Current Topics in Developmental Biology
Volume 37
Meiosis and Gametogenesis
Series Editors Roger A. Pedersen
and
Reproductive Genetics Division Department of Obstetrics, Gynecology, and Reproductive Sciences University of California San Francisco, California 94143
Gerald P. Schatten Department of Obstetrics-Gynecology and Cell and Developmental Biology Oregon Regional Primate Research Center Oregon Health Sciences University Beaverton, Oregon 97006
Editorial Board Peter Gruss Max-Planck-Institute of Biophysical Chemistry, Gijttingen, Germany
Philip lngham University of Sheffield, United Kingdom
Mary Lou King University of Miami, Florida
Story C. Landis National Institutes of Health/National Institute of Neurological Disorders and Stroke, Bethesda, Maryland
David R. McClay Duke University, Durham, North Carolina
Yosh itaka Naga hama National Institute for Basic Biology, Okazaki, Japan
Susan Strome Indiana University, Bloomington
Virginia WaI bot Stanford University, California
Founding Editors A. A. Moscona Alberto Monroy
Meiosis and Gametogenesis Edited by
Mary Ann Handel Department o f Biochemistry, Cellular and Molecular Biology University o f Tennessee Knoxville, Tennessee
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Cover photo credit: Figure 7 of Chapter 7 “Chromosome Cores and Chromatin at Meiotic Prophase” by Peter B. Moens, Ronald E. Perlman. Walther Traut, and Henry H. Q. Heng.
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Contents
Contributors xi ... Preface Xlll
1 Recombination in the Mammalian Germ Line Douglas L . Pittman and John C. Schimenti Introduction 2 Problems Posed by the Mammalian System of Gametogenesis Crossing Over 8 Gene Conversion 12 V. Recombination and Disease 18 V1. Genetic Control of Recombination 22 VII. Conclusion 26 References 26 1. 11. Ill. IV.
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2 Meiotic Recombination Hotspots: Shaping the Genome and Insights into Hypervariable Minisatellite DNA Change Wayne P Wahls
I. Introduction 38 11. General Features of Chromosome Dynamics during Meiosis 111. Genetic Identification of Recombination Hotspots 40
39
IV. Double-Strand DNA Breaks and Open Chromatin SO V. Roles of Protein-DNA Binding in Hotspot Activation 52 VI. Control of Recombination in i i c . and i n fruns. Near and Far 56 VI1. Hotspots as Initiators or Rcsolven of Recombination: Two Models VIII. Summary 65 References 67
57
V
vi
Contents
3 Pairing Sites and the Role of Chromosome Pairing in Meiosis and Spermatogenesis in Male Drosophila Bruce D. McKee 1. Introduction 78 11. Meiotic Pairing Sites in Chromosomes of Drosophila Males: Distribution,
Molecular Composition. and Function
79
111. Chromosome Pairing and Spermiogenesis
IV. Summary and Implications 11 1 References
96
109
4 Functions of DNA Repair Genes during Meiosis W. Jason Cumrnings and Miriam E. Zolan
I. DNA Repair and Organismal Physiology I17 119 123 IV. Relative Abundance of Homology-Based DSB Repair Events 126 V. A Coprinus cinereus Epistasis Group for DNA Repair and Meiosis 128 V1. Conclusions and Perspective 132 References 135 11. Pathways of DSB Repair 111. Genetics of DSB Repair
5 Gene Expression during Mammalian Meiosis E. M . Eddy and Deborah A. O'Brien
I. Introduction
142
11. RNA Synthesis during Meiosis 111. Genes Expressed during Meiosis
IV. Conclusion References
147 148
178 I82
6 Caught in the Act: Deducing Meiotic Function from Protein lmmunolocalization Terry Ashley and Annemieke Plug
I. The Plot
202
11. Setting the Stage: Meiosis Plain and Simple
203
vi i
Contents 111. Surveillance Methods
1V. V. VI. VII. VIII. IX.
207 21 I Reconstructing the Scene Verifying an Alibi (Temporal and Spatial Resolution) Developing a List of Suspects 216 Setting Up a Sting Operation 228 130 Preliminary Conclusions Unsolved Cases 232 References 232
214
7 Chromosome Cores and Chromatin at Meiotic Prophase Peter B. Moens, Ronald E. Pearlman, Walther Traut, and Henry H . Q. Heng
I. Introduction
241
11. SC Structure from Electron Microscopy
242 SC Structure from Immunocytology 245 Chromatin Loop Attachments to the Meiotic Chromosome Cores Sequences Associated with the Core 2.50 DNA Content of the Chromatin Loops 253 Time Course of Chromatin Loop Development at Meiosis 2.56 Alignment of Chromatin Loops 257 IX. Recombination at the SC 257 References 260
111. IV. V. VI. VII. VIII.
247
8 Chromosome Segregation during Meiosis: Building an Unambivalent Bivalent Daniel I? Moore and Terry L. Orr-Weaver
I. Introduction 264 11. Mechanism of Chromosome Orientation
111. IV. V. VI. VII.
266 Chiasmata 269 Homolog Attachment and Segregation without Chiasmata 283 Sister Kinetochore Function 287 Maintaining Attachment between Sister Chromatids for Meiosis I1 292 Summary References 293
9 Regulation and Execution of Meiosis in Drosophila Males Jean Maines and Steven W a s s e r i ~ ~ n
I. Introduction
301
290
...
Contents
Vlll
11. Regulation of the Meiotic Cell Divisions
309
111. Spindle Formation and Function in the Meiotic Cell Divisions
1V. Cytokinesis 319 V. Conclusions and Perspectives References 326
317
325
10 Sexual Dimorphism in the Regulation of Mammalian Meiosis Mary Ann Handel and John J. Eppig
I. Introduction and Overview
333
11. Regulation of the Onset of Meiotic Prophase 335 111. Genetic Events of Meiotic Prophase: A Regulatory Role in
Gametogenesis? 336 IV. Regulating G,/M Transition and Meiotic Divisions 350 V. Gametic Function of Meiotic Prophase VI. Summary and Perspectives 35 1 References 352
339
11 Genetic Control of Mammalian Female Meiosis Patricia A. Hunt and Renee LeMaire-Adkins
I. lntroduction
359
11. The Human Female Meiotic Process Is Error Prone 111. Female Meiosis Is Initiated during Fetal Development
IV. V. VI. VII. V111. IX.
360 361 362 A Quality Control Checkpoint Operates at Pachyrene 363 The Ability to Resume Meiosis Is Acquired during Follicle Growth Chromosomes Play an Active Role in the Formation of the Meiotic Spindle 368 The Metaphase/Anaphase Transition 370 374 Arrest at Second Meiotic Metaphase: Do Chromosomes Play a Role? The Future: Mammalian Meiotic Mutants Will Provide Important Insights 375 into the Control of Mammalian Female Meiosis References 377
12 Nondisjunction in the Human Male Terry J. Hassold
I . Introduction: An Overview of the Problem
383
ix
Contents 11. Approaches to Studying Male Meiotic Nondisjunction: Methodology and Results 384 111. The Etiology of Male Nondisjunction 393 IV. Summary and Future Directions 400 References 402
Index 407 Contents of Previous Volumes
419
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Contributors
Niri~ibt.r\ 111 p ~ i r m r t i i ~ \ t .I\I I ~ I (ire I
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Terry Ashley Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 065 10 (20 1 ) W. Jason Cummings Department o i Biology, Indiana University, Bloomington, Indiana 47405 ( 1 17)
E. M. Eddy Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 (141)
John J. Eppig The Jackson Laboratory, Bar Harbor, Maine 04609 (333) Mary Ann Handel Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville. Tennessee 37996 (333) Terry J. Hassold Department of Genetics and the Center for Human Genetics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106 (3x3) Henry H. Q. Heng Department of Biology, York University, 4700 Keele Street, North York, Ontario, Canada M35 1 P3 (241) Patricia A. Hunt Department of Genetics, Case Western Reserve University, Cleveland, Ohio 44106 (359) RenCe LeMaire-Adkins Department of Genetics and the Center for Human Genetics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 441 06 (359) Jean Z. Maines Department of Molecular Biology and Oncology. University of Texas Southwestern Medical Center, Dallas, Texas 75235 (301) Bruce D. McKee Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996 (77) Peter B. Moens Department of Biology, York University, 4700 Keele Street, North York, Ontario, Canada M3S 1P3 (241) Daniel P. Moore Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142 (263) Deborah A. O'Brien Departments of Cell Biology and Anatomy and Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599 (141) xi
xi i
Contributors
Terry L. Orr-Weaver Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 142 (26.3) Ronald E. Pearlman Department of Biology, York University, 4700 Keele Street, North York, Ontario, Canada M35 1P3 (241) Douglas L. Pittman The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609 ( I ) Annemieke W. Plug Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510 (201) John C. Schimenti The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609 ( 1 ) Walther Traut Institut fur Biologie, Medizinische Universitat Zu Lubeck, Ratzeburger Allee 160, D-23538 Liibeck, Germany (241) Wayne P. Wahls Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 (37) Steven A. Wasserman Department of Molecular Biology and Oncology. University of Texas Southwestern Medical Center, Dallas, Texas 75235 (301) Miriam E. Zolan Department of Biology, Indiana University, Bloomington, Indiana 47405 ( 1 17)
Preface
In complex organisms, meiosis is unique to and, in many respects, defining of gametogenesis. No other cells undergo this form of cell division, which is initiated by a single round of DNA replication and homologous chromosome pairing and recombination and culminates in two division phases, one a reductive division in which homologous chromosomes are segregated, and the other an equational division in which sister chromatids are separated. This volume focuses primarily, though not exclusively, on meiosis in the context of gametogenesis in higher eukaryotes, because it is hoped that insights into meiosis may provide greater understanding of the regulation of gametogenesis and, ultimately, the possibility of exogenous control. Now is a good time for a retrospective and perspective on meiosis during gametogencsis. The recent explosion of new information about the molecular genetics of recombination derives, in large part, from studies on yeasts, where mutation analysis has been especially productive. Additionally, new methods for analysis of proteins essential for meiosis and methods for induction of mutations in candidate genes are both leading to new insights into meiotic mechanisms during gametogenesis. In most, but not all, cases recombination is an essential feature of meiosis. It reassorts genetic linkages, it is a mechanism for DNA-damage repair, and it is required for appropriate segregation of homologous chromosomes. Thus an understanding of the mechanisms of recombination is essential. New information from studies of lower eukaryotes is providing inroads to the study of meiotic recombination in higher eukaryotes, where an inherent problem has been the lack of well-defined spots of recombination characterized at the molecular level. Pittman and Schimenti show how insights from studies of recombination in fungi are guiding experiments in mice, and they describe the utility of clever schemes for measuring recombination, thus overcoming the lack of defined recombination sites. Wayne Wahls illustrates the experimental advantages of hotspots of recombination in both fungi and mammals, thus addressing this same problem. Information on the biochemistry of hotspot activation allows development of models of when and where recombination occurs; these can account for known changes at hypervariable minisatellite DNA sequences in mammals. Cummings and Zolan discuss mechanisms of DNA repair and show how these are likely to be fundamental to mechanisms of meiotic recombination. The model system of the basidiomycete Coprirrus cinrrc.ic.s can be used to explore whether double-strand DNA breaks are an essential feature of meiosis in eukaryotic cells. Not all
...
