulate transmembrane ionic movements that ultimately regulate intracellular pH. For the purposes of this review, work from our laboratory suggests that the release of cholesterol from the sperm membrane leads to the activation of a signal transduction pathway leading to protein tyrosine phosphorylation (see below). The role of cholesterol and its loss in regulating transmembrane signaling will, therefore, remain an area of future interest. Ion Fluxes

As described above, Ca2+and HC0,- appear to play key roles in regulating aspects of capacitation, although the mechanism by which they d o so is presently unclear. Mammalian sperm contain voltage-sensitive Ca2+channels (Florman and Babcock, 1991; Florman et al., 1998) that play a role in acrosomal exocytosis induced by the ZP (Arnoult et al., 1996). The mechanism by which Ca2+influences signal transduction during capacitation, however, is not clear. This, in part, is due to difficulty in dissociating Ca2+events mediating capacitation versus those involved in the acrosome reaction. It is likely that information regarding the role of this divalent cation in capacitation will be further clarified once the identity of the Ca2+transport mechanisms present and functioning in sperm are fully characterized. Several aspects of mammalian sperm function, including capacitation, are regulated by intracellular pH (pH,). Although two acid efflux mechanisms have been identified in mouse sperm and could be involved in this process (Zeng et a1.,1996), the transport mechanisms that control pH, in these cells are not fully understood. One of these pathways shares the characteristics of a somatic cell Na+-dependent CI-/HCO,- exchanger, and the second pathway does not require extracellular ions to function. These authors described an increase in pH, during capacitation, and these data are consistent with reports by Vredenburgh-Wilberg and Parrish (1995) describing an increase in pHi during capacitation of bovine sperm by heparin. Al-

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though the increase in pHi accompanying heparin-induced bovine sperm capacitation is not inhibited by Rp-CAMP(Uguz et al., 1994),this PK-A antagonist can block capacitation, suggesting that a PK-A regulatory pathway(s) functions either parallel to, or downstream of, pathways activated as a consequence of changes in pHi. Hyperpolarization of the sperm plasma membrane has also been shown to accompany capacitation in mouse and bovine sperm (Zeng et al., 1995). This hyperpolarization is due, in part, to an enhanced K+ permeability and could be related to the release of inhibitory moduIation during capacitation (Amoult et al., 1996). Although little is known about the consequences of this hyperpolarization, it is speculated that such membrane potential changes could recruit Ca2+channels from an inactivated state to a closed, but activatable, state from which they could be subsequently opened by an agonist-induced depolarization (e.g., with the ZP) (Arnoult et al., 1996;Florman et al., 1998).Currently, the role of membrane potential in regulating any of the aforementioned aspects of capacitation at the molecular level is not known but remains an important avenue for future investigation.

C.

Transmembrane and lntracellular Signaling

Although the role for cAMP in regulating mammalian sperm motility is well established, its role incapacitation as well as in the acrosomereaction is still uncertain (Yanagimachi, 1994). We and others have suggested a role for cAMP during capacitation (White and Aitken, 1989; Parrish et al., 1994; Visconti et al., 1995b; Leclerc et al., 1996), and our lab has recently demonstrated that an increase in protein kinase A (PK-A) activity accompanies mouse sperm capacitation (Visconti et al., 1997). Measurement of PK-A activity represents the most accurate reflection of steady-state changes in intracellular cAMP concentrations. The mechanism by which sperm cAMP concentrations are regulated during capacitation is of interest, since its regulation may be integrated with the aforementioned changes in Ca2+and HC0,- movements. Ca2+and HC0,- have both been implicated in the regulation of sperm cAMP concentrations through effects to stimulate adenylyl cyclase activity (Hyne and Garbers, 1979; Okarnura et al., 1985; Garty and Salomon, 1987). The mammalian sperm adenylyl cyclase possesses unique properties, and its regulation has been the subject of multiple studies. The sequence and topology of this enzyme, however, has not yet been established and the exact mechanism by which this enzyme is stimulated by these ions is not clear. In attempts to further understand the signal transduction cascades that regulate capacitation, our laboratory has recently correlated mouse, human, and bovine sperm capacitation with an increase in protein tyrosine phosphorylation of a variety of substrates (Visconti et al., 1995a; Carrera et al., 1996; Galantino-Homer et al., 1997). Many of these results have since been corroborated by other labs (Aitken et al., 1995; Leclerc et al., 1996; Luconi et al., 1996; Emiliozzi and Fenichel, 1997). Using the mouse as an experimental paradigm, our laboratory has demonstrated

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that capacitation in vitro of cauda epididymal sperm promotes the tyrosine phosphorylation of a subset of proteins of Mr 40,000 to 120,000. These phosphorylations require the presence of BSA, Ca2+,and HC0,- in the medium, and the concentrations of these media constituents needed for protein tyrosine phosphorylation to occur are correlated with those needed for capacitation (Visconti et al., 1995a).Moreover, caput sperm, which do not possess the ability to undergo capacitation and fertilize eggs (Yanagimachi, 1994), do not display these changes in protein tyrosine phosphorylation when incubated under conditions normally conducive to capacitation (Visconti et al., 1995a);the ability of sperm to display the changes in protein tyrosine phosphorylation are first seen during the caput to corpus transition (Fornes, Visconti, and Kopf, unpublished). Taken together, these data suggest that the ability of mouse sperm to become capacitated as well as their ability to undergo an increase in protein tyrosine phosphorylation are acquired during epididymal transit and may represent an essential component of epididymal maturation in this species. The requirement for BSA, Ca2+,and HC0,- in the extracellular medium to support protein tyrosine phosphorylation represents a novel mode of regulation of the signaling events in sperm leading to these posttranslational modifications. As described above, regulation of capacitation in vitro by BSA is thought to rely on the ability of this protein to serve as a sink for the removal of cholesterol from the sperm plasma membrane. This interrelationship between BSA and cholesterol movement also appears to be important in the regulation of protein tyrosine phosphorylation since abolishing the ability of BSA to serve as an extracellular acceptor for cholesterol inhibits protein tyrosine phosphorylation and sperm capacitation (Visconti, Ning, Fornes, Alvarez, and Kopf, unpublished). These, as well as many other experiments, suggest that cholesterol release/movement is tightly tied to transmembrane signaling events in the sperm that ultimately regulate protein tyrosine phosphorylation. These effects of cholesterol efflux on transmembrane signaling and intracellular signal transduction represent a new mode of cellular signaling, and the mechanism by which this occurs clearly warrants further investigation. For example, might cholesterol efflux-induced signal transduction occur via changes in membrane fluidity thereby indirectly modulating membrane-associated signal transduction enzymes and/or ion channels? Or might these aforementioned signal transduction enzymes and/or ion channels be modulated directly by this sterol? The extracellular Ca2+and HC0,- requirement for both protein tyrosine phosphorylation and capacitation represents a novel regulatory mechanism of cellular signaling since these ions have been shown to be activators of the mammalian sperm adenylyl cyclase (Hyne and Garbers, 1979; Okamura et al., 1985; Garty and Salomon,1987; Visconti et al., 199%). Since there appears to be a relationship between Ca2+,HC0,-, and increased adenylyl cyclase activity, experiments were designed to determine whether the action of these ions on protein tyrosine phosphorylation and capacitation involved a CAMP-mediated pathway. As previously stated, protein tyrosine phosphorylation does not occur when sperm are incu-

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bated in the absence of BSA, Ca2+,or HC0,-. However, sperm incubated in media devoid of any of these components but in the presence of CAMP agonists (e.g., dibutyryl CAMP, 8-bromo CAMP,Sp-CAMPS, isobutyl-methylxanthine) results in an increase in protein tyrosine phosphorylation as well as capacitation (Visconti et al., 1995b). In addition, protein tyrosine phosphorylation is accelerated by active cAMP agonists in complete media that support capacitation. The results of these experiments suggest that (1) the action of cAMP appears to be downstream of the actions of BSA, Ca2+,and HC0,- but upstream of protein tyrosine phosphorylation and (2) protein tyrosine phosphorylation and capacitation are regulated through a PK-A pathway. Further confirmation of this second conclusion is that two inhibitors of PK-A, Rp-CAMPS,and H-89, both of which inhibit this enzyme by completely distinct mechanisms, inhibit both protein tyrosine phosphorylation and capacitation of sperm in complete medium (Visconti et al., 199%). Moreover, PK-A activity increases during capacitation (Visconti et al., 1997).Since the mode of action of BSA appears to be tied to the removal of plasma membrane cholesterol, it is likely that cholesterol release is also upstream of the CAMP-induced protein tyrosine phosphorylation. Whether cholesterol removal is upstream or parallel to the action of Ca2+and/or HC0,- is not presently known. One hypothesis to be tested is that the removal of cholesterol, with a resultant change in sperm plasma membrane fluidity, could modulate Ca2+and/or HC0,-ion fluxes leading to the activation of the adenylyl cyclase. In this regard, it is interesting to note the recent observations of Harrison and colleagues (1996) that HC0,- ion can induce major changes in the lipid architecture of boar sperm that appear to be independent of cholesterol removal and changes in intracellular pH. These investigators demonstrated that HCO, can increase the binding of the impermeant lipophilic probe merocyanine to the sperm membrane; this probe binds to plasma membranes with enhanced affinity as the lipid components of the membranes become more disordered. This effect was very rapid and could be mimicked in the absence of HC0,- by a variety of cyclic nucleotide phosphodiesterase inhibitors, suggesting that these HC0,- effects might be mediated by cyclic nucleotides. It is not clear how this effect on membrane structure might be mediated, but these observations point to the possibility of multiple effects of HC0,- on sperm membrane dynamics as well as signal transkwtion. Taken together, these data suggest that protein tyrosine phosphorylation and capacitation appear to be under regulation of a CAMP/PIC-A pathway. Upregulation of protein tyrosine phosphorylation by PK-A during sperm capacitation is, to our knowledge, the first demonstration of a connection between these signal transduction pathways at this level. Since similar results have now been reported in sperm of other species (Leclerc et al., 1996; Galantino-Homer et al., 1997),it is likely that this unique mode of signal transduction crosstalk may be universal to mammalian sperm. Presently, it is not known whether the resultant increase in protein tyrosine phosphorylation is due to the stimulation of a tyrosine kinase, to an inhibition of a phosphotyrosine phosphatase, or to both. Recently, Berruti and Borgonovo (1996) described what appears to be a male germ cell-specific tyrosine kinase that they

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have termed sp42. This protein was initially purified from boar sperm, but proteins that cross react with sp42 antibodies are also present in human, mouse, and rat sperm. Whether sp42 represents a tyrosine kinase that participates in the aforementioned signaling cascade leading to capacitation is unknown. Recently we (Carrera et al., 1996) and others (Luconi et al., 1996) have demonstrated in human sperm that extracellular Ca2+can exert an inhibitory effect on protein tyrosine phosphorylation, and Carrera and colleagues (1996) have provided evidence that this Ca2+-induced dephosphorylation may be regulated by a calmodulin-dependent mechanism, possibly involving calcineurin. Although calcineurin is a major phosphatase in sperm, the role of this particular enzyme in this particular series of dephosphorylations has not been proven. Thus, the identity of the kinases and phosphatases involved in this aforementioned unique signal transduction pathway is still to be resolved and remains are area of future experimentation. Characterization of the protein substrates that are phosphorylated on tyrosine residues in both a CAMP-dependent and -independent manner during capacitation is also of interest, as their identity may yield a great amount of information regarding their roles in capacitation. We have initiated a systematic approach to the identification and characterization of these proteins in sperm and have identified a major substrate for tyrosine phosphorylation during capacitation in human sperm (Carrera et al., 1996). This protein is associated with the fibrous sheath of the flagellum and is the human homologue of the mouse sperm AKAP82 and pro-AKAP82. A Kinase Anchor Proteins (AKAPs) represent an ever growing family of scaffolding proteins that function in cells to tether the regulatory subunits of PK-A to organelles or cytoskeletal elements, thus permitting precise control of signal transduction in discrete regions of the cell (Pawson and Scott, 1997). It is interesting to note that other members of the AKAP family have been demonstrated to bind a variety of signal transduction enzymes including calcineurin and protein kinase C (Klauck et al., 1996), suggesting that this family of proteins could potentially serve as scaffolding proteins to anchor entire signal transduction complexes to discrete cellular locations. Currently, the role of tyrosine phosphorylation in regulating AKAP82 function and the role of AKAP82 in sperm function are not known, but it is tempting to speculate that posttranslational modifications of this protein might regulate events associated with flagellar bending (e.g., changes in wave amplitude during hyperactivation of motility). Although there is no information to date regarding the regulation of a specific protein or process in sperm by protein tyrosine phosphorylation, recent reports implicating protein tyrosine phosphorylation in the modulation of T-type Ca2+channels in mouse spermatogenic cells has potentially important implications with regard to capacitation and its role in preparing sperm to undergo a ZP-induced acrosome reaction. Using whole-cell patch clamp techniques on dissociated mouse spermatogenic cells, Arnoult and colleagues (1997) have demonstrated that voltage-dependent facilitation of T-currents induced by membrane depolarizaticns or high-frequency stimulations could be n;imicked by protein tyrosine kinase inhibitors. Moreover, an-

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tagonists of protein tyrosine phosphatase activity can block this voltage-dependent current facilitation. The authors hypothesize that T channels may be held in a low conductance state by tonic tyrosine phosphorylation and can convert to a high conductance state by dephosphorylation. If T-type Ca2+-channels were to play a role in capacitation and the ZP-induced acrosome reaction, one could postulate a multistate model of T-channel conductance controlled by protein tyrosine phosphorylation. In such a model, T-type Ca2+channels might be partially active in sperm that are not capacitated since the channel is not phosphorylated, and Ca2+-movement through channels would initiate signal transduction leading to protein tyrosine phosphorylation and capacitation as described above. One of the substrates that could be phosphorylated during capacitation might be the T-type Ca*+-channel that, when phosphorylated, would convert to a low conductance state. This would ensure that changes in Ca2+-conductance necessary for capacitation would not continue unabated and result in a precocious induction of the acrosome reaction prior to initiation of exocytosis by a physiological ligand such as the ZP. Binding of capacitated sperm to the ZP would then trigger a rapid dephosphorylation of the T-type Ca2+-channel, leading to an increase in conductance and an ensuing acrosome reaction. This ZP-induced dephosphorylation of the T-type Ca2+- channel could occur via a phosphoprotein phosphatase. Elements of such a model remain to be tested. Based on the aforementioned observations, a working model for signal transduction during capacitation has been established and is shown in Figure 1. There are several aspects of this model that must be tested experimentally and this general model does not take into account other observations regarding the regulation of capacitation seen in other species. Some of the these observations may be unique to a particular species but ultimately must be considered in developing a unifying hypothesis of capacitation. For example, recent work using human sperm has focused on the role of superoxide anion generation related to capacitation and hyperactivation of motility (De Lamirande and Gagnon, 1993).Leclerc and colleagues (1997) have reported that reactive oxygen species upregulate protein tyrosine phosphorylation of several proteins in human sperm. These results are in agreement with the work of Aitken and colleagues (1995) who have described an increase in protein tyrosine phosphorylation after stimulation of a postulated endcgenous NADPHoxidase or after addition of H202.There are several questions now to be addressed. Firstly, it is not known how free radical generation leads to capacitation. Secondly, the localization of the free radical-generating system(s) in sperm as well as whether the action of superoxide anion is dependent or independent of CAMP are also not presently known. Bovine sperm capacitation in vitro has been shown to be accomplished in media containing either heparin (Parrish et al., 1988) or oviductal fluid (in which the active capacitating agent is thought to be a heparinlike glycosaminoglycan). Heparin (or glycosaminoglycans) does not appear to be essential for capacitation in any of the other species studied thus far. However, it should be emphasized that since most studies are performed in vitro, one cannot rule out the possibility that glycosamino-

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glycans associated with the female tract or the cumulus-enclosed uocyte play an important role in capacitation in vivo. Glycosaminoglycans are postulated to promote capacitation by binding to and removing seminal plasma proteins that are adsorbed to the sperm plasma membrane and that are normally thought to function to inhibit capacitation (Miller et al., 1990; ThCrien et al., 1995). It is of interest that heparin also increases cAMP synthesis (Parrish et al., 1994), elevates pHi (see above), and regulates the capacitation-associated changes in protein tyrosine phosphorylation (Galantino-Homer et al., 1997). The mechanism by which this occurs and its physiological relevance are not clear. Glucose has also been postulated to have effects on capacitation, although this is somewhat controversial and the effects observed are apparently species-dependent. Glucose inhibits heparin-induced bovine sperm capacitation in vitro through an apparent effect on cAMP metabolism, as well as preventing the increase in pHi that accompanies this maturational event (Uguz et al., 1994; Parrish et al., 1994). The capacitation-associated increase in protein tyrosine phosphorylation in bovine sperm incubated in media containing heparin is also inhibited by glucose by a yet unknown mechanism (Galantino-Homer et al., 1997). We have observed that although glucose has these inhibitory effects on protein tyrosine phosphorylation in bovine sperm, capacitation media for mouse sperm, which contains glucose, has no apparent inhibitory effects on protein tyrosine phosphorylation (Visconti et al., 199%). Paradoxically, others have found that glucose is beneficial for capacitation in other species (Rogers and Perreault, 1990; Fraser and Herod, 1990; Mahadevan et al., 1997). The species-dependent differences in response to this saccharide are not understood, nor is its mechanism of action.

IV. CONCLUSIONS AND FUTURE DIRECTIONS We are poised to make significant advances in the understanding of capacitation at the molecular level. The outcome of this knowledge will ultimately translate to new and better techniques for enhancing fertility, identifying and treating certain forms of male infertility, and preventing conception. Several questions of considerable importance must be addressed over the next few years. For example, what is the mechanism by which cholesterol moves from the sperm plasma membrane and how does this movement initiate intracellular signaling? What is the mechanism by which the cAMP/PK-A pathway is stimulated and how does stimulation of this pathway lead to crosstalk and upregulation of protein tyrosine phosphorylation? Finally, what is the nature of the protein substrates that arephosphorylated on tyrosine residues and how does the phosphorylation of these substrates impact the major endpoints of capacitation (e.g., hyperactivation of motility, competence to undergo a regulated acrosome reaction, and fertilization)? Answers to these questions will provide us with a molecular insight into this poorly understood, but extremely important extratesticular maturational event.

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ACKNOWLEDGMENTS W e wish to thank all of the members of our lab who have contributed to the work outlined in this review, and whose hard work and dedication are gratefully appreciated. This work was supported by NIH grants HD-06274, HD34811, HD-22732, and HD- 33052. PEV was supported by HD-06274 and the Rockefeller Foundation; HGH was supported by USDA #9502560 and 5-T32-GM 07170 (NIH).

REFERENCES Aitken, R.J., Paterson, M., Fisher, H., and Buckingham, D.W. (1995). Van Duin M. Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J. Cell Sci. 108:2017-2025 Arnoult, C., Zeng, Y., and Florman, H.M. (1996). ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization. J . Cell Biol. 134: 637-645. Arnoult, C., Lemos, J.R., and Florman, H.M. (1997). Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation. EMBO J. 161593-1599. Austin, C.R. (1951). Observations on the penetration of the sperm into the mammalian egg. Aust. J. Sci. Res. 4:B581-B596. Austin, C.R. (1952). The “capacitation” of the mammalian sperm. Nature 170:326. Bearer, E.L. and Friend, D.S. (1982). Modification of anionic-lipid domains preceding membrane fusion in guinea pig sperm. J. Cell Biol. 92:604-615. Bedford, J.M. (1983). Significance of the need for sperm capacitation before fertilization in eutherian mammals. Biol. Reprod. 28:108-120. Berruti, G. and Borgonovo, B. (1996). sp42, the boar sperm tyrosine kinase, is a male germ cell-specific product with a highly conserved tissue expression extending to other mammalian species. J. Cell Sci. 109:851-858. Boatman, D.E and Robbins, R.S. (1991). Bicarbonate: Carbon-dioxide regulation of sperm capacitation, hyperactivated motility, and acrosome reactions. Biol. Reprod. 44:806-8 13. Carrera, A,, Moos, J., Ning, X.P., Gerton, G.L., Tesarik, J., Kopf, G.S., and Moss, S.B. (1996): Regulation of protein tyrosine phosphorylation in human sperm by . a calciudcalmodulin-dependent mechanism: Identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. Dev. Biol. f80:284-296. Chang, M.C. (1951). Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature 168:697-698. Chang, M.C. (19.55). Development of fertilizing capacity of rabbit spermatozoa in the uterus. Nature 175:1036-1037. Chian, R.C. and Sirard, M.A. (1995). Fertilizing ability of bovine spermatozoa cocultured with oviduct epithelial cells. Biol. Reprod. 52:156-162. Cohen-Dayag, A., Eisenbach, M. (1994). Potential assays for sperm capacitation in mammals. Am. J. Physiol. Cell Physiol. 267:C1167-C1176. Cross, N.L. (1996). Human seminal plasma prevents sperm from becoming acrosomally responsive to the agonist, progesterone: Cholesterol is the major inhibitor. Biol. Reprod. 54:138- 145. Cross, N.L. (1998). Role of cholesterol in sperm capacitation. Biol. Reprod. 59:7-11. Cross, N.L. and Overstreet, J.W. (1987). Glycoconjugatesofthe human spermsurface: Distribution and alterations that accompany capacitation in vitro. Gamete Res. 16:23-35. Cross, N.L. and Razy-Faulkner, P. (1997). Control of human sperm intracellular pH by cholesterol and its relationship to the response of the acrosome to progesterone. Biol. Reprod. 56:1169-1174.

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DasGupta, S., Mills, C.L., and Fraser, L.R. (1993). Ca"-related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay. J. Reprod. Fertil. 99:135-143. Davis, B.K. (1978). Inhibition of fertilizing capacity in mammalian spermatozoa by natural and synthetic vesicles. In: Symposium on the Pharmacological Effects of Lipids, Champaign, IL. (Kabara, J., Ed.), pp. 145-157. The American Oil Chemist's Society. Davis, B.K., Byme, R., and Bedigan, K. (1980). Studies on the mechanism of capacitation: Albumin-mediated changes in plasma membrane lipids during in vitro incubation of rat sperm cells. Proc. Natl. Acad. Sci. USA 77:1546-1550. Davis, B.K. (1981). Timing of fertilization in mammals: Sperm cholesteroVphospholipid ratio as a determinant of the capacitation interval. Proc. Natl. Acad. Sci. USA 78:7560-7564. De Lamirande, E. and Gagnon, C. (1993). A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa. Int. J. Androl. 16:21-25. Ehrenwald, E., Parks, J.E., and Foote, R.H. (1988). Cholesterol efflux from bovine sperm. I. Induction of the acrosome reaction with lysophosphatidylcholineafter reducing sperm cholesterol. Gamete Res. 20:145-157. Ehrenwald, E., Foote, R.H., and Parks, J.E. (1990). Bovine oviductal fluid components and their potential role in sperm cholesterol efflux. Mol. Reprod. Dev. 25:195-204. Emiliozzi, C. and Fenichel, P. ( 1997)Protein tyrosine phosphorylation is associated with capacitation of human sperm in vitro but is not sufficient for its completion. Biol. Reprod. 56:674-679. Florman, H.M. and Babcock, D.F. (1991) Progress towards understanding the molecular basis of capacitation. In: CRC Uniscience-Chemistry of Fertilization. (Wassarman, P., Ed.), pp. 105-132. CRC Press Inc. Boca Raton, FL. Florman, H.M., Lemos, J.R., Arnoult, C., Kazam, I., Li, C., and O'Toole, C.M. (1998). A tale of two channels: A perspective on the control of mammalian fertilization by egg-activated ion channels in sperm. Biol. Reprod. 59:12-16. Fraser, L.R. and Herod, J,E. (1990). Expression of capacitation-dependentchanges in chlortetracycline fluorescence patterns in mouse spermatozoa requires a suitable glycolysable substrate. J. Reprod. Fertil. 88:611-621. Galantino-Homer, H., Visconti, P.E., and Kopf, G.S. (1997). Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3',5'-monophosphate-dependent pathway. Biol. Reprod. 56:707-719. Garty, N. and Salomon, Y. (1997). Stimulation of partially purified adenylate cyclase from bull sperm by bicarbonate. FEBS Lett. 218:148-152. Go, K.J. and Wolf, D.P. (1985). Albumin-mediated changes in sperm sterol content during capacitation. Biol. Reprod. 32:145-153. Harrison, R.A.P. (1996). Capacitation mechanisms, and the role of capacitation as seen in eutherian mammals. Reprod. Fertil. Dev. 8:581-594. Harrison, R.A.P. and Gadella, B.M. (1995). Membrane changes during capacitation with special reference to lipid architecture. In: Human Sperm Acrosome Reaction. (Fenichel, P. and Parinaud, J . , Eds.), Vol. 236, pp. 45-65. Colloque INSERM/John Libbey Eurotext, Montrouse, France. Harrison, R.A.P., Ashworth, P.J.C., and Miller, N.G.A. (1996). Bicarbonate/CO, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membranes. Mol. Reprod. Dev. 45:378-391. Heffner, L.J. and Storey, B.T. (1982). Cold lability of sperm binding to zona pellucida. J. Exp. Zool. 219:155-161. Hoshi, K., Aita, T., Yanagida, K., Yoshimatsu, N., and Sata, A. (1990). Variation in the cholesterol/phospholipid ratio in human spermatozoa and its relationship with capacitation. Human Reprod. 5:7 1-74. Hunter, A.G. and Nomes, H.O. (1969). Characterization and isolation of a sperm-coating antigen from rabbit seminal plasma with capacity to block fertilization. 1. Reprod. Fertil. 2#:419-427.

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Hyne, R.V. and Garbers, D.L. (1979). Regulation of guinea pig sperm adenylate cyclase by calcium. Biol. Reprod. 21:1135-1142. Inoue, M. and Wolf, D.P. (1975). Sperm binding characteristics of the murine zona pellucida. Biol. Reprod. I3:340-348. Klauck, T.M., Faux, M.C., Labudda, K., Langeberg, L.K., Jaken, S., and Scott, J.D. (1996). Coordination of three signaling enzymes by AKAP79, a mammalian scaffolding protein. Science 271: 1589-1592. Koehler, J.K. and Gaddum-Rose, P. (1975). Media induced alterations of the membrane associated particles of the guinea pig sperm tail. J. Ultrastruct. Res. 51:106-1 18. Langlais, 1. and Roberts, K.D. (1985). A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res. 12:183-224. Langlais, J., Kan, F.W.K., Granger, L., Raymond, L., Bleau, G., and Roberts, K.D. (1988). Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation. Gamete Res. 20:185-201. Leclerc, P., de Lamirande, E., and Gagnon, C. (1 996). Cyclic adenosine 3 ' 5 ' monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod. 55:684-692. Leclerc, P., de Lamirande, E., and Gagnon, C. (1997). Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives. Free Rad. Biol. Med. 22~643-656. Lee, M.A and Storey, B.T. (1986). Bicarbonate is essential for fertilization of mouse eggs; mouse sperm require it to undergo the acrosome reaction. Biol Reprod. 34:349-356. Lee, M.A., Trucco, G.S., Bechtol, K.B., Wummer, N., Kopf, G.S., Blasco, L., and Storey, B.T. (1987). Capacitation and acrosome reactions in human spermatozoa monitored by a chlortetracycline fluorescence assay. Fea. Steril. 48:649-658. Luconi, M., Krausz, C., Forti, G., and Baldi, E. (1996). Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa. Biol. Reprod. 55:207-216. Mahadevan, M.M., Miller, M.M., and Moutos, D.M. (1997). Absence of glucose decreases human fertilization and sperm movement characteristics in vitro. Human Reprod. 12:119-123. Miller, D.J., Winer, M.A., and Ax, R.L. (1990). Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol. Reprod. 42:899-9 15. Moore, H.D.M. (1995). Modification of sperm membrane antigens during capacitation. In: Human Sperm Acrosome Reaction. (Fenichel, P. and Parinaud, J., Eds.), Vol. 236, pp. 35-43. Colloque INSERWJohn Libbey Eurotext, France. Neill, J. and Olds-Clarke, P. (1987). A computer-assisted assay for mouse sperm hyperactivation demonstrates that bicarbonate but not bovine serum albumin is required. Gamete Res. IR:121-140. Okamura, N., Tajima, Y., Soejima, A,, Masuda, H., and Sugita, Y . (1985). Sodium. bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa through the direct activation of adenylate cyclase. J. Biol. Chem. 260:9699-9705. Okamura, N., Tajima, Y . , and Sugita, Y. (1988). Decrease in bicarbonate transport activities during epididymal maturation of porcine sperm. Biochem. Biophys. Res. Commnn. 157:1280-1287. Olds-Clarke, P. (1989). Sperm from tw32 + mice: Capacitation is normal, but hyperactivation is premature and nonhyperactivated sperm are slow. Dev. Biol. 131:475-482. Parkkila, S., Rajaniemi, H., and Kellokumpu, S. (1993). Polarized expression ofaband 3-related protein in mammalian sperm cells. Biol. Reprod. 49:326-331. Panish, J.J., Susko-Parrish, J., Winer, M.A., and First, N.L. (1988). Capacitation of bovine sperm by heparin. Biol Reprod. 38: 1171- 1180. Panish, J.J., Susko-Panish, J.L., Handrow, R.R., Sims, M.M., andFirst, N.L. (1989). Capacitation of bovine spermatozoa by oviduct fluid. Biol. Reprod. 40:1020-1025.

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OVARIAN NITRIC OXIDE A LOCAL REGULATOR OF OVULATION, OOCYTE MATURATION, AND LUTEAL FUNCTION

Lisa M. Olson

...

................................ 111. NO and Ovulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................... A. Ovarian NO Synthesis . . . . . B. Expression, Location, and Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Inhibitor Studie D. Mechanisms/Fu .......................................... E. Isoform-Specific Functions IV. NO and Oocyte Maturation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. NO and Luteal Function VI. Summary.. . . . . . Acknowledgments ......................................... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Developmental Biochemistry Volume 5, pages 109-127. Copyright Q 1999 by JAI Press Inc. All right of reproduction in any form reserved. ISBN: 0-7623-0202-X

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110 110 112 113 114 116 119

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INTRODUCTION

1.

One of the first papers reporting that immature ovarian cells synthesized the unique signaling molecule, nitric oxide (NO), was published in 1993 (Ellman et al., 1993). Since that report, several investigators have shown that ovarian cells synthesize NO and that multiple isoforms of NO synthase (NOS) are expressed in the ovary in a developmental and cell-specific fashion. This review will present the current understanding of ovarian NO as a local regulator of ovulation, oocyte development, and luteal function.

II.

NOANDNOS

NO, a lipid and water-soluble gas, is synthesized from the stoichiometric oxidation of one of the guanidino nitrogens of arginine by NO synthase yielding NO and citrulline (Nathan, 1992; Moncada et al., 1991; Ignarro, 1990). This reaction utilizes 1.5 molecules of NADPH and 2 molecules of molecular oxygen as cosubstrates and also requires five other cofactors: heme, tetrahydropbiopterin, calmodulin, and the flavins FMN and FAD (MacMicking et al., 1997; Griffith and Stuehr, 1995) (Figure 1). NOSs function as homodimers, and a family of NOS isoforms with different biochemical characteristics have been isolated and cloned (Xie et al., 1992; Lamas et al., 1992). Two isoforms with common biochemical characteristics were first identified in the brain and endothelium and were originally termed ncNOS and ecNOS, respectively (Bredt et al., 1991; Lamas et al., 1992). These isoforms require calcium and calmodulin for activity, produce small amounts of NO over short periods, are often constitutively expressed, and are generally involved in maintaining vital homeostatic responses such as neurotransmission and vasodilation (Table 1). A third isoform, termed inducible NOS (iNOS) was initially isolated from macrophages (Xie et al., 1992). This isozyme is calcium- and calmodulin-independent, is primarily transcriptionally regulated, results in a sustained synthesis of NO over long periods and is correlated with cytotoxickytostatic immune responses (Nathan, 1992; Moncada et al., 1991;MacMicking et al., 1997). It is now clear that all three NOS isoforms are expressed in a variety of cell types and tissues, thus a numerical nomenclature has been promoted as a more accurate means of referring to the three isoforms (Table 1). Furthermore, it is becoming apparent that the “constitutive” isoforms are highly regulated both by transcriptional (Yoshizumi et al., 1993; Weiner et al., 1994; Uematsu et al., 1995) and post-transcriptional mechanisms (Robinson et al., 1996) and that the “inducible” isoform is often constitutively present in tissues (Conrad et al., 1993; Bryant et al., 1995; Huang et al., 1995). NO exerts many of its functions by reacting with metal- and thiol-containing proteins, which can result in both activation and inhibition of target proteins (Nathan, 1992; Moncadaet al., 1991; Stamler, 1994; MacMicking et al., 1997;Griffith

I

1

Arginine

NO,,NO,

cNOS

Cilline

NOm affects internal target proteins

.

NO =

I i

Hemoglobin

target proteins

P450 etuyrnes Aconitase Cytochrorne C reductase

Guanylcyclase MAP Kinase

cox

p21 ras

Figure 7. Schematic diagram of the N O pathway. N O isformed in the generator cell via the action of either cNOS (ncNOS or ecNOS) or iNOS. N O can act in an autocrine manner or diffuse out of the generator cell into a target cell where it may interact with a number of metal- and/or thiol-containing proteins resulting in its biological effect(s).After diffusion out of the generator cell, N O can react with oxygen to form the water-soluble metabolites, nitrite (NO;) and nitrate (NO;). N O can also interactwith oxygen radical to form peroxynitrite (OONO-), which is associated with tissue damage. The induction of iNOS is suppressed by glucocorticoids. Both isoforms of the enzyme are inhibited by analogues of L-arginine such as L-NAME and L-NIL. The pathway is also inhibited by hemoglobin. 111

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Table 1. Nomenclature and Characteristics of NOS Isoforms Calcium and Numerical Nomenclature

Human Chromosomal Location

,NOS

I II

17cen-q12

ecNOS

111

7q35-36

Descriptive Nomenclature

Calmodulin Required for Activrtv

Amounts of N O Svnthesized

Yes no ws

small

~~

ncNOS

12q24.2

large small

and Stuehr, 1995) (Figure 1). For example, endothelial cellderived NO has been shown to mediate vasodilation by diffusing into surrounding smooth muscle cells, binding the heme moiety of guanylate cyclase leading to increases in cGMP and smooth muscle cell relaxation (Nathan, 1992; Moncada et al., 1991; Stamler, 1994). Direct activation of metal-containing enzymes such as cyclooxygenase 1 and 2 leads to increases in prostaglandin synthesis (Salvemini et al., 1993; Salvemini et al., 1994). Furthermore, NO directly activates the guanine nucleotide-binding protein p21 rils, which leads to increased mitogen-activated protein kinase activity as well as greater nuclear translocation of the transcription factor, NFKB (Lander et al., 1996; Lander et al., 1993). The direct cytotoxic actions of NO are also due to inactivation of heme and iron-sulfur+ontaining active sites of enzymes, some examples of which include aconitase and the proteins of the mitochondrial electron transport chain (Henry et al., 1993; MacMicking et al., 1997). In addition, NO is highly reactive with superoxide anion forming the very potent free radical peroxynitrite, which can covalently modify tyrosine residues of proteins and inactivate them (Stamler, 1994) (Figure 1). Indeed, high levels of nitrotyrosine are associated with tissue damage (Haddad et al., 1994). It is clear that NO is a ubiquitous signaling molecule with several potential targets. As agas, it also has the ability to quickly diffuse out of cells and thus may act in both autocrine and paracrine ways (Figure 1). The specificity of NO’S functions must therefore rely on regulation of the expressed isoform of NOS, the amount of NO produced, and the variety of target proteins present.