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Preface
meioses are associated with recombination, and the male Drosophila is the best known exception. McKee examines this unique situation, where chromosome pairing is separable from recombination, to probe genetic and molecular requirements for pairing and to develop a model for how insufficiencies in pairing lead to spermatogenic defects, thus again linking meiotic mechanisms to the process of gametogenesis. Gene expression also is inextricably linked to both meiosis and gametogenesis; sometimes it is difficult to separate the two. Eddy and O’Brien tackle this tough task and provide an encyclopedic compendium of genes expressed during meiosis in mammals. This information forms the foundation that will ultimately help us to separate genes expressed for meiotic function from genes expressed for gametogenic function. Among gene products that are expressed during meiosis, those proteins associated with paired chromosomes and the synaptonemal complex are the most likely candidates for unique meiotic function. Ashley and Plug show how techniques of immunolocalization and creation of specific knockout mutations can provide temporal and spatial information indicating the meiotic function of a number of proteins. Although these studies alone cannot define the function of these proteins, they do lay the foundation for elucidating the function of these interesting candidates and lead to testable hypotheses about function. Moens and coauthors address the nature of both the proteins and the DNA sequences associated with the synaptonemal complex in reviewing what we know of structure of the synaptonemal complex as well as the interesting variability in the nature of chromatin loops attached to SC cores. They present evidence for discriminate utiliLation and differential packaging of DNA sequences in chromatin loops, suggesting that this may be related to differences in rates of recombination along the length of the chromosome. How does the germ cell know that the genetic business of recombination is completed and that it is time for the meiotic division phases’? This puzzle deals with the cell biology of meiosis and the misnamed meiotic “cell cycle,” which is, of course, a terminal pathway and not a cycle at all. Prior genetic events of recombination are indeed essential for execution of the division phase, and Moore and Orr-Weaver address issues of chromosome segregation (reductional segregation of homologs at MI anaphase and equational segregation of chromatids at MI1 anaphase). They focus on how chromatids are tied together in the bivalent to ensure their proper segregation, which is of great importance since the consequences of malsegregation are unbalanced gametes and aneuploidy. They also discuss the evidence for chiasma binding substance and sister-chromatid cohesion and the role of kinetochores in segregation. Maines and Wasserman analyze the regulation of the meiotic divisions in the context of the developmental program of DrosophilLi spermatogenesis, covering the role of cell cycle regulators as well as that of spindle assembly, critical for proper segregation. How are these stages regulated in mammalian gametogenesis‘? Less is known here because of the paucity of informative mutations, but Handel and Eppig consider rcgula-
Prcfacc
xv
tion of the onset of meiotic division phase and specifically how it differs between mammalian oocytes, which are characterized by a discontinuous meiotic process, and spermatocytes, which are characterized by a continuous meiotic process. They also probe the essentially unknown territory of exactly how the temporal pattern of the genetic events of meiotic prophase regulates the onset of the division phase during mammalian gametogenesis. This is important because errors in the genetic events can give rise to aneuploidy, the etiology of which is discussed in the final two chapters of this volume. Hunt and LeMaire-Adkins address the puzzle of why, in mammals. female meiosis should be so extraordinarily error-prone. They consider the contributing roles of the tempo of meiotic progress and a spindle assemhly/chromosome-niediated checkpoint, which may be absent in mammalian oocytes. Although autosomal chromosome aneuploidy is less commonly derived from male gametes, sex chromosome aneuploidy is not, and Hassold considers the origins of nondisjunction during spermatogenesis (a process seemingly not as error-prone ;is oogenesis in mammals) and, in particular, the possible roles of recombination, aging, and environmental factors. In its focus on events of chromosome pairing and recombination as well as on the control of the division phases, this book hits the high points of'the direction that meiosis research is now taking. Particularly satisfying is that information derived from different organisms and from a variety of techniques converges to provide new insights into meiotic mechanisms. There is certainly much more to come that will be instructive about both meiosis and gametogenesis. Mary Ann Handel
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1 Recombination in the Mammalian Germ Line Douglas L. Pittman and John C. Schimenti The Jackson Laboratory Bar Harbor, Maine 04609
1. Introduction 11. Problems Posed by the Maniinalm System of Gametogenesis A. Inability to Recover All Meiotic Products
B. Mitotic Expansion of the Gcrm Lineage C. The Number Problem D. Phenotypic Markers 111. Crossing Over A. Sex Differences in Crossing Over B. Physical versus Genetic Differences C. Recombination Hotspots IV. Gene Conversion A . Evolutionary Evidence B. The MHC C. Strategies for Measuring Gene Conversion V. Recombination and Disease VI. Genetic Control of Recornbination A. Early Exchange Genes B. Early Synapsis Genes C. Late Exchange Genes VII. Conclusion References
Elucidation of meiotic recombination mechanism, in mammals faces many obstacles. Much of our understanding ha5 been built upon studies in the fungi, which have served to guide experimental design in mammalian cells and mice. A clearer picture is now emerging which reveals that many of the general principles of recombination are conserved across this evolutionary divide. A number of genes critical to meiotic recombination in yeast also exist in mammals. Transgenic technologies, in addition to advances in molecular biology, now provide several strategies to investigate the properties and regulation of mammalian recombination. Thi\ chapter reviews the current state of knowledge regarding recombination in the mammalian germ line, covering topics such as gene conversion, recombination mechanics, recombination-based genetic mutation, crossing over, and genes involved in meiotic recombination. Copyright 0 1998 by Academic Press.
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Douglas L. Pittman and John C. Schimenti
1. Introduction The mechanisms of mammalian meiotic recombination are not well understood. The nature of mammals presents many challenges to investigation, such as a long reproductive cycle, germ cells that cannot be propagated in culture, and an inability to recover the products of individual meioses. Even though genetics is built on meiotic recombination, our understanding of recombination processes in mammals is largely restricted to basic phenomena: crossing over occurs during meiosis at a relatively predictable rate, it is subject to interference, and unequal recombination and gene conversion occurs at some frequency that is difficult to measure and detect. Although human and mouse geneticists generally do not concern themselves with mechanisms of recombination-and especially not with forms of recombination other than crossing over-we are absolutely dependent on exploiting it to map traits and mutate genes via homologous recombination. Much of what we know about meiosis and recombination in eukaryotes has come from studies of fungi, and there is substantial evidence that mammalian recombination is fundamentally similar. Experiments in mammalian cells have demonstrated an association between conversion and crossovers (Bollag and Liskay, 1988),the formation of heteroduplex DNA during recombination (Bollag et ol., 1992), and the association of gene conversion with adjacent crossovers-all critical hallmarks of fungal meiosis that led to unifying models of recombination (Bollag and Liskay, 1988; Metzenberg et al., 1991). In fact, the successful development of gene targeting technology borrowed heavily from the characteristics of recombination and transplacement parameters in yeast (Thomas and Capecchi, 1986).Targeting experiments in mouse embryonic stem (ES) cells have shown that repair and homologous integration of transfected plasmids occurs in a manner consistent with the double-strand-break (DSB) repair model that was developed from studies of fungi (Valancius and Smithies, 1991). Although there are some notable distinctions in recombination between these vastly different species-for example, gene targeting in yeast is nearly 100%efficient, unlike in mammalian cellsit appears that basic recombination mechanisms are shared. Many key proteins involved in recombination, such as RecA, topoisomerases, helicases, and DNA repair molecules, are highly conserved from yeast to man, and the exact functions in mammals are now being elucidated in cultured cells and mice. Although exploitation of cross-species homology is a powerful means of identifying genes involved i n mammalian recombination, the nature of gametogenesis presents a challenge for studying the mechanisms and characteristics of this process. One limitation is that mammals do not have asci; hence, it is not possible to recover and examine all the products of a mammalian meiosis. Our current understanding, then, still depends on inference from molecular data and the development of clever assays to deduce the nature of meiotic events. A second limitation is that there is currently no way to select for mutations that
1. Recombination in the Mammalian Gerni Line
3
affect meiosis or recombination in mice. Nevertheless, meiosis in yeast and mammals appears to be basically similar; homologous chromosomes align, form synaptonemal complexes, display interference, and generally undergo at least one crossover per chromosome to ensure proper disjunction. It is our challenge to overcome the limitations inherent in the organism to uncover the extent of the similarities with fungi, and to understand the mechanisms controlling mammalian meiosis and gametogenesis. In this chapter, we discuss ( 1 ) the characteristics of mammalian meiosis in relation to studying recombination, ( 2 ) the forms, properties, and mechanisms of recombination in mammalian cells, (3) relevance to human disease and genome evolution, and (4) genetic control of recombination. Because it is often impossible to distinguish between meiotic recombination and events that occur in precursor cells, we take the precaution of using the term “germ line” recombination. Despite the obvious biological distinction between mitotic and meiotic recombination, the outcome is teleologically identical; all that matters is what winds up in the gametes. Because of this uncertainty, we do not limit our discussion to what is absolutely clear about meiotic recombination but also discuss experiments in cultured cells that have been essential in elucidating the mechanisms and properties of recombination in mammals.
II. Problems Posed by the Mammalian System of Cametogenesis A. Inability to Recover All Meiotic Products
The ability to recover all the products of a meiosis is the single most important characteristic of fungal meiosis that enabled a fundamental understanding of recombination. Gene conversion between alleles is manifested by non-Mendelian segregation. For example, whereas a heterozygous diploid yeast ( A l a ) will be expected to produce two A spores and two N spores, interchromosomal gene conversion results in 3: 1 segregation in favor of either allele. Conversions are often associated with an adjacent reciprocal crossover, an observation central to the development of the Holliday model of recombination. Conversions and crossovers were envisioned as alternative outcomes of Holliday junction resolution (Holliday, 1964). Some fungi undergo a single division after meiosis, leading to the discovery of postmeiotic segregation (PMS). This is manifested as a 5:3 segregation pattern in eight spored asci. In yeast, wherc individual spores can be grown in the haploid state, PMS results in “sectored” colonies, a 5050 mix of two genotypes at a locus. The existence of PMS implies that heteroduplex DNA is an intermediate in recombination events, and when mismatches are not corrected, the two strands replicate and segregate the nucleotide differences to daughter cells. The models
Douglas L. Pittman and John C. Schimenti
4
of genetic recombination that were developed over the years were designed to explain gene conversion, crossing over, the association between them, and PMS (Holliday, 1964; Meselson and Radding, 1975; Szostak et a/., 1983). Although there is solid evidence of gene conversion and heteroduplex intermediates in mammalian cultured cells, direct proof for these phenomena in meiosis is lacking. Because segregation patterns in individual meioses cannot be followed, it is essentially impossible to detect events such as gene conversion between alleles. Allelic conversion is indistinguishable from a double crossover (although double reciprocal crossovers within subcentimorgan intervals is exceedingly unlikely). The evidence for gene conversion between nonallelic genes in the germ line is relatively strong and much easier to detect (see Section lV, part A), but still circumstantial; absolute proof is formally lacking in the absence of the ability to recover all meiotic participants. Detection of PMS poses a very difficult experimental problem. The occurrence of such an event would lead to a mosaic animal. For example, if a mouse heterozygous for a mutation at the albino locus ( C I S )was crossed to a homozygous albino mouse (cIc),half the animals would be pigmented and the rest would not. However, if a recombination event within the albino locus (the tyrosinase gene) in the heterozygote yielded an unrepaired heteroduplex, and that gamete fused with an albino gamete, the resulting animal would be a niosaic containing both pigmented and unpigmented cells visible in the coat. Indeed, there are several reports in the early mouse literature of mosaic animals (Gruneberg, 1952). The problem in attempting to identify PMS from the appearance of mosaic offspring is that de n o w mutation, either in early development or in meiotic cells, can produce similar phenotypes. For example, ENU mutagenesis of embryos causes a substantial amount of mosaic progeny (Russell ef al., 1988). Single-sperm PCR analysis provides another avenue for the identification of heteroduplex DNA. The ability to amplify both strands of an individual gamete has provided evidence for unrepaired heteroduplex at the HLA locus in human sperm (Huang et al., 1995). However, the uncertainties associated with PCR, such as in virro mutagenesis or contamination, make it difficult to rule out experimental artifact in such analyses. Nevertheless, the single-sperm PCR approach, if coupled with a means to specifically amplify and analyze genes that have simultaneously undergone a recombination event, currently offers the best hope for detailed molecular characterization of recombination in mammals. B. Mitotic Expansion of the Germ Lineage
1. Spermatogenesis The production of mature spermatozoa is the culmination of a series of events called spermatogenesis. Males possess a self-renewing pool of spermatogonial stem cells (type a, spermatogonia) that appear about 3-5 days post partum. These
I . Recombination in the Mammalian Germ Line
5
stem cells then undergo division both to renew themselves and to give rise to cells destined for meiosis (type A I spermatogonia). After three additional mitotic divisions producing morphologically distinct type A,, A,, and A, spermatogonia, subsequent rounds of divisions produce intermediate spermatogonia and type B spermatogonia. The type B spermatogonia divide to create primary 2n spermatocytes that undergo meiosis to yield four haploid spermatids. These then undergo spermiogenesis to become mature spermatozoa. Based on the number of divisions, as many as 256 spermatids may arise from a single type A1 spermatogonium in one round of spermatogenesis (Handel, 1987).
2. Oogenesis The primordial germ cells give rise to about 20,000-25,000 oocytes by about Day 14 of mouse gestation, which is the peak number in the lifetime of an animal (Mintz and Russell, 1957; Tam and Snow, 1981). At that time, mitotic divisions cease, meiosis begins, and the oocytes arrest in the first meiotic prophase. Prior to ovulation, meiosis I is completed, but the final division is dependent on fertilization. The relevance of these processes for recombination is that both involve extensive phases of mitotic expansion prior to actual formation of the gametes. A recombination event in primordial germ cells, their precursors, or gonial cells can result in the production of multiple gametes containing products of the same recombination event. These are sometimes referred to as “jackpots.” Jackpots appear to be responsible for some gene conversions found in the mouse H-2 complex (Geliebter, Zeff, Melvold, r t cil., 1986), where multiple progeny in litters were found to possess identical variant alleles that arose via a conversionlike process. The events could be traced to the maternal parent (Loh and Baltimore, 1984). Similarly, this type of phenomenon was documented in the case of Myk-103 transgenic mouse, in which a herpes simplex virus thymidine kinase transgene (TK), flanked by duplicated sequences at the insertion site, underwent frequent deletion via intrachromosomal recombination in spermatogonia (Wilkie et al., 1991). As the males aged, a higher proportion oftheir germ cells contained the deleted chromosome. Examples such as these demonstrate that unequal recombination occurs in the premeiotic germ lines of both males and females. This must be taken into account when measurement of recombination rates in mammals is done on the aggregate of progeny without extensive molecular analysis to identify the exact points of exchange; it always remains a possibility that similar recombinants in different offspring are in fact derivatives of an identical event. Although there has not been much effort to document such phenomena as they relate to crossing over, Rosemary Elliot and Verne Chapman have identified “litter effects” in mice, whereby multiple offspring appear to have inherited very similar crossovers (R. Elliot,
6
Douglas L. Pittman and John C . Schimenti
personal communication, 1997). It is not clear that mitotic recombination is involved in these cases, but it indicates the possibility that some crossing over occurs premeiotically or is influenced by events in cells prior to meiosis.
C. The Number Problem A practical issue facing studies of recombination in mammals concerns the perlocus frequency of events. To screen thousands of animals for rare reconibinations, and to get statistically significant numbers, is an impractical task for most researchers. The discovery of gene conversion in the murine major histocompatibility complex (MHC) using graft rejection as a screen (even though the intention was not to investigate gene conversion) was a monumental achievement (see Section IV, part B). Even with an assay that may not be so labor-intensive, identification of rare events might simply not be feasible. Not only is raising enough animals to detect particular recombination events ;I major issue, but the rarity of an event can preclude more detailed molecular investigations. For example, molecular support for the idea that DSBs initiate recombination came from studies in yeast showing that breaks occur at the beginning of gene conversion gradients (Sun e/ al., 1989). Such experiments depend on the existence of a gene that undergoes an extremely high rate of conversion (greater than 5- 10% of meioses), allowing the molecular detection of the DSBs in a pool of meiotic cells, and recovery of products to reveal co-conversion of flanking alleles (Nicolas et d . , 1989). At present, the existence of a locus that undergoes frequent allelic conversion in mammals has not been identified, and the ability to recover the meiotic products is still a formidable technical constraint. One promising strategy to overcome this problem is to induce DSBs in a locus-specific manner. Several groups have utilized the rare-cutting I-Sce I endonuclease to induce break formation in mammalian cells and to show that this markedly induces homologous recombination over spontaneous levels (Brenneman et d., 1996; Choulika et al., 1995; Rouet et ul., 1994a,b; Smih r t al., 1995). Whereas spermatogenesis provides an ample number of meiotic events to screen for rare types of recombination using either PCR (Jeffreys et al., 1994; Zangenberg et al., 1995) or expression of reporter transgenes (Murti rt d., 19921, oogenesis does not. In human studies, it is simple to obtain unlimited amounts of sperm from men, but recovery of oocytes from woman is not an option. Mice have upward of 20,000 oocytes in late gestation, but they do not complete meiosis until fertilization. I t is therefore impractical to study large numbers of events directly i n haploid oocytes. On the other hand, the one advantage that female meiosis affords is the potential to analyze all the products of a meiosis; this is because the oocyte, which is only one of the four meiotic products, remains physically associated with the polar bodies. The first polar body is extruded at the first meiotic division. and this divides on some occasions
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( J . Eppig. personal communication, 1997). The second polar body is released following completion of meiosis 11, which occurs upon fertilization. Because it is rouline to recover dozens o f oocytes from a superovulated female mouse, it would be reasonable to examine hundreds or thousands that have undergone the first meiotic division. To screen each one by a PCR-based assay might be quite a feat, but given the appropriate promoters and reporter constructs (such as the lacZ system employed by the authors; see subsequent discussion), it may be possible to examine only those oocytes that are phenotypically converted to determine if the recombination was reciprocal. The prospects for doing such experiments on spermatogenic cells are bleaker. Because mitotic expansion as well as meiotic divisions occur in a syncytium, there is no physical separation of the products from different meioses. One possibility is the development of adequate culture systems that could accurately recapitulate the events of meiosis. If it were possible to isolate individual primary spermatocytes, physically separate them, and induce them to undergo meiosis in culture, this would provide the mammalian equivalent of tetrad analysis. Several investigators are working toward developing culture systems that might ultimately be exploited in this fashion (Rassoulzadegan er al., 1993; Handel er al., 1995).
D. Phenotypic Markers
Another limitation to the investigation of recombination in mice is the lack of visible markers. In yeast, gene conversion experiments use selectable loci such as HIS4 or LEU2. Experiments can be designed in which spores produced from a meiotic event grow only if a planncd recombination event has occurred. Furthermore, the ability to create contrived loci via transplacement in yeast makes it simple to construct paradigms for the visualization, quantitation, and molecular analysis of recombination events. The advent of gene targeting technology in ES cells now permits the design of analogous experiments in mice. The range of phenotypic markers is more limiting, however, as mice or their gametes cannot be plated out and subjected to selection. Obvious phenotypic markers are coat color genes, some of which can be detected shortly after birth. For example, one can imagine setting up a screen for conversion events at the albino locus, in which the rare recombinant would be pigmented. Although such a strategy would allow one to capture an apparent recombination event, the success of such a strategy would depend on the frequency ( 1 in 10,000? 1 in lOO,OOO'?) and the determination of the investigator. If events are rare, the number of animals required for statistical analysis and a solid understanding of recombination properties (such as conversion tract length) could be prohibitive. To overcome the problems of visible markers and numbers, experiments have been conducted to score rare events in sperm. These are discussed in detail in a subsequent section.
8
Douglas L. Pittman and John C. Schimenti
111. Crossing Over Two unique events that distinguish meiosis from mitosis are high levels of genetic recombination and the reductional division that takes place during meiosis. The independent assortment of nonhomologous chromosomes at the reductional division and genetic recombination between homologous chromosomes ensure genetic variation among the meiotic products. However, the truly essential role of meiotic recombination is to ensure proper pairing and segregation of the homologous chromosomes at the reductional division. Failure of homologous chromosomes to recombine results in random chromosome distribution and the production of aneuploid gametes. Therefore, it is important to have at least one recombination (crossover) event per homologous chromosome pair in each meiosis (Carpenter, 1984). Crossing over is defined as a reciprocal genetic exchange between homologous chromosomes. In eukaryotes, this phenomenon was first demonstrated in studies of sex-linked and autosomal genetic markers in Drosophila melanogaster (Morgan, 191 1; Morgan and Lynch, 1912). Linkage in mammals was first described by Castle and Wright in studies of the Norway rat (Castle and Wright, 1915). In the modem era, studies in humans and mice have yielded the most information about the characteristics of crossing over. In the past few years, the genomes of these organisms have become saturated with polymorphic markers that have been mapped at high resolution (Dib et al., 1996; Dietrich er al., 1996). Mouse backcross mapping panels provide a relatively “clean” set of information generated in a genetically controlled manner. The backcross mapping panel at The Jackson Laboratory has yielded an overview of the frequency and distribution of crossovers in the female germ line (Rowe et ul., 1994). With over 2500 markers on the 94 animal (Spretus X C57BL/6J) X C57BL/6J backcross map, on average, about 13.5 crossovers are detected per offspring (L. Rowe, personal communication, 1997). Each backcross animal represents one of four products from a meiosis of either parent (in this case, we consider only the F, parent). For any one crossover event, two of the four gametes produced in a meiosis would contain reciprocal recombinant products, representing two of the four chromatids in a homologous chromosome pair. Thus, the average of 13.5 recombinant chromosomes/F, backcross animal reflects only half of the chromatid pairs that could participate in recombination during meiosis, predicting an overall average of 27 crossovers per meiosis. This correlates very well with cytological studies that determined a mean chiasma frequency of 25.4 in female mouse cells (Lawrie et al., 1995). It is generally accepted that chiasmata represent sites of recombination. If these 27 crossovers were distributed randomly across the 20 chromosome pairs in a Poisson distribution, an average of 5.1 pairs per meiosis would be nonrecombinant. If this were true, extensive aneuploidy would be predicted in mammalian meiosis. Alternatively, it is likely that a mechanism is in operation, most likely interference, that causes the meiotic recombination events to be
I . Recombination in the Mammalian Germ Line
9
distributed nonrandomly such that each chromosome pair generally undergoes at least one crossover. Indeed, each chromosome pair in meiotic cells generally contains at least one “obligate” chiasma (Lawrie et al., 1995). Genetic evidence for a mechanism that enables distribution of recombinations to all chromosomes comes from analysis of the yeast synaptonemal complex protein Zip1 (discussed later). Zip1 mutants show a loss of interference, with an attendant increase in aneuploidy (Sym and Roeder, 1994). Despite the large array of genetic tools now at our disposal, such as the highdensity linkage maps, they have not been specifically exploited to provide a comprehensive understanding of the mechanisms or regulation of crossing over. In this section, we summarize observations that have yielded some insight into meiotic crossing over in mammals. In particular, we concentrate on sex differences in recombination rate, the distribution of crossovers along chromosomes, and hotspots for meiotic recombination.
A. Sex Differences in Crossing Over
Sex differences in recombination in mammals have been reported since the 1920s (Cooper, 1939; Dunn, 1920; Murray and Snell, 1945). Dunn and Bennett (1967) accumulated the genetic data available at that time and noted that recombination frequencies were generally higher in females. Twenty-four of 54 intervals examined had a sex difference, and 19 intervals were larger in females. For this reason, females are often used in mapping experiments in order to maximize the amount of data generated (Silver, 1996). Modern, molecular marker-based genetic maps continue to demonstrate that in most intervals, recombination frequencies are significantly higher in females (Roderick rt a/., 1996). The genetic map along chromosome 17 illustrates this well. Crossing over in males occurs at 63% of the frequency observed in females. and the recombination frequency is higher in females in 19 of the 23 intervals measured in both sexes. However, one example of a recombination frequency higher i n males than in females is the H2-t+f interval; the frcquency of recombination in males is nearly twofold that in females. Sex-specific differences in recombination frequency also occur in humans and were first described by Renwick and Schulze ( 1 965). Taking advantage of restriction fragment length polymorphicms (RFLPs) as genetic markers, Donis-Keller et a/. (1987) constructed the first comprehensive genetic map of the human genome. Even though the number of meioses in this study was small, it illustrated that sex-specific differences are prominent. The female genetic map was approximately 90% larger than the male map. As in mice, however, a small number of intervals were genetically larger i n males. One such interval was on chromosome 15 in the approximately 4-Mb region associated with Pradcr-Willi/AnFelman syndrome. The map distance in males was 17.2 cM in this region and 12.7 cM in females (Robinson and Lalande, 1995). A physical map of pig chromosoine I , which is the largest chromosome in
10
Douglas L. Pittman and John C. Schimenti
pigs, revealed unusual sex-specific crossover distributions (Ellegren ef a/., 1994). In one interval near the terminus of chromosome 1, the genetic distance in females was 41.4 cM, compared to 6.9 cM in males. However, a segment in the center of the chromosome had a significant excess of recombination in males; one interval had a map distance of 3 1 .O cM, compared to 7.8 cM in females. More studies of individual chromosomes are necessary to establish if this is a common trend in the pig. The opposite distribution pattern was observed in a study of human chromosome 19 (Weber et al., 1993). Even though the genetic maps are similar (128 cM for females and 114 cM for males), an increase in recombination at the distal end of chromosome 19 occurs in males. For example, D19S180-Dl9S254 is 27.4 cM in males, compared to 6.8 cM in females. In the interior region, female recombination was substantially higher. Cytological examination of meiotic cells has yielded some clues to the basis for differences in crossover rates and locations between sexes. During the diplotene stage of meiosis I, the synaptonemal complex breaks down, and contact between homologous chromosomes is maintained by the chiasmata, which represent the sites of crossing over (Carpenter, 1994). On mouse chromosomes 1 and 14, the mean number of chiasma per bivalent did not differ between males and females (e.g., chromosome 1: males 1.62, females, 1.67), but a difference in chiasma distribution was observed (Gorlov et a/., 1994). In males, chiasmata were formed more often at the terminal regions and rarely in the middle region of the two chromosomes. In females, there appeared to be an even distribution of chiasmata. Lawrie et a/. ( 1 995) confirmed these differences for all of the autosomes in the mouse. These distribution patterns were originally noted by Polani (1972) and Speed (1977). Speed also reported that chiasma frequency in oocytes decrease with age, but aging in the male did not affect the chiasma frequency. These differences in chiasma (crossover) distribution may help explain the differences between the two sexes. Crossing over may occur at preferred regions along the chromosome and these regions may differ between the sexes. Therefore, one would expect an increase in the genetic map distance at the distal end of chromosomes in males, and the available data suggest this is the case. In human studies, the chiasma counts also agree with the genetic data (Morton, 1991), but the only difference that has been noted between meiotic chromosomes in males and females is the length of the synaptonemal complex (during late zygotene and pachytene). In females, the SC length was observed to be nearly double the length observed in males (Wallace and Hulten, 1985).