111.

NO AND OVULATION

One of the first papers on the potential role of NO in the ovary demonstrated that cells dispersed from immature rat ovaries synthesized NO in response to IL- 1 (Ellman et al., 1993). As IL-Ip is known to play a significant role in the ovulatory process (Kol and Adashi, 1995) and has been shown in other cells to transcriptionally induce iNOS (Perrella et al., 1994; Kunz et al., 1994), these authors speculated that IL1 P-induced NO functioned as a cytotoxic agent in the rupture of the follicular wall during ovulation (Figure 2). Several investigators have shown that NO mediates several but not all of IL- 1p’s functions in the ovulatory process (Ben-Shlomo

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et al., 1994; Bonello et al., 1996; Nakamura et al., 1996). The data that address the role of NO in ovulation and its possible mechanism(s) of action are presented below. A.

Ovarian NO Synthesis

NO has a short half-life (2-30 s) and therefore the direct measurement of NO production is impractical. In aqueous solutions, however, it rapidly reacts with oxygen to form the stable water-soluble metabolites nitrite and nitrate (Figure 1). The concentration of these ions, which can be measured via a variety ofmethods, is used as a measure of the tissue content of NO and/or the synthesis of NO by cultured cells (Hevel and Marletta, 1994; Archer, 1993). An alternative method used to quantify NOS activity is to directly measure the catalytic activity of a cell or tissue extract (i.e., the conversion of radioactive arginine to citrulline; Hevel and Marletta, 1994; Archer, 1993). While both methods are sensitive measurements of the overall capacity of a cell or tissue extract to synthesize NO, neither one provides a true kinetic

I

LHlhCG

Activates Collagenase

Blood Flow Follicular Rupture

Inhibits Atresia

Figure 2. Putative role for NO in the ovulation cascade. In response to LH/hCC, ovarian NOS isoforms and NO synthesis increase. In some studies, IL-I p has been shown to be downstream of LH/hCG and to mediate its effects on NO synthesis. Inhibition of NOS results in lowered ovulations that may be due to the requirement for N O for optimum blood flow, prostaglandin production, and inhibition of atresia.

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measurement of NO production, that is, the synthesis of NO over a discrete amount of time. Nonetheless, utilizing these methods, ovarian NO levels have been shown to increase during the periovulatory period. Several studies in women have reported that plasma nitrites and nitrates are highest during the follicular phase and peak at midcycle in both normally cycling women (Rosselli et al., 1994; Cicinelli et al., 1996) and in women undergoing hormonal stimulation of their cycles (Rosselli et al., 1994). In addition, follicular fluid nitrite and nitrate content correlated positively with IL- 1P concentration and with the numbers of oocytes retrieved (Tao et al., 1997). Follicular fluid nitrite and nitrate content has also been reported to positively correlate with follicular volume in one study (Anteby et al., 1996) but to have no correlation with ovum diameter in another study (Sugino et al., 1996). While these data are correlative, these studies support a relationship between IL- 1P-induced NO synthesis and ovulation. Studies in rodents have focused on the induction of ovarian NO synthesis primarily by cytokines. Rat ovarian NO production increases in response to hCG and IL-1P in the perfused ovary (Bonello et al., 1996) and in vivo (Nakamura et al., 1996). Cultured immature rat ovarian cells (Ben-Shlomo et al., 1994; Ellman et al., 1993) and isolated rat granulosa cells (Tabraue et al., 1997) synthesize NO in a dose-dependent manner in response to IL- 1 p. Furthermore, TNFa stimulates the NO pathway in isolated bovine thecal cells, causing increased cGMP levels, the significance of which is currently not clear (Tabraue et al., 1997). Taken together, these data demonstrate that the NO pathway is operative and sensitive to cytokine stimulation during the periovulatory period and suggests that NO is a downstream mediator of LWhCG and IL- l p in the ovulatory process. B.

Expression, Location, and Regulation

The expression of all three NOS isoforms during the periovulatory period has been investigated using a variety of methods and animal models. In the superovulated rodent model, utilizing immunoblotting and RT-PCR techniques, several investigators have identified ecNOS and iNOS in the ovary (Van Voorhis et al., 1995; Jablonka-Shariff and Olson, 1997b; Zackrisson et al., 1996; Powers et al., 1996; Matsumi et al., 1998). Using isoform-specific antibodies, ecNOS was localized to the thecal and stromal layers of the preovulatory follicle (Van Voorhis et al., 1995; Jablonka-Shariff and Olson, 1997b; Zackrisson et al., 1996; Powers et al., 1996). Results from our laboratory have also demonstrated ecNOS expression in rat granulosa cell layers and on the oocyte surface (Jablonka-Shariff and Olson, 1997b). In addition to cell-specific expression of ecNOS in the rat ovary, the overall levels of ovarian ecNOS significantly increased in response to exogenous gonadotropin stimulation, particularly during the periovulatory period following hCG injection (Jablonka-Shariff and Olson, 199713; Van Voorhis et al., 1995; Zackrisson et al., 1996). Similarly, ecNOS mRNA levels increased following hCG administration in rat ovaries (Van Voorhis et al., 1995). In addition to superovulated rodents, adult cy-

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cling female rats have been reported to show ecNOS-positive immunostaining in primary and secondary follicles during proestrus and estrus (Chatterjee et al., 1996). The collective evidence demonstrates that cells within the follicular unit express ecNOS during both normal and enhanced follicular development. Furthermore, ecNOS mRNA and protein are induced by gonadotropins. The pattern of iNOS expression differs from that of ecNOS and is less consistent between laboratories. In our laboratory, iNOS protein was unchanged in response to PMSG administration but increased sixfold by 12 h following hCG administration and was localized exclusively in the stromal and thecal layers of preovulatory follicles of the rat. Similarly, Zackrisson and colleagues utilized immunohistochemical techniques to localize iNOS in rat ovarian thecal and stromal cells. However in the same study he detected iNOS by Western blotting in granulosa cells of preovulatory follicles (Zackrisson et al., 1996). In contrast to iNOS protein levels, amounts of iNOS mRNA are highest in unstimulated rat ovaries and decline following PMSG and hCG administration (Van Voorhis et al., 1995). Furthermore, iNOS mRNA was detected only in granulosa cells of primary follicles (Van Voorhis et al., 1995). In another study, iNOS mRNA levels were reported to significantly decline within 6 h of PMSG administration but returned to control untreated levels by 48 h following PMSG (Matsumi et al., 1998). In this case, both iNOS mRNA and protein were identified in granulosa cells of immature follicles, although the intensity of the signals were reported to fluctuate considerably (Matsumi et al., 1998). In normal cycling rats, iNOS mRNA has been found to be highest during early proestrus (Srivastava et al., 1997). In light of these differences, it is difficult at this time to make firm conclusions concerning the localization and regulation of ovarian iNOS. Certainly iNOS is basally expressed in the ovary and its level is regulated by gonadotropins. Furthermore, while the disparity between iNOS mRNA and protein levels during the periovulatory period could be explained by a complex posttranscriptional form of regulation by hCG, the dissimilarity in localization are difficult to interpret and are most probably due to technical differences. Expression of ncNOS protein or mRNA has not been found in the superovulated rat ovary (Van Voorhis et al., 1995; Zackrisson et al., 1996; Jablonka-Shariff and Olson, 1997b). In contrast however, ncNOS mRNA was detected using RNase protection assays and immunoblotting in the cycling rat (Srivastava et al., 1997), bovine (Majewski et al., 1995), porcine (Majewski et al., 1995), and rabbit (Hesla et al., 1997) ovary.

C.

Inhibitor Studies

The direct involvement of NO in the ovulatory process has been most clearly defined by examining the influence of NOS inhibitors (Figure 1) such as N-o-nitro-L-arginine methyl ester (L-NAME) or L-N6-( 1-iminoethy1)lysine (L-NIL) on the number of oocytes ovulating in response to hCG. The first report identified that administration of NOS inhibitors either intraperitoneally or in-

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tracbursally significantly lowered the number of oocytes retrieved from the ampullae of hyperstimulated rats (Shukovski and Tsafriri, 1995). The specificity of the response was further strengthened utilizing a drug that generated NO and ameliorated the lowered ovulations observed with the administration of NOS inhibitors (Shukovski and Tsafriri, 1995). This primary report has been corroborated by a number of studies that demonstrate that administration of NOS inhibitors to perfused ovaries and in vivo reduce the number of oocytes released in rats, mice, and rabbits (Powers et al., 1995; Bonello et al., 1996; Yamauchi et al., 1997; Kugu et al., 1995). These studies provide compelling evidence that NO plays a major role in maintaining an efficient rate of ovulation. D. Mechanisms/Functions Regulation of Blood Flow

Dissociation of the follicle wall and eventual rupture is known to be dependent on the hydrostatic pressure within the follicular antrum (Reynolds et al., 1992; Niswender et al., 1976; Espey, 1994), which in turn is dependent on the hydrostatic pressure within the capillary vasculature of the theca interna (Espey, 1994). Endothelial cell-derived NO is a recognized signal for vasodilation of smooth muscle in homeostatic blood pressure regulation (Nathan, 1992; Moncada et al., 1991). As the theca expresses both iNOS and ecNOS, it follows that one mechanism through which NO may affect ovulatory efficiency is via its modulation of ovarian blood flow. Utilizing the perfused rat ovary, Bonello and colleagues have shown that LH/hCG or IL-1 P-induced ovulations are significantly lowered when NOS inhibitors are also perfused (Bonello et al., 1996). Furthermore, they noted that administration of NOS inhibitors resulted in a significant reduction in perfusion flow rate. When they equalized the flow rates between treatment groups, they partially ameliorated the lowered ovulation rate observed with NOS inhibitors. Thus, changes in blood flow rate in response to the vasodilatory action of NO may have a profound influence on ovulation efficiency in vivo. Regulation of Prostaglandin Biosynthesis

It is now very clear that prostaglandins are key regulators of the ovulatory process and function in both follicular rupture and regulation of vascular permeability (Orczyk and Behrman, 1972; Tsafriri et al., 1972; Tsafriri et al., 1993; Espey, 1994). Indeed, LH increases the inducible isoform of prostaglandin synthase (PGHS-2) (Sirois, 1994; Sirois et al., 1992), and mice lacking the PGHS-2 gene are anovulatory and therefore infertile (Lim et al., 1997; Dinchuk et al., 1995). NO directly activates both the constitutive and inducible isoforms of prostaglandin synthase (Salvemini et al., 1993) and modulates prostaglandin synthesis in the rat

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ovary (Olson et al., 1996a), the oocyte-cumulus complexes (Jawerbaum et al., 1997), and the perfused rabbit ovary (Yamauchi et al., 1997). Therefore, an additional means through which NO may function in the ovulatory process is via its regulation of prostaglandin synthesis. Steroidogen esis

NO is known to interact with heme or iron-containing proteins including those in the P450 family (Wink et al., 1993). Since all the steroidogenic enzymes belong to the P450 family, several studies have examined whether steroidogenesis is altered by NO. While NO appears to definitely regulate luteal steroidogenesis (discussed in greater detail later), its regulation of follicular steroidogenesis is less conclusive. In women, plasma nitrite and nitrates have shown a positive correlation with plasma estradiol concentration (Anteby et al., 1996; Rosselli et al., 1994), and estradiol increases ecNOS activity and mRNA in guinea pigs (Weiner et al., 1994). Thus, the reported positive correlation between estradiol and NO is likely due to regulation of NO synthesis by estradiol rather than an effect of NO on steroidogenesis. Treatment of perfused ovaries with NOS inhibitors has been reported to increase (Yamauchi et al., 1997) and decrease (Bonello et al., 1996) estradiol concentrations in the rabbit and rat respectively, while progesterone synthesis was unaffected (Yamauchi et al., 1997; Bonello et al., 1996). In cultured porcine granulosa cells, NO has been implicated as a negative regulator of steroidogenesis (Masuda et al., 1997). Treatment of purified granulosa cells with an NO donor resulted in decreased aromatase activity (Masuda et al., 1997). Furthermore, inhibition of NOS resulted in a dose-dependent increase in estradiol synthesis from both basal and gonadotropin-stimulated cells (Masuda et al., 1997). Atresia

Most studies designed to examine the role of NO in ovulation have utilized pharmacological inhibitors of NOS that are administered either beforc.or concurrently with the ovulatory dose of hCG. Therefore, NO synthesis would be inhibited throughout the last 8 to 10hours of follicular growth and development. An alternative explanation for the reduction in ovulated oocytes is that a lack of NO increases the rate of follicular atresia involving apoptotic cell death. Utilizing a model of atresia in which preovulatory follicles are cultured in vitro, Chun and colleagues (1995) have demonstrated that apoptotic granulosa cell death was prevented by IL- 1p. Follicular NO synthesis was also increased by IL-lp, and both effects were abrogated by the IL- 1p receptor antagonist. This effectively linked IL- 1p stimulation of NO synthesis and inhibition of apoptosis. Further proof of this function for NO arose by treating follicles with pharmacological donors of NO or with cGMP, a known second messenger of NO, both of which significantly suppressed follicular death.

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NO has also been implicated in the regulation of the receptor for epidermal growth factor (Hattori et al., 1996), a cytokine that has been shown to be an effective inhibitor of ovarian atresia (Hsueh et al., 1994). NO donors significantly increased the numbers of '251-EGFbinding sites in rat granulosa cells (Hattori et al., 1996). While NO donors also increased cGMP levels, dibutyryl-cGMP was ineffective in regulating EGF binding sites, suggesting that the effect of NO was independent of cGMP (Hattori et al., 1996). These studies demonstrate that NO plays an important role in preventing ovarian atresia and that NO is a downstream mediator of IL-1p. This signal transduction pathway utilizes both cGMP- dependent and cGMPindependent mechanisms for its actions. E.

lsoform Specific Functions

The ovary expresses multiple isoforms of NOS that are expressed in a cell-specific manner and are regulated by gonadotropins in distinct ways. Due to the potential multiple roles for ovarian NO, one may wonder if each isoform synthesizes NO for a particular function. Some hints regarding the isoform specific functions of ovarian NO have been derived from experiments utilizing NOS inhibitors, which vary in their selectivity for the NOS isoforms. Two studies have documented that aminoguanidine, at doses that selectively inhibit iNOS (Misko et al., 1993),was as effective at reducing the number of ovulated oocytes as nonselective NOS inhibitors (Shukovski and Tsafriri, 1995; Yamauchi et al., 1997). These inhibitor studies imply that iNOSderived NO may be more critical than ecNOS-derived NO for the ovulatory process. The relative importance of ecNOS and iNOS for ovulation has been clarified by examining the fertility of mice in which the ecNOS or iNOS gene has been disrupted (Jablonka-Shariff and Olson, 1998). In contrast to the inhibitor studies, our experiments with the ecNOS and iNOS knockout mice suggest that ecNOS-derived rather than iNOS-derived NO is the critical player for ovulation. We have observed that ecNOS-deficient breeding pairs had significantly fewer pups in each litter than either wild-type or iNOS-deficient breeding pairs (Jablonka-Shariff and Olson, 1998). Surprisingly, iNOS-deficient and wildtype breeding pairs were equally productive (Beltsos et al., 1997). We also superovulated immature wild-type, ecNOSdeficient, and iNOS-deficient female mice and counted the number of oocytes in the ampullae 16 h following hCG administration. While both ecNOS-deficient and iNOS-deficient mice showed a lowered ovulation rate compared with wild-type mice, the deficiency in ecNOS resulted in more profound suppression of ovulation than that observed in iNOS knockout mice (Beltsos et al., 1997; Jablonka-Shariff and Olson, 1998). We must keep in mind that the single knock-out phenotypes may be somewhat misleading because the ovary expresses both ecNOS and iNOS. In the iNOS-deficient mice, ecNOS-derived NO may be very effective in compensating for the lack of iNOS-derived NO, since eNOS is widely expressed throughout the ovary. The ecNOS-deficient mice may show a more severe reproductive phenotype because iNOS is expressed in only the theca and stroma. iNOS-dervied NO may not

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be able to diffuse into the follicle and compensate effectively for the lack of ecNOS. The definitive answer to this caveat must await the generation of the double knockout, which will be deficient in both ecNOS and iNOS. Thus, our studies support the hypothesis that NO is an important modulator of ovulation. Currently, however, our evidence favors a central role for eNOS rather than iNOS-derived NO.

IV.

NO AND OOCYTE MATURATION

Our observation of the expression of ecNOS on the oocyte surface and the lowered ovulations associated with a lack of NO suggested that NO may play a role in oocyte maturation. Oocytes, which are arrested in prophase I, normally resume meiosis in response to the LH surge and release the first polar body before arresting again at metaphase I1 (Tsafriri, 1978). We examined oocytes obtained 17 h following an ovulatory dose of hCG, either from rats treated with NOS inhibitors or from mice in which the ecNOS gene had been disrupted (Jablonka-Shariff and Olson, 1999; Jablonka-Shariff and Olson, 1998). Ovulated oocytes were counted and classified as being at the following stages of meiosis: Germinal Vesicle [GV; intact germinal vesicle), Metaphase I (GV breakdown without release of the first polar body), Metaphase I1 (1st polar body released), or showing atypical morphology. We found fewer than 5% of all oocytes were at the GV stage, regardless of treatment, indicating that the majority of oocytes had resumed meiosis. Similarly, while perfusion of rabbit ovaries with NOS inhibitors lowered the number of ovulated oocytes, no influence of NOS inhibition on germinal vesicle breakdown was reported (Yamauchi et al., 1997). However, we documented that a lack of NO, specifically ecNOSderived NO, resulted in abnormal meiotic development. When NO was lacking, fewer ovulated oocytes were at metaphase 11, the normal stage of meiosis, with significantly greater number of oocytes either present at metaphase I or showing atypical morphology. The results were very similar regardless of whether NO was absent during both follicular development and the periovulatory period (as was the case with ecNOS KO mice) or just during the periovulatory period (as was the case with rats treated with NOS inhibitors) (Jablonka-Shariff and Olson, 1397a; JablonkaShariff and Olson, 1998). We also found that oocytes expressed ecNOS during follicular development and retained their expression of ecNOS following ovulation (Jablonka-Shariff and Olson, 1998). These data suggest that NO is required for optimum oocyte maturation and may be playing an essential role for the transition through metaphase. Oocytes showing atypical degenerative morphology may arise either by failing to undergo the transition through metaphase I or failing to arrest at metaphase I1 (Figure 3). It is clear that the atypical oocytes are dying since they show strong positive staining utilizing the TUNEL method, which indicates cells undergoing cell death (Jablonka-Shariffand Olson, 1998). It is unknown whether NO is regulating internal events within the oocyte or whether oocytes utilize NO to communicate with

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Prophase I

GVBD

Metaphase I

Metaphase 2

Atypical

Figure 3. Model for the influence of NO on oocyte meiotic maturation. Oocytes arrested in prophase of meiosis I are induced to resume meiosis following the LH surge. The resumption of meiosis involves germinal vesicle breakdown (GVBD), progression through metaphase I, and a secondary arrest at metaphase II following release of the first polar body. When NO is lacking, we have observed a decrease in oocytes at metaphase II (normal stage following ovulation) and increases in the numbers of oocytes at metaphase I and showing atypical morphology. These data suggest that NO may be essential for both the transition through rnetaphase I as well as arrest at metaphase II.

their surrounding granulosa cells. Clearly, while these data are promising, much remains to be clarified regarding the role of NO in oocyte maturation.

V.

NO AND LUTEAL FUNCTION

A study designed to determine the amount of NO synthesized at different stages of the rat ovarian cycle demonstrated that cells derived from the corpus luteum synthesized the greatest level of NO relative to cells obtained at any other stage (Olson et al., 1996b). Using the pseudopregnant rat model, we have demonstrated that luteal cells expressed ecNOS very strongly throughout pseudopregnancy with a homogenous distribution within the corpus luteum (Jablonka-Shariff and Olson, 1997b). In contrast, in early pseudopregnancy, iNOS was strongly expressed only by stromal cells surrounding the corpus luteum (Jablonka-Shariff and Olson, 1997b). Only in late pseudopregnancy (days 8-10), did we observe positive staining for iNOS within the corpus luteum (Jablonka-Shariff and Olson, 1997b). Its known that leukocytes and macrophages infiltrate the aging corpus luteum (Lei et al., 1991; Bagavandoss et al., 1990) and thus we suspected that the iNOS-positive cells present in the late corpus luteum may be of that lineage.

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IL-1P and tumor necrosis factor are powerful inducers of iNOS in a variety of cell types (Drapier et al., 1988; Ben-Shlomo et al., 1994; Ellman et al., 1993) and have potent luteolytic actions in bovine (Benyo and Pate, 1992; Roby and Terranova, 1990) and rat (Ahsan et al., 1997) luteal cells. The high level of nitrite production and the changes in expression of iNOS in late pseudopregnancy supports a model where cytokine-induced, iNOS-derived NO may function as a luteolytic agent. Indeed, NO has profound effects on steroidogenesis of luteal cells (Olson et al., 1996b; Snyderet al., 1996; Van Voorhis et al., 1994; Ahsan et al., 1997; Dave et al., 1997). Progesterone synthesis of cultured rat granulosa cells obtained following estrus was lowered with NO donors (Dave et al., 1997). Furthermore, co-cultures of rat luteal cells with activated peritoneal macrophages synthesized larger amounts of NO and less progesterone than rat luteal cells cultured alone. The reduced progesterone synthesis in the co-cultures could be prevented by the iNOS-specific inhibitor, aminoguanidine. These experiments support the idea that NO synthesized by macrophages inhibits progesterone synthesis and therefore functions as a luteolytic agent. However, these experiments were conducted utilizing activated peritoneal macrophages and it is unknown whether these macrophages reflect the physiological function of recruited or resident ovarian macrophages during the luteal phase. In contrast to the previous experiments, culturing human granulosa-lutein and rat luteal cells with NOS inhibitors resulted in increased estradiol synthesis with no effect on progesterone synthesis (Van Voorhis et al., 1994; Olson et al., 1996b). However, addition of drugs that generated NO in vitro decreased both estradiol and progesterone synthesis by human and rat luteal cells (Van Voorhis et al., 1994; 01son et al., 1996b). Taken together, these data suggest that NO primarily inhibits luteal estradiol synthesis andlor secretion. Indeed, Van Voorhis and colleagues have shown that NO inhibits the catalytic activity of aromatase by measuring the release of 'H,O from [ 1P-3H] androstenedione (Van Voorhis et al., 1994). NO has been reported to alter protein function via a number of mechanisms including binding to heme moieties of proteins as well as interacting with sulfhydryl groups of cysteines with subsequent formation of nitrosothiols (Stamler, 1994). Snyder.and colleagues have suggested that the latter mechanism is the route on which NO inhibits aromatase activity because addition of mercaptoethanol, which provided an excess of sulfydryl groups, effectively blocked the NO-induced inhibition of aromatase (Snyder et al., 1996). In the rat corpus luteum, estradiol is required to maintain optimum progesterone synthesis and adequate luteal function (Gibori et al., 1988; Keyes et a]., 1980;Gibori et al., 1984). Thus, inhibition of estrogen synthesis by nitric oxide may be an important regulator of luteal lifespan. Luteal NO synthesis has also been reported to increase rat luteal PGF,, levels with no effect on PGE, (Motta et al., 1997).Furthermore, culturing ovaries obtained from pseudopregnant rats with oxytocin specifically increased ovarian PGF,, synthesis, which was inhibited by L-NAME, a NOS inhibitor (Motta et al., 1997).

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Since, PGF,, is a very potent luteolytic agent, these data also support a role for NO in luteal regression and suggest that NO is a downstream regulator of oxytocindependent production of PGF,, and subsequent luteal demise. A complicating twist in this scenario is the high expression of ecNOS throughout pseudopregnancy. What is the function for ecNOS-derived NO and does it also regulate estradiol synthesis? It is possible that ecNOS synthesizes small amounts of NO within the luteal cell, which diffuses to the smooth muscle surrounding the ovarian blood vessels to maintain adequate blood flow. Indeed, blood flow to the ovary is significantly increased with pregnancy and is reduced when NOS is inhibited (Kassab et al., 1998). Furthermore, NO release is a prerequisite for epithelial and endothelial cell motility and plays essential roles in angiogenesis (Noiri et al., 1997). Thus, luteal NO synthesis may also be an important modulator of luteal angiogenesis and blood flow.

VI.

SUMMARY

While still a relatively new area, there is compelling evidence that NO is a local regulator of a number of key ovarian processes. Clearly, NO is a key regulator of the ovulatory process. The ovary expresses ecNOS and iNOS in a cell- and development-specific pattern. NO synthesis increases during the periovulatory period in response to both LH/hCG and IL- 1p. When NOS is inhibited with drugs or is genetically disrupted, ovulation and oocyte maturation are significantly impaired. Periovulatory NO synthesis has been implicated in the regulation of ovarian blood flow, prostaglandin synthesis, steroidogenesis, atresia, and the meiotic cell cycle. NO also has key functions during the luteal phase. The corpus luteum expresses the highest level of ecNOS found during the ovarian cycle and iNOS expression also increases significantly during the late luteal phase. Three important potential functions for luteal NO are regulation of steroidogenesis, angiogenesis, and blood flow. While several functions for NO have been identified, the signal transduction pathway(s) that are utilized by NO at critical stages of ovarian development have not yet been definitively elucidated. The ecNOS- and iNOS-deficient mice provide excellent tools for the dissection of the relative functions for each isoform in the ovary. Furthermore, the study of ovarian NOS provides an experimental system in which the roles of ecNOS and iNOS in development (follicular and oocyte maturation) and differentiation (formation of the corpus luteum) can be studied.

ACKNOWLEDGMENTS The author acknowledges and appreciates the dedication and hard work of Dr. Albina Jablonka-Shariff,Dr. Angeline Beltsos, Ms. Sapna Ravi, and Ms. Charlotte Jones.

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Espey, L.L. (1994). Current Status of the hypothesis that mammalian ovulation is comparable to an inflammatory event. Biol. Reprod. 50:233-238. Gibori, G., Chen, Y.-D.I., Khan, I., Azhar, S . , and Reaven, G.M. (1984). Regulation of luteal cell lipoprotein receptors, sterol contents, and steroidogenesis by estradiol in the pregnant rat. Endocrinology 114:609-617. Gibori, G., Khan, I., Warshaw, M.L., McLean, M.P., Puryear, T.K., Nelson, S., Durkee, T.J., Azhar, S., Steinschneider, A., and Rao, M.C. (1988). Placental-derived regulators and the complex control of luteal cell function. Recent Prog. Horm. Res. 44:377-429. Griffith, O.W. and Stuehr, D.J. (1995). Nitric oxide synthases: Properties and catalytic mechanism. Ann. Rev. Physiol. 57707-736. Haddad, I.Y., Pataki, G., Hu, P., Galliani, C., Beckman, J.S., and Matalon, S. (1994). Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J.Clin.Invest. 94~2407-2413. Hattori, M.-A,, Sakamoto, K., Fujihara, N., and Kojinia, 1. (1996) Nitric oxide: A modulator for the epidermal growth factor receptor expression in developing ovarian granulosa cells. Am. J. Physiol. 270:C812-C8 18. Henry, Y., Lepoivre, M., Drapier, M. C., Ducrocq, C., Boucher, J.L., and Guissani, A. (1993). EPR characterization of molecular targets for NO in mammalian cells and organelles. Faseb J. 7:1124-1134. Hesla, J.S., Preutthipan, S., Maguire, M.P., Chang, T.S.K., Wallach, E.E., and Dharmarajan, A.M. (1997). Nitric oxide modulates human chorionic gonadotropin-induced ovulation in the rabbit. Fertil. Steril. 67548-552. Hevel, J.M. and Marletta, M.A. (1994). Nitric-Oxide Synthase Assays. Meth. in Enzymol. 233:250-258. Hsueh, A.J.W., Billig, H., and Tsafriri, A. (1994). Ovarian follicle atresia: A hormonally controlled apoptotic process. Endocr. Rev. 15:707-724. Huang, J . , Roby, K.F., Pace, L., Russell, S.W., and Hunt, J.S. (1995). Cellular localization and hormonal regulation of inducible nitric oxide synthase in cycling mouse uterus. J . Leukoc. Biol. 57:27-35. Ignarro, L.J. (1990). Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu. Rev. Pharmacol. Toxicol. 30:35-60. Jablonka-Shariff, A. and Olson, L.M. (1997a). Ovarian nitric oxide is necessary for optimum ovulation and oocyte meiotic maturation. Biol. Reprod. 56:232. Jablonka-Shariff, A. and Olson, L.M. (1997b). Hormonal regulation of nitric oxide synthases and their cell-specificexpression during folliculardevelopment in the rat ovary. Endocrinology 138:460-468. Jablonka-Shariff, A. Basuray, R., and Olson, L.M. (1999). Inhibitors of Nitric Oxide synthase influence oocyte maturation in rats. J. SOC.Gynecol. Investig. (In press.) Jahlonka-Shariff, A. and Olson, L. M. (1998) The role of nitric oxide in oocyte meiotic maturation and ovulation: Meiotic abnormalites of endothelial nitric oxide synthase knock-out mouse oocytes. Endocrinology 139:2944-2954. Jawerbaum, A., Gonzalez, E.T., Faletti, A,, Novaro, B., and Gimeno, M.A.F. (1997). Nitric oxide mediates human chorionic gonadotrophin-induced prostaglandin E generation in rat oocyte-cumulus complexes. Reprod. Fertil. Dev. 9:39 1-394. Kassab, S., Miller, M.T., Hester, R., Novak, J., and Granger, J.P. (1998). Systemic hemodynamics and regional blood flow during chronic nitric oxide synthesis inhibition in pregnant rats. Hypertension 31:3 15-320. Keyes, P.L., Gibori, G., Possley, R.M., and Brown, J.M. (1980). Early changes in luteal function associated with the luteotropic effect of testosterone in the pregnant rat. Biol. Reprod. 22: 1142-1148. Kol, S . and Adashi, E.Y. (1995). lntraovarian factors regulating ovarian function. Cum. Opin. Ob. Gyn. 71209-213.

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Kugu, K., Dharmarajan, A.M., Preutthipan, S., and Wallach, E.E. (1995). Role of calciudcalmodulin-dependent protein kinase I1 in gonadotrophin-induced ovulation in in-vitro-perfused rabbit ovaries. J. Reprod. Fert. 103:273-278. Kunz, D., Muhl, H., Walker, G., and Pfeilschifter, J. (1994). Two distinct signaling pathways trigger the expression of inducible nitric oxide synthase in rat renal mesangial cells. Proc. Natl. Acad. Sci. USA 9115387-5391. Lamas, S., Marsden, P.A., Li, G.K., Tempst, P., and Michel, T. (1992). Endothelial nitric oxide synthase: Molecular cloning and characterization of a distinct constitutive enzyme isofom. Proc. Natl. Acad. Sci. USA 89:6348-6352. Lander, H.M., Ogiste, J.S., Pearce, S.F.A., Levi, R., and Novogrodsky, A. (1993) Nitric oxide-stimulated guanine nucleotide exchange on p2lras. J. Immunol. 151:7182-7187. Lander, H.M., Jacovina, A.T., Davis, R.J., and Tauras, J.M. (1996). Differential activation of mitogen-activated protein kinases by nitric oxide-related species. J. Biol. Chem. 271: 19705-19709. Lei, Z.M., Chegini, N., and Rao, C.V. (1991). Quantitative cell composition of human and bovine corpora lutea from various reproductive states. Biol. Reprod. 44:1148-1156. Lim, H . , Paria, B.C., Das, S.K., Dinchuk, J.E., Langenbach, R., Trzaskos, J.M., and Dey, S.K. (1997). Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91 : 197-208. MacMicking, J., Xie, Q.-W., and Nathan, C. (1997). Nitric oxide and macrophage function. Annu. Rev. Immunol. 15:323-350. Majewski, M., Sienkiewicz, W., Kaleczyc, J., Mayer, B., Czaja, K., and.Lakomy, M. (1995). The distribution and co-localization of immunoreactivity to nitric oxide synthase, vasoactive intestinal polypeptide and substance P within nerve fibres supplying bovine and porcine female genital organs. Cell Tissue Res. 281:445-464. Masuda, M., Kubota, T., Kamada, S., and Aso, T. (1997). Nitric oxide inhibits steroidogenesis in cultured porcine granulosa cells. Mol. Human Reprod. 3:285-292. Matsumi, H., Yano, T., Koji, T., Ogura, T., Tsutsumi, O., Taketani, Y., and Esumi, H. (1998). Expression and localization of inducible nitric oxide synthase in the rat ovary: A possible involvement of nitric oxide in the follicular development. Biochem. Biophys. Res. Commun. 243:61-12. Misko, T.P., Moore, W.M., Kasten, T.P., Nickols, G.A., Corbett, J.A., Tilton, R.G., McDaniel, M.L., Williamson, J.R., and Currie, M.G. (1993). Selective inhibition of the inducible nitric oxide synthase by aminoguanidine. Eur. J. Pham. 233:119-125. Moncada, S., Palmer, R.M., and Higgs, E.A. (1991). Nitric oxide: Physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43:109-142. Motta, A.B., Franchi, A.M., and Gimeno, M.F. (1997). Role of nitric oxide on uterine and ovarian prostaglandin synthesis during luteolysis in the rat. Prostaglandins, Leukotrienes, and Essential Fatty Acids. 56:265-269. Nakamura, Y., Ono, M., Nakata, M., Tamura, H., Takiguchi, S., Sugino, N., Shimamura, K., and Kato, H. (1996). Nitric oxide synthase (NOS) activity in the ovary during ovulation in immature rats injected pregnant mare serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG). BioLReprod. 54:68. (Abstract). Nathan, C. (1992). Nitric oxide as a secretory product of mammalian cells. Faseb J. 6:3051-3064. Niswender, G.D., Reimers, T.J., Diekman, M.A., and Nett, T.M. (1976). Blood flow: A mediator of ovarian function. Biol. Reprod. 14:64-81. Noiri, E., Hu, Y., Bahou, W.F., Keese, C.R., Giaever, I., and Goligorsky. M.S. (1997). Permissive role of nitric oxide in endothelin-induced migration of endothelial cells. J. Biol. Chem. 272: 1741-1752. Olson, L.M., Jablonka-Shariff, A., Salvemini, D., and Masferrer, J. (1996a). Inhibitors of nitric oxide synthase (NOS) lower ovulation rate: Interaction between nitric oxide and prostaglandin synthesis. Xlth Ovarian Workshop 43 (Abstract).

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THE REGULATION AND REPROGRAMMING OF GENE EXPRESSION IN THE PREIMPLANTATION EMBRYO

Richard M. Schultz

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Maternal-to-Zygotic Transition: Functions of ZGA 111. Time of Initiation of ZGA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Differences in the Transcriptional Activity between the Male and Female Pronuclei . . . . . . . . . . . . . . . . . V. Role of DNA Replication in Reprogramming Gene Expression VI. Enhancer and TATA-Box Requirements for Gene Expression A. Changes in Enhancer Utilization. . . . . . . . . . . . . . . . . . . . . B. Changes in TATA-Box Utilization . . . . VII. Development of a Transcriptionally Repres VIII. Nature of the Transcriptionally Repressive IX. Potential Uncoupling of Transcription and Translation in the One-Cell Embryo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Developmental Biochemistry Volume 5, pages 129-164. Copyright 0 1999 by JAI Press Inc. All right of reproduction in any form reserved. ISBN: 0-7623-0202-X

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X. Molecular Basis for the Time of Onset of ZGA . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Clinical Ramifications of the Timing of ZGA . . . . . . . . . . . . . . . . . . . . . . . . . . . XII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

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INTRODUCTION

Preimplantation development is characterized by three major developmental transitions that occur following fertilization. The first is the maternal-to-zygotic transition, which is also referred to as zygotic gene activation (ZGA) or embryonic genome activation (EGA), in which maternal transcripts that direct early development are replaced by transcripts expressed from the zygotic/embryonic genome. The expression of these embryonic transcripts is essential for further develop, since inhibiting their expression (e.g., by the addition of a-amanitin) results in cleavage arrest usually within one cleavage division following addition of the inhibitor of transcription. The second transition is compaction. Prior to compaction, the individual blastomeres of the developing embryo loosely adhere to one another and are clearly discernible under the light microscope (Johnson and Maro, 1986). During compaction the blastomeres flatten on one another such that following compaction it is difficult to clearly delineate the boundaries between the blastomeres. The loss of clearly definable cell boundaries is due to the formation of adherens junctions (E-cadherinbased), tight junctions, and gap junctions. The blastomeres of the compacted embryo are also highly polarized (Johnson and Maro, 1986). For example, the apical surface contains numerous microvilli, whereas the basolateral surface is relatively amicrovillar; this constitutes surface polarity. A cortical domain of filamentous actin concentrates under the apical surface, microtubules align parallel to the basolateral surface, and endocytotic vesicles lie between the apical region and the basolaterally located nucleus; these constitute cytoplasmic polarity. Thus, compaction results in the formation of a communicating polarized epithelium. The third transition is the differentiation of the morula into the blastocyst, which is composed of totipotent cells of the inner cell mass (ICM) that will give rise to the embryo proper, and the differentiated cells of the trophectoderm (TE), which is a fluid-transporting epithelium and that will give rise to extraembryonic tissue. Fluid accumulation in the blastocoel is due to an energy-dependent transepithelial sodium flux (Biggers et al., 1988). Sodium ions enter the trophectoderm cells via numerous apically located channels that are permeant for sodium (e.g., an amiloride-sensitive sodium channel; Manejwala et al., 1989; Zhao et al., 1997). A basolaterally located sodiudpotassium ATPase then actively pumps the intracellular sodium into the blastocoel. The presence of the tight junctions between the trophectoderm cells retains the sodium ions in the blastocoel. An osmotically-driven,

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passive movement of water across the trophectoderm results in blastocoel formation. The movement of other ions such as chloride and bicarbonate also contributes to blastocoel formation. The formation of the blastocoel is essential for the further development of the ICM cells. Each of these developmental transitions is accompanied by major changes in the pattern of gene expression. While cell-cell interactions appear to be critical for the changes in gene expression that occur during compaction and the formation of the blastocyst, little is known regarding the molecular basis underlying these changes in gene expression. In contrast, our understanding of the molecular basis of the maternal-to-zygotic transition has deepened dramatically during the past decade. This review will focus almost exclusively on this first transition during preimplantation development in the mouse.

11.

MATERNAL-TO-ZYGOTIC TRANSITION: FUNCTIONS OF ZGA

During oocyte growth, the oocyte synthesizes and accumulates organelles and macromolecules (e.g., mitochondria, mRNAs, and proteins) that will constitute the maternal contribution to early development. Following fertilization these maternal components direct early development prior to ZGA. ZGA must be initiated and successfully executed in order for further development to continue, and in fact, if this transition does not occur, the embryo will arrest development usually within one cleavage division. For example, one-cell mouse embryos cultured in medium containing the transcription inhibitor a-amanitin will cleave to the two-cell stage but not to the four-cell stage. There are some strains of mice, however, in which about 15% of such a-amanitin-treated embryos cleave to the three- to four-cell stage (Rambhatla and Latham, 1995), and this may reflect a greater maternal endowment in these strains. Nevertheless, the maternal control over early development in the mouse appears to be limited to the first two cell divisions. The function in further development, if there are any, of maternally derived, oocyte-specific proteins and mRNAs that survive beyond he time of ZGA is not known. ZGA has at least two functions that are required for the continued progression of development. Oocyte maturation initiates the destruction of maternal RNA and this destruction proceeds through the two-cell stage (Schultz, 1993 and references therein). For example, the amount of rRNA decreases from about 260 pg/oocyte to about 200 pg/egg, and the amount of poly(A+) RNA decreases from about 85 pg to 35 pg during this period. The decrease in poly(A+)RNA is due to both degradation (50 pg) and deadenylation (20 pg). The degradation of maternal mRNA continues into the two-cell stage such that, for many mRNAs, about 90% of the maternal transcript is degraded (e.g., actin). One function of ZGA is therefore to replace maternal transcripts that are common to the oocyte and early embryo (e.g., actin) with zygotic transcripts.

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The second function of ZGA is to bring about a dramatic reprogramming in the pattern of gene expression that is coupled with the generation of novel transcripts that are not expressed in the oocyte. This reprogramming of gene expression is reflected in marked differences in the patterns of protein synthesis following highresolution, two-dimensional gel electrophoresis of metabolically radiolabeled proteins (Latham et al., 1991). Results of these studies that monitored the synthesis of some 1,500polypeptides indicated that about 85% of the polypeptides synthesized in the two-cell embryo revealed changes in their rate of synthesis that differed by at least twofold. Moreover, ZGA resulted in the synthesis of polypeptides that were not expressed in the oocyte. These changes are most pronounced by the mid twocell stage and essentially complete by the four-cell stage. While some of the changes reflect the mobilization of maternal mRNAs, many are due to the generation of zygotic transcripts since a-amanitin inhibits their synthesis. This reprogramming of gene expression is likely the molecular underpinning for the transformation of the differentiated oocyte into the totipotent blastomeres that are present at the two-cell stage. The recent successful cloning of the sheep named Dolly from a transplanted nucleus that was likely obtained from a differentiated mammary epithelial cell is a spectacular example of the early embryo’s capacity to reprogram the pattern of gene expression (Wilmut et al., 1997). Nevertheless, the very low success rate (1 out of 277 embryos transferred) to date implies that this is a very complex process with little margin for error. Consistent with this proposal is the observation that following transplantation of cleavage-stage nuclei to enucleated one-cell mouse embryos, the embryos arrest development at the two-cell stage (McGrath and Solter, 1984). Analysis of gene expression following transplantation of an eight-cell nucleus to an enucleated one-cell embryo revealed pronounced differences in the pattern of gene expression in the resulting two-cell embryos when compared to the normal pattern of expression, as assessed by 2D gels (Latham et al., 1994). These differences, which may reflect an underlying inability of the reconstructed embryos to develop, have been attributed to the inability of the transplanted nucleus to remodel its chromatin during this critical period of ZGA (Latham et al., 1994 and references therein).

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TIME OF INITIATION OF ZCA

To understand the molecular basis for ZGA and the reprogramming of gene expression, it is imperative to know when ZGA occurs. A large body of evidence indicates unequivocally that ZGA has occurred by the two-cell stage. For example, the synthesis of a paternally-derived variant of P,-microglobulin (Sawicki et al., 198l), the formation of paternally-derived intracisternal particles (Szollosi and Yotsuyanagi, 1985), or the expression of a paternally-derived, P-actin promoter-driven luciferase reporter transgene (Matsurnoto et a]., 1994) are first observed in the two-cell

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embryo. As mentioned in section 11, quantitative analysis of high-resolution, twodimensional gels reveals a marked reprogramming in the pattern of gene expression, and moreover, the expression of polypeptides whose synthesis is inhibited by a-amanitin is first observed in the two-cell embryo. Screening of cDNA clones obtained from a two-cell cDNA subtraction library (the library was subtracted with an egg and eight-cell library) reveals the existence of cDNAs that are only expressed during the two-cell stage (i.e., their expression is stage-specific; Rothstein et al., 1992). This result is consistent with the observation that the expression of 38 polypeptides out of the 1,500 polypeptides analyzed transiently increases during the two-cell stage (Latham et al., 199 1). However, the identify of only a few of these polypeptides has been determined for example, transcripts encoding the splicing factor U2afbp-rs (Latham et al., 1995), hsp 70 (Christians et al., 1995), and the translation initiation factor eIF-1 A (formerly called eIF-4C; Davis et al., 1996). Lastly, the expression of a variety of plasmid-borne reporter genes bearing a range of different promoter elements is readily detected in the two-cell embryo. These reporter genes encompass several viral promoters such as SV40 early promoter (Bonnerot et al., 1991; Ram and Schultz, 1993), polyomavirus early promoter (Mtlin et al., 1993), herpes simplex virus thymidine kinase promoter (Wiekowski et al., 1991), adenovirus EIIa (Dooley et al., 1989), and promoters of cellular genes (e.g., hypoxanthine ribosyl transferase promoter, p-actin promoter, and hydroxmethylglutaryl CoA reductase promoter; Vernet et al., 1992; Matsumoto et al., 1994). Conflicting results were obtained in early studies directed toward assessing the transcriptional activity of the one-cell embryo. Assaying endogenous RNA polymerase by monitoring the incorporation of [3H]UTP by lightly fixed and permeabilized one-cell embryos did not reveal any incorporation, as detected by autoradiography, in either the male or female pronucleus (Moore, 1975). Incorporation, however, was observed in the nuclei of two-cell embryos. This suggested that the one-cell embryo was transcriptionally inactive. Initial attempts to radiol-abe1 metabolically one-cell embryos with l3H]uridine were hampered by the poor uptake of L3H]uridine,its metabolism to [3H]UTP, and subsequent incorporation into RNA by the one-cell embryo. Metabolic radiolabeling, however, was achieved with 13H]adenosine,which is taken up around 1,000times faster than uridine, and electrophoretic analysis of the radiolabeled material detected a low level of incorporation into heterogeneous poly(A+) and poly(A-) RNA (Clegg and Pik6, 1982). The ['Hladenosine was incorporated internally and not solely into the poly(A+) tail since one-third of the radioactivity was released as AMP following RNase A and T, digestion under conditions that would leave poly(A) tracts intact. The identity of these transcripts, however, was not determined and thus the question of whether the one-cell embryo is capable of generating functional transcripts remained unresolved (See section IX for further discussion of this point.) More recent studies using far more sensitive assays demonstrated that the onecell embryo is both transcriptionally competent and transcriptionally active. A series of nuclear transplantation experiments revealed that the one-cell embryo has a

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functional RNA polymerase I1 (Latham et al., 1992)(Figure 1). In these experiments, a nucleus from a two-cell donor that has been treated with a-amanitin to inhibit endogenous RNA polymerase I1 was transferred to an enucleated one-cell embryo (Figure 1);under these experimental conditions it was known that the effect of a-amanitin treatment was irreversible. The embryo was then assessed for the expression of an accepted marker of ZGA in G2 of the one-cell embryo. The marker used in these studies was the transcription-requiring complex (TRC), which is a family of structurally-related polypeptides of Mr=68,000, 70,000, and 73,000, whose synthesis transiently increases during the two-cell stage (Conover et al., 1991). (The synthesis of the TRC, which can constitute about 5% of total protein synthesis in the two-cell embryo, gives rise to three polypeptides that are readily detected following 1D SDS-PAGE. The TRC was mistakenly referred to in the earlier literature as corresponding to HSP70 proteins, which were the first products of ZGA to be identified; Bensaude et al., 1983). 1-Cell

+2-Cell

TRC synthesis

Control

Donor

(8 @-

($J '(I Analyze for TRC synthesis in G2

Figure 7. Demonstration of transcriptional competence of one-cell embryos by nuclear transplantation. Donor two-cell nuclei from a-amanitin-treated embryos are transferred to enucleated one-cell embryos in which both the male and female pronuclei have been removed. Following transplantation, the cells are analyzed for TRC synthesis was shown to occur at a time that corresponds to C2 of the one-cell stage. Cytochalasin D, which has no effect on ZGA, is used to generate these donor cells since it is easier to remove nuclei from such treated embryos.

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The rationale of this experiment was that active RNA polymerase I1 in the onecell embryo would complement the inhibited RNA polymerase I1 present in the transplanted two-cell nucleus, which brings with it all of the other transcription factors required for the expression of the TRC. Results of these experiments indicated that TRC expression was readily observed in G2 of the one-cell embryo. Moreover, the level of expression was 30 to 40% that observed when two-cell embryos were radiolabeled for the same length of time. Thus, the one-cell embryo contains functional RNA polymerase 11, that is, it is transcriptionally competent. Results of several studies using plasmid-borne reporter genes also suggested that the one-cell embryo was transcriptionally active. For example, luciferase activity was detected in G2 of the one-cell embryo following injection of the male pronucleus with an Sp 1-dependent-driven luciferase reporter gene during early S phase (Ram and Schultz, 1993); the reporter gene did not contain an enhancer. The level of expression was about 20% of that observed when the reporter gene was injected into the nucleus of a mid-two-cell blastomere. These results indicated that a functional transcription machinery was present in the one-cell embryo. Interestingly, little, if any, expression was detected in G2 if the female pronucleus was injected during S phase (See section IV for further discussion.). Functional RNA polymerase I and I11 are also present in the one-cell embyro (Nothias et al., 1996). Injection of a chloramphenical acetyl transferase reporter gene under the control of the RNA polymerase IMependent ribosomal DNA promoter into the male pronucleus of S phase-arrested, one-cell embryos (the embryos were incubated in the presence of aphidicolin, which inhibits DNA polymerases a and 6) revealed accumulation of the appropriate transcript by G2 of the one-cell embryo. The amount of this transcript was about 20% of that maximally accumulated when the cleavage-arrested embryos were cultured to a time that corresponded chronologically to the two-cell stage and then were analyzed for expression. A similar result was obtained when the S phase-arrested, one-cell embryos were injected with a plasmid bearing the RNA polymerase 111-dependent adenovirus VA1 RNA gene. In this case, the amount of transcript accumulated by G2 of the S phase-arrested, one-cell embryo was around 30% of that maximally accumulated. While this approach using plasmid-borne reporter genes has -furnished many valuable insights into the molecular basis for ZGA, the inherent drawback of the experimental design is that the plasmids are injected as naked DNA. Even though, following injection, the plasmids are assembled into chromatin, as assessed by the formation of superhelical DNA (Martinez-Salas et al., 1989), whether the assembled chromatin structure is the same as that for an endogenous gene in its native chromatin context is not known. Moreover, the male pronucleus inefficiently assembles the injected DNA into chromatin when compared to chromatin assembly following injection of either the oocyte or cleavage-stage nucleus (Martinez-Salas et al., 1989). Thus, the initial production of transcripts could have occurred from plasmids not yet assembled into chromatin and in fact there was an inverse correlation between the efficiency of transcription and chromatin assembly.

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Although the ability of the embryo to transcribe genes in the appropriate chromatin context is not assessed by the use of plasmid-borne reporter genes, the expression of either transgenes or the total pool of endogenous genes, each of which is in a bonafide chromatin context, can also bedetected during the one-cell stage. For example, transcripts for a paternally-derived luciferase reporter gene that is driven by the p-actin promoter were readily detected by G2 of the one-cell embryo (Matsumot0 et al., 1994), as was luciferase activity for a paternally derived HSP70 promoter-dependent luciferase transgene (Christians et al., 1995). For the HSP70 promoter-driven transgene, the amount of luciferase activity detected in the late one-cell embryo was about 30% of the amount maximally expressed during the two-cell stage. The expression of the total endogenous pool of genes was assessed by monitoring the incorporation of BrUTP, which is a substrate for RNA polymerase I1 (Jackson et al., 1993), by antibodies to BrdU that also crossreact with BrU incorporated into RNA. Epifluorescent microscopy of one-cell embryos injected with BrUTP revealed an a-amanitin- and RNase-sensitive incorporation into both pronuclei (Bouniol et al., 1995). Confocal microscopy of permeabilized embryos incubated in a transcription cocktail containing BrUTP also indicated incorporation of BrUTP in an a-amanitin- and RNase-sensitive manner (Aoki et al., 1997); this assay can be viewed as a nuclear run-on assay that measures RNA polymerases actively engaged in transcription at the time of permeabilization. For the BrUTP incorporation by permeabilized one-cell embryos, incorporation was first observed by earlyhid-S phase. Moreover, the amount of incorporation by the male pronucleus was about four to five times greater than that by the female pronucleus (see section IV for further discussion). Quantification of the signal revealed that the amount incorporated by both pronuclei of the G2-stage, one-cell embryo was around 40% of that of the two-cell blastomere in G2 (Aoki et al., 1997); the chromosomal content for both the G2 one-cell embryo and a G2 two-cell blastomere is 4C. In both assay systems (i.e., microinjection of BrUTP or incorporation by permeabilized embryos), the incorporation was punctate but uniform throughout the nucleoplasm. Also, transcription was first detected i n the male pronucleus before it was detected in the female pronucleus (Bouniol etal., 1995; Aoki et al., 1997). This temporal difference in the onset of transcription may be linked to the observation that DNA replication in the male pronucleus initiates prior to that DNA replication in the female pronucleus (Bouniol-Baly et al., 1997: Ferreria and Carmo-Fonseca, M., 1997; Aoki and Schultz, unpublished observations).Arole for DNAreplication and initiation of transcription is discussed in section V. In summary, there is remarkable agreement for each of the aforementioned experimental approaches that measured the expression of a particular reporter gene or transgene or that assessed global transcription in that each approach indicated that the one-cell embryo was transcriptionally active. Moreover, when the results could be quantified, the data suggested that the transcriptional capability of the one-cell

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embryo in G2 was about 20 to 40% that of the two-cell embryo. Thus, the one-cell embryo is capable of supporting a fairly robust level of transcription.

IV.

DIFFERENCES IN THE TRANSCRIPTIONAL ACTIVITY BETWEEN THE MALE AND FEMALE PRONUCLEI

As mentioned above, the male pronucleus appeared to be transcriptionally more active than the female pronucleus in that it supported higher levels of expression of a reporter gene (Ram and Schultz, 1993) and endogenous genes (Aoki et al., 1997). Such differences were also observed in cleavage-arrested aphidicolin-treated embryos that were chronologically at the two-cell stage but were injected with the plasmid-borne reporter gene at the one-cell stage (Wiekowski et al., 1993). Moreover, a similar four- to five-fold difference in reporter gene expression was observed in these embryos, as was also found for BrUTP incorporation by permeabilized one-cell embryos (Aoki et al., 1997). These differences in transcriptional activity between the male and female pronuclei likely reflect the different origins of the male and female pronuclei that may lead to intrinsic underlying differences in nuclearkhromatin structure. Such differences may account for the observation that fusion of zygotic halves containing either a female or male pronucleus with a metaphase 11-arrested egg resulted in a more rapid premature chromosome condensation of the maternal chromatin, which also achieved a greater degree of condensation than its paternal counterpart (Ciemerych and Czolowska, 1993). As previously mentioned, the male and female pronuclei have different origins. In contrast to the female chromosomes that are already chromatinized, the DNA of the sperm is complexed with protamines, which have replaced the histones during spermatogenesis. The high degree of DNA condensation fostered by protamines permits the DNA to be tightly packaged into the sperm head. Following insemination, a protamine-histone exchange occurs in which the sperm DNA becomes chromatinized (Nonchev and Tsanev, 1990). Immunofluorescent studies using antibodies to protamines and histones revealed that the male pronucleus retained its protamine complement for up to 2 to 4 h following fertilization. Subsequently, the protamines were exchanged for histones and this exchange was completed prior to DNA replication. Glutathione that is generated during oocyte maturation is required for reduction of protamine disulfide-bonds, protamine dissociation, and male pronucleus formation (Perreault, 1992 and references therein). The hyperphosphorylated forms of nucleoplasmin, which is a histone-binding protein, can facilitate this exchange in lower species (Ohsumi and Katagiri, 1991; Len0 et al., 1996). Whether nucleoplasmim is present and functions as such in mammalian eggs is not known. This protamine-histone exchange could provide a window of opportunity for maternally derived transcription factors to gain access to their cis-cognate

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DNA-binding sequences before these sequences become sequestered into nucleosomes, which are inherently transcriptionally repressive (Wolffe, 1991, 1994). Consistent with this proposal is the observation that confocal microscopy indicated that the nuclear concentration of transcription factors such as TATA-box binding protein (TBP) and Spl (Worrad et al., 1994) as well as oct-4 and etl-1 (Worrad and Schultz, unpublished observations) was higher in the male pronucleus than the female pronucleus. Such a difference could account for greater level of transcription supported by the male pronucleus. Nevertheless, it was also possible that the transcriptional capacity of the female pronucleus was inherently less than that of the male pronucleus. This does not seem to be the case, however, since BrUTP incorporation by the female pronucleus in a parthenogenetically activated egg was equivalent to that of the combined BrUTP incorporation of the male and female pronuclei in a fertilized egg (Aoki et al., 1997). Likewise, the amount of TBP sequestered by the female pronucleus in a parthenogenetically activated egg was much greater than that of a fertilized egg and essentially equivalent to that present in both the male and female pronuclei. Thus, when the female pronucleus does not have to compete with the male pronucleus for transcription factors, the female pronucleus can readily sequester the maternally derived transcription factors. Differences in the acetylation state of histones between the male and female pronuclei may also contribute to the differences in the transcriptional activity of the two pronuclei (Adenot et al., 1997). Acetylation of histones is now widely recognized as a posttranslational modification that is highly correlated with the presence of transcriptionally permissive chromatin (O’Neill and Turner, 1996 and references therein). The acetylation of the amino terminal residues in the histones is catalyzed in a specific sequence. For histone H4 in humans, the sequence is lysine 16, then lysine 8 and/or 12, and then lysine 5 (Turner et al., 1989); fully acetylated histone H4 is acetylated on lysines, 5, 8,12, and 16. The transcriptional permissiveness of nucleosomes containing hyperacetylated histones is likely linked to their ability to interact with transcription factors. For example, the binding of the transcription factor TFIIIA to nucleosomes that were assembled on the 5s RNA gene was greater when the nucleosomes were assembled with acetylated core histones (Lee et al., 1993). Likewise, nucleosomes containing hyperacetylated histones that were assembled on DNA containing a single, GAL4 binding site bound GAL4 better than nucleosomes containing hypoacetylated histones (Vettese-Dadey et al., 1996). Thus, acetylation appeared to relieve the repressive properties of the histone amino terminal tails since protease removal of these tails stimulates binding of transcription factors (e.g., Lee et al., 1993). The recent observation that histone acetyltransferases can be integral components of the transcription initiation complex (Brownell et al., 1996), where they may facilitate transcription factor binding during the initiation phase (Vettesse-Dadey et al., 1996) and/or nucleosome displacement during transcriptional elongation (Brownell and Allis, 1996), affords

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an exciting explanation to link gene-specific chromatin remodeling with gene expression. Intriguingly, hyperacetylated histone H3 and H4, and RNA polymerase I1 display an enhanced localization at the nuclear periphery in the two-cell embryo, as detected by confocal microscopy (Worrad et al., 1995; Stein et al., 1997). Although there is no evidence for enhanced transcriptional activity at the nuclear periphery on the basis of detecting RNA polymerase-mediated incorporation of BrUTP (Aoki et al., 1997), this domain could perhaps direct the expression of a subset of genes. Using antibodies that recognize specific acetylated lysine 5 of histone H4 (i.e., hyperacetylated histone H4, since lysine 5 is the last lysine to be acetylated) and confocal microscopy, the male pronucleus of the one-cell embryo was found to contain a higher concentration of hyperacetylated histone H4 than the female pronucleus (Adenot et al., 1997). Since diacetylated histone H4 acetylated at lysines 5 and 12 is deposited during DNA replication and because this isoform would be recognized by the antibody, it was possible that the difference reflected the fact that the male pronucleus initiates DNA replication prior to the female pronucleus (Bouniol-Daly et al., 1997; Ferriera and Carmo-Fornseca, 1997; Aoki and Schultz, unpublished observations) and that the deposition-isofom, and not the hyperacetylated form, of H4 was what was detected. This interpretation is unlikely, however, since the difference was observed before S phase initiated. Moreover, the difference, which was lost by G2 of the first cell cycle, was also lost in S phase-arrested, one-cell embryos. This time-dependent loss of the difference between the male and female pronuclei likely accounts for the report that no apparent differences between the pronuclei were observed for hyperacetylated H4 content since these measurements were made in late one-cell embryos (Wiekowski et al., 1997). Thus, the paternal chromatin appears to outcompete the maternal chromatin for hyperacetylated histones. This initial difference in chromatin structure could also facilitate the preferential binding of transcription factors to paternal chromatin and contribute to the attainment of higher concentrations of transcription factors in the male pronucleus. The biological significance of these differences in transcriptional activity remains to be elucidated. It should be noted that this is the last time that the two genomes are physically separated from each other because once the first mitosis is completed, both parental sets of chromosomes reside in a common nucleus. Differential methylation of imprinted genes during spermatogenesis and oogenesis is a prime candidate for the imprinting mark (Bartolomei and Tilghman, 1997 and references therein). It is conceivable that the differences in paternal and maternal chromatin structure in the pronuclei could provide the final opportunity for modifications of imprinted genes. This could be achieved by differential accessibility of transcription factors and DNA methylases and demethylases. The ability of Spl , which is preferentially localized in the male pronucleus, to protect against methylation of CpG islands could provide such a mechanism (Brandeis et al., 1994; Macleod et al., 1994).

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V.

ROLE OF DNA REPLICATION IN REPROGRAMMING GENE EXPRESSION DURING ZGA

The observation that the one-cell embryo is transcriptionally active suggests that some event(s) following fertilization but prior to mitosis must be critical in the initial reprogramming of gene expression. DNA replication could provide such a locus of regulation since DNA replication with consequent nucleosome displacement would serve to facilitate the access of maternally-derived transcription factors to their cis-acting DNA-binding sequences prior to the formation of a nucleosome (Felsenfeld, 1992; Wolffe, 1991, 1994) (Figure 2). The basis for the

I

DNA Replication

n

I

Competition

Figure 2. Schematic diagram depicting coupling of DNA replication with transcription. The chromatin template is transcriptionally inactive because transcription factors cannot gain access to the DNA-binding element (black rectangle on third nucleosome). DNA replication disrupts the nucleosomes and hence makes the DNA-binding element accessible to both the histone octarner core [nucleosornes](open circle) and a transcription factor (shaded oblong). Since 145 bp of DNA wrap around the octarner core, amounts of free DNA less than this cannot support nucleosome formation and favor binding of transcription factors. If the amount of free DNA is sufficient to foster nucleosome formation, then a competition for this DNA, which harbors a DNA-binding element, will ensue. As shown in the bottom panel, in one case a nucleosome was formed, whereas in the other instance, the transcription factor out-competed nucleosorne formation and hence could initiate the formation of a productive transcription complex. The diagram has been oversimplified in order to convey the central components of the model.

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competition between transcription factor binding or nucleosome assembly is that some 145 bp of DNA are required for nucleosome formation. Thus, there will be a period of time during DNA replication in which transcription factors will have access to their binding sites before a transcriptionally repressive nucleosome could assemble and occlude that binding site. The spectrum of genes whose expression is linked to DNA replication will reflect the array of promoter and enhancer elements for each gene and the complement of maternal transcription factors and enhancer proteins that can bind to these elements. The first round of DNA replication is essential for the expression of the TRC and eIF-1A (Davis et al., 1996). Addition of aphidicolin to one-cell embryos in GI arrests the embryos at the GUS boundary. Analysis of such aphidicolin-treated, cleavage-arrested embryos that were chronologically at the two-cell stage revealed a pronounced decrease in the synthesis of the TRC, as assessed from 1D gels, or reduction in eIF-1A transcript abundance, as assessed by RT-PCR, when compared to control two-cell embryos of the same age (Davis et al., 1996). Whereas TRC synthesis constituted about 4 to 6% of total protein synthesis in control two-cell embryos in G2, its level was reduced to less than 1% in the S phase-arrested embryos. Similarly, the increase in eIF-1A transcript abundance in the aphidicolin-treated embryos was reduced to about 20% that of control two-cell embryos in G2. If the inhibitory effect of aphidicolin was solely attributable to reducing the number of genes (i.e., there are twice the number of gene copies in the untreated embryos in G2 than the aphidicolin-treated embryos), the inhibitory effect would expected to be around 50%. The significantly greater degree of inhibition argues against this explanation. Global transcription of endogenous genes was also coupled to DNA replication. Aphidicolin-treated, I-cell embryos assayed for BrUTP incorporation in G2 of the first cell cycle revealed a 35% decrease when compared to untreated control embryos (Aoki et al., 1997). Interestingly, the effect of aphidicolin on transcription by the male pronucleus was greater than that for the female pronucleus. While the basis for this difference is not known, it may be yet another manifestation of the differences in chromatidnuclear structure between the two pronuclei. While inhibiting DNA replication did inhibit global transcription of endogenous genes, what was also striking was that a very robust level of transcriptional activity still occurred; that is, the amount of total transcription in the S-phase-arrested embryos was 65% that of the untreated embryos. This implied the existence of two classes of genes-namely, genes whose expression was independent of DNA replication and genes whose expression was linked to DNA replication. This prediction was confirmed by 2D gel electrophoresis of metabolically radiolabeled proteins, which demonstrated that the level of expression of some a-amanitin-sensitive polypeptides synthesized by the two-cell embryo was also inhibited in S-phase-arrested, one-cell embryos radiolabeled when they were chronologically at the twocell stage, while the expression of other a-amanitin-sensitive polypeptides was essentially uninhibited in these S-phase-arrested embryos (Davis and Schultz, 1997).

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Results of experiments examining the spatiotemporal dynamics of DNA replication and transcription sites in one-cell embryos were also consistent with the presence of replication-dependent and replication-independent genes (Bouniol-Baly et al., 1997). DNA replication sites were detected by incorporation of digitoxinmodified dUTP (detected with antibodies to digitoxin) and transcription sites were detected by incorporation of BrUTP (detected with anti-BrdU antibodies) following microinjection of both of these nucleotides. While most of the transcription sites did not co-localize with replication sites, there were sites of colocalization. Several different mechanisms, which are not mutually exclusive, could be used to activate the expression of the replication-independent class of genes. The removal of histones by histone-binding proteins such as nucleoplasmin could generate nucleosome-free regions of DNA that would then be accessible for transcription factor binding. For example, the binding of Spl and USF to nucleosome core particles assembled on a DNA template is stimulated by nucleoplasmin (and not other highly acidic molecules such as polyglutamate, which could also bind nonspecifically to histones and serve as a histone sink; Chen et al., 1994). While the transcription factor GAL4 can bind to nucleosomes, this binding does not displace the histones associated with the nucleosome to which GAL4 binds. Nucleoplasmin addition, however, promotes the release of these core histones and the formation of a nucleosome-free region (Owen-Hughes and Workman, 1996). These types of processes could occur in the one-cell embryo to promote the binding of transcription factors in the absence of DNA replication. Chromatin-remodeling machines, such as members of the SWUSNF family, that were initially identified by their requirement for the transcription of some but not all genes can bind to chromatin and remodel chromatin in an ATP-dependent manner and thus generate binding sites for the transcription machinery in the absence of DNAreplication (Kingston et al., 1996and references therein; Kadonaga, 1998 and references therein). This remodeling is detected as the translational repositioning of nucleosomes that were initially positioned over a specific DNA sequence, a loss of the 10-bp DNase I digestion ladder of a rotationally positioned nucleosome, or an enhanced DNase sensitivity of chromatin to nucleases (e.g., increase in DNase I hypersensitivity). In fact, preimplantation mouse embryos contain SWUSNF homologs. etl-1, has recently been identifiedduing an enhancer trap screen (Soininen et al., 1992; Schoor et al., 1993). etl-1 is related to the Drosophila brahma gene, which is a transcriptional regulator of homeotic genes, and to the yeast transcriptional activator SNF2/SW12. The action of such chromatin-remodeling machines could account for the expression of the class of replication-independent genes. The requirement of DNA replication for gene expression is at first glance at odds with the results of experiments obtained using plasmid-borne reporter genes (e.g., Wiekowski et al., 1991, 1993; Henery et al., 1995). In these experiments, the plasmids were injected into the pronucleus of an aphidicolin-treated, one-cell embryo and these cleavage-arrested embryos were then assayed for luciferase activity at a time that corresponded to the two-cell stage. The level of expression in these treated

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embryos was in fact much greater than if the plasmid was injected into the nucleus of a two-cell blastomere (see section VII for further discussion). Thus, there was no obvious role or requirement for DNA replication for their expression. It must be emphasized, however, that these plasmids lacked an origin of replication, and as carefully pointed out by Henery and colleagues (19951, “expression of embryonic genes reveals the ability to express genes that undergo DNA replication, whereas expression of plasmid-encoded genes reveals the capacity of cells to transcribe DNA in the absence of replication.” Thus, the use of these types of plasmid-borne reporter genes is most suitable and appropriate to define promoter and enhancer requirements for transcription but not to analyze the connection between DNA replication and gene expression. Mitosis is associated with the loss andor displacement of transcription factors from their promoters (Martinez-BalbBs et al., 1995; Segil et al., 1996). While this could provide another window of opportunity for resetting the pattern of gene expression, it does pose the questions: (1) if the reprogramming of gene expression that is clearly evident in the two-cell embryo is initially established in the one-cell embryo, how does this reprogramming survive the first mitosis and (2) what could be the basis for this molecular memory? Although the expression of eIF-1A was tightly linked to the first round of replication, its expression was independent of the second round of DNA replication (Davis et al., 1996). The amount of eIF- 1A transcript was the same for two-cell embryos harvested within 1 h following cleavage of one-cell embryos and then cultured in the presence of aphidicolin to the mid-2-cell stage when compared to untreated embryos (Davis et al., 1996). Thus, the eIF-1A promoter that was apparently activated during S phase of the first cell cycle somehow had to be marked so that following the first mitosis this promoter was poised to support transcription in the absence of DNA replication. Retention of transcription factors in mitotic chromosomes could provide one component of this molecular memory. Immunocytochemical and subcellular fraction of mitotic Hela cells indicated that some TFIID remained associated with mitotic chromatin (Segil et al., 1996). Thus, upon entry into interphase, TFIID could nucleate the formation of a productive transcription complex for a gene expressed in G2 of the prior cell cycle. The retention of other transcription factors or enhancer proteins could promote the same effect. The DNase I hypersensitivity of some promoters is retained in mitotic chromatin even though transcription factors required for the expression of the gene are not found associated with chromatin. For example, HSFl, which is required for the expression of the hsp70 gene, was absent from mitotic chromatin that nevertheless retained the characteristic DNase I hypersensitivity profile characteristic for the hsp70 promoter (Martinez-BalbBs et al., 1995). Thus, the promoter for this gene was somehow marked. Analysis of transcription start sites during mitosis in vivo by ligation-mediated PCR was also consistent with a conformationally distorted chromatin conformation for genes that would normally be transcribed following entry into interphase. In contrast, genes not destined for expression were present in a nor-

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ma1 chromatin conformation (Michelotti et al., 1997). Although protein(s) associated with mitotic chromosomes may provide the mitotic mark (Michelotti et al., 1997), their identity remains unknown. Chromatin remodeling mediated by SWVSNF complexes could also contribute to marking mitotic chromatin because following removal of SWVSNF complexes, which evict histones from Gal4-bound nucleosomes, DNase I-hypersensitive sites still remained (Owen-Hughes et al., 1996). Lastly, acetylated histones could also furnish such a molecular memory (Jeppesen, 1997 and references therein). As mentioned above, histone hyperacetylation is highly linked with transcriptionally permissive chromatin (i.e., chromatin that is not necessarily transcriptionally active but is poised for to be transcribed under the appropriate set of conditions). Metaphase chromosomes display a characteristic immunostaining for hyperacetylated histones, and these regions could signify regions of chromatin that will be transcriptionally active following entry into interphase. In fact, R bands, which contain active genes, are enriched with hyperacetylated histones, whereas G bands, which contain either untranscribed or poorly transcribed genes, contain hypoacetylated histones. Hyperacetylated histone H4 is associated with mitotic chromosomes of the one-cell embryo and moreover the immunostaining is heterogeneous (Adenot et al., 1997). Thus, during the first cell cycle of the one-cell embryos, promoters that become incorporated within or near nucleosomes containing hyperacetylated histones may become marked such that either these promoters in mitotic chromatin retain transcription factors or, if these factors are displaced, the hyperacetylated nucleosomes could facilitate transcription factor binding following entry into interphase.