B. Physical versus Genetic Distances
Extensive molecular cloning of mammalian genomes has permitted comparisons of physical and genetic map distances. The approximate relationships (sexaveraged) have been calculated to be 0.5 cM/Mb in mice, > 1 cM/Mb in humans,
1 . Recombination in the Mammalian Germ Line
11
and about 0.55 cM/Mb in pigs (Weissenbach et a/., 1992; Copeland et d., 1993; Ellegren et al., 1994). These recombination frequencies are extremely low compared to that in S. cerevisiar (370 cM/Mb) (Petes et a]., 1991). It has generally been assumed that recombination can occur anywhere along the chromosomes and that an increase in genetic distance between two markers correlates with an increase in physical distance. Direct comparisons of the physical and genetic maps demonstrate that the frequency of crossing over is not random across the mouse genome. For example, some regions along a chromosome may have the same physical distance but different genetic map distances (see reviews by Fischer-Lindahl, 1991; Shiroishi et al., 1995; Silver, 1996; Steinmetz et a/., 1986). This unequal distribution pattern of crossovers is also observed in S. cerevisiac (Petes et a/., 1991), so nonrandomness of crossing over is not specific to mammals. Regions that undergo high levels of recombination in yeast are generally found near promoter regions and correspond with the positions of DSB sites (Ohta et a/., 1994; Wu and Lichten, 1994). Chromatin structure studies indicate that these DSB sites are hypersensitive to DNase I and micrococcal nuclease (MNase), which suggests that promoter regions of yeast genes not only are accessible to the transcription machinery but also are more accessible to meiotic recombination proteins. The positions of the hypersensitive sites remain constant between mitosis and meiosis, but those that correspond to DSB positions increased in MNase sensitivity (by 2- to 4-fold) prior to DSB formation (Ohta et al., 1994). As described below, this correlation may not hold true for mammals.
C. Recombination Hotspots
Even with the limited amount of’ data currently available, it is clear that recombination hotspots are also present in mammals. One such region is the MHC in mouse. This entire region along chromosome 17 has been cloned, allowing direct comparisons of physical and genetic distances (Steinmetz et a/., 1982). These studies have demonstrated that recombinational preferences (“hotspots”) in the MHC are clustered in four regions (Shiroishi et al., 199.5). Of the two that are best characterized, the first is located at the 3’ end of the second intron in the Eb gene. The region encompassing the hotspot has been narrowed to approximately 1 kb (Bryda e t a / . , 1992; Kobori rt a/., 1986; Sant’Angelo et al., 1992; Zimmerer and Passmore, 1991). A second hotspot is located adjacent to the Lmp2 gene, which has been delimited to approximately 2 kb (Shiroishi et al., 199.5). The Eb hotspot was the first to be characterized, and several candidate sequences exist that may influence recombinational activity. An AGGC sequence repeated 10- 18 times is present in the Eb hotspot region. This sequence has weak homology ( 5 / 8 bases) to the bacteriophage crossover hotspot instigator, chi. A minisatellite core sequence is also present in this region (Bryda et al., 1992;
12
Douglas L. Pittman and John C. Schimenti
Kobori et al., 1986), as well as sequences similar to a retrotransposon long terminal repeat (LTR), env, and pol genes. The LTR, env, and pol sequences most likely evolved through a retrotransposon insertion (Zimmerer and Passmore, 199 I). Two DNase I-hypersensitive sites (DHSSs) have been identified in the vicinity of the hotspot, and one is specific to pachytene stage meiotic cells (Mizuno et al., 1996; Shenkar et al., 1991). Two potential transcription factorbinding sites are present in the hotspot region, a B motif that may bind H2TFI /KBFl and NF,P, and an octamer-like binding domain. Gel retardation experiments demonstrated that proteins bind to each of these sites (Shenkar et al., 199 1 ), and expression studies indicate that these motifs enhance transcription in a tissue-specific manner (Ling et id., 1993). This is consistent with transcription factors influencing recombination activity in specific regions. By comparing crossover rates to physical length, it was determined that the recombination frequency at the Lmp2 hotspot was nearly 2000 times higher than the average (Shiroishi et al., 1995). Several candidate sequences are also associated with the hotspot in Lmp2, including a (CAGA),-, repeat, an LTR-like sequence, and a middle repetitive sequence (Shiroishi et al. 1990). Shiroishi et al. (1991) mapped the “recombination instigator” to within 395 bp proximal to the hotspot, but no DHSSs have been identified in this region during spermatogenesis. The tentative conclusion is that high-frequency recombination sites in mouse are not necessarily associated with hypersensitive sites. This is in contrast to the studies in S. cerevisiae, but clearly more DHSS studies must be performed at other recombination hotspot sites.
IV. Gene Conversion As described earlier, inability to recover the products of a meiosis makes it formally impossible to prove the occurrence of meiotic gene conversion in mammals. For this reason, mammalian geneticists either qualify descriptions of recombinants as “conversion-like” events or simply refer to anything that looks like a conversion event as a gene conversion. Although the latter may seem sloppy, such conclusions are, for the most part, probably correct. Heritable gene conversion events can occur both meiotically and mitotically. Although the frequency of gene conversion in yeast mitosis is three or four orders of magnitude less than during meiosis (Om-Weaver and Szostak, 1985), mitotic recombination in the germ line can effectively amplify the apparent “frequency” of a particular conversion event. It would result in multiple identically recombinant gametes. This has been observed at the murine H-2K locus, in which multiple indistinguishable (presumed) conversion-generated mutants were recovered within a sibship (see Section IV, part B). In the next section we discuss the evidence for germ line gene conversion in mammals and experiments to measure its frequency.
I . Recombination in the Mammalian Germ Line
13
A. Evolutionary Evidence
Gene conversion can play two seemingly paradoxical roles in the evolution of a gene family. On one hand, related gene family members are subject to sequence homogenization by gene conversion, in effect stunting divergence and evolution. On the other hand, microconversions can rapidly generate diversity by introducing multiple sequence changes in a single event (Baltimore, 1981). Several factors influence whether gene conversion promotes sequence homogeneity or diversity within a gene family. These include frequency of conversion, gene copy number, directional bias, conversion tract size, and preferential recombination start/stop points. If a gene family underwent continual, directionally biased homogenization by gene conversion, individual members would not diverge and evolve. This is obviously not always the case, which suggests several possibilities: ( 1 ) conversion between nonallelic duplicated genes is too infrequent to counteract sequence drift, (2) a mechanism can be invoked to somehow protect duplicated genes from conversion, (3) relatively infrequent conversion events fail to become fixed in populations, and (4) the conversion tract sizes are very small. Conversion frequency would appear to be the most significant determinant of evolutionary impact. Much of the evidence for germ line gene conversion in mammals has been generated by comparative sequence analysis of duplicated genes. A duplication unit containing a patch of near sequence identity within a larger stretch of considerable divergence is the kind of observation best explained by gene conversion. The classic example of such evidence exists in the human fetal globin genes, G, and A,, which arose via duplication of a 5-kb DNA sequence over 30 million yrs ago. Although regions flanking the genes have diverged significantly, in some alleles a 1.5-kb region within the genes is virtually identical, leading to the conclusion that a recent gene conversion event occurred at this locus (Slightom et ul., 1980). There are now numerous such examples in the literature, and some are listed in Table I. The pervasive effects of gene conversion-like activities in the history of gene families are recognized as a serious factor to consider when assessing the evolutionary history of duplicated genes. Several theoretical studies have addressed the confounding effects of gene conversion-mediated homogenization on the evolutionary analysis of repeated genes, presenting mathematical models on the role of gene conversion in evolution (Dover, 1982; Gutz and Leslie, 1976; Lamb and Helmi, 1982; Nagylaki and Petes, 1982; Walsh, 1987).
B. The MHC
The single largest body of data concerning gene conversion in mammals has emerged from studies of the murine MHC. The earliest observations that were
14 Table I.
Dougla5 L. Pittman and John C. Schimenti Exaniplea of Gene Convermn
Example
in
Mammals
Mammal group
Reference
Primates, rodents cows, goats
Erhart er ol. (1985), Fitch et ul. (1990), Hardies P/ a/. ( 1984), Schimenti and Duncan (1984), Schimenti and Duncan (1985), Shapiro and Moshirfar ( 1989). Slightom et nl. (1980) Hess et (I/.(1983), Michelson and Orkin (19831, Schon er crl. (1982). Wernke and Lingrel (1986), Zimmer et trl. (1980) Hammcr el ul. (1991) Kudo and Fukuda ( 1994)
a-Clobins
Humans, goats
Hemoglobin-u pseudogene Glycophorins (blood group antigens) DR-P loci. HLA MHC class I T-cell antigen receptors H-2 class I
Mice Humans
ImmunOglObuhS Lysozymes TcplO genes Cardiac myosin heavy chain Steroid 2 I -hydroxylase
Mice Mice Mice Humans
Opsins Aldosterone synthase Spiral motor neuron gene
H u in an s Humans Humans
Humans Humadchimps Humans Mice
H umaii s
Gorski and Mach ( 1986) Kuhner ('I a/. ( I99 I ) Tunnacliffe et a/. (1985) Geliebter, Zeff, et d.(1986), Kuhner et ( I / . (1990). Mellor ~f ul. (1983). Wcixs r / ul. (1983) Ollo and Rougeon ( 1983) Cross and Renkawitz (1980) Pilder et ul. ( I 992) Tanigawa er (11. (1990) Donohoue et a/.(1986), Higashi r t rrl. (1988). Morel P I crl. (1989), Urabe C/ ul. ( 1990) Reyniers rt ( I / . (1995) Fardella et ul. (1996) Bussaglia er 01. (1995)
eventually interpreted as evidence for gene conversion were based on the recovery of several spontaneous mutants of the H - 2 K class I gene. In heroic studies, these events were recovered on the basis of graft rejection. DNA sequence analysis of the mutant alleles showed that clusters of nucleotide substitutions had been introduced by gene conversion-like events with nonallelic class I genes in the same haplotype (Geliebter, Zeff, ef al., 1986; Mellor et al., 1983; Nathenson et al., 1986). Many of these events result in the transfer of less than 100 bp of DNA. They are referred to as microconversions. One study revealed evidence for 25 fixed microconversions in a survey of inbred mouse strains (Kuhner et al., 1990). Spontaneous mutations at the H-2Kb locus occur at a frequency of 2 X per gamete (Klein, 1978), which is far more frequent than typical mutation rates due to point changes. Since most novel mutants appear to be the result of gene conversion-like events, the frequency of recombinant gametes appears to be as high as 0.0270, an estimate in the range of those obtained by PCR analysis of sperm, as described below.
15
1. Recombination in the Mammalian Germ Line
C. Strategies for Measuring Gene Conversion
The first attempts to measure gene frequencies in mammalian cells utilized tissue culture systems in which a pair of mutated selectable genes (such as thymidine kinase) were introduced, followed by selection for cells that recreated functional marker gene activity by virtue of gene conversion (Liskay and Stachelek, 1983, 1986; Liskay et ul., 1984; Rubnitx and Subramani, 1986). This type of assay, as diagrammed in Fig. I , is an adaptation of recombination screens in yeast. The general frequency of intrachromosomal conversion was on the order of 1 X 10 - 6 . More recently, a duplication of Cp. genes created by gene targeting was found to undergo intrachromosomal gene conversion at the rate of 0.5-0.8% of cells (Baker and Read, 1995). The disparity in frequencies can be attributed to a number of factors, including cell type, sequence composition, nature and degree of heterology, and size of homologous sequences. Although experiments in mitotic cells permit more direct evaluation of gene conversion and the parameters that affect it, it is unclear whether all the lessons learned can be applied to meiotic recombination. Mitotic cells do not undergo synapsis and homologous chromosome pairing (although homologs do sometimes interact to yield crossovers). Furthermore, studies in yeast show that the frequency of meiotic gene conversion is higher by orders of magnitude (Szostak et al., 1983). Our laboratory modified the basic strategy for measuring conversion in tissue culture cells to enable the determination and quantitation of gene conversion in the germ line (Murti et al., 1992). Two major problems facing investigations into germ line gene conversion recombination in mice were solved: scoring enough
w
TK-
x
Y
z
TK-
I
I
I
I
I
I
I I
Fig. 1 A construct containing two differentially mutated thymidine kinase (TK) genes is introduced into cells (either transiently or as a chroniownial integration). The separate mutations are indicated by black vertical lines, and polymorphic resti-ictioii enzyme sites, are shown flanking the mutations (WZ). I n this example, a recombination event has transferred “good” sequence information Ii-om thc copy on the right to that on the left, raulting i n correction of the mutation and ability to grom in HAT medium. Tran\fer of the flanking markci Y has also occurred (coconversion), and W ha\ been eliminated. This unilateral transfer of DNA \equence i \ a gcne conversion.