VI.

ENHANCER A N D TATA-BOX REQUIREMENTS FOR GENE EXPRESSION A.

Changes in Enhancer Utilization

The regulation of transcription can simply be viewed as correctly localizing RNA polymerase to the appropriate position to initiate transcription. From this vantage, the function of promoters (elements located proximal to the transcription start site) and enhancers (elements located more distal to the transcription start site) are essentially the same in that they recruit in a cooperative fashion and localize RNA polymerase to its initiation site. In the absence of these elements, the binding of RNA polymerase to DNA is both of low-affinity and promiscuous. Another function proposed for enhancers is to relieve chromatin-mediated repression of a weak promoter (Majumder and DePamphilis, 1995). It should be noted that these two functions are not mutually exclusive. Our understanding of the how promoters and enhancers are used during the course of ZGA is derived from studies analyzing the expression of plasmid-borne

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reporter genes. As mentioned above, their expression reflects the ability of the embryo’s transcription machinery to activate the plasmid’s promoter and not how their expression is directly coupled to DNA replication because the plasmids do not replicate. Two assumptions underlie the interpretation of the data obtained with these reporter genes. The first is that the level of activity measured for the reporter gene reflects the abundance of the mRNA that encodes for that activity. The second is that the utilization of the promoter under study reflects the utilization of endogenous promoters. Each of these assumptions appears to be valid in the preimplantation mouse embyro. The reporter gene used in these studies typically encodes luciferase, which is readily quantifiable in single embryos. Injection of known amount of luciferase into embryos indicated a half-life of less than 2 h (Christians et al., 1995). This short half-life indicates that levels of luciferase activity will fairly accurately reflect levels of luciferase mRNA. Others studies monitoring the time course of expression of luciferase activity demonstrated luciferase activity paralleled the expression of endogenous genes like the TRC (Wiekowski et al., 1991). The seminal observation that the requirement for an enhancer is developmentally acquired came from quantifying the expression of a luciferase reporter gene [which was driven by the thymidine kinase (tk) promoter and was also coupled to the FlOl enhancer] following injection of either one-cell or two-cell embryos (Wiekowski et al., 1991, 1993; Henery et al., 1995) (Figure 3). Injection of the male pronucleus of a one-cell embryo that was then cultured in the presence of aphidicolin to a time corresponding to the two- to four-cell stage revealed a high level of expression regardless of the presence or absence of the enhancer. In contrast, if the embryos were cultured to the two-cell stage in the absence of aphidicolin (i.e., they cleaved to the two-cell stage) and then injected, high levels of expression required the presence of the enhancer (i.e., in a zygotic nucleus the promoter was only efficiently utilized if it was coupled to the enhancer). Interestingly, the level of expres: sion of the enhancer-driven stimulation in the two-cell nucleus was essentially the same as the expression obtained in the S-phase-arrested one-cell embryos (Wiekowski et al., 1991)(Figure 3). The ability of the enhancer to stimulate the promoter increased between the two-cell and four-cell stages. The fold stimulation following injection of a two-cell nucleus was about seven- to 20-fold (Figure 3). In contrast, cleavage of injected two-cell embryos to the four-cell stage that were then assayed for luciferase activity revealed a 20 to 100-fold stimulation in response to the enhancer. This increase was a consequence of both a two- to three-fold lower level of expression from the basal promoter in the four-cell embryo, as well as a two-fold increase in enhancer-driven expression when compared to the two-cell embryo (Henery et al., 1995). These results suggest that the role of the enhancer may be to relieve the repression that accompanies the formation of the two-cell embryo. This repression, however, was apparently absent in S phase-arrested embryos since the level of expression in these embryos was the same regardless of the presence or absence of the enhancer and,

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Nucleus Injected

0

Male PN

Promoter

Enhancer

Promoter+Butyrate Enhancer+Butyrate

Female PN

0

0.5

1

1.5

2

2.5

3

3.5

Relative Amount of Gene Expression

Figure 3. Role of enhancers and histone acetylation in regulating gene expression. A plasmid-borne reporter gene was either driven by the tk promoter (Promoter) or was coupled with the F l O l enhancer (Enhancer). In some cases the embryos were also cultured in the presence of butyrate, which induces histone hyperacetylation (Promoter+Butyrate, Enhancer+Butyrate). Either male pronucleus (Male PN), female pronucleus (Female PN), or zygotic nucleus (two-cell) was injected. When one-cell embryos were injected, they were cultured in the presence of aphidicolin to a time that corresponded to the mid to late two-cell stage. The data are expressed relative to the amount of luciferase activity detected in the male pronucleus of an S-phase-arrested, one-cell embryo and were taken from Wiekowski et al., 1993.

moreover, was the same as that of the case where two-cell nuclei are injected with the enhancer-driven reporter gene (Wiekowski et al., 1991)(Figure 3). Thus, the first round of DNA replication appeared critical for establishing this repression (see section VII for further discussion). Lastly, the increasing severity of this repressive environment with further development, as measured by a decrease in expression from a basal promoter and an increased stimulation in response to the enhancer, is also observed in rabbit embryos (DeLouis et al., 1992; Christians et al., 1994). Thus, this phenomenon may be a general property of developing preimplantation embryos. There are, however, a number of alternative explanations for these results and the following conclusions that were drawn: (1) a transcriptionally repressive state develops by the two-cell stage and (2) the role of the enhancer is to relieve this inhibition. For example, the plasmid could be lost during the course of the first mitosis and hence not available for transcription in the newly formed two-cell embryos; different promoter elements may be used by the cleavage-arrested one-cell embryos and the two-cell embryos; the transcriptional capacities may differ in the cleavage-

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arrested one-cell embryos and the two-cell embryos; aphidicolin treatment somehow affects the expression of the reporter gene. As described below, these explanations are unlikely. Enhancer-dependent expression is observed following transplantation of an injected pronucleus into a two-cell blastomere (Henery et al., 1995) (Figure 4). The first mitosis-and concomitant loss of the plasmid from the injected pronucleus-was circumvented by the transplantation procedure and hence cannot account for the enhancer requirement observed in the two-cell embryo. The luciferase reporter gene is driven by the tkpromoter, which has two Spl binding sites (termed the proximal and distal sites)-a CAAT box binding site that binds CTF and a TATA box that binds TBP. Analysis of the expression of a set of linker scanning mutations that inactivated each of these sites without altering the distances between each of the promoter elements revealed that each mutation had the same relative effect on tk Protocol Nucleus Injected

Transplanted Assayed To At

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Demonstration of the development of a transcriptionally repressive state by nuclear transplantation. In the first experimental protocol, the injected nucleus was transplanted t o an S-phase-arrested, one-cell embryo that was then analyzed at a time that corresponded to the mid-late two-cell stage. In the second protocol, the injected nucleus was transplanted to an S-phase-arrested, two-cell embryo thatwas analyzed at a time that corresponded to the four-cell stage, whereas in the third protocol the injected nucleus was transplanted to a two-cell blastomere that divided. In the last protocol, the injected two-cell blastomere in C2 was transplanted to an S-phase-arrested, one-cell embryo that was then at a time that corresponded to the mid-late two-cell stage. The data are expressed relative to the amount of activity observed for the tk promoter-containing reporter gene in the S-phase-arrested one-cell embryos, and were taken from Henery et al., 1995.

Figure 4.

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promoter activity in both the aphidicolin-treated and two-cell embryos (Majumder et al., 1993). This suggested that the same promoter elements were used for both Sphase-arrested and control, untreated embryos. The transcriptional capacities of the two types of embryos also appeared similar. Spl is essential for the expression of the reporter gene and a series of competition experiments using injection of the reporter gene together with aplasmid containing 12 tandem Spl DNA-binding sites revealed that the amount of Spl in fact increased two- to six-fold between the oneand two-cell stages (Majumderet al., 1993).This suggested that the requirement for an enhancer in the two-cell embryo was not to compensate for lower levels of critical transcription factors. The lack of a requirement for an enhancer in the aphidicolin-treated embryos was unlikely due to simply inhibiting DNA replication, since aphidicolin-treated, two-cell embryos still required an enhancer for efficient reporter gene expression (Wiekowski et al., 1991); recall that S-phase-arrested, one-cell embryos did not require the enhancer. In totu, these results suggest that a transcriptionally repressive state develops following ZGA (see sections VII and VIII for further discussion) and that the primary function of an enhancer is to relieve this repression, in particular for genes with weak promoters. B.

Changes in TATA-Box Utilization

In general, promoters that are stimulated by an enhancer contain a TATA box, which binds TBP and recruits RNA polymerase to the promoter. Analysis of TATA-containing and TATA-less forms of the luciferase reporter gene driven by the tk promoter and coupled to the FlOl enhancer revealed that the TATA box was not required for efficient enhancer-driven expression in undifferentiated cells such as two- to eight-cell blastomeres and embryonic stem (ES) cells (Majumder and DePamphilis, 1994). In contrast, differentiated cells, such as the oocyte and 3T3 cells, required a TATA box for enhancer-driven expression. Moreover, in the undifferentiated cells, enhancer stimulation of the TATA-less promoter was mediated by the distal Spl DNA-binding site. One developmental consequence of such a change in promoter utilization would be to favor high levels of expression of housekeeping genes, which are often regulated by a TATA-less promoter, at the beginning of development but then to maintain low constitutive levels of expression as differentiation ensues. A change in TATA box utilization may also serve as a major locus of regulation of gene expression that is inextricably coupled with cell specification and the initiation and/or maintenance of spatially restricted patterns of gene expression that are observed in the two cell lineages present in the blastocyst. In fact, 2D gel analysis of inner and outer cells of the morula indicated that these changes initiate in the morula (Handyside and Johnson, 1978). A change in the pattern of promoter utilization that is coupled with the allocation of inner and outer cells during the morula stage, such that inner (undifferentiated) cells do not require a TATA box for efficient,

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enhancer-driven expression whereas outer cells develop a TATA-dependency for efficient, enhancer-driven expression, could account, at least in part, for these spatially restricted patterns of gene expression that are readily apparent in the blastocyst. Although such a change in promoter utilization is clearly an oversimplification of what must be a complex process in the initiation and maintenance of spatially restricted patterns of gene expression present in the blastocyst, there is evidence that the expression of some ICM-specific genes is modulated by such amechanism (i.e., enhancer-coupled expression does not require a TATA-box in undifferentiated cells). For example, oct-4 , which is preferentially expressed in the ICM cells and downregulated in the TE cells, does not have a TATA box; oct-4 expression only requires an enhancer and Spl (Yeom et al., 1996 and references therein). Perhaps S p l , which is present in the ICM (Worrad and Schultz, 1997), replaces the function of the TATA box and is required for enhancer action. It should be noted that oct-4 is strongly implicated in establishment of the germline and maintenance of the totipotent state (Yeom et al., 1996) since embryos homozygous for a null allele of oct-4 fail to form a true ICM (Nichols et al., 1998). Likewise, the retinoic acid-regulated, zinc-finger gene rex-1 is preferentially expressed in the ICM,and lacks a canonical TATA box (Rogers et al., 1991; Hosler et al., 1993). The ICM-specific expression of fgf-4, which contains a TATA-box, may be a consequence of the requirement for oct-4 for fgf-4 expression.

VII.

DEVELOPMENT OF A TRANSCRIPTIONALLY REPRESSIVE STATE

The developmental acquisition of a requirement for an enhancer for efficient reporter gene expression suggests that a transcriptionally repressive state develops during the one- to two-cell transition. This repression, as manifested by a requirement for the enhancer, was not observed in aphidicolin-treated, cleavage-arrested embryos in which the plasmid was injected into the male pronucleus (Figure 3). The extent of BrUTP incorporation by S-phase-arrested, one-cell embryos was also consistent with the development of a transcriptionally repressive state (Aoki et al., 1997) (Figure 5). The total amount of incorporation of BrUTP by the two pronuclei of such S-phase-arrested embryos was about four times that of a two-cell blastomere in G2 when expressed on a per chromosome basis. The strength of this repressive state apparently increases with development. As discussed in section VILA., promoter activity of the reporter gene was dramatically reduced following injection of a two-cell blastomere nucleus when compared to S-phase-arrested, one-cell embryos (Henery et al., 1995). This repression was around five times more severe when the plasmid was injected into arecipient in G2 than in S . A similar increase in the repressive environment with development was also observed following transplantation of a pronucleus microinjected with the enhancerless promoter to

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c

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aphM

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Figure 5. Role of DNA replication in the development of a transcriptionally repressive state. BrUTP incorporation of membrane-permeabilized embryos was conducted on two-cell embryos in C2 (2C) or S-phase-arrested, one-cell embryos that were analyzed at a time that corresponded to C2 of the two-cell stage. Both the male (aph M) and female (aph F) were analyzed. Transcription was also analyzed at a time that corresponded to G2 of the two-cell stage for S-phase-arrested, two-cell embryos (aphidicolin added after the first round of replication; aph 2C) or in two-cell embryos treated with trapoxin (trap). The data are expressed relative to the amount of BrUTP incorporated by a two-cell blastomere nucleus in C2. (Data taken from Aoki et al., 1997).

an enucleated early two-cell blastomere recipient (Figure 4). When compared to Sphase-arrested embryos, the level of expression was about 40% following transfer to a two-cell blastomere in S phase and only 15% following transfer to a two-cell blastomere in G2 that then cleaved to the four-cell stage (Henery et al., 1995). In both of these instances, this repression was relieved if the promoter was coupled to an enhancer. NucIear transpIantation experiments also indicated that the repression of expression from a plasmid-borne reporter gene that was observed following injection of the plasmid into a two-cell blastomere nucleus was essentially irreversible (Henery et al., 1995)(Figure 4). Transfer of a two-cell blastomere nucleus that was injected in G2 with the enhancerless promoter to an enucleated S-phase-arrested, one-cell embryo resulted in only 5% the level of expression when compared to S-phase-arrested, one-cell embryos, and this expression was ineffectively stimulated by the presence of an enhancer. This irreversible repression seemingly contradicted a previous report that transplantation of a nucleus from an eight-cell embryo or nuclei from differentiated cells to an enucleated one-cell embryo resulted in the reexpression of the TRC when examined at the two-cell stage (Latham et al., 1991); the TRC is apparently irreversibly repressed since its expression is not observed in

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eight-cell embryos or later stage embryos. The basis for this difference may reflect differences in the abilities of the reporter gene and the endogenous TRC gene(s) to undergo DNA replication following transplantation. The inability of the plasmid to undergo DNA replication may prevent its reprogramming that would be coupled to its reactivation. In contrast, DNA replication may permit the reprogramming and hence re-expression of the previously repressed endogenous TRC gene(s). While it was not determined whether the transplanted nucleus underwent a round of replication, the observation that the recipient enucleated one-cell embryos cleaved to the two-cell stage is consistent with this suggestion. The second round of DNA replication may be involved in the repression of expression of specific endogenous genes whose expression transiently increases during the two-cell stage and total endogenous transcription. Inhibiting the second round of DNA replication prevented the decrease in both the synthesis of the TRC and hsp70, and the decline abundance of the eIF-1A mRNA (Davis et al., 1996; Christians et al., 1995).The total amount of BrUTP incorporated (as expressed on a per chromosome basis) by a two-cell blastomere nucleus in G2 was about threefold less than that incorporated by a blastomere obtained from a two-cell embryo that was placed in aphidicolin just prior to the second S phase (Aoki et al., 1997) (Figure 5). The molecular basis for this could be that the second round of DNA replication would displace productive transcription complexes assembled on their promoters. The factors that constitute the basis for the transcriptionally repressive state (see section VIII) would then prevent stable transcription complexes from reforming and hence reduce the expression of these genes. It should be noted that these same factors could account for the repression of the plasmid-borne reporter genes, which do not undergo DNA replication, following injection of two-cell blastomere nuclei. Finally, as described above, inhibiting the first round of DNA replication apparently prevented the generation of these factor(s) because S-phase-arrested, one-cell embryos did not require an enhancer for high levels of reporter gene expression and supported higher levels of transcription of endogenous genes when compared to two-cell embryos.

VIII.

NATURE OF THE TRANSCRIPTIONALLY REPRESSIVE STATE

Several converging lines of evidence based on the expression of reporter genes and endogenous genes suggest that changes in histone acetylation and the expression of histone H 1 may underlie the development of the transcriptionally repressive state. Thus, changes in chromatin structure may be intimately involved in the reprogramming of gene expression that occurs during ZGA. As discussed above, the expression of a reporter gene injected into the female pronucleus of an S-phase-arrested, one-cell embryo was about four- to five-fold less than the expression observed if the male pronucleus was injected (Wiekowski

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et al., 1993). The expression obtained from the female pronucleus, however, was equal to that of the male pronucleus if the embryos were cultured in butyrate, which is an inhibitor of histone deacetylases and inducer of histone hyperacetylation. (Wiekowski et al., 1993)(Figure 3). Thus, histone acetylation and the attendant modifications to chromatin structure could relieve the repression that was inherent to the female pronucleus. Histone acetylation also appeared to relieve the repression that was present in the two-cell embryo since butyrate treatment stimulated by at least 20-fold the expression of an enhancerless promoter following injection of a two-cell blastomere nucleus but only by about three-fold for the enhancer-driven reporter gene (Figure 3). Moreover, the stimulation attributed to the enhancer was about 10 to 12-fold in the absence of butyrate but only about two-fold in the presence of butyrate. Thus, histone hyperacetylation relieved the repression observed in the two-cell embryo as revealed by an increased expression of the enhancerlesspromoter and by a reduced stimulation whcn the enhancer was present (Wiekowski eta]., 1993). Transplantation of pronuclei injected with the enhancerless promoter reporter gene into two-cell blastomeres that were experimentally manipulated to alter the final ploidy indicated that the degree of repression was inversely correlated with the ploidy of the recipient cell. Thus, the cytoplasm of the two-cell embryo appeared to contain factor(s) capable of repressing the promoter, and this factor(s) appeared to be titrated out by cellular DNA. The experimental stimulation of histone acetylation also prevented the repression of both the TRC and eIF-1A expression (Davis et al., 1996). Addition of trapoxin, which is an irreversible inhibitor of histone deacetylases (Kijima et al., 1993), to mid-2-cell stage embryos inhibited the decrease normally associated with cleavage to the four-cell stage and in fact increased the expression of each of these genes (Wiekowski et al., 1993;Davis et al., 1996). Trapoxin also stimulated BrUTP incorporation of two-cell embryos (Aoki et al., 1997)(Figure 5). Thus, altering chromatin structure overcame the transcriptionally repressive state that develops in the two-cell embryo and represses endogenous transcription. The expression of a somatic form of histone H1 could constitute one component of the transcriptionally repressive state. The biogenesis of a mature nucleosome is associated with the formation of transcriptionally repressive chromatin. While the association of DNA with histones H3 and H4 can generate a nucleosomelike structure, the DNA can still readily interact with transcription factors. This ability is diminished with the further incorporation of histones H2A and H2B that results in the formation of a nucleosome that can exclude the binding of basic components of the transcription machinery (e.g., RNA polymerase 11).The formation of a truly transcriptionally repressive chromatin structure is brought about by the addition of histone HI, which can be incorporated only after the addition of histones H2A and H2B and causes chromatin to condense into a 30-nm fiber (Bouvetet al., 1994). The requirement for an enhancer for efficient promoter utilization in zygotic nuclei, but not the pronuclei, could reflect the developmental acquisition of chromatin con-

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taining histone H1 since enhancers only stimulate their promoters if the chromatin contains H1 (Paranjape et al., 1994). Moreover, the reduced affinity of histone H1 for hyperacetylated nucleosomes (Turner, 1991) coupled with facilitated binding of transcription factors by hyperacetylated core nucleosomes (Lee et al., 1993; Vettese-Dadey et al., 1996) could account for the following: (1) the ability of histone deacetylase inhibitors to obviate the need for an enhancer for efficient promoter utilization and (2) the stimulatory effect on the expression of specific endogenous genes as well as the total endogenous gene pool. During oogenesis, somatic subtypes of histone H1 are replaced by the variant H1" subtype, as determined from immunocytochemical studies using antibodies specific to these forms of histone H 1 (Clarke et al., 1997).Following fertilization, somatic histone H1 was first detected by either immunoblotting or immunocytochemistry at the early four-cell stage (Clarke et al., 1992). The accumulation of histone HI was due to zygotic expression and not recruitment of maternal mRNAs since it was inhibited by a-amanitin. Metabolic radiolabeling and electrophoretic fractionation of acid-soluble proteins suggested that the synthesis of histone H3 and H4 began in the early one-cell embryo. In contrast, the synthesis of histones H2A, H2B, and H1 was not detected until the late one-cell or early twocell stage (Wiekowski et al., 1997). It was possible that somatic histone HI was synthesized at these early stages because these embryos contain transcripts for histone H1 subtypes (Clarke et al., 1998), but that the protein had not accumulated to sufficient levels that could be detected by immunoblotting or immunocytochemistry. The accumulation of histone H 1 could promote the repression of transcription and a requirement for an enhancer for efficient gene expression in zygotic nuclei. Nevertheless, this requirement for an enhancer was not observed when the reporter gene was injected into the male pronucleus of S-phase-arrested embryos that were assayed at the chronological two-cell stage (Wiekowski et al., 1993). Moreover, if the plasmid-borne reporter gene was injected into the male pronucleus of aphidicolintreated, cleavage-arrested embryos when they were chronologically at the two-cell stage, high levels of expression were observed regardless of whether an enhancer was or was not present (Wiekowski et al., 1997).Thus, the repressive state apparently did not develop in the presence of aphidicolin. A similar finding was observed for total gene expression. BrUTP incorporation, when expressed on a per unit chromosome basis, by S-phase arrested embryos that were assayed at the chronological two-cell stage, was greater than that for their two-cell counterparts (Aoki et al., 1997), again suggesting that the DNA replication was coupled with the formation of a transcriptionally repressive environment. The observation that the synthesis of histone H1 was dramatically reduced in aphidicolin-treated embryos (Wiekowski et al., 1997) could account, at least in part, for the failure of the transcriptionally repressive state to develop in S-phase-arrested, one-cell embryos. Consistent with this proposed role for histone H1 is the observation that injected histone H1, but not H2A, inhibits the expression of the TRC (Stein and Schultz, unpublished observations).

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Changes in histone hyperacetylation may also contribute to the transcriptionally repressive state. One- and two-cell embryos have functional histone deacetylases because treatment of these embryos with histone deacetylase inhibitors resulted in an increase in the amount of hyperacetylated histones, as detected by antibodies specific for different acetylated isoforms of different core histones (Worrad et al., 1995; Stein et al., 1997; Adenot et al., 1997; Wiekowski et al., 1997). Development from the one-cell stage to the eight-cell stage was accompanied by a decrease in the synthesis of hyperacetylated histone H4, as determined following gel electrophoresis (Wiekowski et al., 1997). Although the histone mass was not measured directly (e.g., by immunoblotting), this decrease in synthesis could reflect a decrease in the mass amount of hyperacetylated histones, assuming that metabolism acetylated isofoms of the maternal-derived histones is the same as that of newly synthesized histones. In summary, the reciprocal decrease in histone acetylation coupled with an increase in histone H1 expression could, in principle, promote the conversion of transcriptionally permissive chromatin into a transcriptionally repressed form and hence serve as the foundation for the development of the transcriptionally repressed state that is first observed at the two-cell stage. The second round of DNA replication may also contribute to the development of this state by displacing transcriptionally productive complexes and thus providing an opportunity for the assembly of a mature, transcriptionally repressive chromatin structure.

IX.

POTENTIAL UNCOUPLING OF TRANSCRIPTION AND TRANSLATION IN THE ONE-CELL EMBRYO

Although the one-cell embryo is clearly transcriptionally active, there are suggestions that the transcripts made are not efficiently translated. For example, following addition of a-amanitin to one-cell embryos in G2, the synthesis of a-amanitin-sensitive polypeptides that are the hallmark of ZGA was not observed in the developing two-cell embryos (Schultz, unpublished results). Thus, if these transcripts were produced during the this initial period of genome activation, they were poorly, if at all, translated. Analysis of the expression of transgenes also led to a similar conclusion; whereas the expression of a paternally-derived luciferase transgene driven by the p-actin promoter was detected in the one-cell embryo, luciferase activity first became detectable in the two-cell embryo (Matsumoto et al., 1994). Additional experiments that assess expression of a luciferase reporter gene at both the protein and mRNA levels also suggested an uncoupling of transcription from translation (Nothias et al., 1996). Injection of the reporter gene into the nucleus of a two-cell blastomere was followed by the rapid expression of both luciferase mRNA, as detected by a quantitative RT-PCR assay, and luciferase activity. In contrast, analysis of S-phase-arrested, one-cell embryos revealed that, while luciferase mRNA was readily detected at a time that corresponded to G2 of the one-

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cell embryo (and while it reached about 20% the maximal level found at a time that corresponded to the mid-to-late two-cei: stage), luciferase activity was only readily detectable starting at a time that corresponded to early-to-mid two-cell stage. The detection of luciferase activity paralleled a second phase of luciferase mRNA accumulation in these S-phase-arrested embryos. Also in support of the conclusion that the nascent transcripts expressed in the one-cell embryo were poorly translated was that addition of a-amanitin, shortly after injection of the plasmid, inhibited the expression of luciferase activity. Nevertheless, if such an uncoupling was present in the one-cell embryo, coupling must be rapidly established following the first mitosis. Although low levels of expression of a luciferase transgene driven by the hsp7O promoter were observed in small fraction of one-cell embryos in G2, almost all of the embryos exhibited a robust increase in luciferase activity immediately after cleavage (Christians et al., 1995). What could be the basis for this apparent uncoupling of transcription and translation as reflected by low levels of translation of nascent mRNAs? Several possibilities come to mind. One is that the nascent transcripts are rapidly degraded upon entry into the cytoplasm and hence do not accumulate to levels that can support translation of sufficient amounts of protein to be detected. In~fact,injected mRNAs are rapidly degraded in one-cell embryos when compared to growing oocytes (Ebert et al., 1984). Nevertheless, this proposal does not account for the observation that TRC expression is readily observed in G2 of the one-cell embryo following transplantation of a two-cell nucleus to an enucleated one-cell embryo (Latham et al., 1992). Hence, the newly synthesized transcripts encoding the TRC are capable of achieving translatable cytoplasmic concentrations in the one-cell embryo. This also suggests that the translation of nascent mRNAs is not suppressed by complexing with proteins present in the one-cell embryo (i.e., the transcripts become sequestered as “masked” mRNAs). A second possibility is that the nascent transcripts present in the pronuclei are not exported to the cytoplasm. This could be due to the inability of the pronuclei to splice precursor mRNAs. The splicing machinery maybe improperly assembled in pronuclei since the protein detected by an antiserum that recognizes a protein component that co-localizes with snRNPs is not detected by immunocytochemistry in the pronuclei but is detected in the nuclei of the two-cell embryo (Vautier et al., 1994). It should be noted that all of the luciferase plasmid-borne reporter genes and transgenes used to direct the expression of luciferase contain an intron in the 3’UTR. Whether splicing of this intron is essential for export is not known. Regardless of whether the intron was spliced or not spliced, if the retention of transcripts by the pronuclei serves as the mechanism for the uncoupling of transcription and translation, the rapid appearance of luciferase activity directed by the hsp70 promoterdriven transgene following cleavage to the two-cell stage could be a consequence to their release into the cytoplasm and availability for immediate translation. Are nascent transcripts retained by the pronuclei and not exported to the cytoplasm? Following injection of BrUTP into living, one-cell embryos, transcripts are

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not observed in the cytoplasm following a three-hour culture (Bouniol et al., 1995). While this is consistent with the failure of export, export to the cytoplasm may result in a dilution of these transcripts below the limits for their detection. Another possibility is that since transcripts containing incorporated BrUTP are not spliced in vitro (Sierakowska et al., 1991), these transcripts containing incorporated BrUTP are not processed and hence are not exported even though the pronuclei are competent to export nascent mRNAs. The ability of the pronuclei present in the one-cell embryo to export nascent transcripts to the cytoplasm remains unresolved. Such pronuclei in S-phase-arrested, one-cell embryos, however, must be able to execute successfully this export since luciferase expression is observed by a time that corresponds to the mid-two-cell stage (Nothias et al., 1997). What could be the biological significance of this possible uncoupling of transcription from translation? The transcription observed in the me-cell embryo may be promiscuous in that it is simply a consequence of the dramatic chromatin remodeling that must occur during the protamine-histone exchange for the paternallyderived chromosomes as well as during the first round of DNA replication for both sets of chromosomes. It is easy to visualize how the precocious expression of certain genes could be deleterious for further development. The potential uncoupling of transcription from translation could provide a protective mechanism that guards against any inappropriate gene expression that occurs while the maternally and paternally derived chromatin is extensively remodeled into a chromatin structure that represses transcription following formation of a zygotic nucleus. Once this chromatin structure is established, genes would now be selectively expressed as a function of their enhancer and promoter requirements (Nothias et al., 1997).

X.

MOLECULAR BASIS FOR THE TIME OF ONSET OF ZGA

In organisms such as Xenopus laevis, whose eggs harbor an extremely large pool of maternally-derived histones-the Xenopus laevis egg contains enough histone to support the formation of 15,000 to 20,000 nuclei (Woodland and Adamson, 1977)-a competition between chromatin assembly and transcription complex assembly is strongly implicated in regulating the timing for the onset of ZGA during early development. For example, although Xenopus embryos possess a functional transcription apparatus (Newport and Kirschner, 1982; Prioleau et al., 1994, 1995), transcription and the reprogramming of gene expression do not occur until after the mid-blastula transition that happens after 12 rounds of DNA replication (Newport and Kirschner, 1982). The large maternal stockpile of histones, by rapidly driving the newly replicated DNA into transcriptionally repressive chromatin, prevents the formation of stable basal transcription complexes (Prioleau et al., 1995). Once the maternal histone pool is titrated out by the exponential increase in DNA following 12 rounds of DNA replication, stable basal transcription complexes can form and transcription and the reprogramming of gene expression can initiate.

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In contrast to Xenopus luevis, the maternal histone pool in the mouse one-cell embryo (based on synthetic rates for histones) is probably sufficient for only one to two rounds of DNA replication (Wassarman and Mrozak, 1981). Consistent with such a small histone pool is the observation that polyspermic eggs have the capacity to transform up to three to four sperm nuclei into metaphase chromosomes (Clarke and Masui, 1986); a similar capacity was also determined from experiments that manipulated the cytoplasmic volume by either bisection or cell fusion (Clarke and Masui, 1987). This small pool of maternal histones may hence be insufficient to prevent effectively the assembly of stable basal transcription complexes. Thus, titration of the maternal histone pool by an increase in the mass of DNA due to blastomere proliferation may not be a critical factor in regulating the onset of transcription in the mouse embryo and other mammalian eggs; this is because the maternal transcription factors may be able to outcompete successfully maternal histones for the newly replicated chromatin. This could, at least in part, account for the early onset of transcription in mammalian embryos ranging from rodents to humans (Telford et al., 1990). Moreover, the lack of a rapid S phase in the mouse embryo and other mammalian embryos would permit sufficient time for productively assembled transcription complexes to generate full-length transcripts. In contrast to mammalian embryos, S phase is very short prior to the midblastula transition in Xenopus luevis (Newport and Kirschner, 1982) and hence these rapid rounds of DNA replication could prematurely terminate the transcription of genes for which transcription had initiated.

XI.

CLINICAL RAMIFICATIONS OF THE TIMING OF ZGA

The time during which embryos undergo ZGA may be very susceptible to perturbations that may upset the fidelity of the reprogramming of gene expression. While it is commonly assumed that such changes will lead to death of the preimplantation embryo prior to implantation, it should be noted that a brief exposure of one-cell embryos to mutagens whose time course of action is restricted to the one-cell stage results in a high incidence of congenital abnormalities in postimplantation embryos (e.g., neural tube closure defects and limb abnormalities; Generoso et al., 1987, 1988). Furthermore, results of recent experiments suggest that there may be subtle longterm developmental and behavioral consequences. While cryopreservation of two-cell embryos does not induce major congenital abnormalities-a situation similar to that for babies obtained from cryopreserved embryos generated by conventional IVF (Wada et al., 1994)-the incidence of changes in morphophysiological and behavioral features of offspring derived from these cryopreserved embryos was significantly elevated (Dulioust et al., 1995). For example, while the postnatal weights showed no difference when compared to controls, adult male B6CBA mice generated from cryopreserved two-cell embryos displayed an increased body weight independent of either maternal weight or litter size. Moreover, for C3D2

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mice, seven of nine preweaning developmental traits were perturbed, and performance in behavioral tests was reduced for female B6CBA mice generated from cryopreserved two-cell embryos. Thus, while these mice displayed no gross abnormalities, they may not be totally “normal” and the abnormalities may manifest themselves later in life. Whether these subtle abnormalities can be attributed to cryopreservation during the course of ZGA remains to be established, but nevertheless potential concerns are raised regarding the cryopreservation of human embryos at the four- to eight-cell stage-a time when these embryos are undergoing the maternal-to-zygotic transition.

XI1 SUMMARY Shortly after pronucleus formation and entry into S phase, transcription initiates in both pronuclei of the one-cell embryo (Figure 6). The male pronucleus supports higher levels of transcription than the female pronucleus, and this difference is attributed to their differences in chromatin structure that reflects their different hstories (e.g., the protamine-histone exchange that occurs in the male pronucleus affords an opportunity for the sequestration of maternal transcription factors that is not available to the female pronucleus whose DNA is already packaged into chromatin). The first Time (h post insemination) PB

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4 Synthesis of histone H3 and H4 Synthesis of histone HZA, HZB, and H1

Schematic diagram depicting the developmental changes in gene expression during the first two cell cycles. See text for further description.

Figure 6.

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round of DNA replication also presents an opportunity for the binding of maternal transcription factors to both parental sets of chromosomes and is required for the expression of certain genes. Nevertheless, there is also a set of genes whose transcription is independent of DNA replication. How the promoters that are activated during this period of time are marked so that following the first mitosis transcription may rapidly start following entry into interphase is not known. It is also not certain that the transcripts generated in the one-cell embryo are actually exported to the cytoplasm and efficiently translated. The biological significance of the onset of ZGA in the onecell embryo is thus unresolved. The formation of zygotic nuclei containing both parental sets of chromosomes is associated with an extensive reprogramming in the pattern of gene expression as well as the progressive development of a transcriptionally repressive state. The transcriptionally repressive state may be a consequence of further chromatin remodeling that may occur during the second round of DNA replication. Furthermore, a decrease in the amount of hyperacetylated histones coupled with the synthesis of somatic histone H1 may also contribute to the transcriptionally repressive state. This repression could constitute a major basis underpinning the reprogramming for gene expression since it will ensure that only the genes that are required to be expressed will be expressed, this expression reflecting the cellular complement of transcription factors at each particular stage of development.