16
Douglas L. Pittman and John C. Schimenti
meioses (progeny) and detecting the events. The solution to the first problem was to score gametes rather than progeny, and the solution to the second problem was to employ a transgene (lacZ) whose product is easily visualized when a planned conversion occurs. Constructs analogous to the tissue culture versions in Fig. 1 were used to detect the conversion events between lacZ genes in spermatogenic cells (Fig. 2). A gene conversion event that corrected the mutation in the protamine-driven recipient lacZ gene with sequence from the donor would enable the production of functional P-galactosidase in spermatids (Fig. 2). "Blue" spermatids were observed in all transgenic lines at frequencies of up to 2%, and correction of a restriction site in the recipient could be observed on PCR ampli1994). This assay was later adapted to fication of sperm (Murti, Schimenti, et d., show that ectopic conversion could also be observed between recipient and donor sequences located on different chromosomes (Murti, Bumbolis, et a/., 1994). Although this transgenic assay afforded, for the first time, a means to measure gene conversion rates in vivo,these experiments did not enable a distinction to be made between the relative levels of premeiotic and meiotic conversion. In fact, because clusters of positive spermatids were observed in the seminiferous tu-
A.
Recipient
Prm 1
Donor
L a c Z
B.
Functional iacZ in testis Fig. 2 Transgene construct for measuring iiitriichrom(~bOrnii1gene conversion in the germ line of mice. ( A ) The black hoxea represent inouse protamine I sequences, and patterned boxe\ are lor2 sequences. The recipient IncZ gene is under the transcriptional contrnl of the Prni I promotei-. and the distal Prm sequences contain a polyadenylation \ignal. Ti-anscriptional orientations of thc lorZ genes are to the right. The black vertical stripe in the recipient ItrcZ gene is ii donor IncZ gene is truncated for q u e n c e s encoding the first 36 and enzyme. (Adapted lroin Murti ef t r l . 1992). ( B ) An intrachromosomnl intact lucZ gene. Ilouble crossovers can also restore function, but this
Z-hl? insertion mutation. The last 136 amino acids of the gene conversion rc\tores iiii is highly unlikely.
I . Recombination in the Mamni;iliaii Germ Line
17
bules, this was taken as evidence that at least some proportion of the converted spermatids were derived from c ~ c n t sthat occurred during the mitotic expansion of the germ line. Three studies have exploited PCR analysis of sperm to detect and quantify prior gene conversion events. Hogstrand and colleagues examined the conversion frequency of MHC class 1 genes in mice. Conversion between the nonallelic ternplates on homologous chroniosonies was observed at a rate of about 0.0024 (Hogstrand and Bohme, 1994). Remarkably, these MHC templates were very small (186 bp) and highly divergent (79% identical to each other). Evidence for gene conversion between HLA class I1 genes in humans has also been obtained by sperm analysis (Zangenberg c’t u / . , 1995). I n these studies, about 0.01% of sperm carried a novel allele that was attributed to gene conversion. Even more remarkable, investigations into human microsatellite loci revealed gene conversionlike events at a frequency of 0.4% at the MS32 locus (Jeffreys et ( I / . , 1994). In coinparing the various studics of gene conversion rates that are now emerging, it is important to consider that multiple factors can influence the recombination frequency. For mammalian cultured cells, it is known that ( 1 ) a pair of sequences must be highly homologous for efficient recornbination (Liskay et a/., 1987; teRiele e t a / . . 1992), ( 2 ) recombination rates decrease linearly with size of the shared homologies from 2 k b down to 295 bp (Liskay et L I ~ . ,1987; Rubnitz and Subramani, 1984), and (3) at least 134-232 bp of perfect, uninterrupted homology is required for efficient initiation of recombination (Liskay et d . , 1987). In gene targeting experiments i n ES cells, divergence of less than 1% results in a greater than 10-fold decrease in homologous recombination frequency (teRiele et d . , 1992). The highest levels of conver5ion were observed between the /acZ teinplates used in the original transgenic studies of Murti er 01. (1992). The donor and recipient genes shared 2.5 kb ot’ homology, were situated immediately adjacent to one another, and satisfied all thc criteria for high-efficiency recombination outlined earlier. The minisatellite repeats in the study by Jeffreys er d . (1994) are examples of allelic (not intrachromosomal ) conversion, in which case homology was high between donor and recipicnt and overall homology length was at least several hundred base pairs. These elements also showed a high level of gene conversion. However, the MHC gene templates did not match any of the criteria. This is a possible explanation lor the greater than 100-fold difference i n rates observed. Recently, a genetic background elfect has been observed in the case of the /ocZ transgenes. Remarkably, when they were rendered congenic on the CS7BL/6J inbred strain, the conversion rates dropped precipitously to about 0.001c/r ( J. R. Murti and J. Schimenti, unpublished ohcrvations), which is the lowest frequency of all the cases discussed above. The effect is revcrsible: treatment with DNAdamaging agents or breeding into the ”permissive” background was found to increase frequency. These observations are reason for caution in the interpreta-
18
Douglas L. Pittman and John C. Schimenti
tion of recombination data at certain loci. Different mouse strains or people may possess different capacities for illegitimate recombination. A dramatic example is the case of ataxia telangiectasia cells, in which intrachromosomal recombination occurs at a frequency up to 200-fold greater than in normal cells (Meyn, 1993). Given the large body of sequence data demonstrating the wide array of genes being affected by gene conversion and the studies that have actually obtained frequencies at particular loci, it is clear that gene conversion is an active recombinational mechanism in the mammalian germ line. It has served to create diversity in productive ways, and also to maintain sequence heterogeneity in some gene families. Ultimately, selection acts to sort out the “good” from the “bad” events, and the neutral events take the form of polymorphism in populations, as exemplified by the classic example of the human fetal globin genes (Slightom et al., 1980). With the development of transgenic strategies and PCR assays for detecting gene conversion in gametes, it will be possible to look more closely at the nature of gene conversion events in mice. For example, it has been possible to PCRamplify gene-converted transgenes from individual spermatids obtained from the IacZ transgenic system described in Fig. 2 (W. Hanneman and J. Schimenti, unpublished results). With knockouts now being generated for many DNA repair and recombination genes (see section VI), it will be possible to examine the role of various genes on gene conversion in mice. In the next few years, it is rcasonable to expect that these technologies will answer questions relating to conversion tract length, conversion-associated crossovers, effects of homology on recombination rate, and postmeiotic segregation.
V. Recombination and Disease There are biological pros and cons to unequal recombination. On one hand. most types of unequal recombination events, such as translocations, deletions, and inversions, are deleterious. The consequences range from mutations of a single gene to chromosomal aberrations and aneuploidy. On the other hand, i t is clear that unequal recombination is a major form of genome evolution. One of the most important consequences of unequal recombination is gene duplication, a critically important phenomenon that enables utilization of preexisting genetic material as a substrate for functional change and adaptation. Extra gene copies created through duplication may ultimately diverge to perform related but specialized developmental and biochemical function. An example is the human P-globin gene family, which has evolved a highly coordinated process of tissueand stage-specific expression of developmentally specialized genes. While it can be argued that the evolutionary optimum would be higher or lower in any given mammal, clearly nature has arrived at a reasonable compromise. As it stands, a
1. Recombination in the Mammalian Germ Line
19
considerable proportion of all mammalian genes exist as members of gene f a m lies or even superfamilies. In mammals, there is evidence for a wide range of unequal homologous recombination events. The sequences involved in such exchanges can be on the same chromosome, sister chromatids, homologous chromosomes, or entirely different chromosomes. In short, regions of homology can serve as recombination templates anywhere in the genome. as in yeast. However, proximity of the homologous sequences appears to be a much more critical factor in the mammalian genome than it is in yeast. A major catalyst of illegitimate exchange is repetitive sequences. The mammalian genome is replete with such elements, the most common of which are the Ah-like repeats. These are approximately 300 bp in size, and about one-half million exist in the human genome. Hence, there is an Alu sequence every 6 kb, on average. The L1 long interspersed repeat elements are present in fewer copies (about 100,000) but are up to 7 kb in length. Because of their prevalence and the relatively high levels of homology between elements within a class, unequal recombination between them is a potentially major form of mutagenesis. Indeed, recombination between repetitive elements has been documented in several human diseases, including Lesch-Nyhan syndrome (Marcus et al., 1993), familial hypercholesterolemia (Lehrman et ( I / . , 1985), Tay-Sachs disease (Myerowitz and Hogikyan, 1987), P-thalassemia (Gilman, 1987), and human growth hormone deficiency (Vnencak-Jones rt d., 1988). There is also substantial evidence that translocations resulting in leukemias can be catalyzed by repetitive sequences (Stallings et al., 1993). Mutations can also occur by unequal recombination between members of a gene family. A dramatic example is afforded by the human color vision genes. Unequal recombination in this family alters the copy number of green and red pigment genes and generates hybrid genes. Depending on the rearrangements, a person’s ability to discern colors can vary (Nathans, Piantanida, et d., 1986; Nathans, Thomas, et al., 1986). About 10% of men experience some degree of color blindness. This indicates the enormous potential for mutagenesis via recombination. Because regional duplications involving entire genes or chromosome segments provide much larger tracts of homology, the frequency of illegitimate recombination may be higher than events mediated by repetitive elements. Finally, gene conversion has been implicated in the etiology of several disease-causing mutations. Examples of human diseases that were ostensibly generated by gene conversion-like events include steroid 2 1 -hydroxylase deficiency (Collier et a/., 1993; Higashi, Tanae, Inoue, and Fuju-Kuriama, 1988; Higashi, Tanae, Inoue, Hiromasa, et d., 1988; Morel et a/., 1989) and congenital adrenal hyperplasia (Amor et al., 1988; Rheaume et al., 1994). In these cases, the functional genes appear to have been converted by highly homologous, nearby pseudogenes. The human glycophorin genes A and B, which encode the MNS blood
20
Ilouglas L. Pittman and John C. Schimenti
group antigens, appear to contain a hotspot of recombination. Hybrid glycophorin genes have been formed by unequal recombination events, including gene conversion (Huang et a/., 1993: Kudo and Fukuda, 1994). There is also evidence that one cause for familial hypertrophic cardiomyopathy is the formation of a hybrid myosin heavy chain as a consequence of gene conversion between the closely linked a- and P-chain genes (Tanigawa c't d., 1990). Although the human genome is replete with repetitive elements and duplicated genes, it does not experience catastrophic instability due to recombination. Why is this'? First, although repetitive sequences are homologous, they are far from identical. As discussed earlier, it is known that the frequency of recombination is directly related to the degree of similarity and overall length of' homology. Alu sequences, which are only 300 bp long in humans (and about half that size in the mouse), d o not provide particularly efficient templates for homologous recombination, from both the homology and the length viewpoints. It is thought that the strict homology requirements are controlled in part by DNA repair genes. For example, a knockout of the mutS homolog Msh2 gene in mouse cells improves the efficiency of homeologous recombination to levels equivalent to that between isogenic sequences (de Wind et d.. 1995). Another hypothesis is that organisms increase heterogeneity between duplicated sequences through mechanisms such as MIPing (methylation-induced polymorphism) and RIPing (repeat induced point mutation) (Kricker et 01.. 1992). Both processes accelerate the accumulation of nucleotide differences, which in turn inhibit recombination. Finally, it has been proposed that genetic events that reduce unequal recombination, such as insertion of repetitive elements that break up extended regions of sequence homology, can uncouple duplicated genes from concerted evolution (Hess et a / . , 1984: Murti et d., 1992: Schimenti and Duncan, 1984; Walsh, 1987). Mutations caused by recombination offer unique opportunities to investigate the means by which events have occurred. In determining whether a recombination event was meiotic or premeiotic, the same caveats as described earlier apply. Again, it is always difficult to prove meiotic recombination, but mitotic recombination can be shown if a parent who transmitted the mutation carries the recombinant locus in somatic cells has more than one child that is affected in the same way. This aside, meiotic unequal recombination events (other than gene conkersion) can be classified into the following categories: ( I ) intrachroniosomal recombination, either between sister chromatids or within the same chromatid, and ( 2 ) interchromosomal recombination between nonallelic honiologs. This is illustrated in Fig. 3. The outcomes can be nearly identical, except for markers flanking the crossover point. So, to distinguish between the two. polymorphisnis must be identified to sort out which parent donated the mutation. and further to determine the chromosomal linkage of the donor's alleles. In the example shown, sequences must be identified (at positions X and Y ) that can be physically associated with the rearranged gene. One of these must be unique to either parent
a.
b.
a ya
xa
Gene1
Gene2
SCE exchange
Mating
+
ya
Xa
ya Y"
lnterchromosomal exchange
+ Fig. 3 linequal recombination between homologous chrorno\otnc\ versus sister chromatid exchange (SCE). ( A ) Genes 1 and 2 are duplicated homolog\. X and Y are anonymous loci flanking the genes. In this example. recomb~nationoccurs between genes 1 and 2 on \i\tcr chromatids of one chrornosornal homolog. The resulting hybrid gene remains flanked by the "a" alleles of X and Y. The "n" alleles of X and Y are unique to the other parent. ( B ) An unequal crossover occur\ between the hornologous Chroino\omes o l t h e donor parent. The \ariatit chromosorne exhibit:, the exchange o f polymorphhms at the X and Y loci.
22
Douglas L. Pittman and John C. Schirnenti
(the “a” alleles, as shown, as opposed to the unmutated “n” alleles contributed by the other parent). Either the same loci or other loci flanking the gene for which the donor parent is heterozygous must then be identified. It must then be determined whether Xa and Ya are on the same chromosome (and Xb + Yb on the homolog), or if the linkage is Xa-Gene I-Gene 2-Yb (and the converse on the homolog). This might require typing of grandparents. Once the linkage is known, recombination between homologous chromosomes or sister chromatids can be distinguished. Evidence for sister chromatid exchange exists in humans; an intragenic duplication in the dystrophin gene has been identified in the etiology of a Duchenne muscular dystrophy case (Hu et uf., 1989).
VI. Genetic Control of Recombination Meiotic recombination is essential in mammalian organisms for the proper segregation of homologous chromosomes during the first meiotic division. Even though recombination is such a critical process, very little is known about the genes required for chromsomal recombination during meiosis because of the sterility resulting from such defects (Baker et ul., 1976). At present, no mammalian genes have been directly demonstrated to be required for meiotic recombination. The purpose of this section is to discuss genes that may be involved in recombination in the mammalian germ line. Studies in fungi have identified several genes required for normal recombination during meiosis. In Sacchavomyces cerevisicre, these can be divided into three groups, based on their mutant phenotypes: early exchange, synapsis, and late exchange (Mao-Draayer et al., 1996). The early exchange group includes genes essential for the initiation of meiotic recombination, and act before DSB formation. The second group consists of genes required for chromosome synapsis that, when mutated, alter levels of meiotic recombination. The late exchange group includes genes required for processing and resolving recombination intermediates, and act after DSB formation. Mammalian homologs of genes in the early and late exchange classes have been identified, generally on the basis of sequence similarity to the yeast relatives. The current state of knowledge with regard to the mammalian genes in each class is outlined below.
A. Early Exchange Genes
Mammalian homologs for two of the early exchange genes, RADSO and M R E I I , have been identified (Dolganov et ul., 1996; Kim et al., 1996; Petrini el al., 199.5). In yeast, these two genes are required for mitotic DNA recombinational repair and initiation of meiotic recombination (Game, 1993; Johzuka and Ogawa, 199.5; Petes et al., 1991). The yeast RadSO protein contains an ATP-binding
I . Recombination in the Mammalian Germ Line
23
domain and requires ATP to bind double- and single-stranded DNA (Raymond and Kleckner, 1993). Mutation o f the ATP-binding domain (rud%s) does not inhibit DSB formation during mciosis but does inhibit the subsequent 5' to 3' processing of the double-stranded ends (Alani et ul., 1990). The human RADSU homolog (hRAD.50) was identitied during a positional cloning effort to identify a gene responsible for acute myeloid leukemia (Dolganov et ul., 1996). Consistent with its role in DNA repair and meiotic recombination, it contains consensus nucleotide-binding domains, expression is increased in the testes, and the protein is localized within the nucleus. The human M R E l l homolog (hMRE11) was isolated in a two-hybrid screen for genes interacting with DNA ligase 1 (Petrini et ul., 1995). It shares extensive homology with yeast MRE11 and is ubiquitously expressed. However, it is found at higher levels in the spleen and testes. Like the hRad50 protein, hMre11 is localized in the nucleus. Two hybrid studies have shown that Rad50 interacts with Mre 1 1 and Xrs2 in yeast (Johzuka and Ogawa, 1995). The hMre1 1 and hRad50 proteins also interact, forming a complex with at least three other proteins (Dolganov et d.,1996). It is possible that one of the three unidentified proteins is a homolog of the yeast XRS2, another early exchange gene known to interact with the M R E l l and KAD.50 gene products.
B. Early Synapsis Genes Early synapsis genes are required for chromosome pairing, and mutants in this group of genes reduce recombination. For example, crossing over and gene conversion in hnpl, redl, and riwkl yeast mutants occur at approximately 1025% of wild-type levels (Hollingsworth and Byers, 1989; Rockmill and Roeder, 1991; for review, see Roeder, 1995). Mutations result in failure of homologous chromosomes to synapse properly, and the synaptonemal complex is either altered or absent (Hollingsworth and Byers, 1989; Leem and Ogawa, 1992; Rockmill and Roeder, 1991; Sym ct ul., 1993). The Hop1 protein has a zinc-finger DNA-binding motif (Hollingsworth et d., 1990) and displays nonspecific, Zn2+dependent DNA-binding activity (Friedman et a/., 1994). I t binds along the length of meiotic chromosomes during pachynema (Hollingsworth et a/., 1990). Mammalian homologs to the early synapsis yeast genes have yet to be identified. Nevertheless, given the fundamental similarity of meiosis across species, it is probable that functional homologs of these genes exist in mammals. Perhaps their activity or function allows considerable flexibility, and the modern orthologs have diverged beyond experimental recognition. Several synaptonetnal complex proteins have been identified in rodents by classic biochemical techniques; they include C o r l , S y n l , Ubc9, Scpl, and Scp3 (Dobson e t a / . , 1994; Kovalenko et a/., 1996; Lammers et a/., 1994; Moens et d., 1992; Schmekel et ul., 1996). Although we currently know litlle about their functions, antibodies to these
24
Douglas L. Pittman and John C. Schimenti
proteins provide useful reagents for studying the effects of knockout mutations on meiotic progression. For example, mice deficient in the ataxia telangiectasia gene are sterile from severe defects in meiosis. Antibodies to Cor1 were used to reveal that defects in synapsis appear in midzygotene sperinatocytes (Xu et al., 1996). An intriguing synaptonemal complex protein in yeast is Zip1 (Sym et nl., 1993). Mutations in this gene do not markedly alter overall recombination rates but abolish interference (Sym and Roeder. 1994). The consequence is an attendant increase in aberrant disjunction. Crossing over in niamnials is subject to strong negative interference, but ZIP1 homologs have not yet been identified. As indicated above, it is possible that one of the many novel synaptonemal complex proteins identified in rodents might represent a functional homolog. Targeted mutagenesis of the mouse P m 2 gene (the homolog of yeast P M S I ) , which is involved in mismatch repair in both yeast and mice, resulted in male (but not female) sterility characterized by defects in chromosome synapsis (Baker er u/., 199.5).This was not predicted from the phenotype of yeast mutants, which can sporulate but exhibit higher levels of postmeiotic segregation-an indicator of mismatch repair deficiency.
C. Late Exchange Genes
All but one of the yeast genes in the late exchange group were originally identified as being required for DNA repair. This observation led to the conjecture that the mitotic DNA repair genes were recruited for meiotic recombination (Game, 1993). An outstanding feature of late exchange genes is their similarity to the E. coli RecA protein, which is involved in homologous recombination and DNA repair. RecA coats single-stranded DNA, forming a helical filament, and promotes synapsis and strand transfer between homologous DNA molecules in an ATP-dependent manner (for reviews. see Radding. 199 1 ; West, 1992). Late exchange proteins show their strongest similarity to the RecA domain that interacts with ATP (Lovett, 1994). Strand exchange activity has been demonstrated for the human and yeast RadS 1 yeast proteins (Baumann et d . ,1996: Ogawa ct al.. 1993: Sung, 1994). Similarity of DMCl. RAD.55, and RAD57 to RecA suggests they also bind DNA and promote strand exchange. Human and mouse homologs have been identified for RAD.51, RAD52, and DMCI (Habu r t d . , 1996; Morita et d . , 1993; Sato, Hotta, rf ml., 199.5; Sato, Kobayashi, et ol., 199.5; Shen et (11.. 1995; Shinohara et d . , 1993). The MmRAD51 gene is expressed at high levels in ovary and testes, and the protein is associated with the axial/lateral element in synaptonemal complexes i n mouse sperinatocytes and oocytes (Ashley er al., 199.5; Haaf ef LII., 1995; Plug or ul., 1996). It appears early in meiosis as small, evenly dispersed foci (270 in sper-
I . Recombination in the Marnmalian (ierm Line
25
matocytes, 350 in oocytes). By the end of leptotene, 32-38 larger foci arc detected in both sexes, suggesting that the protein complex with which RadSI associates becomes larger during chromosomal condensation (Plug et (11.. 1996). Several of the late exchange gene products appear to interact, including the yeast and mammalian Rad52 and RadSI proteins (Donovan et d., 1994; Shen et ul,, 1996; Shinohara et a/., 1992). Targeted mutagenesis of the mouse RurlSl gene resulted in early embryonic lethality (Tsuzuki et d . , 1996). The mutation appears to be a cell lethal; this stands in contrast to yeast, in which diploids are viable. The mouse knockout data are even more surprising in light of the fact that mouse RadSI could partially rescue a yeast rad51 mutant (Morita et d., 1993), which suggested a conserved role for this gene between species. The drastic phenotype of Rad.51 complicates analysis of its role in mammalian meiosis. For this and other genes that prove to have confounding phenotypes, conditional mutations in germ cells will be required to understand their roles in meiotic recombination. The DMCl yeast gene is the only known gene required late in recombination that is not expressed during mitosis in yeast. Like the other members of the late class, DMCl is required for processing recombination intermediates (Bishop et a/., 1992). The Dmcl protein is bound to more than 64 sites along the chromosomes during meiosis (Bishop. 1994) and co-localizes with the RadSI protein. Binding of Dmc 1 to meiotic chromosomes requires Rad5 I , but not vice versa, suggesting that RadS 1 binds to the chromosomes prior to Dmc 1 binding. These results and the results of the two-hybrid studies support the idea that the Rad5 I , RadS2, and Dmcl proteins are part of a meiotic recombination complex that acts after recombination initiation. Mammalian DMCl homologs were recently isolated from mouse and human cDNA libraries (Habu et al., 1996; Sato, Hotta, et id., 1995; Sato, Kobayashi, et ul., 1995). Both mouse and human genes code for a 340-amino acid protein that contains the two nucleotide-binding motifs (GEFRTGKT and LLIID) important for binding single- and double-stranded DNA. Transcription of the mouse D M C l gene appears to be testes-specific, consistent with its proposed role i n meiotic recombination. In contrast, the human homolog is expressed in every tissue examined. The genes described in this section (as well as a host of other yeast genes and mammalian homologs not mentioned) are likely to be involved in mammalian meiotic recombination. Sequence homologies with known yeast genes. protein interactions, and cellular localization are consistent with this idea. However, as in the case of R a d S / , it appears that the exact roles may not be strictly conserved. This has already been observed to be the case with other genes. Mutations in P m 2 (as described earlier) and M l h l . which causes meiotic arrest at the pachytene stage in mouse spermatogencsis (Baker et al., 1996; Edelmann et a / . , 1996). causes phenotypes that appear to reflect a gain of function since the divergence of yeast and mammals. Remarkably, in the case of the Pins2 knockout, the novel function is limited to spermatogenesis, not oogenesis. With Mil?!, there is also a
26
Douglas L. Pittman and John C. Schimenti
dichotomy, in that oocytes can complete the first meiotic division, but spermatocytes cannot. Additional experimentation with targeted mutants in mice will be required to fully understand the role of the yeast homologs in mammals. Because it is likely that mutations in several of these genes will result in sterility (or worse!). methods other than classic breeding will be required to measure the effects of mutations on the initiation and resolution of recombination events. Transgenic constructs, such as the lacZ system described earlier, or PCR of defective gametes may be useful in this regard.