ACKNOWLEDGMENTS The research conducted in the author’s laboratory was supported by a grant from the NIH (HD 22681). The author would also like to thank Me1 DePamphilis and Bryan Turner for many stimulating discussions regarding regulation of transcription in mouse embryos and the role of histone acetylation in regulating gene expression.

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ROLES OF METALLOPROTEASEDISINTEGRINS IN CELL-CELL INTERACTIONS, IN NEUROCENESIS, AND IN THE CLEAVAGE OF TNFa

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I. Introduction and Outline. . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Snake Venom Proteins . . . . ... 111. Role of the Metalloprotease-Disintegrin Fertilin in Fertilization . . . . . . . . . . . . . 170 173 IV. Evidence for a Role of Integrins in Fertilization V. Processing of Fertilin during Sperm Maturation in the Testes and Epididymis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 VI. Evidence for a Potential Role of Metalloprotease-Disintegrins . . 175 as Integrin Ligands in Somatic Cell-Cell Interactions . . . . . . . . . . . . . . . . . . . . . VII. Are Metalloprotease-Disintegrins Membrane Fusion Proteins?. . A. Evidence for a Role in Membrane Fusion. . . . . . . . . . . . . . . . B. Alternative Interpretations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 VIII. Metalloprotease-Disintegrin Substrates . . . . . . . . . . . . . 179

Advances in Developmental Biochemistry Volume 5, pages 165-198. Copyright 0 1999 by JAI Press Inc. All right of reproduction in any form reserved. ISBN: 0-7623-0202-X 165

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A. ADAM 10 and Drosophila Kuzbanian May Play a Role in the Activation of Notch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Identification of the TNFa Convertase Suggests that Metalloprotease-Disintegrins Play a Role in Protein

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EctodomainShedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 IX. Do other Metalloprotease-Disintegrins also Mediate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Protein Ectodomain Shedding?. . X. Intracellular Maturation and Prote Processing of Metalloprotease-Disintegrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 XI. Metalloprotease-Disintegrins Lacking a Membrane Anchor . . . . . . . . . . . . . . . . 186 . 189 XII. Metalloprotease-Disintegrin Expression Patterns during Development XIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

1.

INTRODUCTION AND OUTLINE

Metalloprotease-disintegrin proteins are a family of membrane-anchored glycoproteins that are related to snake venom integrin ligands and metalloproteases. They have been implicated in such diverse physiological functions as fertilization, muscle fusion, TNFa processing, and neuronal differentiation. All currently known membrane-anchored metalloprotease-disintegrins are composed of several distinct and conserved protein modules: an N-terminal signal sequence is followed by a pro-domain, metalloprotease domain, disintegrin domain, cysteine rich region, EGF repeat, transmembrane domain, and cytoplasmic tail (see figure 1). Due to this unique and highly conserved domain organization, metalloproteasedisintegrins are also referred to as MDC proteins (metalloprotease/ disintegrid cysteine-rich proteins) or ADAMs (a disintegrin and metalloprotease). Historically, the first protein sequence information for disintegrin proteins was obtained from short soluble snake venom peptides which bind to the platelet integrin aIIbp3 with high affinity (see Figure 1). Snake venom disintegrins therefore function as competitive inhibitors of platelet aggregation (Gan et al., 1988; Huang et al., 1989; Huang et al., 1987; Shebuski et al., 1989). These soluble snake venom peptides were named disintegrins because of their ability to disrupt integrin binding to the extracellular matrix, in this case the fibrin clot (Dennis et al., 1990; Gould et al., 1990; Kini and Evans, 1992; Musial et al., 1990; Niewiarowski et al., 1994). In membrane-anchored metalloprotease-disintegrins, the name disintegrin refers to the sequence similarity with snake venom disintegrins. In contrast to soluble snake venom disintegrins, their membrane-anchored counterparts are predicted to promote cellkell adhesion by binding to an integrin. Viperidue venoms also contain hemorrhagic metalloproteases, which are derived from larger precursors (see Figure 11,many of which contain adisintegrin domain (Fox and Bjarnason, 1995; Jia et al., 1996; Kini and Evans, 1992; Paineet al., 1992; Takeyaetal., 1992; Takeyaet al., 1990a; Takeya et al., 1990b). Of the 21 distinct membrane-anchored

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metalloprotease-disintegrins that have been identified to date, 11 have a catalytic site consensus sequence (HEXXH) (Wolfsberg and White, 1996) in their metalloprotease-domain, while the other 10 do not (see Figure l), even though their metalloprotease-domain is otherwise quite conserved. Metalloproteasedisintegrins thus fall into at least two groups-one, which presumably has catalytic activity, and the second which most likely does not. The purpose of this review is to discuss what is currently known about the different functions of metalloprotease-disintegrins, and to highlight some of the most interesting and pressing questions that have now been raised about this intriguing protein family. A brief summary of the known properties of snake venom disintegrins and metalloproteases is followed by a discussion of the role of membraneanchored metalloprotease-disintegrins in cell-cell interactions and proteolysis of extracytoplasmic or extracellular protein domains. Most of the data that directly implicate metalloprotease disintegrins in cell-cell interactions has emerged from studies of fertilization. Taken together, the fertilization studies support a model where sperm-egg binding involves two or more metalloprotease-disintegrins on the sperm and one or more integrin(s) on the egg. Metalloprotease-disintegrins have also been proposed to function as membrane fusion proteins in sperm-egg and muscle membrane fusion. While there is little doubt that they are important for membrane fusion to occur, the currently available evidence does not distinguish between a direct role in membrane fusion as a bona fide membrane fusion protein and a role in a process that is a prerequisite for fusion. The other main function of metalloproteasedisintegrins appears to be in the specific cleavage of luminal or extracellular protein domains. Firstly, a Drosophila metalloprotease-disintegrin termed Kuzbanian (KUZ) modulates the function of Notch, which is involvedin several distinct cell fate decisions during development. Secondly, two. metalloprotease-disintegrins, the T N F a convertase and the putative mamma€ian KUZ orthologue ADAM 10, have been linked to shedding of T N F a from the plasma membrane. This raises the possibility that other metalloproteasedisintegrins also play a role in protein ectodomain shedding or processing. Metalloprotease-disintegrins are themselves subjected to proteolytic processing in the secretory pathway. Removal of the prodomain by proprotein convertases is most likely needed to activate the protease domain, and removal of the metalloprotease domain may in some cases affect the protein's role in cell-cell interactions. Recent evidence suggests that catalytically active metalloprotease-disintegrins may fall into two subgroups: proteases that function at or near the cell surface, and proteases the reside in the trans-Golgi network or endosomal compartment. The latter may process other proteins traveling through the secretory or endocytic pathway. Finally, analysis of the expression of metalloprotease-disintegrins in developing embryos and in adult animals has unveiled distinct and interesting expression patterns that point towards specific functions at different stages of development.

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II.

SNAKE VENOM PROTEINS

The first identified disintegrins were purified from snake venom as potent anticoagulants. Their primary sequences were determined by protein sequencing instead of cDNA cloning and included, in most cases, an RGD sequence within a short soluble peptide of 50 to 80 amino acid residues (Dennis et al., 1990; Gan et al., 1988; Gould et al., 1990; Huang et al., 1989; Huang et al., 1987; Kini and Evans, 1992; Musial et al., 1990; Niewiarowski et al., 1994; Shebuski et al., 1989). NMR structures of several disintegrins point toward one critical structural feature that these toxins have in common: an RGD sequence residing at the tip of a hairpin loop of 11 amino acid residues in length (Adler et al., 1993; Adler et al., 1991; Calvete et al., 1991; Calvete et al., 1997; Saudek et al., 1991). This hairpin loop is apparently essential for high affinity binding to the platelet integrin gpIIb/IIIa, and similar RGD sequence-containing hairpin loops have been found in other integrin ligands such as fibronectin (Leahy et al., 1996;Main et al., 1992), the leech anticoagulant decorsin (Krezel et al., 1994), and the structurally unrelated snake venom protein dendroaspin (McDowell et al., 1992; Sutcliffe et al., 1994). Although most snake venom disintegrins have an RGD sequence, distinct disintegrins display clear preferences for specific RGD-binding integrins (Lu et al., 1994;Marcinkiewiczetal., 1997;McLaneetal., 1996;Pfaffet al., 1994;Scarborough et al., 1993). These preferences are most likely due to amino acid residues flanking the RGD sequence, a concept that was independently established by using a phage display library to select sequences with a preference for specific integrin receptors (Koivunen et al., 1993; Koivunen et al., 1995; O’Neil et al., 1992). The toxic cocktail of peptides and proteins in snake venom also includes hemorrhagic metalloproteases ranging from about 25 to 90 kD in size (Kini and Evans, 1992), which are related to membrane-anchored metalloprotease-disintegrins (Figure 1). The different molecular weights of these snake venom metalloproteases reflect differences in how they are processed from larger precursor proteins. The shortest hemorrhagic proteases are composed only of a metalloprotease domain, whereas longer ones also include a disintegrin domain and cysteine-rich region (Fox and Bjarnason, 1995; Hite et al., 1992; Kini and Evans, 1992; Paine et al., 1992;Takeyaet al., 1992; Takeyaet al., 1990a; Takeyaet al., 1990b).In some cases, a C-type lectin-domain is linked covalently to the cysteine-rich region via a disulfide bond (Kini, 1996; Takeya et al., 1992). It has been proposed that snake venom metalloproteases aggravate bleeding by attacking the basement membrane of blood vessels. Recent evidence suggests that they also contribute to an inflammatory response by releasing cytokines such as TNFa from the plasma membrane (Mourada-Silva et al., 1996). This is particularly intriguing in light of the finding that the TNFa convertase is apparently a member of the metalloprotease-disintegrin protein family (Black et al., 1997; Moss et al., 1997; Rosendahl et al., 1997) (see below).

I

zY

Examples01 Snake venom dislnfegnn~ and metalloproleases

Figure 7.

The domain organization of membrane-anchored metalloprotease-disintegrin proteins is shown in the left panel, and examples of the domains found in snake venom disintegrins and metalloproteases are shown in the right panel. Left Panel: Metalloprotease-disintegrins can be divided into two classes-proteins with a catalytic site in their metalloprotease domain (Zn++-binding consensus sequence: HEXXH) and proteins without a catalytic site. Of the 21 distinct metalloprotease-disintegrinsthat are currently known (JudyWhite, personal communication), the following 11 proteins contain an HEXXH sequence: Fertilin a /ADAM1 (Wolfsberget al., 1993), ADAM 8 (Yoshida eta[., 1990), MDC9/ADAM9 (Weskamp et al., 1996), KUZ/SUP-l7/ADAM 10 (Howard et al., 1996; Rooke et al., 1996; Rosendahl et al., 1997; Wen et al., 1997), meltrin a/ADAM 12 (Yagami-Hiromasaet al., 1995), MDC 15/Metargidin/ADAM15 (Kratzschmaret al., 1996), xMDC 16/ADAM16 (Shillinget al., 19971, TACE/ADAM 17 (Black et al., 1997; Moss et a!., 1997), meltrin !3/ADAM19 (Inoue et al., 1998), and ADAM 21 (Hooft van Huijsduijnen, 1998). The pro-domains of these proteins have an additional cysteine compared to the pro-domains of the noncatalytic metalloprotease-disintegrins.This cysteine is predicted to bind to the active site and keep the protease inactive until the pro-domain is removed (see also figure 5). Therefore the pro-domain of the catalytically active protein on the left is depicted as an inhibitorywedge, whereas the pro-domain of a non-catalytic protein on the right is not. Metalioprotease-disintegrins with or without a catalytic site may both be involved in cell-cell or cell-matrix interactions via their disintegrin domain, cysteine-rich domain, or ECF repeat. The disintegrin domain of several snake venom disintegrins contains an RCD sequence in a conserved position. In membrane anchored metalloprotease-disintegrins, the sequence found in lieu of the snake venom RCD is predicted to function as an integrin ligand (indicated as XYZC in the figure). Right Panel: A short, soluble RCD-containing snake venom disintegrin is shown binding to an integrin (allbp3) as a competitive inhibitor of platelet aggregation. Snake venom also contains metalloproteases (SVMPs), which may be composed of only a metalloprotease domain, but can also contain an additional disintegrin domain, a cysteine-rich region, and in some cases a C-type lectin attached via a disulfide bond (not shown in the model) (Fox and Bjarnason, 1995; Kini and Evans, 1992; Wolfsberg and White, 1996). These proteins are secreted as soluble proteins and, in some cases, precursors with multiple domains are further processed to yield individual protein domains. 169

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111.

ROLE OF THE METALLOPROTEASE-DISINTEGRIN FERTlLlN IN FERTILIZATION

The first recognized membrane-anchored metalloprotease-disintegrins were the a and p subunit of the guinea pig sperm protein fertilin (originally referred to as PH30) (Blobel et al., 1990; Blobel et al., 1992; Primakoff et al., 1987; Wolfsberg et al., 1993). PH-30 emerged from a screen for monoclonal antibodies against sperm membrane proteins that reside in specific membrane domains on guinea pig sperm (Myles and Primakoff, 1984; Myles et al., 1981; Primakoff and Myles, 1983). The initial purpose of this screen was to gather reagents for studying the function of sperm membrane proteins as well as how different plasma membrane domains of mammalian sperm are established and maintained. The resulting monoclonal antibodies were named according to where they bind on sperm (anterior-, posterior-, and whole head: AH, PH, and WH respectively; whole-, and posterior tail: WT, PT, respectively). The sorting of sperm membrane proteins to these distinct membrane domains is a fascinating cell biological question in its own right, and the monoclonal antibodies provided a wealth of information about the temporal and spatial relocation of sperm surface antigens (Cowan et al., 1986; Myles et al., 1987; Myles and Primakoff, 1984; Myles et al., 1984; Phelps et al., 1990; Phelps et al., 1988). Because proteins present on the sperm surface are likely to have a role in fertilization, Primakoff and Myles also tested whether any of the monoclonal antibodies blocked specific aspects of fertilization or sperm function (Flaherty et al., 1993; Lathrop et al., 1990; Lin et al., 1994; Primakoff et al., 1988a; Primakoff et al., 1985; Primakoff et al., 1987; Primakoff et al., 1988b). A particularly intriguing finding was that an antibody that reacted with the posterior head antigen (fertilin) PH-30 blocked sperm-egg membrane fusion (Primakoff et al., 1987). The significance of this finding was underscored by the observation that a second antibody (PH- 1) against fertilin did not affect fusion, even though both antibodies bound to live sperm. This argued against simple steric inhibition as an explanation for how the PH-30 antibody blocks sperm-egg fusion and instead suggested binding to a functional epitope. In these experiments, the zona pellucida was removed from the eggs, a procedure that allows a better evaluation of sperm-egg membrane binding and fusion by bypassing the zona pellucida binding step. Since the posterior sperm head participates in membrane fusion, but the anterior head does not, the localization of fertilin on the posterior head is consistent with a role in sperm-egg membrane binding and/or fusion. However, by immuno-electron microscopy, fertilin was not found in the equatorial region of guinea pig sperm, where fusion with the egg is thought to begin based on electron micrographs of fusing sperm and egg (Primakoff et al., 1987). Nevertheless, the observation that the PH30 monoclonal antibody inhibited sperm-egg fusion without blocking sperm-egg binding suggested that fertilin plays a role in the fusion of sperm and egg and may thus also be part of the sperm-egg fusion machinery.

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Exciting additional clues about the function of fertilin emerged from its primary structure (Blobel et al., 1992; Wolfsberg et al., 1993). Fertilin is a heterodimeric complex of two membrane-anchored glycoproteins-fertilins a and p (Blobel et al., 1990) (see Figure 2). Both subunits were affinity purified from sperm with the PH-30 monoclonal antibody, and peptide sequence information was then used to clone and sequence the corresponding cDNAs (Blobel et al., 1992). The most revealing insight was the discovery that the N-terminus of mature fertilin p is related to snake venom disintegrins. The apparent relationship with these snake venom integrin ligands immediately suggested that fertilin interacts with an integrin on the egg, even though fertilin does not harbor an RGD sequence. Furthermore, a potential fusion peptide was found in the a subunit. This short hydrophobic sequence resembles certain viral fusion peptides in that it can be modeled as an amphipathic alpha helix with a kink introduced by two proline residues (Blobel et al., 1992; White, 1992). The initial N-terminal protein sequence information for the mature fertilin a subunit indicated that it does not contain an intact disintegrin domain secretory

Cell surface

Cell surface

pathway PH.30 epltope CCPOSed

[tunclion blocking mAb)

Figure2. Processingandfunctional maturation of the guinea pig sperm protein fertilin. In the testis, fertilin is first detectable as a heterodimeric complex of two metalloprotease-disintegrins, fertilin a and p (Blobel et al., 1990). Fertilin a has a catalytic site in its metalloprotease domain, whereas fertilin p does not (Blobel et al., 1992; Wolfsberg et al., 1993). Fertilin a is processed in the secretory pathway of testicular cells, most likely by a pro-protein convertase such as PC4 before emerging on the cell surface (Lum and Blobel, 1997; Mbikay et al., 1997). This releases the metalloprotease domain of fertilin a. The fate of the metalloprotease domain after cleavage is presently unclear. Fertilin p is then processed in two steps during sperm maturation in the epididymis, and the final processing correlates with the acquisition of fertilization competence in the distal corpus epididymis (Blobel et al., 1990). Processing of fertilin exposes an epitope that is recognized by the function-blocking monoclonal antibody PH-30. The current working hypothesis for the function of fertilin is that the disintegrin domains of fertilin a and/or p mediate sperm-egg membrane binding and fusion by interacting with an integrin on the egg.

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(Blobel et al., 1992). However, a recent reevaluation of the N-terminus of mature fertilin a showed clearly that this subunit also contains an intact disintegrin domain (Lum and Blobel, 1997). Thus mature fertilin is a heterodimeric complex of two membrane-anchored glycoproteins that both contain an N-terminal disintegrin domain and therefore most likely interact with an integrin on the egg (Figure 2). This hypothesis has since been tested via several different approaches. Because fertilin does not contain an RGD sequence, the sequence found in lieu of the snake venom RGD was proposed to bind to integrins (Blobel and White, 1992; Blobel et al., 1992). By this definition, the predicted binding sequence of fertilin is TDE. An additional cysteine residue is present next to the TDE sequence in fertilin but not in RGD-containing snake venom disintegrins. Peptides containing the predicted binding sequence of fertilin and the additional cysteine residue (TDEC) were tested for their ability to inhibit guinea pig fertilization (Myles et al., 1994). Indeed, short cyclic or linear peptide containing the predicted binding sequence of fertilin p were able to block fertilization in a concentrationdependent manner, with half-maximal inhibition between 5 and 50 pM. A control peptide with the identical amino acid residues in a different order had no effect, even at the highest concentration tested. This type of experiment has since been repeated in several different species. Peptides corresponding to the predicted binding sequence of mouse fertilins a and p and of another mouse metalloprotease-disintegrin called cyritestin inhibit mouse in-vitro fertilization (Almeida et al., 1995;Evans et al., 1997a;Evans et al., 1995; Linder and Heinlein, 1997; Yuan et al., 1997).Fusion of human sperm with hamster eggs can be blocked by addition of peptides mimicking monkey fertilin p but also by RGD peptides (Gichuhi et al., 1997). The RGD-containing snake venom disintegrin echistatin blocks binding of human sperm to zona-free hamster eggs, suggesting that integrins that recognize RGD may also play a role in fertilization in hamsters (Bronson et al., 1995). Finally, several metalloproteasedisintegrin genes have been identified in Xenopus laevis testis, and peptides corresponding to the predicted binding sequence of three out of five deduced protein sequences inhibit X . laevis fertilization (Shilling et al., 1997). Clearly, there are certain caveats about using peptides to inhibit fertilization such as that peptides might act via a different mechanism than blocking binding of a metalloproteasedisintegrin to an integrin. Yet, in most cases, the conclusions of the peptide inhibition studies are supported by the finding that scrambled control peptides or peptides with specific amino acid replacements were not inhibitory. Taken together, these results support a model in which interactions of several distinct metalloprotease-disintegrins on sperm with integrins on the egg are necessary for fertilization to occur. To corroborate the peptide inhibition studies, it will now be essential to analyze the function of expressed metalloprotease-disintegrinprotein domains or of intact proteins and to evaluate their ability to bind to specific integrins. Employing expressed protein domains or purified proteins may also reveal if other parts of a metalloprotease-

Roles of Metallopro tease-Disintegrins

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disintegrin besides the predicted binding sequence are involved in interactions with a cell surface receptor on the egg or with proteins in the extracellular matrix. A first step in this direction has been taken by Evans and colleagues who used bacterially expressed fertilin a and p fusion proteins to block fertilization (Evans et al., 1997a; Evans et al., 1997b). One drawback of bacterially expressed fusion proteins is that they may not faithfully reproduce the folding and disulfide-bond formation of a disintegrin domain or cysteine-rich region that is normally expressed in a eukariotic cell. Therefore, Evans and colleagues reduced the fusion proteins and allowed them to refold in a dilute solution before use. This resulted in a monomeric and presumably properly folded population of fusion-proteins. The refolded fertilin p fusion-protein binds to the region of the plasma membrane where sperm bind, and this binding can be reduced with peptides corresponding to the predicted binding sequence of mouse fertilin. Interestingly, the fertilin a construct, which lacked most of the disintegrin domain, also binds to eggs, suggesting that other parts of a metalloprotease-disintegrin may also be involved in cell-cell interaction. Binding of the fertilin a and p fusion proteins could also be decreased by treating eggs with chymotrypsin, arguing that the fusion proteins bind to a receptor protein on the egg.

IV.

EVIDENCE FOR A ROLE OF INTEGRINS IN F E RT ILIZAT I O N

Which integrins are candidate receptors for fertilin and other metalloproteasedisintegrins on the egg? Immunofluorescence analysis revealed that the a6pl and the a v p 3 integrin subunits are expressed on the surface of mouse eggs (Almeida et al., 1995; Hierck et al., 1993; Tarone et al., 1993). Addition of a function-blocking monoclonal antibody against the a6 integrin (GoH3) to zona-free mouse eggs substantially reduces sperm binding (Almeida et al., 1995). Furthermore, mouse sperm can bind to tissue culture cells expressing a6 integrin but not to control cells and this binding can be prevented by fertilin binding-sequence peptides. Taken together, these results provide good evidence that the a6 integrin plays an important role in fertilization. Interestingly, Evans and colleagues could prevent binding of the bacterial fertilin p fusion protein to eggs with an antibody against p l integrins but not with the GoH3 antibody against a6 integrin (Evans et al., 1997a). Since at least 10 metalloprotease-disintegrin proteins are expressed in mouse testis, it is possible that other proteins related to fertilin are also involved in binding to integrins or other proteins on theegg (Linder andHeinlein, 1997; Shillinget al., 1997; Weskamp and Blobel, 1994; Wolfsberg et al., 1995a; Wolfsberg et al., 199%; Yuan et al., 1997). It will now be important to determine which of the metalloproteasedisintegrins expressed in testis are actually present on mature sperm and then to understand which integrins on the egg these proteins interact with and whether there is a temporal andor spatial order to these interactions.

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V. PROCESSING OF FERTILIN DURING SPERM MATURATION IN THE TESTIS AND EPIDIDYMIS Before fertilin can act in sperm-egg binding and fusion, both of its subunits are proteolytically processed during sperm maturation in the male reproductive tract (Blobel et al., 1990;Lum and Blobel, 1997; Phelps etal., 1990) (Figure 2). This processing is thought to be an important component of fertilin’s functional maturation. The precursor of fertilin a is cleaved intracellularly in testicular cells before the fertilin heterodimer emerges on the cell surface. In guinea pigs, the fertilin heterodimer can first be detected on the sperm surface in late spermatids (Carroll et al., 1995).The cleavage of fertilin a occurs next to four arginine residues, a known cleavage site of the proprotein convertase furin (Lum and Blobel, 1997). Furin is a serine protease in the transGolgi network that cleaves and activates several different proteins, including prohormones, viral fusion proteins, and certain adhesion proteins (Chretien et al., 1995; Seidah et al., 1994). A targeted deletion of the related proprotein convertase PC4, which is only expressed in spermatocytes and round spermatids, results in infertility, without affecting sperm count or sperm motility (Mbikay et al., 1997). Therefore, a defect in fertilin a processing could affect the function of fertilin a and/or might also interfere with the subsequent processing and activation of fertilin p. Fertilin p processing occurs while sperm are in transit through the epididymis (Blobel et al., 1990). In vitro studies indicate that one or more serine proteases derived from testicular sperm can mimic the processing of fertilin j3 in the epididymis (Lum and Blobel, 1997). It is thus possible that one or more of the proteases responsible for fertilin processing may derive from sperm instead of the epididymis, although this does not rule out the role of epididymal proteases in processing fertilin directly. In vivo, removal of the fertilin p prodomain and metalloprotease-domain occurs between the distal corpus and proximal cauda epididymis, which is where sperm acquire motility and fertilization competence (Hunnicutt et al., 1997; Lum and Blobel, 1997). It is noteworthy that the monoclonal antibody which blocks sperm-egg fusion (PH-30), only binds to epididymal sperm that have passed the distal corpus epididymis and therefore carry processed fertilin p (Blobel et al., 1990; Hunnicutt et al., 1997;Phelps et al., 1990). The mAb PH-30 epitope of fertilin is not accessible on testicular sperm, but it can be exposed by removing the prodomain of fertilin p with trypsin in vitro (Blobel et al., 1990). In vitro trypsinization of testicular sperm also triggers the relocalization of fertilin from the whole head to the posterior head domain (Hunnicutt et al., 1997; Phelps et al., 1990).Evidently, proteolytic processing appears to be an important aspect of the maturation and functional activation of fertilin and may serve to expose an epitope involved in sperm-egg fusion, and to relocalize and concentrate fertilin in the posterior sperm head. Proteolytic processing may also be important for several other sperm surface proteins that are cleaved and change their localization on the sperm membrane in concert with fertilin (Hunnicutt et al., 1997; Myles et al., 1987; Myles and Primakoff, 1984; Phelps et al., 1990).

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VI. EVIDENCE FOR A POTENTIAL ROLE OF METALLOPROTEASE-DISINTEGRINSAS INTEGRIN LICANDS IN SOMATIC CELL-CELL INTERACTIONS Since metalloprotease-disintegrins are apparently essential for sperm-egg membrane binding to occur, it is tempting to speculate that metalloprotease-disintegrins expressed in somatic cells also mediate cell-cell interactions, perhaps by binding to integrins (Blobel, 1997;Blobel and White, 1992;Blobel et al., 1992;Wolfsberg and White, 1996). The first evidence that metalloprotease-disintegrin proteins are also expressed in somatic cells and indeed the first evidence that they comprise a protein family came from a search for proteins related to fertilin by PCR using cDNA from different mouse tissues as a template (Weskamp and Blobel, 1994). Additional family members have been cloned from a variety of different cells and tissues (Black et al., 1997;Emi et al., 1993; Heinlein et al., 1994; Herren et al., 1997; Hooft van Huijsduijnen, 1998;Howard et al., 1996;Inoue et al., 1998; Kratzschmaret al., 1996;Moss et al., 1997; Perry et al., 1994; Perry et al., 1995; Perry et al., 1992; Rosendahl et al., 1997; Shilling et al., 1997; Weskamp et al., 1996; Wolfsberg et al., 199%; Wolfsberg and White, 1996; Yagami-Hiromasa et al., 1995; Yoshida et al., 1990). Among the widely expressed metalloprotease-disintegrins, metargidin (MDC 15) is particularly intriguing because it is the only currently known membrane-anchored family member with an RGD sequence in its predicted integrin binding domain (Kratzschmar et al., 1996). Metargidin is therefore a candidate binding partner for integrins that bind the RGD sequence. All other membrane-anchored metalloprotease-disintegrins besides MDC 15 do not contain an RGD sequence in their predicted integrin binding site, although an acidic glutamate residue is often present in lieu of the aspartate in the RGD sequence. An alignment of most of the presently known predicted binding sequences is shown in a recent review by Wolfsberg and White (Wolfsberg and White, 1996). Interestingly, the predicted integrin binding sequence of some soluble snake venom metalloprotease-disintegrins resemble the predicted binding sequence of the widely expressed metalloprotease-disintegrin MDC9 (Weskamp et al., 1996). Two of these snake venom proteins, Jararhagin (De Luca et al., 1995; Karniguti et al., 1996; Kamiguti et al., 1997; Paine et al., 1992; Usami et al., 1994) and Atrolysin (Fox and Bjarnason, 1995;Jia et al., 1997; Shimokawa et al., 1996) are able to block collageninduced platelet aggregation with an IC,,,of about 0.1 pM, presumably by binding to the a2p1 collagen receptor. A disintegrin domain and cysteine-rich region of atrolysin, expressed in a baculovirus system, inhibits collagen-induced platelet aggregation with an IC,,, of around 0.5 pM. Peptides corresponding to the 12 amino acid loop, which includes the predicted binding sequence of atrolysin, ESEC, also inhibit collagen-induced platelet aggregation although only with an ICsoof 200 to 550 pM. These results provide the first indication that metalloprotease-disintegrins with a sequence other than RGD in their predicted binding site can interact with integrins. The difference in inhibitory potential between the binding site peptides and the purified

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expressed protein implies either that the peptides do not properly mimic the conformation of the binding sequence in atrolysin, or that other parts of the disintegrin domain or cysteine-rich regions contribute to blocking collagen-induced platelet aggregation. Because of the similarity in the predicted integrin binding sequence of MDC9 and atrolysin or jararhagin, it will be interesting to determine whether MDC9 also interacts with the a 2 p l integrin. The metalloprotease domain of atrolysin may contribute to a role in blocking platelet aggregation, as atrolysin with an intact metalloprotease domain is a somewhat more potent inhibitor of collagen-induced platelet aggregation than the expressed protein lacking the metalloprotease domain (see above; Jia et al., 1997). The related jararhagin can cleave purified p l integrin subunit in vitro and blocks collagen-induced phosphorylation of pp72(syk) in platelets (Kamiguti et al., 1996; Kamiguti et al., 1997). The authors suggest that the disintegrin domain might target the metalloprotease domain to the a 2 p l integrin and that the resulting cleavage of the p l integrin blocks signal transduction via the a 2 p l integrin. From the available data, it is not clear whether pp72(syk) phosphorylation in platelets is blocked because p l has been cleaved or because binding to collagen has been prevented. Nevertheless, the concept of targeting the metalloprotease domain to an integrin via the disintegrin domain is attractive (Blobel, 1997) (Figure 3). This may provide one of

I

Substrate

Figure 3.

I&_

lntegrin

Examples of different models for targeting .the metalloprotease to its substrate. The metalloprotease could conceivably be targeted to its substrate directly or indirectly, in cis (on the same membrane as the substrate), or in trans (on opposing membranes). The Left Panel depicts possible variations of direct targeting in cis: the cleavage site alone may be the main determinant of cleavage site specificity, or alternatively, the disintegrin domain, cysteine-rich domain, ECF repeat, or cytoplasmic domain could deliver the protease to its substrate. The Middle Panel shows an example of indirect targeting in cis: The protease may be targeted to a substrate in the vicinity of integrins, such as in focal contacts or adherens junctions, via binding of the disintegrin-domain to an integrin. In the Right Panel, the metalloprotease is targeted indirectly to a substrate in trans by binding to an integrin.

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several means that metalloprotease-disintegrins could use to gain direct or indirect access to the appropriate substrates. By direct access, aprocess is meant where, for example, an integrin itself is both targeted by the disintegrin domain and cleaved by the metalloprotease domain, whereas indirect access refers to a process where proteins in proximity to an integrin are cleaved (see below and Figure 3).

VI I.

ARE METALLOPROTEASE-D IS1NTEGRINS MEMBRANE FUSION PROTEINS?

Metalloprotease-disintegrins have been implicated in two distinct membrane fusion events: Fertilin is thought to play a role in sperm-egg binding and fusion (Primakoff et al., 1987), and a protein called meltrin a has been linked to the fusion of myoblasts into myotubes (Yagami-Hiromasa et al., 1995). However, despite the evidence linking fertilin and meltrin a to membrane fusion events, the currently available data do not allow the crucial distinction between a direct role in fusion as part of the membrane fusion machinery and an indirect role such as in providing a binding step or signaling event that is a prerequisite for fusion to occur. A.

Evidence for a Role in Membrane Fusion

Fertilin was initially implicated in sperm-egg membrane fusion by a monoclonal antibody, which blocks fusion without blocking sperm-egg binding (Primakoff et al., 1987). Furthermore, peptides mimicking the fertilin p predicted binding sequence block sperm-egg fusion without blocking spermegg binding (Myles et al., 1994; Yuan et al., 1997). Judging by its protein sequences, fertilin shares two hallmark features with bona fide viral fusion proteins: specific binding domains and a hydrophobic fusion peptide (Blobel et al., 1992). The specific binding domains are the disintegrin domains of fertilins a and the j3,which are predicted to bind to integrins on the egg (Figure 2). A sequence closely resembling viral fusion peptides exists within the cysteine-rich region of the a subunit. Viral fusion peptides are short hydrophobic sequences that can be modeled as an amphipathic alpha helix and that are thought to trigger membrane fusion by inserting into a target membrane (White, 1990; White, 1992). There is no apparent sequence conservation among different viral fusion peptides, suggesting that the presence of an amphipathic a helix is more important than the primary sequence. Because the potential fusion peptide of fertilin a can be modeled as an amphipathic alpha helix kinked around two prolines, it has been proposed that fertilin may be the functional equivalent of a viral fusion protein (Bigler et al., 1997; Huovila et al., 1996; Myles and Primakoff, 1997; Snell and White, 1996; White, 1992).The localization of fertilin in the posterior sperm head is also consistent with a role in membrane fusion. As outlined above, the posterior head ofmammalian sperm participates in fusion with the egg, and fertilin migrates to the posterior head during sperm maturation. By analogy, viral fusion proteins are also known to require

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a certain density on the cell surface to become functional (Danieli et al., 1996). Thus the relocalization of fertilin to the posterior head could serve to concentrate fertilin in this region. Several other cell types besides sperm and egg are capable of membrane fusion, most notably myoblasts, monocytes, and cytotrophoblasts (Huovila et al., 1996; White, 1992; White and Blobel, 1989). By searching for homologues of fertilin that might mediate the fusion of C2C12 mouse muscle cells, A. Fujisawa-Sehara and colleagues discovered a metalloprotease-disintegrin termed meltrin a (YagamiHiromasa et al., 1995). Overexpression of a truncated form of meltrin a lacking the metalloprotease domain increased muscle fusion, whereas overexpression of a full length form and also of antisense mRNA resulted in a decrease in muscle fusion. Like fertilin a, meltrin a also has a short hydrophobic region in its cysteine-rich region. These results led to the suggestion that meltnn a might have a direct role in the fusion of myoblasts into multinucleated myotubes. Thus, two metalloprotease-disintegrins, fertilin and meltrin a, have been implicated in the process of cell-cell fusion. B.