VII. Conclusion In the past several years, great strides have been made in the characteriLation of recombination in the mammalian germ line. Technical barriers posed by mammals are being circumvented by the implementation of transgenic and molecular approaches that permit analysis of individual gametes. Such experiments serve to elucidate the types and rates of recombination events i n humans and mice. These observations in turn provide insight into the role of recornbination in molecular evolution and disease. Nevertheless, we anticipate that important progress in the next few years will come from targeted mutagenesis of genes involved in meiotic recombination. These experiments have been, and will continue to be, guided by the extensive studies done in yeast. Another major advance would be the establishment of a culture system for mammalian gametes that could accurately reproduce the salient events of mammalian meiosis. Finally, as the genome project proceeds to identify all the genes present in mammals, and as techniques are established for examining the regulated expression of all genes on a genomewide scale, it is likely that we will begin to decipher the genetic control of events in mammalian meiosis.
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Shen. Z., Denison, K.. Lobb, R., Gatewood, J. M., and Chen, D. J. (1995).The human and mouse homologs of the yea\[ RADS2 gene: cDNA cloning, sequence anal) sis, assignment to human chromosome 12p12.2-pl3, and mRNA expression in mouse tissues. Geironiics 25, 199-206. Shenkar, R., Shen, M . H., and Arnheim, N. (1991 ). DNase I-hypersensitive \ites and transcription factor-binding motifs within the mouse E beta meiotic recombination hotspot. Mol. Cell. Biol. 11, 1813-1819. Shinohara, A,, Ogawa, H., Matsuda, Y., Ushio. N., Ikeo, K., and 0 g a u a . T. ( 1993). Cloning of human, mouse and fission yeast recombination genes homologous to RADS I and recA Nrrture Get7rr. 4, 239-243. Shinohara, A,, Ogawa, H., and Ogawa, T. (1992). RadSI protein involvcd i n repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69, 457-470. Shiroishi, T., Hanmwa, N., Sagai, T.. Ishiura, M., Gojobori, T., Steinmetr, M., and Moriwaki, K. ( 1990). Recombinational hotspot specific to female meiosis in the mouse major histocornpatibility complex. Imrtiuno~enrtics31, 79-88. Shiroishi, T., Koidc. T., Yoshino, M., Sagai, T., and Moriwaki, K. (1995). Hotspots of honiologous recombination in mouse meiosis. A d . . Biophyr. 31, I 19- 132. Shiroishi, T., Sagai, T., Hanzawa, N., Gotoh, H.. and Moriwaki, K. (1991). Genetic control of sex-dependent meiotic recombination in the major histocompatibilit) complex of the mouse. EMBO ./. 10, 68 1-686. Silver, L. M . (1996). “Mouse Genetics: Concepts and Applications. Oxford University Press, New York. Slightom, J. L., Blechl, A. E., and Smithies. 0. (1980). Human fetal Gy- and Ay-glohin genes: Complete nucleotide sequences suggest that DNA can he exchanged between these duplicated gene\. Cell 21, 627-638. Smih, F., Rouet. P., Ronianienko, P. J., and Jasin, M. (1995). Double-strand break5 at ihe target lociis stimulate gene targeting in embryonic stem cells. Nucleic. Acid! Res. 23, 5012-5019. Speed, R. M. (1977). The effects of aging on the meiotic chromosomes of male and fcmale mice. Ctirntnosoina 64, 24 1-254. Stallings, R., Doggett, N., Okumura, K., Matera, A,, and Ward, D. (1993). Are repetitive DNA sequences involved with leukemia chromosome breakpoints? h i “Genome Rearrangement and Stability” ( K . Davies and S. Warren, Eds.), Cold Spring Harbor Laboratory Press, pp. 59-78. Cold Spring Harbor. Steinmetz, M., Stephan, D., and Fischer Lindahl, K. (1986). Gene organiution and recombinational hotspots in the niurine major histocompatibility complex. Cell 44, 895-904. Steinmetz, M., Winoto, A,, Minard, K.. and Hood, L. ( 1982). Clusters of genes encoding mouse transplantation antigens. Cell 28, 489-498. Sun. H., Treco. D., Schultes, N. P., and Szostak, J . W. (1989). Double-btrand breaks at an initiation site for meiotic gene conversion. NNIIOP338, 87-90. Sung, P. (1994). Catalysis of ATP-dependcnt pairing and strand exchange by yeast RADSI protein. Science 265, I24 I - 1243. Sym, M., Engebrecht, J., and Roeder, G. S. (1993). ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis. Cell 72. 365-378. Sym. M., and Roder, G. S . (1994). Crossover interference is abolished In the absence of a \ynaptonemal complex protein. Cell 79, 283-292. Szostak, J., Orr-Weaver, T., Rothstein, R., and Stahl, F. ( 1983). The double-strand break repair model for recombination. Cell 33, 25-35, Tam, P., and Snow, M. (1981). Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos. J . Ernhryd. &p. Morphol. 64, 133- 147. Tanigawa, G., Jarcho, J. A,, Kass, S., Solomon, S . D., Vosherg, H. P., Seidman, J. G., and Seidman, C. E. (1990). A molecular basis for familial hypertrophic cardioniyopathy: An alpha/beta cardiac myosin heavy chain hybrid gene. Cell 62, 991 -998.
I . Recombination in the M a m m a l i a n Germ Line
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teRiele, H., Maandag, E., and Berns. A . ( 1992). Highly efficient gene targeting in embryonic stem cells through homologous recombination with icogenic DNA constructs. Proc. Null. A u d . Sci. USA 89, 5 128-5 132. Thomas, K., and Capecchi, M. (1986). High frequency targeting of genes to specific sites in the mammalian genome. Cell 44, 419-428. Tsuzuki, T., Fujii, Y., Sakumi, K., Toiiiinga. Y., Nakao, K., Sekiguchi, M., Matsushiro, A., Yoshimura, Y., and Morita, T. (1996) Targeted disruption of the RadSI gene leads to lethality in embryonic mice. Proc. Nnrcil. A ~ u r lSci. USA 93, 6236-6240. Tunnacliffe, A,. Kefford, R., Milstein, C., Forster, A,, and Rabbitts, T. H. (1985). Sequence and evolution of the human T-cell antigen receptor beta-chain genes. Pmc. Ncrtl. Accrd. %i. USA 82, 5068-5072. Urabc. K., Kimura, A., Harada, F., Iwnnaga, T., and Sarazuki, T. (1990). Gene conversion in \teroid 21-hydroxylase genes. AM. ./. Hrrrrr. G e w f . 46, 1178-1 186. Valancius, V.. and Smithies, 0. (199l ). I)ouble-strand gap repair in a maininalian gene targeting reaction. Mol. Cell. Biol. 11, 438')-4397. Vnencak-Joneq, C. L., Phillips, J . A. 11.. Chen, E. Y., and Seeburg, P. H. (1988). Molecular basis of human growth hormone gene dclctions. I-'r(~t.. Nrrtl. Acad. Sci. USA 85, 5615-5619. Wallace, B. M., and Hulten, M. A. (19x5). Meiotic chromosome pairing in the normal human female. Ann. Hum. Genet. 49, 215-226 Walsh, J . ( 1 987). Sequence-dependent gene conversion: Can duplicated genes diverge fast enough to escape conversion? Genetics 117, 543-557. Weber, J. L., Wang, Z.. Hansen, K.. Stephenson, M., Kappel, C., Salzman, S.. Wilkie, P. J., Keats, B., Dracopoli, N. C., Brandriff, B. F.. ut trl. ( 1993). Evidence for human meiotic recombination interference obtained through con\trtiction of a short tandem repeat-polymorphism linkage map of chromosome 19. Am. J . H u m (;cvret. 53, 1079-1095. Weiss, E., Mellor, A., Golden, L., Fiihrncr, K., Simpson, E., Hurst, J., and Flavell, R. (19x3). The structure of a mutant H-2 gene stiggc\t\ that the generation of polymorphism in H-2 genes may occur by gene converrion-lihc C\ents. NOILIIV301, 67 1-674. Weissenbach, J., Gyapay, G., Dib, C.. Vignal, A,, Morissette, J.. Millasseau, P.. Vaysseix, G., Lathrop, M. ( 1992). A second-generation linkage map of the human genome. Ntrrure 359, 794-801 Wernke. S. M., and Lingrel, J . B. ( I9Xh). Nuclcotide \equence of the goat embryonic alpha globin gene (zeta) and linkage and evolutionary analysi\ of the complete alpha globin cluster. J. M ol. Biol. 192, 457-471 We\t. S. C. ( 1992). Enzymes and molecular mechanisms of genetic recombination. Annu. Rns. Rioc.hern. 6 1, 603-640. Wilkie, T. E., Braun, R. E., Ehrman, W.. Palmiter, R., and Hammer, R. (1991). Germ-line intra chromosomal recombination restore\ lertility i n transgenic MyK- 103 male mice. Geric,.\ Dri.. 5, 38-48. Wu, T. C., and Lichten. M. (1994). Mciosis-induced double-strand break \ites determined by yeast chromatin structure. Science 263, 5 15-5 18. Xu, Y., Ashley, T., Brainerd, E., Bronson. K., Meyn, S., and Baltimore, D. (1996). Targeted disruption of ATM leads to growth rctmhtion, chromosomal fragmentation during meiosis, i n mune defects, and thymic lymphoma. G r r m Deli.. 10, 241 1-2422. Zangenberg, G., Huang, M. M., Arnhcim, N., and h-lich, H . ( 1995). New HLA-DPB I alleles generated by interallelic gene conversion detected by analysis of sperm [see cotnments]. Nurure Genet. 10, 407-414. Zimmer, E. A,, Martin, S. L., Beverley, S. M., Kan, Y. W., and Wilson, A. C. (1980). Rapid duplication and loss of gene coding t o r the alpha chain\ of hemoglobin. Pmc. N(7//. Actrd. Sci. USA 77, 2158-2162. Zimmerer, E. J., and Passtnore, H. C?. ( 1991). Structural and genetic properties of the E h reconi33, 132- 140. binational hotspot in the mouse. /,/r,rrrciro~urr~~rrc~
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2 Meiotic Recombination Hotspots: Shaping the Genome and Insights into Hypervariable Minisatellite DNA Change Wayne I? Wahls Department of Biochemistry Vanderbilt University School of Medicine Nashville, Tennessee 37232-0146
I. Introduction 11. General Features of Chromosonic Dynamics during Meiosis 111. Genetic ldcntification of Recomhination Hotspots
A. B. C. D.
Disparity between Genetic and Phy\ical Maps Marker Effects and Polarity 01 Recombination Recombination Hotspots of the Mouse MHC Mammalian Recombination Hotspot\ Revealed in Plasmid Recombination Assays
IV. Double-Strand DNA Breaks and Open Chromatin A. Double-Strand DNA Break\ Activate Recombination Hotspots B. DNA Accessibility within C‘hroiiiatin V. Roles of Protein-DNA Binding 111 Hotspot Activation V1. Control of Recombination in cis and rrmix, Near and Far VII. Hotspots as Initiators or Resol\.cn oi Recombination: Two Models A. Recornhination Hotspots Enh;iiicing an Early Step in Recombination: Convergence of Genetics and t3iochemi\(ry B. Recombination Hotspots Enliaiicing a Late Step in Recombination: Hypervariable Sire of Resolution of Holliday Junctions VIII. Summary References
Meiotic homologous recombination serves three principal roles. First, recombination reassorts the linkages between newly-arising alleles to provide genetic diversity upon which natural selection can act. Second, recombination is used t o repair certain types of DNA damage to provide a mechanism of gciiomic homeostasis. Third, with few exceptions homologous recombination is required for the appropriate segregation of homologous chromosomes during meiosis. Recombinatioii rates are elevated near DNA sites called “recoiiibination hotspots.” These sites influence the distribution of recombination along chromosome< and the timing of recombination during the life cycle. Recent advances have revealed biochemical Tteps of hotspot activation and have suggested that hotspots may regulate when and where recombination occurs. Two models for hotapot activation, one in which hotspots act early in the recombination pathway and one in which hotspots act late in the recombination pathway, are presented. The latter niodel can account for changes at hypervariable mini-
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Wayne P. Wahls satellite DNA in metazoan genomes by invoking resolution of Holliday junctions at minisatellite DNA repeats. Copyright 0 1998 by Academic Press.