Alternative Interpretations

Despite the compelling evidence for a role of fertilin a and meltrin a in membrane fusion, all currently available data are equally consistent with a role for these proteins in a step that is a prerequisite for fusion to occur. Such a function must be distinguished from a direct role in triggering membrane fusion as part of the actual fusion machine. Hydrophobic sequences that can be modeled as amphipathic fusion peptides are also present in a conserved position in other metalloproteasedisintegrin proteins, including MDC9 (Weskamp et a]., 1996) and MDCll (Emi et al., 1993; Katagiri et al., 1995). While fertilin a,meltrin a,MDC9, and MDCI 1 are all widely expressed, any role in fusion would have to be restricted to fusing cells, either by only activating the fusion peptide in fusing cells, or by keeping it inactive in nonfusing cells. An alternative possibility is that these hydrophobic sequences are a structural feature of metalloprotease-disintegrins that are important for protein folding or for the interaction with other proteins. In the context of understanding membrane fusion, an interesting observation regarding meltrin a was its expression in fusing muscle cells and in bone (YagamiHiromasa et al., 1995), where osteoclasts are a prominent cell type. Osteoclasts are formed by the fusion of cells derived from monocytic precursors and therefore the expression of meltrin a in muscle and bone suggested a possible role in both muscle fusion and osteoclast fusion. However, an analysis of the expression of meltrin a and the related meltrin p in bone cells revealed strong expression of both proteins in osteoblasts, but no detectable expression in osteoclasts (Inoue et al., 1998). While these results do not rule out expression in early osteoclast precursors, they strongly suggest that meltrins cx and p are involved in osteoblast function, but not in the fusion of osteoclasts. This result argues against a conserved role of meltrin a in cell-cell fusion in different cell types.

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One criterion for a bona fide membrane-fusion protein is that membrane fusion can be reconstituted by transfection of the candidate fusion protein into nonfusing cells or by reconstitution into lipid vesicles (White, 1990; White, 1992; White and Blobel, 1989). Transfection of meltrin a into fibroblasts did not lead to an increase in cell fusion (Yagami-Hiromasa et al., 1995). Clearly, failure to reconstitute fusion does not rule out arole in fusion because the cellular fusion machinery may be more complex than viral fusion proteins. Muscle cells might contain other proteins that are required for meltrin cx to promote membrane fusion but that are not expressed or active in fibroblasts. Ultimately, the identity of a bona fide cell-cell fusion protein or protein machine will only be determined by reconstituting membrane fusion with defined components. In the interim, it will be important to more accurately understand the roles of meltrin a and fertilin in the cascade of steps that result in membrane fusion and thereby perhaps distinguish between a direct and indirect role in fusion. Another possibility that must be considered in this context is that cellular membrane fusion may not actually require a fusion peptide to fuse membranes. In a fascinating new development in the field of intracellular membrane fusion, the fusion of Golgi vesicles has been reconstituted in liposomes in vitro with purified SNARE proteins, which do not have any apparent fusion peptides. The proposed model is that the coiled-coil domains in the membrane-anchored V-SNAREin a vesicle and t-SNARES in the target membrane anneal thereby forcing the membranes into very close apposition. This is proposed to destabilize both lipid bilayers and trigger fusion (Weber et al., 1998). If such a model is applied to cell-cell fusion reactions, then membrane fusion proteins might indeed be a specialized form of cell-cell adhesion proteins. In this scenario, proteins functioning only in cell-cell adhesion would allow cells to bind to each other, without bringing the opposing membranes close enough together to trigger fusion. Perhaps only certain highly specialized cell-cell adhesion/fusion proteins are able to undergo a conformational change following the initial binding step, which could conceivably force the opposing membranes so close together that they fuse. A direct role in mediating membrane fusion-by insertion of a fusion peptide in analogy to viral fusion proteins or by bringing opposing transmembrane domains into very close proximity, perhaps via a conformational change-must then be distinguished from other cell-cell binding steps that do not directly trigger membrane fusion.

VIII. A.

METALLOPROTEASE-DISINTEGRINSUBSTRATES

ADAM1 0 and Drosophila Kuzbanian May Play a Role in the Activation of Notch

The first evidence for metalloprotease activity of a membrane-anchored metalloprotease-disintegrin was provided by P. Glynn and colleagues, who purified

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a protein termed MADM (membrane anchored disintegrin metalloprotease, also referred to as ADAM10) from bovine brain based on its ability to cleave the myelin basic protein (Chantry et al., 1989; Howard and Glynn, 1995; Howard et al., 1996). The myelin basic protein is a cytoplasmic protein and may therefore not be a substrate for the extracellular protease domain of MADM/ADAM10 on intact cells in vivo. Recent evidence suggests that MADM/ADAM I0 is the human orthologue of Drosophila Kuzbanian (Fambrough et al., 1996; Pan and Rubin, 1997; Rooke et al., 1996; Sotillos et al., 1997) and C. elegans SUP17 (Wen et al., 1997). Mutations in KUZ and SUP17 affect cell fate decisions, most likely by interfering with the cleavage and activation of Notch (Pan and Rubin, 1997; Sotillos et al., 1997; Wen et al., 1997). The Drosophila metalloprotease-disintegrin KUZ was initially identified in a genetic screen of mosaic flies for genes with a role in neurogenesis (Rooke et al., 1996). In both embryos and in the adult fly, kuz is important for lateral inhibition of the neuronal phenotype. Therefore, embryos lacking maternal and zygotic KUZ have a severe neuralizing phenotype. Furthermore, KUZ plays a role in several additional developmental processes. It is required for neuronal extension (Fambrough et al., 1996), and in mosaic adults, the mutation disrupts the regular array of ommatidia in the eye (Rooke et al., 1996). In the epidermis of mosaic flies, supernumerary sensory bristles are observed in mutant cell clusters instead of the single sensory bristle in wild-type epidermis. Rooke and colleagues report observing the supernumerary bristles only at the boundary of mutant and wild-type cell patches. This would imply that only the wildtype cells can send a signal promoting neural development to the mutant cells. However, in an independent report on the phenotype of kuz-/-flies by Sotillos and colleagues, supernumerary bristles were also observed within a mutant cell patch indicating that neural induction can be promoted by kuz-/-cells (Sotillos et al., 1997). Once neural development is initiated in one cell, adjacent cells receive a signal inhibiting neural differertiation in a process referred to as lateral inhibition (Simpson, 1990). kuz -/-clones are evidently unable to receive this signal and, as a consequence, clusters of sensory bristles develop instead of single bristles. The cell-autonomous role of KUZ in lateral inhibition is apparently due to a defect in an extracellular cleavage of Notch (Pan and Rubin, 1997). Notch is a protein of about 300 kD that plays arole in cell fate decisions in several different tissues (Artavanis-Tsakonas et al., 1995; Kimble and Simpson, 1997). Through an elegant genetic analysis, Pan and Rubin determined that KUZ may be acting upstream in a pathway that affects the function of Notch. Pan and Rubin further suggested that KUZ has arole in the constitutive cleavage of Notch (Pan and Rubin, 1997), which occurs constitutively in the secretory pathway (Blaumueller et al., 1997). However, a more recent study presents compelling evidence that furinlike proprotein convertase cleaves Notch in the secretory pathway (Logeat et al., 1998). A current model of KUZ function is that it cleaves Notch in a membrane-proximal position subsequent to the constitutive processing by a fur-

Roles of Metalloprotease-Disintegrins

181

inlike protease. The membrane-proximal cleavage performed by KUZ is most likely triggered when Notch interacts with one of its ligands such as Delta (Bray, 1998; Chan and Yan, 1998). As a consequence of Notch processing by KUZ, an additional cleavage of Notch within its transmembrane domain by a distinct and yet to be identified protease may be triggered. Cleavage of Notch within the transmembrane domain has been shown to release the cytoplasmic tail of Notch from the plasma membrane (Schroeter et al., 1998; Struhl and Adachi, 1998). The soluble cytoplasmic tail of Notch then interacts with the CSL (CBFl/Suppressor of Hairless/Lag- 1) family of DNA binding proteins and activates genes that respond to Delta- /Notch-dependent signaling. This model is consistent with the genetic phenotype of kuz-/-flies in that a defect in KUZ could prevent both the membrane proximal cleavage and the subsequent cleavage in the transmembrane domain of Notch, thus preventing Notch signaling via CSL proteins. In addition, KUZ may have other yet to be identified substrates including other proteins that may play a role in the Notch signaling pathway. The availability of a dominant negative mutation provided an opportunity for Pan and Rubin to test the functional conservation of KUZ in other species. Overexpression of a mouse dominant negative construct in Xenopus laevis also increased the number of neuronal cells, most likely by interfering with lateral inhibition (Pan and Rubin, 1997). Furthermore, the dominant negative KUZ had a similar effect on neuronal extension as mutant KUZ. Dominant negative KUZ could perhaps interfere with the function of endogenous wild-type KUZ by binding to substrates without cleaving them or by disrupting a complex with another protein, perhaps even another metalloprotease-disintegrin. Mutation of a short stretch of KUZ cytoplasmic tail residues that is conserved between species weakens the dominant negative phenotype of KUZDN. This result raises the possibility that interaction of KUZ with a cytoplasmic protein or a cytoplasmic domain of a membrane protein may have a role in the dominant negative phenotype. The C. elegans protein LIN-l2/Notch is also involved in cell fate decisions during hermaphrodite gonadogenesis where it plays a role in determining which of two precursor cells turns into an anchor cell or into a uterine precursor cell (Greenwald et al., 1983). A screen for suppressers of a specifii lin-I2 mutation led to the identification of sup-1 7 (Ferguson and Horvitz, 1985; Tax et al., 1997), which is a putative orthologue of Drosophila kuz (Wen et al., 1997). A genetic analysis of sup-17 suggested that it acts cell-autonomously in a parallel pathway or upstream of lin-12 and further that sup-I 7might affect the function of the extracellular domain of LIN-12 (Wen et al., 1997). Thus the role of KUZ/SUP-l7/ADAM10 as a mediator in NOTCH signaling appears to be remarkably conserved in different organisms. In both C. elegans and in Drosophila, the phenotype of KUZ/SUP- 17 null mutants is not as severe as the phenotype of LIN- 12/Notch null mutants (Sotillos et al., 1997; Wen et al., 1997). Evidently, LIN-l2/Notch may have some residual signaling activity in the absence of an extracellular cleavage, or alternatively, other proteases including other metalloprotease-disintegrins

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might be able to activate LIN-12/Notch and thus partially compensate for loss of KUZ/SUP- 17/ADAM 10 (see below). B. Identification of the T N F a Convertase Suggests that Metalloprotease-Disintegrins Play a Role in Protein Ectodomain Shedding

Membrane-anchored metalloproteases are known to mediate the regulated release or shedding of several physiologically relevant proteins from the plasma membrane. These proteins include cytokines, cytokine receptors, adhesion proteins, and other molecules such as the p amyloid precursor protein (Arribas et al., 1996; Arribas and Massague, 1995; Hooper et al., 1997; Mullberg et al., 1997). A major breakthrough in the field of protein ectodomain shedding resulted from the purification the tumor necrosis factor a (TNFa) convertase by Marcia Moss and colleagues at Glaxo Wellcome (Moss et al., 1997) and by Roy Black and colleagues at Immunex (Black et al., 1997). Using an assay for T N F a cleavage, both groups independently purified the identical novel membrane anchored metalloproteasedisintegrin, which was named TACE (Black et al., 1997; Moss et al., 1997) (or ADAM 17, J. White, personal communication). The function of the purified protease was evaluated and confirmed by several different criteria. Firstly, its cleavage kinetics for a soluble peptide substrate matched the kinetics of cell-based cleavage assays. Secondly, purified TACE showed the same sensitivity to inhibition with hydroxamate-based metalloprotease inhibitors as the T N F a convertase on cells (Moss et al., 1997). Thirdly, neutrophils expressing only a form of TACE with a targeted deletion of the catalytic site have a severe defect in TNFa shedding (Black et al., 1997). It should be noted that a targeted deletion of the catalytic site exon may result in expression of dominant negative protein (see above), which could affect other proteins including metalloprotease-disintegrins directly or indirectly. Nevertheless, the combined evidence that TACE is responsible for processing TNFa in vivo is very compelling. Despite the strong evidence linking TACE to T N F a shedding, the purification of TACE by two independent groups (Black et al., 1997; Moss et al., 1997) did not definitively resolve the issue of which protease processes T N F a in vivo (Lunn et al., 1997; Rosendahl et al., 1997). Rosendahl and colleagues found ADAM10 highly enriched in a protein preparation from monocytic THP- 1 cells that specifically cuts a TNFa cleavage site peptide (Rosendahl et al., 1997). When expressed recombinant ADAM 10 was incubated with different peptides spanning the cleavage site of eight other potential sheddase substrates, only the T N F a peptide was cleaved. This result raises several interesting questions about the substrate specificity of metalloprotease-disintegrins. If an expressed soluble protease displays cleavage specificity for a soluble substrate peptide, will this also be the case for the membrane-anchored protease and a membrane-anchored substrate? Or could there be additional levels of regulation, such as by targeting of the protease to the substrate, that determine which enzyme acts on T N F a (Figure 3).

Roles of Metalloprotease-Disrntegrins

IX.

183

DO OTHER METALLOPROTEASE-DISINTEGRINSALSO MEDIATE PROTEIN ECTODOMAIN SHEDDING?

A fascinating prospect raised by the apparent role of metalloprotease-disintegrins in cleaving TNFa is that other members of this protein family may also have a role in protein ectodomain shedding. A growing number of membrane proteins is known to be released from the plasma membrane by membrane-anchored metalloproteases. The list of known “sheddase” substrates includes cytokines, cytokine receptors, adhesion proteins, leukocyte antigens, and other proteins such as the p amyloid precursor protein (Ehlers and Riordan, 1991; Hooper et al., 1997;Massague and Pandiella, 1993). In many cases this processing can be triggered by the phorbol ester PMA, an activator of protein kinase C. Indeed, when cell surface biotinylated CHO cells are treated with PMA, about 2 to 4% of all labeled proteins on the cell surface are released into the medium by metalloproteases (Arribas et al., 1996). Several independent observations support the hypothesis that metalloproteasedisintegrins are involved in some or perhaps even all protein ectodomain shedding events. Tissue inhibitors of matrix metalloproteases (T1MPs);which inhibit certain matrix-type metalloproteases, do not effectively inhibit protein ectodomain shedding of the IL-6 receptor and the p60 TNF receptor (Hooper et al., 1997; Mullberg et al., 1995). Furthermore, small molecule inhibitors of matrix-type metalloproteases (MMPs) do not inhibit shedding at concentrations that are known to block MMP activity. These results do not rule out the involvement of an MMP that is less sensitive to TIMP or other inhibitors. However, together with the fact that metalloproteasedisintegrins have already been linked to the processing of TNFa, these observations suggest that other membrane-anchored metalloprotease-disintegrins may have roles in protein ectodomain shedding. Finally, several metalloproteasedisintegrins have cytoplasmic signaling motifs (Weskamp et al., 1996; Wolfsberg and White, 1996), and at least one family member, MDC9, is phosphorylated when cells are treated with PMA (Roghani et al., 1999). Interaction with cytoplasmic proteins and phosphorylation by protein kinase C could conceivably play a role in inside-out signaling and activation of the metalloprotease domain. The hypothesis that metalloprotease-disintegrins may be involved in protein ectodomain shedding raises a number of additional issues for consideration. There are currently 1 1 identified metalloprotease-disintegrins that are predicted to be catalytically active because they contain the catalytic site consensus sequence HEXXH (Figure 1). On the other hand, there are over 40 potential substrates that are known to be shed from the plasma membrane (Hooper et al., 1997). It seems likely that additional substrate proteins exist that have not been characterized (see Figure 7 in reference; Arribas and Massague, 1995). Is there overlap in the substrate specificity of metalloprotease-disintegrins, or are there specific substrates or groups of substrates that are only cleaved by one protease? What is the substrate specificity of metalloprotease-disintegrins, and how is it determined? Are substrates cleaved on

1a4

CARL P BLOBEL

the same membrane as the protease resides on, that is in cis, or are they cleaved in trans on the plasma membrane of an opposing cell? Is the substrate specificity determined only by a membrane proximal cleavage site (Arribas et al., 1997; Brakebusch et al., 1994; Migaki et al., 1995; Mullberg et al., 1994; Sisodia, 1992;Tang et al., 1996), or are parts of the metalloprotease-disintegrin, such as the disintegrindomain, the cysteine-rich region,-theEGF repeat, or the cytoplasmic domain-used to target the metalloprotease to its substrate (Figure 3)? Is there a common factor regulating protein ectodomain shedding as suggested by CHO cell mutants that apparently have a general defect in all PMA-induced shedding by metalloproteases (Arribas et al., 1996; Arribas and Massague, 1995)? Recent evidence indicates that L-selectin shedding by a metalloprotease may be negatively regulated by calmodulin, which binds to the cytoplasmic tail of L-selectin (Kahn et al., 1998). Thus both the protease as well as the substrate might be subjected to regulation by cytoplasmic factors.

X. INTRACELLULAR MATURATION AND PROTEOLMIC PROCESSING OF METALLOPROTEASE-DISINTEGRINS Metalloproteases and most other proteases are made as inactive precursors or zymogens that require proteolytic processing to become active. Several metalloprotease-disintegrins with a catalytic site consensus sequence contain a cleavage site for furin between their prodomain and metalloprotease domain (Kratzschmaretal., 1996;Weskampet al., 1996). Therefore furinorrelatedproprotein convertases are likely involved in maturation and activation of metalloprotease-disintegrins. Evaluation of the intracellular maturation of MDC9 and MDC15, both of which contain a furin cleavage site between the pro- and metalloprotease-domains, revealed that both proteins are made as precursors that are cleaved in the late secretory pathway (Lum et al., 1998; Roghani et al., 1999). Prodomain removal can be mimicked by furin in vitro, confirming that furin or related proprotein convertases are responsible for processing these two proteins in the secretory pathway. All metalloprotease-disintegrins with a catalytic site (HEXXH) also contain an unpaired cysteine residue in their prodomains. This cysteine residue has been predicted to bind to and inactivate the catalytic site via a cysteine-switch mechanism (Van Wart and Birkedal-Hansen, 1990). Indeed, the protease activity of snake venom adamalysin (Grams et al., 1993) and of MDC9 (Roghani et al., 1999) can be inhibited by a short peptide containing the cysteine-switch residue and a small number of adjacent amino acids. Therefore it is likely that the prodomain of membrane-anchored metalloprotease-disintegrins must be removed by a proprotein convertase before the metalloprotease can become active (Figure 5). After the inhibitory prodomain is removed, the metalloprotease may still be subjected to other means of regulation such as by yet-to-be-identified cytoplasmic factors (see above).

185

Roles of Metalloprotease-Drsrntegrrns

-

TACE, MDC9 Regulated function at or near cell surface’

0

0

I

O

I

0

Golgi in TGNIendosome?.

Figure 4. Model depicting two different subcellular localizations and putative functions for catalytically active metalloprotease-disintegrins: Constitutive processingof substrates in the secretory pathway, and regulated processingof substrates on or near the cell surface. TNFa and several other substrates are shed from the plasma membrane on or near the cell surface in a regulated fashion (Hooper et al., 1997). In COS-7 cells, the metalloprotease-disintegrin MDC9 is localized predominantly on or near the cell surface (Weskamp et al., 1996), whereas M D C l 5 (metargidin) is localized predominantly to the trans-Golgi network (TGN) or endosomes (Lum et al., 1998). Furthermore, MDC9 is phosphorylated after stimulation with PMA (Roghani et al., 19991, whereas M D C l 5 is constitutively phosphorylated (1. Arribas and C. Blobel, unpublished observation). One possible scenario, therefore, is that metalloprotease-disintegrins and their respective substrates might be sorted into the regulated and constitutive secretory pathway and that the metalloprotease itself might be regulated or constitutively active.

After prodomain removal in a late secretory compartment, the predominant subcellular localization of MDC9 and MDCl.5 in transiently transfected COS-7 cells is different (Lum et al., 1988; Roghani et al., 1999). Immunofluorcscent labeling of MDC 15 revealed a primarily perinuclear staining pattern consistent with an accumulation of MDCIS in the trans-Golgi network or the endosome. In contrast, MDC9 is found mainly in the periphery of the cell, an observation consistent with a localization at or near the cell surface. While the localization of these two proteins in other cells and tissues remains to be determined, their distinct subcellular localizations in COS-7 cells are consistent with the idea that certain metalloproteasedisintegrins, such as MDC1.5 might function mainly inside the cell, whereas others such as MDC9 and TACE might function mainly on or near the cell surface (Figure 4). Intracellular functions could include processing of proteins in the TGN, whereas cell surface functions could include regulated protein ectodomain shedding, or a role in cell-cell or cell-matrix interactions.

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Figure 5. Regulation of the metalloprotease via a cysteine-switch mechanism (adapted from Woessner, 1991). Several metalloprotease-disintegrins, including MDC9 and MDCl5, are synthesized as precursors. The pro-domains of MDC9 and M D C l 5 are apparently removed by pro-protein convertases such as furin in the trans-Colgi network, resulting in a membrane anchored metalloprotease- and disintegrin-domain Lum et al., 1998; Roghani et al., 1999). The pro-domain contains an unpaired cysteine residue, which is thought to bind to the active site, thus preventing access to substrates. Specifically, the free cysteine residue in the pro-domain likely provides a fourth coordination site for the Zn++ ion, which is also bound by three histidine residues in the catalytic site. The pro-domain must therefore be removed from the active site for the protease to become active. This could occur via proteolytic removal of the pro-domain (the boxed RRRR attached to the pro-domain indicates a furin cleavage site), or by a conformational change of a pro-domain that remains attached to the protease. After removal of the prodomain, additional steps may be necessary to activate the protease such as inside-out activation in the case of regulated protein ectodomain shedding.

XI.

METALLOPROTEASE-DISINTEGRINS LACKING A MEMBRANE ANCHOR

Proteolytic processing of metalloprotease-disintegrins such as fertilin, MDC9, and MDC15 is likely to be an important aspect of their maturation and activation. In the case of fertilin a, processing releases a soluble metalloprotease domain, which could conceivably act at a distance from the cell or could be sorted into the acro-

Roles of Metalloprotease-Disintegrins

187

some. If other metalloprotease-disintegrins are found to be processed such that a soluble metalloprotease-domain, a disintegrin-domain, or other functional extracellular protein domains are released, the resulting soluble protein domains would have very different predicted functions compared to the membrane-anchored forms. Rather than mediating cell-cell interactions, a soluble disintegrin domain could compete with the membrane-anchored form, or with other membraneanchored or extracellular matrix proteins for binding to an integrin (Figure 6). This might result in de-adhesion. Alternatively, soluble disintegrin protein domains could be immobilized on the extracellular matrix and therefore provide sites for

With membrane anchor:

:ell-cell bindingkignaling

Without membrane anchor:

De-adhesionkignaling

A

Cell-matrix bindingkignaling

e l XYZC

Figure 6. Models of metalloprotease-disintegrin functions depending on the presence or absence of a membrane anchor. Only functions of the disintegrin domain are considered, although the presence or absence of a membrane anchor will also affect the targeting and function of the metalloprotease or other protein domains. On the left, a membrane-anchored metalloprotease-disintegrin is shown engaged in a cell-cell interaction. This could be accompanied by a signal transmitted either via the integrin, or via the disintegrin cytoplasmic tail. In the Center, a soluble disintegrin domain is modeled as a competitive inhibitor of an integrin-dependent cell-matrix interaction potentially resulting in de-adhesion. This is conceptually similar to the function of snake venom disintegrins (see figure 1). O n the Right, the disintegrin domain is shown as an integrin ligand that is immobilized on the matrix. Soluble or matrix-bound protein domains could be created in three different ways: (a) release by membrane proximal cleavage of membrane anchored proteins, (b) expression of genes that do not encode a membrane anchor, or (c) expressionof splicevariants of membrane-anchored proteins.

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cell-matrix attachment, anchor the metalloprotease on the matrix, or target it to another cell. While there is currently no evidence for release of soluble disintegrin-domains from a membrane-anchored precursor, a few examples of metalloproteasedisintegrins that are synthesized without a membrane anchor have been described. These proteins are either encoded by a gene that lacks the transmembrane domain and cytoplasmic sequences, or are splice variants of membrane-anchored proteins. The ADAMTS 1 gene encodes a metalloprotease-disintegrin lacking a membrane anchor and cytoplasmic domain (Kuno et al., 1997). Instead of a membrane anchor, a thrombospondin (TSP) type-1 motif and several TSP submotifs are appended to the disintegrin domain of ADAMTS-1. ADAMTS-1 was identified through a screen for genes that are selectively expressed in a cachexigenic tumor cell line, and its expression is upregulated in vivo by triggering an inflammatory response in mice. Once ADAMTS-1 is secreted, the TSP motifs may serve to immobilize it on the extracellular matrix. A second soluble metalloprotease-disintegrin named decysin was isolated by PCR from germinal center dendritic cells, and is upregulated upon maturation of these cells (Mueller et al., 1997). Decysin consists only of a metalloprotease domain and a truncated disintegrin domain and may therefore be secreted as a soiuble protease. The first example of splice variants of metalloprotease-disintegrin genes was the human ADAMl 1 protein, which was initially reported as a potential breast cancer tumor suppressor just before the BRCAl sequence was identified (Emi et al., 1993; Katagiri et al., 1995). The wild-type ADAMl 1 reportedly exists in at least two forms, one with a transmembrane domain and a very short cytoplasmic tail, the other lacking a transmembrane domain and parts of the cysteine-rich domain and EGF- repeat. The ADAM 11 gene maps close to BRCAl, and mutations in the ADAMl 1 gene have been found in two out of 650 patients with breast cancer. The two reported mutations would result in a truncated protein lacking most of the disintegrin domain. Given that several proteins with a role in cell adhesion also function as tumor suppressors, one possibility is that the lack of a functional disintegrin domain in ADAMl 1 might contribute to the development of breast cancer. The second metalloprotease-disintegrin with an interesting splice variant is human meltrin a (ADAM12) (Gilpin et al., 1998). Human placenta and certain tumor cell lines express a splice variant of meltrin a consisting of nearly the entire extracellular domain but no transmembrane domain or cytoplasmic tail. A minigene encoding a truncated meltrin a lacking both the metalloprotease domain and the transmembrane domain was expressed in human rhabdomyosarcoma cells, and these cells were injected into nude mice. Tumors resulting from the transfected cells contained ectopic mouse muscle cells, whereas tumors resulting from nontransfected control cells did not contain these ectopic host-derived cells. Evidently, the soluble form of meltrin a is somehow capable of recruiting mouse muscle cells into the human tumor. Since the spliced form of meltrin a is expressed at

Roles of Metalloprotease-Disintegrins

189

high levels in placenta, it may have some function in assembling the interdigitating tissue and blood vessels of the placenta and the uterus.

XII.

METALLOPROTEASE-DISINTECRINEXPRESSION PATTERNS DURING DEVELOPMENT

To date, the only metalloprotease-disintegrin with a clearly defined role in development is Drosophila KUZ (see above). Because cell-cell and cell matrix interactions and specific proteolytic processing events are known to have important roles during development, it should be interesting to evaluate the potential role of other metalloprotease-disintegrins in development. Analysis of the expression patterns of certain family members during development has already provided important clues about their potential functions. In Xenopus luevis, three metalloproteasedisintegrin genes are currently known to be expressed in early embryos (Alfandari et al., 1997; Cai et al., 1998). xMDC9, a putative orthologue of mouse and human MDC9, is expressed as a maternal message in the egg, and is found in all developmental stages and adult tissues that have been analyzed to date. xMDC9 and its mammalian homologues may thus have a function that is utilized or required by most or all cells and tissues (Cai et al., 1998). Two other genes,ADAM13 (Alfandari et al., 1997) andxMDCIla, have a highly specific expression pattern during development. ADAM13, which is related to meltrin a and p, is expressed in the developing neural crest and somitic mesoderm. The ADAM13 protein has been localized to areas of cellkell contacts in myocyte end junctions, suggesting a role in cell-cell interactions in muscle development and in the neural crest. xMDC1 l a expression is detectable only in the neural crest beginning at embryonic stage 18. The expression of both ADAM13 and xMDC1 l a in the neural crest suggests that metalloproteasedisintegrins might play a role in cells derived from this structure, including sensory neurons, cartilage, and bone. ADAM 13 has an active metalloprotease domain, whereas xMDC 11a does not. Therefore the functions of ADAM 13 might include cleavage of membrane proteins and/or a role in cell-cell or cell-matrix interactions, whereas xMDC1 l a is likely to function in cell-cell and cell-matrix interaction but not as a protease. In C. elegans, a metalloprotease-disintegrin termed ADMI (ADAM14) is expressed in sheath cells of sensory organs and in the hypodermis, pharynx, vulva, and mature sperm. Since cells in the hypodermis, vulva, and sperm cells are capable of membrane fusion, it has been suggested that ADM-1 may have a role in somatic and gamete cell fusions (Podbilewicz, 1996). The distinct expression patterns of metalloprotease-disintegrins during development in different organisms have thus provided crucial preliminary insights into possible functions. These insights will guide the further functional analysis by, for example, expression of dominant negative proteins or by targeted deletions. Additional insights into functions of metalloprotease-disintegrins might emerge from the analysis of mutations in

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metalloprotease-disintegrin genes and their modifiers in genetic model systems as has been demonstrated by the elegant analysis of KUZ in Drosophilu and SUP17 in C. eleguns. Systems that allow evaluation of protein function during development are thus likely to become a fertile ground for future studies of metalloproteasedisintegrin proteins.

XIII.

CONCLUSION

Over the last decade, metalloprotease-disintegrins have been implicated in a variety of important, yet quite distinct functions. While they were initially recognized as potent snake venom toxins, it has become clear that these proteins make up a family of more than 20 members that are expressed in different cells and tissues in organisms ranging from C. eleguns to humans. We only know the functions of a small number of these proteins, but the insights gained from these proteins provide important clues about the potential roles of other members of this protein family. Because metalloprotease-disintegrins on sperm have been linked to sperm-egg binding and fusion, it appears likely that they will also mediate cell-cell interactions in somatic cells. Furthermore, those family members with an active metalloprotease could function in protein ectodomain shedding or in protein maturation in the secretory pathway. With respect to the metalloprotease activity, the challenges lying ahead will be to establish what the substrates of the different metalloproteases are, how they are recognized, and whether or not their cleavage is regulated. In addition, it will be important to understand the functions of the disintegrin-domain, cysteine-rich region, and EGF-repeat in cell-cell interaction, and to identify receptors. It should then be possible to analyze whether the cytoplasmic domains have a role in inside-out regulation of the protease, or in outside-in signaling upon binding other cell surface proteins. Finally, targeted deletions or expression of dominant negative proteins will provide important insights into the functions of metalloprotease-disintegrins in development and in adult animals. These efforts should ultimately produce a clearer picture of how different cells and tissues utilize metalloprotease-disintegrins to promote cell-cell or cell-matrix interactions, proteolysis, and perhaps signaling and how these functions are integrated with the role of other cell adhesion proteins, proteases, and signaling pathways.

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A N INTIMATE BIOCHEMISTRY ECC-RECULATED ACROSOME REACTIONS OF MAMMALIAN SPERM

Harvey M. Florman, Christophe Arnoult, Imrana G. Kazam, Chungqing Li, and Christine M.B. O’Toole

I. Introduction. . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . .200 11. Gametes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,201 A. Sperm and the Acrosomal Granule ........... . . . . . . . . . . ,201 B. Eggs and the Zona Pellucida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 111. Overview of Mammalian Fertilization and the Role of the Acrosome Reaction . . . . . . . . . . . . . . . . . . . . . ,203 A. Sperm Capacitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 B. Through the Cumulus Oophorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 C. Zona Pellucida Contact .......... D. Through the Zona Pellu E. Role of the Acrosome Reaction in Regulating Gamete Interaction. . . . . . . . . 207 Advances in Developmental Biochemistry Volume 5, pages 199-233. Copyright 0 1999 by JAI Press Inc. All right of reproduction in any form reserved.

ISBN:0-7623-0202-X 199

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IV. Mechanisms of the ZP3-Dependent Acrosome Reaction . . . . . . . . . . . . . . . . . . .208 A. ZP3 Signals and Signal Reception by Sperm . . . . . . . . . . . . . . . . . . . . . . . . ,208 B. Primary Signal Transducers and Targets. . . . . C. Ionic Mediators of ZP3 Signal Transduction. . . . . . . . . . . . . . . . . . . . . . . . . . 214 . . . . . ,218 D. Downstream Elements of ZP3 Signal Transduction V. Logic of Sperm Signal Transduction and the Example of Capacitation. . . . . . . . 219 VI.Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

The union of ovum and spermatozoon is nota process in which the sperm penetrates by virtue of its mechanical properties, but one in which a particularly intimate and specific biochemical reuction p l a y the chief role. -F.R. Lillie, 1912

1.

INTRODUCTION

The acrosome reaction is a Ca2’-dependent secretory event that occurs during the early stages of fertilization in the sperm of many animal species, including all mammals. The morphological alterations associated with this process were probably noted as early as 1927 (Popa, 1927), although a more thorough appreciation of these events was delayed until the development of both phase contrast optics and the transmission electron microscope (Dan, 1952,1956; Colwin and Colwin, 1955, 1956; Dan and Wada, 1955; Austin and Bishop, 1958; Barros et al., 1967; Franklin et al., 1970). These early studies also provided the first insights that the acrosome reaction is regulated by egg-associated factors (Dan, 1952). Subsequent isolation of acrosome reaction-inducing agonists from eggs then set the stage for the examination of the sperm signal transduction mechanism that controls secretion during fertilization. Sperm signaling pathways are central to models of fertilization, in that the acrosome reaction and the attendant membrane changes must be completed prior to gamete fusion. In addition, the presence of a single secretory granule in sperm and the requirement that granule release occur in the vicinity of eggs imposes a low tolerance of spontaneous exocytosis. In contrast, somatic secretory cells often exhibit a low levels of basal exocytosis that are amplified by stimulatory signals. Thus, the acrosome reaction may provide a unique model in which to examine positive and negative controls of exocytosis. Lytton Stracey once observed of a review that “in having no point of view, it resembled nothing so much as a large heap of sawdust” (Conquest, 1994). The acrosome reaction has been reviewed frequently during the last decade and to proffer another general review would risk our being quashed by Stracey’s admonition. To

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dodge this critique, the present review is organized around two notions. First, intracellular Ca2+(Ca2+,)plays a central role in the control of secretion in somatic cells (Katz and Miledi, 1967; Douglas, 1968) and in sperm (Yanagimachi, 1994). The coupling mechanisms that link stimulatory signals from agonists to sperm Ca*+,as well as the downstream effectors of Ca2+iaction form a focus of this discussion. Second, a cardinal feature of mammalian sperm is that fertility is modulated within the female reproductive tract through the process of “capacitation.” We will consider how the efficiency of signal transduction through agonist-activated pathways is regulated during capacitation and the consequences of this control pathway on the incidence of spontaneous acrosome reactions. The reader seeking a general understanding of acrosome reactions is referred to Yanagimachi’s comprehensive and pellucid discussion (1994). In addition, focused reviews have addressed aspects of acrosomal biogenesis, the mechanism of acrosome reactions, and the role of secretion in the fertilization process (Shapiro, 1987; Fraser and Ahuja, 1988; Green, 1988; Garbers, 1989; Schackmann, 1989; Wassarman, 1990; Kopf and Gerton, 1990; Storey, 1991; Storey and Kopf, 1991; Ward and Kopf 1993; Wassarman, 1995; Darszon et al., 1996; Snell and White, 1996).

II. GAMETES A.

Sperm and the Acrosomal Granule

Sperm arise from a spermatogonial stem cell population through a process of terminal differentiation within the seminiferous epithelium of the testis. The end product is a highly polarized cell consisting of a flagellum and a tapered head, although the dimensions of these components vary among the mammalian species (Cummins and Woodall, 1985). The motility machinery is housed within the flagellum, including a 9 plus 2 arrangement of microtubules and a constellation of associated structural elements. Mammalian sperm are further specialized in that their complement of mitochondria are sequestered in the middle piece of the flagellum. The sperm head contains two membrane-bound compartments: a nucleus located in the basal portion of the head and an acrosome, a secretory vesicle capping the nucleus in the rostra1 region of the head. The acrosome is surrounded by narrow streams of cytoplasm. The structure and differentiation of mammalian sperm has been reviewed recently by Eddy and O’Brien (1994). The acrosome is a secretory granule that is assembled in spermatids by the coalescence of Golgi apparatusderived vesicles. This process produces a large, membrane-limited organelle with an inner acrosomal membrane apposed to the nucleus, an outer acrosomal membrane subjacent to the plasmalemma, and an equatorial segment where these two membrane domains intersect. Our inability to obtain highly purified preparations of isolated acrosomes, despite earlier claims to the contrary (Hartree, 1977), has limited our understanding of the function of tbis or-

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ganelle. This situation can be appreciated by considering the case of a wellcharacterized secretory system, the neuronal presynaptic terminal. The ready availability of isolated synaptic vesicle preparations has facilitated the characterization of both the membrane proteins, which participate in exocytosis, as well as the secretory products (Jessell and Kandel, 1993). Despite this limitation, a constellation of proteins are localized within the acrosome, based on immunocytochemical or subcellular fractionation studies. These acrosomal proteins include a number of protease, glycosidase, and phospholipase activities. In particular, the serine protease acrosin is expressed only in the male germ lineage, is localized within the acrosome, and has been assigned a role in sperm penetration of the zona pellucida (Stambaugh and Buckley, 1968; Meizel, 1984). Hyaluronidase is also present within the acrosome and on the sperm surface. In addition, the acrosome contains signaling molecules in active or precursor forms, including proenkephalin (Kew et al., 1990) cholecystokinin (Persson et al., 1989) and gastrin (Schalling et al., 1990). The role of these molecules in fertilization is unknown, although plausible speculation has considered paracrine effects on either the egg or on the female reproductive tract, as well as autocrine effects on sperm. Other acrosomal contents may play either structural roles or serve yet undefined functions during fertilization. Additional details of acrosomal content and morphology are available in a comprehensive review (Eddy and O’Brien, 1994). We will return to the role of the acrosome and of the acrosome reaction in mammalian fertilization in subsequent portions of this discussion. B.

Eggs and the Zona Pellucida

The mammalian ovum, or egg, is produced within the ovarian follicle through the process of oogenesis. Differentiation is punctuated. An initial series of cellular transformations occurs within the fetal ovary during which oogonia curtail mitosis, initiate meiosis, and arrest cell division at the diplotene stage of meiosis I. Oocytes are stored in this nongrowing state of dictyate arrest until sexual maturity at which time they are recruited in response to ovarian signals into a pool of growing oocytes. The growth phase is characterized by the completion of meiosis, which, in most mammals, proceeds to metaphase I1 and is only completed following sperm fusion (Wassarman and Albertini, 1994). The growth phase is also characterized by an increase in oocyte diameter and by the elaboration of an extracellular matrix, or zona pellucida. The zona pellucida is composed of three glycoproteins, designated ZPl,ZP2, and ZP3, which are synthesized and secreted by the oocyte (Bleil and Wassarman, 1980a, b). These components assemble into an reticular structure consisting of linear filaments. A model of zona pellucida structure has been proposed, based on biochemical and electronmicroscopic analysis, in which linear filaments composed of ZP2 and ZP3 are crosslinked into amatrix by ZP1 (Greve and Wassarman, 1985). This structure surrounds the oocyte during the final stages of growth, is retained following ovulation

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and fertilization, and is shed by the embryo at the time of uterine implantation. Two biological roles are attributed to the zona pellucida: (i) regulation of the early events of sperm-egg interaction and (ii) protection of the preimplantation embryo (Wassarman and Albertini, 1994; Wassarman, 1995). This review focuses on the first of these two general functions and particularly upon the mechanisms of adhesionsecretion coupling that Link zona pellucida contact to acrosome reactions.

111.

OVERVIEW O F MAMMALIAN FERTILIZATION A N D THE ROLE OF THE ACROSOME REACTION

Mammalian fertilization is the result of a precisely regulated series of cellular interactions. This process can be divided into a number of component stages, including the following: (i) the early events that precede egg fusion, consisting of the preliminary event of sperm capacitation, sperm penetration of the cumulus oophorus, zona pellucida adhesion, initiation and completion of acrosome reactions, penetration of the zona pellucida, egg plasma membrane contact and fusion; and (ii) the late events that occur within the egg that consist of sperm nuclear decondensation followed by pronuclear consolidation and syngamy. A.

Sperm Capacitation

The early stages of fertilization begin prior to gamete contact with the process of sperm capacitation. Sperm that are released from mammalian caudae epididymides are able to carry out only a restricted set of physiological processes and are unable to fertilize eggs. Capacitation describes the time-dependent development of fertility. This process was first identified during in-vivo experiments as a temporal delay in fertilization when animals were mated after the time of ovulation (Austin, 1951, 1952; Chang, 1951). Intensive study during the last five decades has produced several generalizations regarding capacitation. 1. This process initially encompassed those physiological alterations required for the development of fertility. However, only 0.1 to 1% of sperm fertilize eggs under typical in vitro conditions and thus a focus on that subpopulation of sperm posed specific experimental difficulties. The definition of capacitation has subsequently been narrowed twice: first, by the recognition that capacitation was restricted to those alterations essential for sperm to undergo an acrosome reaction, and not fertilization per se (Yanagimachi, 1994) and, second, by the observation that sperm signal transduction mechanisms are specifically regulated by capacitation (Ward and Storey, 1984; Florman and First, 1988; Visconti et al., 1995a). Refocusing on well-defined biochemical endpoints has permitted progress toward an understanding of the molecular basis of capacitation.

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H.M. FLORMAN, C. ARNOULT, I.C. KAZAM, C. LI, and C.M.B. O'TOOLE

2. A wide range of physiological processes are altered during capacitation. For example, ZP3-signal transduction is modulated at the level of ligand-receptor binding (Saling et al., 1978; Shur and Hall, 1982; McLaughlin and Shur, 1987) as well as at several downstream sites (see below). Other aspects of sperm physiology that are unlikely to relate to the control of acrosome reactions are also altered during capacitation, including sperm motility patterns. Thus, sperm functional maturation during capacitation includes aspects related to gamete interaction, as well as other aspects of sperm behavior. 3. Inhibitory factors are present in epididymal plasma and in the secretions of the male accessory glands and bind to sperm and impose a state of functional repression. One aspect of capacitation is the dissociation of inhibitors from sperm, resulting in derepression. Observations that capacitation occurs with moderate efficiency under defined conditions in vitro led to the suggestion that it may be a spontaneous process that is exclusively the result of derepression. However, positive regulators are produced by male accessory glands and by the female reproductive tract that enhance the ability of sperm to interact with eggs (Florman and Babcock, 1990). Capacitation should then be viewed as the result of both positive modulation and derepression occurring within the environment of the female reproductive tract. 4. Alterations in a wide range of metabolic processes occur during capacitation. In particular, key intracellular mediators of capacitation may include effector proteins that are regulated by CAMP, by a CAMP-dependent protein tyrosine phosphorylation process, by pH, or by Ca2+(Florman and Babcock, 1990; Yanagimachi, 1994). Thus, capacitation reflects a process by which ZP3-dependent signal transduction is positively modulated and, hence, is aprerequisite for gamete interaction. The mechanisms that underlie this regulatory process are considered elsewhere in this volume. B.

Through the Cumulus Oophorus

Capacitated sperm are confronted with a series of structural obstacles in the form of the cumulus oophorus and the zona pellucida. The cumulus oophorus surrounds the ovulated egg and is composed of several concentric layers of ovarian granulosa cells. The cumulus oophorus may act as a physiological filter since sperm that have already completed the acrosome reaction become trapped at its outer edge (Cummins and Yanagimachi, 1986). Previously, it was proposed that sperm underwent the acrosome reaction at the outer margin of the cumulus oophorus based on observations that cumulus cells are embedded within a hyaluronic acid-rich extracellular matrix, which was thought to present a physical barrier to sperm, and that sperm contain a hyaluronidase activity that is released following acrosome reactions (Werthessen et al., 1945; Austin, 1948,1960;Austin and Bishop, 1958). How-

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ever, the presence of PH20, a sperm surface hyaluronidase (Lin et al., 1994), suggests a mechanism by which acrosome-intact sperm may penetrate the cumulus layers and reach the zona pellucida. The cumulus oophorus may also provide signaling molecules that regulate sperm function during the approach to eggs. In particular, cumulus cells produce and secrete progesterone. Sperm possess a nongenomic progestin receptor on the cell surface that may be related to the neuronal GABA, receptor (Wistrom and Meizel, 1993). Activation of this receptor produces a transient rise in intracellular Ca2+(Ca”,), as reported by intracellular, ion-selective fluorescent indicator dyes, and presumably activates Ca2+-dependent effector mechanisms (Thomas and Meizel 1989; Blackmore et al., 1990, 1991; Blackmore and Lattanzio, 1991; Meizel and Turner 1991; Foresta and Rossato, 1997). High concentrations of progesterone (> 1 pM) initiate acrosome reactions (Thomas and Meizel, 1988, 1989). However, several lines of evidence indicate that acrosome reactions are initiated, after cumulus penetration, at the surface of the zona pellucida during the sequence of events leading to fertilization (see below). It may be that progesterone mediates the final stages of sperm capacitation in the vicinity of eggs and that the induction of acrosome reactions are merely a pharmacological effect of.high drug concentrations. Resolution of the physiological role of progesterone may require the development of selective, high-affinity antagonists of the plasma membrane receptor. In conclusion, it is generally believed that the extracellular matrix of the cumulus oophorus functions as a selective filter permitting only those sperm that have not completed the acrosome reaction to penetrate to the zona pellucida. This matrix may also provide a unique microenvironment that regulates the final stages of capacitation.

C. Zona Pellucida Contact Contact between capacitated sperm and eggs occurs at the surface of the zona pellucida and is mediated by ZP3.ZP3-derived oligosaccharides exhibit several of the characteristics of adhesion domains, including the ability to bind to sperm in a saturable fashion and to inhibit gamete adhesion competitively {Shur and Hall, 1982; Florman et al., 1984; Florman and Wassarman, 1985). Adhesive activity is retained following reductive alkylation of ZP3 and was partially purified in a 3.9-kDa oligosaccharide fraction (Florman and Wassarman, 1985) that has a-galactosyl residues at its nonreducing terminal (Bleil and Wassarman, 1988) and that is conjugated to the ZP3 polypeptide by an 0-glycosidic linkage (Horman and Wassarman, 1985) to N-acetylgalactosaminyl:serinyl/threoninyl residues. Exon swapping and proteolysis experiments suggest that the adhesion oligosaccharide is coupled to peptide regions encoded by exon 7 of the mouseZp3 gene (Rosiere and Wassarman, 1992; Kinloch et al., 1995;Litscher and Wassarman, 1996). Studies with synthetic carbohydrate chains provide insight into the molecular nature of this adhesion domain (Litscher et al., 1995). However, other carbohydrate sequences have also been

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H.M. FLORMAN, C. ARNOULT, I.C. KAZAM, C. LI, and C.M.B. O’TOOLE

implicated in adhesion (Shur and Hall, 1982; Miller et al., 1992; Snell and White, 1996; Johnston et al., 1998). Whether such alternative results are due to complex branching patterns on a single carbohydrate chain or to the relative role of multiple chains in sperm recognition will eventually be resolved following purification of these domains. Similarly, the complimentary receptor for ZP3 on the sperm surface has not been identified definitively. Three candidate proteins are reported to associate with ZP3 in solid-phase assays by affinity chromatography, by chemical crosslinking, or by catalytic transfer of radiotracer (Snell and White, 1996). These include a galactosyltransferase (Miller et al., 1992), a tyrosine kinase (Leyton and Saling, 1989a; Burks et al., 1995), and a 56-kDa galactose lectin (Bleil and Wassarman, 1990; Cheng et al., 1994; Bookbinder et al., 1995). However, the functional role of these proteins in gamete adhesion has been complicated in some cases by uncertainty regarding the localization on sperm plasma membrane (Forster et al., 1997) and by unanticipated phenotypes in knock-out mice (Lu and Shur, 1997). An additional sperm protein has been shown to interact with the zona pellucida and have some of the anticipated characteristics of a gamete adhesion molecule (Hardy and Garbers, 1995). Thus, while several sperm proteins bind ZP3 specifically and with high affinity, the role of these proteins in mammalian gamete adhesion is not yet certain. A related situation persists in marine invertebrate model systems. For example, the sea urchin sperm acrosomal protein, bindin, is generally believed to act as an egg adhesion molecule (Glabe and Vacquier, 1978; Trimmer and Vacquier, 1986), however the identity of the “bindin receptor” on the egg remains controversial (Flotz et al., 1993; Just and Lennarz, 1997; Mauket al., 1997).Given the difficulty in identifying partner adhesion molecules that mediate gamete interaction, it is worth considering that these studies assume the presence of a unique mechanism involving a highaffinity, specific interaction. The fertilization pathway in both mammalian and nonmammalian species exhibits a high degree of specificity. Yet, individual stages of the pathway need not exhibit this same degree of specificity and, alternatively, a high degree of pathway specificity may be achieved through a sequence of steps, each of which has only moderate selectivity. D. Through the Zona Pellucida

Sperm penetrate the zona pellucida only after completion of the acrosome reaction. A similar process occurs in nonmammalian species, where sperm must penetrate the vitelline coat. In abalone this is accomplished by release of lysin, an acrosomal protein that disperses the vitelline coat by a noncatalytic mechanism (Lewis et al., 1982; Shaw et al., 1993). In contrast, the generally accepted model for mammalian sperm penetration of the zona pellucida is the “acrosin hypothesis” in which proteolysis of zona pellucida matrix glycoproteins by acrosin, the acrosomal serine esterase, plays a trailblazing role in the sperm penetration process (Yanag-

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machi, 1994). This hypothesis is based on the following evidence: (i) acrosin is present within the acrosome, where it is activated from zymogen form during the acrosome reaction and is accessible to extracellular substrates following the acrosome reaction (Meizel, 1984); (ii) specific zona pellucida glycoproteins are substrates for acrosin-mediated proteolysis (Urch et al. 1985; Dunbar et al. 1985; Urch, 1990); and (iii) acrosin inhibitors prevent sperm penetration of the zona pellucida (Yanagimachi, 1994). The relative contributions of proteolysis and of mechanical forces to zona pellucida penetration have not been resolved (Green and Purves, 1984; Green, 1988) and must be addressed once again following the demonstration of fertility in transgenic mice with targeted disruptions of the acrosin genes (Baba et al., 1994). It may be that acrosin-dependent proteolysis is essential for dispersal of acrosomal contents during exocytosis, rather than for proteolysis of zona pellucida substrates (Yamagata et al., 1998). Sperm that have penetrated through the zona pellucida contact the egg’s surface. Gamete adhesion is mediated by the interactions of fertilin, a sperm surface protein with adisintegrin domain, and an egg surface integrin (Blobel et al., 1992;Snell and White, 1996; Myles and Primakoff, 1997). Adhesion is followed by the fusion of gamete plasma membranes. The mechanisms that regulate. gamete membrane fusion are not well understood, although considerable progress has been made in the related fields of viral fusion with plasma and intracellular membranes (White, 1990, 1992) and of the exocytotic fusion of secretory vesicles in presynaptic neuronal terminals (Sudhof and Jahn, 1991; Calakos et al., 1994). In particular, sequences in the extracellular domain of fertilin are similar to those of the viral fusion peptide domain, first characterized on influenza haemagglutinin (White, 1990; Hughson, 1995). It is an attractive, though unproven hypothesis, that this domain assumes a similar function during fertilization and mediates gamete membrane fusion. Putative fusogenic peptides are also associated with the sea urchin sperm surface (Glabe, 1985 a,b) and may mediate gamete membrane fusion. E.

Role of the Acrosome Reaction in Regulating Gamete Interaction

The morphological changes attending the acrosome reaction represent the first irreversible step during gamete interaction and, as such, offer an ideal control point for the regulation of the overall efficiency of the process. In fact, it appears that sperm-gg contact is controlled by the progress of the acrosome reaction at several points. As has been discussed previously, sperm that have already completed exocytosis are denied access to eggs as they cannot readily enter the cumulus oophorus matrix (Cummins and Yanagimachi, 1986). However, these cells must compete the acrosome reaction before penetrating the zona pellucida. Importantly, when acrosome-intact sperm are permitted direct contact with the egg plasma membrane following the experimental removal of the zona pellucida, it is found that sperm readily bind to the egg surface but that gamete fusion is delayed until the acrosome reaction has been completed (Yanagimachi and Noda, 1970; Yanagimachi, 1994).

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H.M. FLORMAN, C. ARNOULT, I.G. KAZAM, C. LI, and C.M.B. O’TOOLE

The mechanism by which the acrosome reaction confers fusion competence on sperm is not known. Plausible hypotheses include the following: (i) activation of putative fusion peptides on sperm by acrosomal proteases or by relocalization; (ii) autocrine effects on sperm of bioactive peptides released during acrosome reaction; or (iii) intracellular mediators of the acrosome reaction, such as membrane depolarization or Ca2+(see below), express onset of fusion competence. Thus, exocytosis may switch sperm from a storagehransport mode into a fusion-competent state. The cumulus oophorus filter and the presence of acrosome reaction-inducing agonists in the zona pellucida can then be viewed as a timing mechanism, selecting an appropriate subpopulation of sperm in the vicinity of eggs and placing them in a fertilizing state.

IV.

MECHANISMS OF THE ZP3-DEPENDENT ACROSOME REACTION

The general features of ZP3 signal transduction can be summarized as follows: intracellular Ca2+(Ca”,) is elevated and promotes acrosome reactions. This pathway can then be divided into upstream components, which couple ZP3 signals to a Ca”, response, and downstream effectors of Ca’’,. It is useful to focus on the central role of sperm Ca’+,in this process, given that this ion is necessary and sufficient for the initiation of acrosome reactions A.

ZP3 Signals and Signal Reception by Sperm

It is likely that ZP3 initiates acrosome reactions by acting through a sperm surface receptor; the following indirect evidence supports this notion: (i) the speciesselectivity of ZP3 agonist activity (Lee et al., 1987; Moller et al., 1990; Arnoult et al., 1996a), and (ii) dose-response relationships for ZP3 agonist activity demonstrate saturability (Bleil and Wassarman, 1983). Indeed, it is difficult to suggest an alternative mechanism. However, this receptor has not been identified. ZP3 functions both as the primary adhesion site for sperm on the zona pellucida and as an acrosome reaction-inducing agonist. These distinctions are useful when discussing ZP3 action. Yet, there is no convincing evidence that these are separate processes, and the alternative hypothesis, that adhesion and the stimulation of exocytosis reflect a single event that are only differentiated in carefully chosen bioassays, is equally plausible. This raises the question of whether there is a unitary receptor mediating both events, thereby relegating adhesion as simply the process of receptor activation when the ligand (ZP3) is a component of an insoluble matrix, or of whether separate receptors mediate and regulate adhesion and secretion. Extensive proteolysis of ZP3 with pronase or alkaline reduction produces small glycopeptides (1 S 6 . 0 kDa, as determined by size exclusion chromatography) that bind directly to sperm and inhibit gamete adhesion but fail to initiate acrosome re-

Acrosorne Reaction Mechanisms

209

actions (Florman et al., 1984; Florman and Wassarman, 1985). These ancient studies are widely interpreted as indicating separate roles for ZP3 oligosaccharide and polypeptide domains in sperm-zona pellucida interaction. Acrosome reactions can be initiated when sperm binding sites for ZP3 oligosaccharides are subsequently crosslinked with antibodies directed against ZP3 (Leyton and Saling, 1989b). This suggests that the ZP3 polypeptide acts to aggregate sperm receptor sites and supports the notion that a single class of receptors mediates zonapellucida interaction. As discussed previously, there is controversy regarding the molecular nature of the sperm surface site that mediates zona pellucida adhesion. A likely mechanism of adhesion-dependent signal transduction is suggested in the case of one of the three candidate ZP3 binding proteins, the p95 tyrosine kinase (Burks et al., 1995). In contrast, examination of the deduced amino acid sequences of the other suggested receptor molecules has failed to reveal apparent intracellular signal transduction domain (Miller et al., 1992; Bookbinder et al., 1995; Hardy and Garbers, 1995). This may indicate the presence of auxiIiary signaling subunits in a receptor complex. In conclusion, the earliest stages of ZP3 signal transduction remain obscure.

B.

Primary Signal Transducers and Targets

Receptor stimulation by ZP3 leads to the activation of several primary signal transducers including the heterotrimeric, GTP-binding regulatory proteins, or G proteins, a cation channel that regulates sperm membrane potential, and a tyrosine kinase activity. These pathways will be considered separately and then crosstalk between these signaling mechanisms will be discussed.

C Proteins and the Regulation of Sperm Internal pH G proteins typically are activated by a ligand-stimulated receptor and act as a primary signal transducer. The presence of these proteins in mammalian and nonmammalian sperm was first detected in assays of the ribosyltransferase activity of 6. pertussis exotoxin (Bentley et al., 1986; Kopf et al., 1986). A number of a subunits are expressed during mammalian spermatogenesis, including members of the pertussis toxin- sensitive G, group and G,,, as well as the pertussis toxin-insensitive G,,,, and G, (Glassner et al., 1991; Karnik et al., 1992; Ward and Kopf, 1993; Walensky and Snyder, 1995). The participation of G proteins in ZP3 signaling was indicated initially by the inhibition of Ca2+,mobilization (Florman et al., 1989, 1992; Bailey et al., 1994) and of acrosome reactions (Endo et al., 1987, 1988) following treatment of intact sperm with pertussis toxin, and subsequently by the direct demonstration of G protein activation by ZP3 in digitonidcholate-solubilized extracts of sperm (Ward et al., 1992; Wilde et al., 1992; Ward and Kopf, 1993). G,, and G,, are selectively activated by ZP3 and can account for the pertussis toxin-sensitivity of signaling pathway (Ward

21 0

H.M. FLORMAN, C. ARNOULT, I.C. KAZAM, C. LI, and C.M.B. O’TOOLE

et al., 1992). In addition, the participation of a second G protein in this cascade is suggested by the restricted localization of G,,, to the acrosomal region (Walensky and Snyder, 1995). However, direct evidence for the role of G,,, is not yet available. The mechanism of Gi activation by ZP3 has not been determined. A major class of receptors that activate G protein are characterized by a conserved structure based on seven transmembrane domains, as well as conserved sequences (Bourne et al., 1990; Dohlmann et al., 1991). Several members of this class, including genes encoding putative odorant receptors, are expressed during mammalian spermatogenesis (Meyerhof et al., 1991; Parmentieret al., 1992; Vanderhaeghen et al., 1993; Walensky et al., 1995, 1998). Yet, receptor activation by ZP3 has not been demonstrated and, in some cases, receptor proteins are localized in the tail (Vanderhaeghen et al., 1993; Walensky et al., 1995) where they may regulate flagellar motility, and not in the head, as expected of receptors that transmit ZP3 signals. G proteins may also be activated by receptors with noncanonical structures, including the single transmembrane spanning receptors for insulinlike growth factor I1 (Okamoto et al., 1990; Murayama et al., 1990; Nishimoto, 1993) (see also Korner et al., 1990) and by receptor tyrosine kinases (Nair et al., 1990; Liang and Garrison, 1991; Profrock et al. 1991; Yang et al. 1991). The central question remains: Which second messenger generators are activated by sperm G proteins during ZP3 stimulation? Somatic cell G proteins often regulate adenylyl cyclase, phospholipase Cp, and Ca2+channels; however, it is difficult to build a case for any of these effectors as direct targets of ZP3-activated G proteins in sperm. Initial characterization of sperm G proteins failed to demonstrate the regulation of adenylyl cyclase (Hildebrandt et al., 1985), although there is fragmentary support for the production of CAMP following stimulation of mouse sperm by zona pellucida glycoproteins and for the possible regulatory role of G,, subunits (Leclerc and Kopf, 1995). Similarly, phospholipase C activity is present in sperm (Srivastava et al. 1982; Vanha-Perttula and Kasurinen, 1989; Walensky and Snyder, 1995) and may be activated by ZP3 (Tomes et al., 1996). However, it is likely that this enzyme is activated by Ca2+and may function as a downstream element of ZP3 signaling (Roldan and Harrison, 1989; Roldan and Harrison, 1990; Shi and Roldan, 1995). Finally, voltage sensitive Ca2+channels of sperm are not regulated by G proteins in whole cell patch voltage-clamp studies (Arnoult et al., 1997). What then are the targets for sperm G proteins? The acrosome reaction initiated by egg-derived agonists is accompanied by elevations of intracellular pH (pH,) in mammalian sperm (Florman et al., 1989, 1992; Arnoult et al., 1996a) as well as in nonmammalian sperm (Schackmann and Shapiro, 1981; Schackmann et al., 1981; Lee et al., 1983; Trimmer et al., 1985, 1986; Guerro and Darszon, 1989; Schackmann, 1989; Gonzalez-Martinez et al., 1992; Sase et al., 1995). Studies with the intracellular indicator dyes, BCECF and 5/6-carboxyfluorescein agree that pH, in capacitated mammalian sperm is relatively acidic, with calculated values in the range of 6.65 to 6.8 (Parrish et al., 1989; Arnoult et al., 1996a; Cross and Razy-Faulkner, 1997). ZP3 produces a slow, tran-

,

,

Acrosome Reaction Mechanisms

21 1

sient alkalinization, which has the following features: it rises to 6.8 to 7.0 in both the sperm head and tail regions within 1 to 2 min and then returns to baseline levels within 5 to 7 min (Arnoult et al., 1996a). Alkalinization is required for the sustained elevation of sperm Ca2+,that occurs subsequently and that signals the acrosome reactions. The ZP3-dependent acrosome reaction of mouse and bovine sperm is attenuated by pertussis toxin, and this is accompanied by a corresponding inhibition of agonist-evoked alkalinization. The block in signal transduction imposed by pertussis toxin can be bypassed in the presence of ZP3 by experimental alkalinization with NH,+ or with other permeant weak bases, but such treatments are ineffective in the absence of ZP3 (Arnoult et al., 1996a). Such studies are interpreted to indicate that alkalinization is the only essential pertussis toxin-sensitive element of the ZP3 signaling pathway. The molecular mechanism by which ZP3 elevates pHi is not known. Three widely distributed transport proteins regulate pH, in somatic cells: the Na+-H+ exchanger, the Na+-dependent CI--HCO,- exchanger, and the Na+-independent Cl--HCO,- exchanger (also known as the anion exchanger). These mechanisms are reversible. However, the two Na+-dependentpathways typically couple the inward electrochemical Na+ gradient to the export of acid equivalents, whereas the anion exchanger often operates to import acid equivalents (Roos and Boron, 1981; Thomas, 1984, 1989). In addition, a number of other pHi regulators either exhibit a more limited tissue distribution or act as secondary control systems (Bock and Marsh, 1988). Functional studies in the absence of ZP3 have demonstrated that two pHi regulators are active in mouse sperm and mediate recovery when an acid load is imposed: (i) a Na+-dependent Cl--HCO,- exchanger and (ii) a pathway that is inhibited by arylaminobenzoate antagonists of C1- transport systems, but that lacks the other anticipated features of the Na+-independentCl--HCO,- exchange mechanism (Zen.g et al., 1996). The presence of an anion exhanger is suggested by immunocytochemical studies (Parkkila et al., 1993) although the associated transport activity is not detectable (Zeng et al., 1996). Similarly, transport assays failed to detect a Na+-H+antiport activity (Babcock et al., 1983; Zeng et al., 1996). However, the activities of both of these transport proteins are modulated in somatic cells by protein phosphorylation (Grinstein and Rothstein, 1986; Benedetti et al., 1994) and it may be that the protein constituents that account for these transport activities are present in sperm membranes but inactive until ZP3 treatment produces the appropriate posttranslational regulation. Alternatively, ZP3-dependent alkalinization may be due to the activation of an uncharacterized transport pathway. Cation Channels and Membrane Potential Depolarization

In addition to G proteins, an ion channel is present in mouse and bovine sperm that exhibits no apparent voltage-sensitivity but is activated by ZP3. This channel

21 2

H.M. FLORMAN, C. ARNOULT, I.C. KAZAM, C. LI, and C.M.B. O’TOOLE

conducts some monovalent (Na+,K’) and divalent cations (Ca2+,Ba2+,Mn2+,Mg2+, Ni2+),but does not conduct either larger, organic cations (N-methyl-D-glucamine), trivalent cations (La”), or any of the organic and inorganic anions that were tested (Cl-, SO,-, aspartate-, gluconate-) (Florman, 1994; Arnoult et al., 1996a). This pathway has the characteristics of apoorly-selective cation channel. However, it differs in its ion selectivity from other sperm cation channels, including channels gated by cyclic nucleotides (Weyand et al., 1994), by extracellular ATP (Forestaet al., 1994), and by progesterone (Foresta et al., 1993). This cation channel is activated at an early step in ZP3 signal transduction, as indicated in pharmacological studies (Florman, 1994; Arnoult et al., 1996a). Channel activation produces a small (-50 nM), transient (1-2 min duration) elevation of Ca2+,that is restricted to the sperm head. However, this modest rise is not sufficient to initiate acrosome reactions (Florman, 1994). A more plausible role for this channel is to control sperm membrane potential (V,) during ZP3 signal transduction. The patch electrode voltage-clamp is the method of choice to study V, alterations (Hamill et al., 1981). However, the high resistance seals required for these studies are not readily formed on mammalian sperm due both to geometric factors as well as the low compliance of the plasma membrane (Arnoult et al., 1996b). To date, recordings of “whole cell” currents, as is essential for monitoring of V,, have not been reported in sperm, despite the heroic efforts exerted to obtain “excised patches” from these cells (Espinosa et al., 1998). As an alternative, V, has been examined in sperm populations using potential-sensitive fluorescent probes (Babcock and Pfeiffer, 1987; Foresta et al., 1993, 1996; Espinosa and Darszon, 1995; Zeng et al., 1995; Arnoult et al., 1996a). During capacitation, the spatiallyaveraged V, of sperm populations hyperpolarizes from a calculated value of about -30 mV to greater than -60 mV (Zeng et al., 1995). V, in uncapacitated sperm is determined by the permeabilities of both K+ and Ca2+(Babcock et al., 1983; Babcock and Pfeiffer, 1987;Florman et al., 1992; Espinosa and Darszon, 1995; Zeng et al., 1995) and hyperpolarization is due to an enhanced contribution of K+ permeability (Zeng et al., 1995). ZP3 depolarizes sperm V, from less than -60 mV to about -30 mV. Ion substitution and antagonist studies demonstrate that this depolarizing current is carried by the poorly selective cation channel described previously (Arnoult et al., 1996a). Given the calculated transmembrane ion electrochemical gradients (Babcock, 1983; Babcock et al., 1983; Florman et al., 1989;Zeng et al., 1995) and the ion selectivity of the cation channel (Florman, 1994; Arnoult et al., 1996a), it is most likely that this depolarizing current is carried by Na+, with an additional contribution by Ca2+influx. What is the relationship between cation channel opening and G protein activation during ZP3 signaling? Pharmacological studies indicate that these signal transducers mediate parallel processes in a bifurcated signaling cascade. This conclusion is based on the observations that (i) ZP3 evokes cation channel-dependent membrane depolarization in pertussis toxin-treated sperm, where Gi activation

Acrosorne Zeaction Mechanisms

21 3

does not occur (Arnoult et al., 1996a), (ii) experimental depolarization with K+<, does not generally stimulate G protein-dependent pathways (Florman et al., 1992; Arnoult et al., 1996a) as would be expected if depolarization were an upstream activator of G proteins, and (iii) experimental alkalinization of pH, with permeant weak bases does not activate voltage-dependent downstream pathways (Arnoult et al., 1996a). Membrane depolarization acting as an upstream element of ZP3 signaling provides a means of activating a range of voltage-dependent mechanisms. In particular, the role of sperm voltage- sensitive Ca2+channels will be discussed in a later section. Sperm Tyrosine Kinases

Mammalian sperm contain tyrosine kinase activities (Berruti and Martegani, 1989). In addition, the tyrosine phosphorylation of a limited array of sperm proteins is increased during capacitation (Leyton and Saling, 1989; Duncan and Fraser, 1993; Visconti et al., 1995a,b; Morte et al., 1998). In general, the relevant kinase and phosphatase enzymes have not yet been identified (Morte et al., 1998). Saling and colleagues have proposed that a sperm receptor tyrosine kinase activity is stimulated by ZP3 as an early event in the initiation of the acrosome reaction. Their evidence supporting tyrosine kinase activation can be summarized as follows: (i) certain membrane permeable antagonists of receptor and nonreceptor tyrosine kinases (Levitzki and Gazit, 1995) inhibit the zona pellucida-induced acrosome reaction; (ii) enzyme assays performed after resolution of sperm membrane proteins by gel electrophoresis under nondenaturing conditions indicate ZP3 activation of tyrosine kinase activity; (iii) tyrosine kinases are commonly found to autophosphorylate (Ullrich and Schlessinger, 1990) and solid-phase binding assays suggest that a single membrane protein can bind both antiphosphotyrosine antibodies and ZP3; (iv) aggregation of putative ZP3 binding sites on sperm initiates acrosome reactions, reminiscent of receptor tyrosine kinase signaling through receptor dimerization (Ullrich and Schlessinger, 1990); and (v) a cDNA isolated from human testis exhibits the anticipated sequence motifs characteristic of a receptor tyrosine kinase (Leyton and Saling, 1989a,b; Leyton et al., 1992; Burks et al., 1995). However, these conclusions remain controversial in both humans (Bork, 1996; Tsai and Silver, 1996;Saling et al., 1996) and animal models (Kalab et al., 1994). Extracellular agonists enhance sperm protein tyrosine phosphorylation and sperm response to those agonists is inhibited by antagonists of protein tyrosine kinase activity (Tesarik et al., 1993; Murase and Roldan, 1996; Morte et al., 1998). However, the question of whether a receptor tyrosine kinase in sperm is directly activated by ZP3 binding, or whether nonreceptor tyrosine kinases are stimulated as a downstream effector of signal transduction has not been resolved. This uncertainty is compounded by the observation that tyrosine kinase activity in sperm may be activated through a CAMP-dependent mechanism (Visconti et al., 1995b; Camera et al., 1996).

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Conclusions

Varying levels of evidence linkZP3 stimulation to the activation of three signalgenerating mechanisms. G proteins and acation channel appear to participate in the early events of ZP3 action. Tyrosine kinase(s) are also components of ZP3 signaling, although it is not clear at present whether they act as upstream, secondmessenger generators or as downstream effectors.

C. Ionic Mediators of ZP3 Signal Transduction Several signals are generated following ZP3 binding to a sperm receptor. Intracellular Ca2+and pH increase, and CAMPrises are also reported. The mechanisms by which these second messengers are generated will be considered here. Ca2' Responses

The role of Ca2+as a downstream intracellular mediator of exocytosis was first suggested in studies of secretory vesicle release at the neuromuscular junction and in chromaffin cells (Katz, 1966; Katz and Miledi, 1967; Douglas, 1968), and it is agreed that Ca2+,elevations are a universal and essential element of stimulussecretion coupling (Augustine et al., 1987; Penner and Neher, 1988; Petersen, 1992; Burgoyne and Morgan, 1993). A similar role of Ca2+,in the control of mammalian sperm exocytosis is suggested by a requirement for external Ca2+(Yanagimachi and Usui, 1974), by the initiation of acrosome reactions by Ca2+-transporting ionophores (Summers et al., 1976; Green, 1978), and by the similar role of this ion in the acrosome reaction in nonmammalian species (Dan, 1954; Schackmann et al., 1978; Darszon et al., 1988; Schackmann, 1989; Sase et al., 1995). Understanding of sperm Ca2+,regulation had been hindered due to the small size and limited cytosolic volumes in these cells (Hammerstedt et al., 1978) as well as to the functional heterogeneity of sperm populations during capacitation. The introduction of membrane-permeant, ion-selective fluorescent indicator dyes, which can be passively incorporated into sperm cytoplasm without microinjection, and the deployment of low light-detector systems and image analysis algorithms (Inoue, 1986; Wang and Taylor, 1989a,b) has permitted new progress in this area. Resting Ca2+,in capacitated mammalian sperm is 100 to 150 nM throughout both the head and tail regions of sperm. Following application of a sustained ZP3 stimulus, Ca2+,increases uniformly and throughout sperm at about 600 nM min-' to a peak value of about 400 nM. Elevated levels are sustained in the presence of ZP3 agonist following application of ZP3 stimulus and are an essential signal for the acrosome reaction (Florman et al,, 1989,1992;Storey et al., 1992;Tesariket al., 1993; Florman, 1994; Bailey and Storey, 1994; Arnoult et al., 1996a). Cytosolic Ca2+> values reported by dyes reflect steady-state levels and, in general, are determined by the net balance of several ion transport and buffering sys-

21 5

Acrosome R e a c t i o n Mechanisms

tems. Extensive characterization of Ca2+regulatory mechanisms in somatic cells has revealed the contribution of (i) Ca2+efflux from cytosol by ion-transporting ATPases in the plasma membrane and in smooth endoplasmic reticulum membrane as well as by facilitated diffusion through Ca2+/counterion exchange proteins (Lauger, 1991; Carafoli, 1992; Clapham, 1995); (ii) Ca2+entry pathways into the cytosol across the plasma membrane by means of voltage- and ligand-gated channels, across the endoplasmic reticular membrane by means of Ca2+-releasechannels, and from mitochondria (Tsien and Tsien, 1990; Hille, 1992; Rizzuto et al., 1992; Loew et al., 1994; Tsien et al., 1995; Clapham, 1995; Ichas et al., 1997; Babcock et al., 1997); and (iii) Ca2+buffering by cytosolic proteins (Konishi et al., 1988; Zhou and Neher, 1993). It is likely that all of these regulatory mechanisms are also present in mammalian sperm (Ward and Kopf, 1993; Yanagimachi, 1994) and plausibly are modulated by ZP3. It is now accepted that voltage-sensitive Ca2+channels are present in sperm plasma membranes and participate in ZP3 signaling. The biophysical and molecular characteristics of these channels have been studied extensively in somatic cells. A wide range of functional currents have been detected using the patch clamp method and their characteristics are summarized in Table 1: As discussed previously, similar patch clamp experiments have not been successfully performed on mammalian sperm. However, sperm are transcriptionally and translationally quiescent following release from the seminiferous epithelium in the testis (Mann and Lutwak-Mann, 1981; Yanagimachi, 1994). Channels that are to be used during the posttesticular phase of the sperm life cycle must be synthesized during spermatogenesis and then retained in sperm. Patch clamp experiments reveal the presence of functional T-type Ca2+channels in spermatogenic cells of the rat or mouse (Hagiwara and Kawa, 1984; Lievano et al., 1996; Santi et al., 1996; Arnoult et al., 1996b, Table 1. Voltage-sensitive calcium channels of somatic cellsa a, gene Activation range Antagonist

Current

C D

> -20 mV

arylalkylamines, benzothiazapines, 1,4 dihydropyridines (0.5-500 nM)

N P

B A

> -20 mV > -20 mV

Q

A (9

> -20 mV

R T

E (?) G

> -20mV > -60 mV

0-conotoxin GVlA ( < I pM) a-agatoxin IVA (1-10 nM) W-conotoxin MVllC ( 2 1 pM) Q-agatoxin IVA (100-200 nM) a-conotoxin MVllC ( > l pM) Ni" (see footnote h, Ni", pimozide, mibefradil, 1,4 dihydropyridines (0.5-10 pM) (see footnote h,

L

s

(9 E (9

A Notes:

For additional details, see Hille, 1992; Tsien et al., 1991, 1995; Dunlap et al., 1995. " There are no antagonists that inhibit either R or T currents with high specificity and with high affinity. Ni2+.lnhlhlts . ' all types of CaL+channels, however the Rand T channels are somewhat more sensitive. T a

channel antagonists also suffer from lack of specificity.

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1997, 1998). In addition, PCR analysis indicates the expression of the alA and aIE genes during mouse spermatogenesis (Lievano et al., 1996). The P-, Q-, and R-type currents that are the most likely products of these genes are not observed. Several lines of evidence indicate that T-type channels are retained in sperm and are activated by ZP3 during induction of acrosome reactions

1. T channel antagonists inhibit the sustained ZP3-dependent elevation of Ca2+iin a concentration-dependent fashion that is similar to their potency in inhibiting the T current of spermatogenic cells (Amoult et al., 1996b). 2. T channel antagonists also inhibit the ZP3-dependent acrosome reaction in a concentration-dependent fashion that is similar to their potency in inhibiting the T current of spermatogenic cells (Arnoult et al., 1996b). 3. ZP3 depolarizes sperm membrane potential from resting levels of about -60 mV to about -30 mV (Arnoult et a]., 1996a). This is precisely the voltage range for the activation of the spermatogenic cell T channel (Arnoult et al., 1996b; Lievano et al., 1996; Santi et al., 1996), whereas other types of voltage-sensitive Ca2+channels are not likely to be in an open conformation for significant periods of time during this depolarization (Table 1). The proposed mechanism of T channel activation by ZP3 is through the evoked opening of a cation channel and the resultant sperm membrane depolarization, as discussed previously. T channel activation is required for ZP3-evoked Ca2+elevations and for acrosome reactions. As discussed, fluorescent indicators report that ZP3 stimulation produces a sustained elevation of Ca2+,in sperm. It is unlikely that inward Ca2+current through the T channel can completely account for this response. T channels exhibit rapid, voltage-dependent inactivation and do not produce sustained current (Armstrong and Matteson, 1985; Bean, 1985; Hille, 1992; Bean and McDonough, 1998). In fact, the time resolution of the systems utilized to record ZP3-dependent rises in Ca2+iare too slow to report T channel activation (Florman et al., 1989; Arnoult et al., 1996b). A more plausible model is one in which T channel opening produces a sustained release of Ca2+from an acrosomal store (Walensky and Snyder, 1995; Arnoult et a]., 1996b). This hypothesis is based upon several lines of evidence: (i) mammalian sperm contain a nonmitochondrial pool of Ca2+that is released by inositol- 1,4,5trisphosphate (IP3), as demonstrated in radiotracer flux studies using digitoninpermeabilized sperm; (ii) IP3 receptors are present in the acrosomal membrane of mammalian sperm (Walensky and Snyder, 1995); (iii) acrosome reactions are induced in intact sperm by treatment with thapsigargin, the membrane permeant inhibitor of the SERCA (smooth endoplasmic reticulum Ca2+ATPase) transport system (Blackmore, 1993; Walensky and Snyder, 1995); (iv) IP3 production by sperm is induced by ZP3 treatment (Tomes et al., 1996); and (v) the ionic conductance of IP3 receptors is known to be modulated by cytosolic Ca*+,thus establishing

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the feasibility of a Ca2+-inducedCa2+efflux mechanism (Bezprozvanny et al., 199 1; Turner et al., 1991). pH-Dependent Responses

As discussed previously, transient pH increases accompany ZP3 stimulation and are required for acrosome reactions. It is anticipated that pH shifts will have complex effects upon sperm functions that are mediated by a number of target proteins. At least one mediator of pH action is the mechanisms that mediate sustained increases in sperm Ca2+iduring the acrosome reaction. Similarly, sustained Ca2'i elevations can be produced in the absence of ZP3 stimulation by membrane depolarization and the final sustained CaZtilevel is determined by pH, (Babcock and Pfeiffer, 1987; Florman et al., 1992; Arnoult et al., 1996a). It is unlikely that pH, exerts this effect by directly increasing conductance through T-type voltage-sensitive Ca2' channels. The pH dependence of this channel in spermatogenic cells has not been examined directly. However, T channels in somatic cells are not generally modulated by pHi (Tytgat et al., 1990; McDonald et al., 1994), although such regulation is observed with somatic cell L-type Ca2' channel (Moody, 1984; Irisawa and Sato, 1986; Dixon et al., 1993; Klockner and Isenberg, 1994; McDonald et al., 1994). pH may instead act through the control of Ca2+release from intracellular stores. A decreased proton concentration enhances the IP3 sensitivity of Ca2' release through the IP3 receptor in digitonin-permeabilized mouse sperm, as determined by radiotracer flux studies. The net effect of increasing pH from 6.7 to 7, a range that mirrors the effects of ZP3 on sperm pHi (Arnoult et al., 1996a), is to decrease the IP3 concentration required for half maximal pool emptying from around 500 nM IP3 to around 10 nM (O'Toole and Florman, 1998). Increased IP3 sensitivity may reflect several factors including the pH-dependence of IP3 binding to its receptor (Worley et al., 1987) as well as the biophysical characteristics of the release channel. Sperm adenylyl cyclase is another potential target for ZP3-dependent alkalinization. Enzyme activity has been studied extensively in sperm preparations (Garhers and Kopf, 1980; Leclerc and Kopf, 1995) and has been partially purified (Okamura et al., 1991), but its molecular structure remains unknown. It may be among the Caz'-/calmodulin-regulated class of mammalian adenylyl cyclases (Hyne and Garbes, 1979a,b; Garbers and Kopf, 1980; Hyne and Lopata, 1982; Gross et al., 1987). As discussed previously, catalytic activity is not controlled directly by G, proteins (Hildebrandt et al., 198S), although a potential regulation by G,, has been proposed (Leclerc and Kopf, 1995). However, it exhibits the unique property of being regulated by HC0,-, with the anion both lowering K, and increasing V, through adirect mechanism that is independent of pH (Garbers et al., 1982; Okamura et al., 198.5, 1991). The ZP3-dependent alkalinization will raise equilibrium values of [HCO,-], from about 2.3 mM to more than 5 mM, as calculated by the Henderson-Hasselbalch equation for a HC0,-KO, buffered system:

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this is sufficient to double adenylyl cyclase activity (Okamura et al., 1985). Rapid equilibration of this buffer system by sperm carbonic anhydrase activity (Parkkila and Kaunisto, 1991) provides a means whereby ZP3 will increase [HCO,-I,, irrespective of the molecular mechanism of alkalinization. Thus, activation of adenylyl cyclase by ZP3 during induction of acrosome reactions has been documented but may be a downstream consequence of a cytosolic alkalinization. D.

Downstream Elements of ZP3 Signal Transduction

The primary upstream responses evoked by ZP3 stimulation include a sustained Ca *+ rise and an increase in pH,. An array of downstream elements that can be activated by these early signals are present in sperm. High concentrations of calmodulin are present (Garbers and Kopf, 1980; Feinberg et al., 1981) and the distribution of this protein may be modulated during sperm capacitation (Trejo and Mujica, 1990). In addition, a range of calmodulin-binding proteins are present (Noland et al., 1985; Gross et al., 1987; Aitken et al., 1988; Wasco et al., 1989) including a Ca2+/calmodulin-dependentadenylyl cyclase activity (Gross et al., 1987) and a protein phosphatase activity (Tash et al., 1988; Muramatsu et al., 1992). Sperm also contain other signal transduction proteins, including protein kinase A (Garbers et al., 1973; Beebe et al., 1990, 1992), protein kinase C (Chaudhry and Casillas, 1992), casein kinase I1 (Chaudhry and Casillas, 1989), and tyrosine kinase activity (Berruti and Martegani, 1989; Leyton and Saling, 1989; Tesarik et al., 1993;Burks et al., 1995). In addition, phospholipases A,, C,, C,, and D (Srivastava et al., 1981; Vanha-Perttula and Kasurinen, 1989; Roldan and Mollinedo, 1991; Roland and Dawes, 1993; Roland and Fragio, 1993; Walensky and Snyder, 1995). In somatic cells, many of these enzymes are directly regulated by a primary signal transducer such as a G protein or a tyrosine kinase, and analogous roles have been suggested in sperm. However, an examination of the available evidence indicates that in sperm many of these signaling molecules may be regulated by ZP3-dependent alterations in Ca2+1 or pH,. The case of adenylyl cyclase has already been discussed in detail. Similarly, the Ca2+/Mg2+/H+ ionophore, A23 187, stimulates the activities of phospholipases A,, C, and D in sperm (Roldan and Harrison, 1989; Harrison et al., 1990; Roland and Dawes, 1993; Roldan and Fragio, 1993) and the relationship between Ca2+mobilization and activation of a calmodulin-dependent protein phosphatase is evident. It has been suggested that ZP3 directly activates a sperm tyrosine kinase (Leyton et al., 1992). However, these experiments were performed with partially purified preparations and are difficult to interpret since some tyrosine kinase activity in sperm is controlled by CAMP-dependent mechanisms (Visconti et al, 1996b; Carrera et al., 1996) and might be expected to act as a downstream element of ZP3 signaling. Thus, the route of signal transmission in sperm appears to entail a primary generation of intracellular ionic messengers and the subsequent activation of a range of ion-dependent effector enzymes.

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The final stage of transmission is the activation of the membrane fusion machinery that begins the large scale events of the acrosome reaction. Considerable progress has been made in understanding the protein-protein interactions during exocytosis in somatic cells, particularly in the presynaptic neuronal terminal where the availability of purified preparations of secretory vesicles and target membranes has permitted the identification and molecular cloning of the key proteins (Sudhof and Jahn, 1991; Bennett and Scheller, 1993; Burgoyne and Morgan, 1993; Jessell and Kandel, 1993; von Mollard et al., 1994; Scheller, 1995). The strategy of exocytosis in that model can be summarized as follows: (i) a complex protein machinery exists, in large measure to assure the fidelity of vesicle docking; and (ii) Ca2+has several essential roles including the recruitment of vesicles into an active pool through a Ca2+/calmodulin-dependentprotein kinase 11-mediated mechanism as well as a local role in releasing the secretory apparatus from the inhibitory effects of synaptotagmin (Jessell and Kandel, 1993; Popov and Poo, 1993; Kelly, 1995). Some elements of the neurosecretory apparatus have been detected in sea urchin sperm (Schulz et al., 1997), and the role of these proteins in the mammalian acrosome reaction is of great interest. In conclusion, ZP3 evokes a sustainedrisein Ca2+,and a transient increase in pHi. These signals converge at the level of adenylyl cyclase, which is regulated by [HCO,-] concentrations promoted by ZP3-dependent alkalinization and also by a Ca*+/calmodulin-dependentmechanism. The subsequent activation of protein kinase A regulates a range of targets including tyrosine kinase activities. These responses, as well as the stimulation of other signaling elements, lead to the activation of the exocytotic machinery and the acrosome reaction.

V.

LOGIC OF SPERM SIGNAL TRANSDUCTION AND THE EXAMPLE OF CAPACITATION

The foregoing discussion has focused on the key roles of sperm Ca*+,and pH, in coordinating the events of ZP3 signaling. These ionic mediators act as upstream elements of a signaling cascade and are responsible for recruiting downstream effectors. The question of controlling acrosome reactions then devolves to one of regulating these two intracellular ionic mediators. This may be understood in terms of the physiology of secretion in sperm and in somatic cells. Secretory cells often release vesicles spontaneously at a low rate whereas release rates increase when a stimulus arrives at the release site. For example, miniature synaptic potentials represent spontaneous release from the neuronal presynaptic terminal (Katz, 1966; Stevens, 1993). In contrast, sperm have only one secretory vesicle and, as discussed previously, cells that complete the acrosome reaction prior to egg contact are infertile. This problem may be circumvented, in part, by the fact that large numbers of sperm are produced by mammals, thereby minimizing the consequences of premature acrosome reactions in large populations. However,

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these cells also appear to regulate secretion in order to minimize spontaneous events. Stringency is provided by at least two mechanisms. Firstly, mammalian sperm regulate ZP3 signal transduction by the process of capacitation, such that uncapacitated sperm exhibit very low rates of spontaneous acrosome reaction and do not undergo ZP3-evoked secretion. Several factors contribute to signaling quiescence. 1. pHi of uncapacitated sperm is less than 6.4, as determined using intracellular indicator dyes (Parrish et al., 1989; Vredenburgh and Parrish, 1995). The calculated [HC03li is around 1 mM, a concentration that produces a specific activity of sperm adenylyl cyclase that is approximately 50% of that anticipated in capacitated sperm (Okamura et al., 1985). The role of adenylyl cyclase in ZP3 has been discussed previously and inactivation of this enzyme is expected to suppress acrosome reactions. It is also likely that other elements of ZP3 signaling are pH-dependent and are relatively inactivated under the acidic conditions of uncapacitated sperm. 2. The ZP3-dependent cation channel, an essential component of the signaling cascade, cannot be activated in uncapacitated sperm. Ligand sensitivity is acquired as an early event in capacitation (Florman, 1994). 3. VM of uncapacitated sperm is relatively depolarized, with population-avertiged values of about -30 mV (Zeng et al., 1995). These conditions produce a steady-state inactivation of T-type Ca2+channels (Amoult et al., 1996b; Santi et al., 1996). Thus, even a strong depolarization from the resting potential of uncapacitated sperm would not produce a T current, and hence will not promote a high frequency of acrosome reactions. Thus, three of the upstream elements of ZP3 signaling are inactivated in uncapacitated sperm and these elements only operate efficiently after sperm have completed capacitation. Secondly, the upstream elements of the ZP3 signaling pathway are designed as a coincidence circuit. In this case, the emerging model is that a sustained CaZ+,elevation is required to activate the downstream components of the signaling cascade, that this sustained elevation depends upon Caz+release from an internal store through an IP3 receptor pathway, and that the IP3 receptor operates in this system as the integrator of the coincidence circuit. Specifically, ZP3 produces three primary signals: IP3, elevated pHi, and Ca2+influx through T channels. Yet, when alkalinization or T channel activation are produced experimentally in the absence of other signals (as can be accomplished with permeant weak bases and with membrane depolarization, respectively), sustained Ca2+ielevations and acrosome reactions are not observed (Florman et al., 1992; Arnoult et al., 1996a). Similarly, the Ca2+release mechanism is relatively insensitive to IP3 in the absence of pH signals (O'Toole and Florman, 1998). Thus, fidelity of acrosome reaction signaling is assured by requiring reception of several inputs simultaneously. Coincidence circuits not only provide fidelity, but also provide a mechanism for signaling cross talk. Agonists that provide one of the input signals required by the

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integrator might be expected to act cooperatively with ZP3 to enhance acrosome reaction signaling. One example is the case of progesterone, which amplifies secretory signals of ZP3 (Rolden et al., 1994). This interaction can be understood within the context of signaling crosstalk, as progesterone depolarizes sperm membranes (Foresta et al., 1993) but does not seem to alkalinize pHi(Cross and Razy-Faulkner, 1997). Other external signals within the female reproductive tract, such as extracellular ATP (Foresta et al., 1992, 1996), may also act cooperatively with ZP3 to enhance Ca2+release from internal stores thereby stimulating acrosome reactions.

VI.

CONCLUSIONS

This overview of the ZP3-dependent acrosome reaction mechanisms underscores areas of progress and major gaps in our understanding. The principle obstacles to understanding this process have historically followed from the fact that sperm are small, polarized cells. As a result, identification and purification of signaling molecules has been difficult. However, the application of sensitive biophysical and biochemical methods has permitted rapid progress in recent years. It is apparent that the upstream signals have, in some cases, been identified and the molecular mechanism by which ZP3 produces these intracellular ionic messengers is perceived with some degree of resolution. It is equally apparent that the specific components of the signaling cascade as well as their relationship to the secretory machinery are not well understood. In particular, recent studies have characterized the upstream elements of the cascade, focusing on the mechanisms that mediate the rise in Ca2+i.The next objective may be to identify the Ca2+-regulated downstream targets and to tie these target proteins to secretion.

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INDEX

A compartment, 22,24 A domain, 22-24,26, 30 Abalone sperm, 49-8 1 Abd-A gene, 16-17 Acetylated histones chromatin, 144 SWVSNF, 142 transcriptional activity, 138-139 transcriptionally repressive state, 151-154, 159 Achaete-Scute Complex (AS-C), 30 Acrosin, 202,206-207 Acrosomal proteins, 49-81 Acrosome, 201-202 Acrosome reaction (AR) abalone sperm, 52,55-57,59 capacitation, 87-89 cumulus oophorus, 204-205 egg-regulated, 199-233 gamete interaction, 207-208 induction of, 89 mammalian sperm, 199-233 zona pellucida, 87, 89 ZP3 signaling, 208-219 Acrosome vesicle (AV), 53-57, 59 ADAM10,167,180-182 ADAM11,188 ADAM13, 189 ADAMTS-1, 188 ADMI, 189 235

Adenylyl cyclase CAMP,92,97 G proteins, 210 HCO,’ ,92,98 ZP3,217,220 Adh, 15 AKAPs, 100 Amino acid replacement, 73 Anteroposterior patterning, 20-25 midgut visceral mesoderm, 28 Antp, 28 apterous (up) gene, 29 founder cells, 3 1 Arg residues, 64-65,78 armadillo (arm) gene, 24 atresia, ovarian and NO, 117-118 Atrolysin, 176 bagpipe (hap) gene, 23 combinatorial model, 25-26 dorsoventral patterning, 18 dpp gene, 19,26 hedgehog gene, 24 mef2,34,38 mRNA, 14 P domain, 22-23 target genes, 15 tinman, 12. 14 visceral muscle formation, 14- 15 wingless gene, 24

236

bHLH, 4,30 bHLH53F, 7 Bindin, 54,60, 206 Blood flow rate, ovarian and NO, 116 Body wall muscle development, 29-34 Bovine sperm capacitation, 102 hyperpolarization, 97 pertussis toxin, 21 1 pH, 96 tyrosine phosphorylation, 97 ZP3,211 BrUTP DNA replication, 141-142 transcription uncoupling, 155-156 transcriptional activity, 137-139 transcriptionally repressive state, 149,151, 153 ZGA initiation, 136 BSA capacitation, 90-92 tyrosine phosphorylation, 98-99 btn-lacZ, 17 buttonhead (btd) gene, 21 buttonless (btn) gene, 17 combinatorial model, 25 DM cell formation. 20

INDEX

ZP3 signaling, 208, 2 14, 2 19, 220 Caenorhabditis elegans, 180-181, 189190 CAMP adenylyl cyclase, 92, 97 capacitation, 92, 97-102, 204 G proteins, 2 10 tyrosine kinase, 2 18 ZP3 signaling, 214 cAMPPK-A, 99, 102 Capacitation, 201, 203-204 Ca2+, 89,96-101,204 mammalian sperm, 83-107 Cation channel sperm membrane potential, 21 1-213 ZP3 signaling, 220 cGMP, 112, 117-118 Chlorotetracycline (CTC), 88-89 Cholesterol efflux, 93-96,98-99 Chromatin DNA replication, 142, 144 DNase I hypersensitivity, 143 transcriptional activity, 138 transcriptionally repressive state, 152-153 ZGA onset, 156-157 clift (cli) gene, 16 concertina (cta) gene, 4 Cumulus oophorus, 204-205,208

Ca2+ capacitation, 89,96-101, 204 chlorotetracycline, 88-89 DIDS, 92 G proteins, 210-21 1 dishevelled (dsh) gene, 24 pH, 217 DNase I hypersensitivity, 143-144 signal transduction cascades, 96- 101 Dn:Ds ratios, 74 T channel, 216 Dorsal, 3 ZP3 signaling, 208,214-220 Dorsal median (DM) cell formation, Ca2+(Ca2+i) 11,20 acrosome reaction, 20 1 combinatonal model, 25 progesterone, 205 Dorsal vessel ZP3 signaling, 208 diversification, 27-28 Ca2+i formation, 10 acrosome reaction, 201 Dorsoventral patterning, 18-20 pH, 217 decapentaplegic (dpp) gene T channel, 216 combinatonal model, 25-26

Index

dorsal mesoderm induction, 19 founder cells, 3 1 me$?, 37 midgut mesoderm, 28 mRnas, 19 signaling cascade, 26-27 dpp-response element, 26 Drosophila Medea, 19,27 me$?, 34-38 mesoderm development, 1-47 tinman, 39 Drosophila KUZ Notch, 167, 180-182 role in development, 189-190 ecNOS, 110-112 estradiol synthesis, 122 expression, 1-14-120 guinea pig, 117 luteal function, 120 mRNA, 114-115, 117 oocyte maturation, 119 ovarian blood flow, 116 ovulation, 118, 114-115 EGF signaling combinatorial model, 25 founder cells, 3 1 ventral mesoderm patterning, 20 eIF- 1A DNA replication, 141, 143, 151, 152 mRNA, 151 ZGA initiation, 133 engrailed gene, 21, 23 Enhancers, 143-149 mefl, 36-38 even-skipped (eve) gene, 23 expression, 27-28 tinman, 12 Exons, 7 1-73 Fat body formation, 9- 10, 15 Fertilin, 170-174 sperm-egg fusion, 177-178, 186

237

FGF, 5 folded gastrulation Ifog) gene, 4 Foregut muscles, 7-8 forkhead gene, 23 Founder cells, 29-34 Furin, 174,184 G proteins Ca2+,210-21 1 cation channel, 212-214, enzyme regulation, 218 sperm pH, 209-210 GAL4, 138, 142, 144 Gastrin, 202 Gastrulation, 4-5 Gene duplication, 7 1-73 Gene expression regulation, 129-164 Gene expression reprogramming, 132, 140-144 glial cells missing (gcm) gene, 16 Glucose, 102 Glycosaminoglycans, 101- 102 Gonadal mesoderm development, 16-17 formation, 10 guinea pig ecNOS, 117 fertilin, 60, 170 mRNA, 117 [3H]adenosine, 133 Hamster sperm, 92 HC03-,92,96-99 Heart cell diversification, 27-28 heartless gene, 5 , I8 hedgehog (hh) gene, 24,28 Hematocytes, 16 Hemocytes, 10-11 Heparin, 97, 101-102 Hindgut muscles, 7-9 Histones (see also Acetylated histones) protamines, 137 transcriptionally repressive state, 152-153

238

Xenopus laevis, 156-157 Homeobox genes apterous, 29 bagpipe, 14- 15 buttunless, 17 DM cell formation, 17 engrailed, 22, 23 Kriippel, 29 ladybird, 12, 27-28 S59, 29,31 tinman, 11- 14 @ - I , 14 Homeotic genes, 28 [3H]uridine, 133 [3H]UTP, 133 Hyperpolarization, 97 IL- I p induction of, 88-89 luteal function, 121 ovarian atresia, 117-118 ovarian blood flow rate, 116 ovulation, 112-114 iNOS, 110-112 expression, 114-116 luteal function, 120-122 mRNA, 115 ovulation, 118-119 inscutable (insc)gene, 32 a 6 integrin, 173 Intracellular pH (pHi), 204 Ca2+,217-218,220-221 capacitation, 96-97, 102 G-proteins, 209-210 Intracellular signaling, 97- 102 Introns, 7 1-73 Ion fluxes, 96-97 IP3,216-217, 220 jararhagin, 175-176 18K protein, 55,57,58 aromatic residues, 63 Cys residues, 62-63,77-78

INDEX

fertilization, 59-60 positive selection, 74-75 18K sequences, 60-61,63,7 1 Kriippet (Kr) gene, 29 founder cells, 31 numb gene, 32-34 KUZ Notch, 167, 180-182 role in development, 189-190 L-NAME, 115, 121 lacZ, 27 founder cells, 29 ladybird (IbeAbl) gene expression, 27-28 tinman, 12 lethal of scute (l'sc) gene, 30-3 1 LWhCG, 114, 116 Lineage genes, 32 Lipid Transfer Protein-I (LTP-I), 94 Luciferase activity, 144, 147 mRNA, 145, 154-156 transcription uncoupling, 154-155 Luteal function and NO, 120-122 Lys residues, 64-65, 78 Lysin abalone, 55,57, 206 amino acid, 62 aromatic residues, 66 crystal structure, 64-68 mRNA, 60 positive selection, 74-75 species-specificity, 58-59 VERL, 69-70,73 vitelline envelope, 69-70, 73, 206 Lysin dimer, 66-68, 78 Lysin monomer, 64-65,70,78 Lysin sequences, 60-62, 69-73, 71 MADM, 180 Mammalian egg-regulated acrosome reaction, 199-233 Mammalian sperm, 83-107 Maternal histones, 156-157

Index

MDC9 cellular maturation, 184-185 membrane fusion, 178 protein ectodomain shedding, 183184 RGD sequence, 175-176 MDC11,178 MDC15,175 me&?gene, 34-38 Meltrina, 177-179, 188 Meltrinp, 178 Membrane fusion, 177-179 Mesoderm formation, 3-4 invagination, 4-5 Mesoderm patterning combinatorial model, 25-26 molecular aspects, 26-27 tinman expression, 26 Metalloprotease-disintegrins, 165-198 membrane fusion, 177-179 role in fertilization, 170-173 somatic cell-cell interaction, 175177 Midgut muscles, 7 Motility changes in sperm, 87-88 Mouse sperm capacitation, 210 G proteins, 210 HC03-, 92 hyperpolarization, 97 pertussis toxin, 21 1 pH regulation, 96, 21 1 Sp42,lOO tyrosine phosphorylation, 97 ZP3,211 mRNA bagpipe, 14 Dorsal, 3 dpp, 19 ecNOS, 114-115, 117 eIF-lA, 151 guinea pig, 1 17 iNOS, 115

239

luciferase, 145, 154-156 lysin, 60 maternal, 131-132 ncNOS, 115 tinman, 11- 12 @ - I , 16 ZGA, 131-132 msh gene, 3 1 tinman, 12 muscleblind (mlb) gene, 36 nautilus (nau) gene, 3 1 ncNOS, 110 &A, 115 NO luteal function, 120-122 oocyte maturation, 119-120 ovarian, 109-127 ovarian atresia, 117-118 ovarian blood flow rate, 116 ovulation, 112-119 prostaglandin biosynthesis, 116-117 steroidogenesis, 117, 121 synthesis, 113-114 NOS (see ecNOS, iNOS, ncNOS) NOS inhibitors, 115-116 luteal cells, 121 oocyte maturation, 119-121 ovarian atresia, 117-118 Notch signaling KUZ, 167, 180-181 muscle development, 32-34 numb gene, 32-34 Oocyte maturation and NO, 119-120 Ovarian atresia and NO, 117-118 Ovarian blood flow rate and NO, 116 Ovulation and NO, 112-119 P compartment, 22,24 P domain, 22-24,26 Pair-rule genes, 22-24 PDPl, 35 Pertussis toxin, 209, 221

INDEX

240

PGF,,, 121- 122 pH in mammalian sperm (see Intracel1 ~pH[pHil) 1 ~ PH-30,170, 174 Phospholipase C, 210 Phosphotyrosine phosphatase, 99 PK-A, 97, 102 Plasmid-borne reporter genes DNA replication, 142-145 transcription uncoupling, 154 transcriptional activity, 137 transcriptionally repressive state, 149-151 ZGA initiation, 133, 135-136 Poly(A+)RNA,131, 133 Polypeptides, 132-133, 141 Positive Darwinian selection, 74 Preimplanation mouse embryo, 129159 Proenkephalin, 202 Progesterone, 205 Promoters, 143-149 Pronuclei transcriptional activity, 137139 Prostaglandin biosynthesis and NO, 116-117 Protamine-histone exchange, 137-138, 156 Protein ectodomain shedding, 182-184 Rat ovaries, 112, 114-121 Rat sperm, 100 Receptor tyrosine kinase (RTK), 5 RGD sequence, 168, 171- 172, 175 RhoGEF, 4-5 RhoGTPase, 4 RNA polymerase I, 135 RNA polymerase 11, 134-136, 152 RNA polymerase 111, 135 RNA polymerases BrUTP, 136 [3H]UTP, 133 TATA-box, 144, 148 rp298-lacZ, 6

Rp-cAMP, 97

S59 gene founder cells, 29, 34 muscle identity gene, 32 numb gene, 32 Scr,28 Sea urchin sperm acrosome reaction, 52 bindin, 60,206 Segmental patterning, 21-25 serpent (srp) gene anteroposterior patterning, 21-25 combinatorial model, 25 fat body development, 15-16 glial cells missing gene, 16 gonadal mesoderm, 16 hedgehog gene, 24 hematocyte development, 16 hemocyte development, 15-16 P domain, 22-23 wingless gene, 24 Sheep clone, 132 SITS, 92 sloppy-paired (slp) gene, 23 snail gene, 3-4 dorsoventral patterning, 18 Snake venom, 166-167 disintegrins, 171 Snake venom proteins, 168, 171, 175 SNARE proteins, 179 Somatic cells and metalloprotease-disintegrins, 175-177 Somatic muscle development, 29-30 formation, 6 Sp42,lOO Species-specificity,50-5 1 Sperm hyperactivation, 87-88 Sperm membrane potential (V,), 21 1213,220 Steroidogenesis and NO, 117, 121 SUP17, 180, 183, 190 Superoxide anion generation, 101

Index

SWVSNF, 142, 144

241

bagpipe expression, 29 dorsoventral patterning, 18 founder cells, 30 muscle development, 29 Tyrosine kinase tyrosine phosphorylation, 99- 100,213 ZP3 signaling, 209,213-214,218 Tyrosine phosphorylation CAMP,204 signal transduction cascades, 97- 102 tyrosine kinase, 99-100,213

T channels Ca2+,2 16 tyrosine phosphorylation, 101 ZP3,216-217 TACE, 182, 185 tailless (tll) gene, 21 TATA-box, 138, 148-149 TDE, 172 teashirt (tsh) gene, 28 TFIID, 143 Thickveins (Tkv), 19 Ubx, 28 tinman (tin) gene anteroposterior patterning, 2 1 VERL, 75-78 binding sites, 26-27 vimar, 15 btn, 17 Visceral muscles,, 6-9 combinatorial model, 25-26 Vitelline envelope (VE), 52-53 DM cells, 17, 20 acrosome reaction, 56-57 dorsoventral patterning, 18 dissolution of, 58,68-70 18K protein, 59,73 dpp, 19,39 expression, 11-12, 26 lysin, 58,73, 206 genetic function, 12-14 lysin receptor, 74-78 gonadal mesoderm, 16 heart cell diversification, 27 wingless(wg) gene mej2,34, 37-38 A domain, 24 mRNA, 11-12 expression, 28 structure, 11-12 founder cells, 3 1 tinmanflacz, 7 lb expression, 28 TNFa, 167-168, 182-183 Transcription-requiringcomplex Xenopus laevis maternal histones, i56-157 (TRC) DNA replication, 141 metalloprotease-disintegrins, 172, luciferase activity, 145 181, 189 transcriptionally repressive state, 150-152 $4-1 gene ZGA initiation, 134-135 fat body development, 15 Transcription uncoupling, 154-156 gonadal mesoderm, 16 Transcriptionally repressive state, 146mRNA, 16 154, 159 tinman, 14 Transmembrane signaling, 93,97-102 Zona pellucida (ZP), 202-2 10 tropomyosin I (Tml) gene, 35 ZPl, 202 twist gene, 3-4 ZP2,202 A domain, 22 ZP3 signaling, 202,204, 206, 208-221

INDEX

242

Zygotic gene activation (ZGA), 129164 DNA replication, 140-144 functions, 131-132

initiation, 132-137 mRNA, 131-132 timing, 157-158