1. Introduction Meiosis and homologous recombination have vital roles in the evolution of eukaryotes. Meiosis and the subsequent joining of haploid gametes generates new genotypes by shuffling linkage groups (chromosomes), and recombination generates new genotypes by shuffling alleles of genes within individual chromosomes. Both processes supply the genetic diversity on which natural selection may act. Thus, meiotic reassortment and recombination provide eukaryotes with a mechanism for sampling different combinations of newly arising genetic variants. Recombination also has an apparent role in chromosome dynamics during meiosis: in the absence of recombination, homologous chromosomes fail to segregate appropriately, which suggests that recombination is required for proper chromosomal disjunction. Meiotic recombination rates are induced up to IOOOfold relative to mitotic rates, presumably to maximize the amount of genetic variability in the meiotic products or to ensure homolog disjunction, or both. Recombination rates are higher than average in the vicinity of DNA sites called recombination hotspots. Recombination hotspots may contribute a significant fraction of all recombination that occurs in the genome (Wahls et ul., 1990a; Smith, 1991; Wahls and Smith, 1994; Wu and Lichten, 1994; Lichten and Goldman, 1995; N. Kon, M. D. Krawchuk, B. G. Warren, G. R. Smith, and W. P. Wahls, unpublished observations). While the possibility that these sites regulate recombination is speculative, it is clear that hotspots must enhance rate-limiting steps in the pathway of recombination. They thus provide a larger window through which to view the molecular mechanisms of recombination. Although eukaryotic recombination has been studied genetically for most of the twentieth century, study of the biochemistry of eukaryotic recombination is in its infancy. The problem is one of scale. The products of recombination, whole chromosomes, are huge and can differ by as little as broken and reformed phosphodiester bonds. Thus, much of what we know about the machinery of recombination is inferred from genetic studies. Recombination hotspots were first identified in genetically tractable organisms, such as bacteriophages and fungi, but it is now apparent that hotspots are ubiquitous and active in all organisms. Given that recombination occurs at a higher rate near hotspots, it is not surprising that recent advances in understanding the mechanisms of eukaryotic recombination have come from the study of hotspots. This review outlines the identification of eukaryotic recombination hotspots and presents select examples of hotspots in greater detail. It is written for a general audience. For those who want an introductory recombination text, I
2. Recombination Hotspots and Minisatellite Change
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recommend highly the new textbook by David Leach (1996). For those looking for a more in-depth treatment o f specific subject matter, primary references and recent review articles are cited. The aim is to provide a historical framework for the current understanding of hotspot activity. Although recombination hotspots have been studied principally in fungi, there are a few studies of hotspots in multicellular eukaryotes that have progressed beyond the descriptive stage. These are presented. And finally, two models for hotspot activation, one in which hotspots act early in the recomhination pathway and one in which hotspots act late in the recombination pathway. are presented.
I I . General Features of Chromosome Dynamics during Meiosis During meiosis, chromosomes undergo one round of DNA replication, homologous chromosomes somehow find each other to pair with and undergo recombination, and two rounds of chromosome segregation result in haploid meiotic products. The cytogenetic behavior of chromosomes during meiosis is fairly well characterized. Studies by Barbara McClintock on maize and Neumsporci earlier in the century firmly established the general features of pairing and segregation of chromosomes during meiosis. McClintock also provided the original evidence that reciprocal recombination between genes on chromosomes during meiosis is accompanied by a physical exchange of chromosome parts. Although it is generally accepted that there is a relationship between the initial homologous alignment of chromosomes, formation of tightly associated pairs held together by the synaptonemal complex (SC), homologous recombination, and appropriate segregation of chromosomes, the nature of that relationship is not clear. It is within the macromolecular structure of the SC that homologous recombination occurs. Genetic analyses of homologous recombination, conducted principally in various fungi, have led to theoretical models for how recombination occurs (Holliday, 1964; Meselson and Radding, 1975; Szostak et al., 1983). The models share common steps: ( 1 ) some sort of homology search and pairing of chromosomes; (2) a double-strand DNA (dsDNA) or single-strand DNA (ssDNA) break that initiates recombination; (3) processing of those DNA breaks in some fashion; (4) invasion of one homolog with a DNA strand from the other homolog; ( 5 ) repair synthesis and DNA ligation to create one or more Holliday junctions, points where two homologous chromosomes are covalently linked by crossed DNA strands; (6) branch migration of the Holliday junction; (7) cleavage of the crossed strands in the Holliday junction to separate the homologous chromosomes; (8) ligation of the resulting nicks to produce recombinant chromosomes; and (9) mismatch repair of heteroduplex DNA. Physical and biochemical analyses have confirmed some of these theoretical steps in the pathway. For example, processed dsDNA breaks (steps 2 and 3) have been detected (Sun et d., 1989; Cao et ul.,
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Wayne I? Wahls
1990); a nuclease that produces those breaks has been identified (Bergerat et a/., 1997; Keeney e t a / . , 1997); enzymes likely involved in strand exchange (step 4) have been characterized (Bishop et d., 1992; Shinohara et al., 1992; Benson et a/., 1994; Bishop, 1994); and an exonuclease with a role in mismatch correction (step 9) has been purified (Szankasi and Smith, 1992, 1995). Many of these advances have been made by studying recombination hotspots.
111. Genetic Identification of Recombination Hotspots A. Disparity between Genetic and Physical Maps
The first indication that rates of recombination are not a uniform function of physical distance along the DNA came from mapping experiments. Mosig (1966) first pointed out such discrepancies from studies of the bacteriophage T4. Although in most areas of the T4 genome the measured distances in DNA were proportional to genetic map distances determined from recombinant frequencies, the region including genes 34 and 35 was especially prone to recombination. Mosig stated, “The discrepancies suggest that genetic recombination frequency in this region is increased by local factors other than distances between markers.” As contiguous physical maps of chromosomes become available, invariably there are expansions and contractions of the genetic maps relative to the physical maps (Fig. 1). These differences, which can be quite pronounced (Anderson et al., 1988; Zimmerer and Passmore, 1991; Bryda et a/., 1992; Shiroishi et ul., 1993), reflect variable frequencies of recombination in different regions of chromosomes. In addition to regional variability, the strain (Soper et al., 1988; Zimmerer and Passmore, 1991; Shiroishi et d., 1993) and sex (Shiroisi et al., 1990, 1991) in which meiosis is occurring can markedly influence regional recombination frequencies. Both cis-acting and trans-acting factors are implicated. It has become clear that regional differences in reciprocal recombination between any two genetic markers are common. These recombination hotspots are inferred to contain some feature that promotes nearby recombination.
B. Marker Effects and Polarity of Recombination The second indication that rates of recombination are not a uniform function of physical distance along the DNA came from the analysis of how individual genetic markers behave during recombination. After premeiotic DNA replication there are four chromatids (eight ssDNA strands) between which recombination may occur (Fig. 2A). Two rounds of chromosome segregation separate the chromatids into the four meiotic products. In some organisms, such as Sacchuromyces cerevisiae, the four products of individual meioses are spores held together in an
41
2 Recombination Hotspots and Minisatellite Chdnge Male genetic
----3
a'
Physical map
Female genetic map .a
'b
b .C
.d C
d Fig. 1 Schematic diagram of difference\ between physical and meiotic genetic maps. Expansion and contraction of genetic intervals relativc to physical intervals identifies regions of the genome with rates of recombination higher (interval I ) and lower (interval 3) than average, respectively. I n this idealized example, recombination is Iiighci- i n interval I during inale meiosis relative to female meiosis, revealing sex-hpecific differeiice\ o t recombination rate\. Ci.,-acting elcrnents within or near the intervals are inferred to influencc thcir rate\ o f recornbination.
ascus, collectively called a tetrad. I n other organisms, such as Neurosporii c r ( i . w i or A.sc~~bn/iis irnrnersus, a single round of mitosis occurs after meiosis and eight spores are formed in the ascus, called an octad. It is possible to dissect individual asci, isolate all the spores, and grow individual spore colonies. This permits phenotypic analysis of all four ( o r eight) products of a single meiosis. By studying the products of a single meiosis, it is possible to determine whether recombination is conservative or nonconservative (Fig. 2A). Reciprocal exchange events are conservative; genetic information is neither lost nor gained and the alleles are inherited i n a Mendelian 2:2 ratio. In contrast, during gene conversion the genetic identity of one allele is lost and is replaced by that of the opposite allele, leading to non-Mendelian inheritance with 1:3 or 3: 1 ratios (also called 2:6 or 6:2 ratios for eight-spored fungi). A second type of non-Mendelian inheritance occurs when two spores of a tetrad yield colonies with phenotype A , one spore colony has phenotype t i , and one spore colony has thc sectored phenotype A / o . These sectored colonies provide evidence for heteroduplex DNA
42
Wayne P Wahls
A
Recombining Chromosomes
B
Reciprocal Exchange
0 '
Postmeiotic Segregation a
b
a a
b b
a A
I3 B
A
B B
A
I3
Gene Conversion
a
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a
b
a a
b B
a a
b B
a A
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B B
A
b
A
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A A
B B
A
I3
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I3
Relative Position of Mutant Allele DED81
ARG4
D-
4DSB Fig. 2 Polarity of recombination. ( A ) Reciprocal exchange is conservative and switches the linkage between markers Banking the point of recombination, Gene conversion and postmeiotic segregation (PMS) are nonconservative (aberrant) recombination events that result froin transfer 0 1 genetic information from one allele to the other. Other types of aberrant segregation (not shown) may also
2. Recombination Hotspots and Minisatellite Change
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that contains one DNA strand from each of two parental chromatids. During the first mitotic division those two strands separate from each other and each of the two daughter cells receives a different allele. This is called postmeiotic segregation (PMS). Tetrads exhibiting PMS are classified as 5:3 or 3:5 tetrads (using the nomenclature of eight-spored fungi). Some tetrads, aberrant 4:4 tetrads, have two spore colonies with PMS, indicating the presence of symmetrical heteroduplex DNA in two of the four chromatids. In general, the frequency of gene conversion is much higher than that of PMS for most markers, suggesting that the heteroduplex DNA is efficiently repaired. This repair leads either to gene conversion or to restoration of the original allele. Thus, a combination of heteroduplex formation and mismatch correction can lead to gene conversion events. The relative proportions of PMS, gene conversion, and symmetrical or asymmetrical heteroduplex DNA in different organisms arc accounted for by several related models of recombination (Holliday, 1964; Meselson and Radding, 1975; Szostak et ul., 1983). For a comparison of recombination models, see the review by Petes et a / . (1991). Because all models propose that reciprocal exchange and gene conversion are alternative resolutions of a common intermediate in the pathway of recombination, it is possible to infer overall recombination rates at individual markers within any given locus by determining their frequencies of gene conversion. The frequency of meiotic gene conversion varies depending on the locus and alleles being studied. In S. cereiisine, about 1-50% of tetrads will have a gene conversion event at any given locus ( Fogel et al., 198 1; Petes et al., I99 1 ). When multiple different alleles within a gene are studied there is usually a gradient of conversion frequencies across the gene (Fig. 2B). This is known as polarity. Polarity was discovered simultaneously in two fungi, Ascobo/lt.s immersus (Lissouba and Rizet, 1960) and N<.irro.vporu crassci (Murray, 1960). Murray also showed that polarity was conferred by elements close to the gene; chromosomal inversions also inverted the polarity gradient (Murray, 1968). Polarity has been demonstrated for several genes in S. rwetisiae, including ARC4 (Fogel rt a/., 1981; Nicolas et al., 1989), CYS3 (Chaudhuri and Messing, 1992), DED81 (Schultes and Szostak, 1990), H I S / (Fogel et al., 1967; Savage and Hasting, 1981), HIS2 (Malone et al., 1992), and HIS4 (Detloff et al., 1992). Polarity gradients generally have a high frequency at one end of the gene, a low frequency at the other end, and a relatively linear range in between, but there are some
occur. Aberrant segregation events in unselected tetrads can be used to gauge recombination rates at individual genetic markers. (B) An example o f polarity resulting from different frequencies of aberrant segregation for multiple marker5 at the /IED8/ and ARC4 loci in S. crwvisiae. Regions of increased accessibility of DNA within meiotic chromatin (striped ovals) and meiosis-specific dsDNA breaks (DSB arrows) map to the high-frequency end of polarity gradients, suggesting that reconibination initiates at those sites. (Data are froin Fogel P / d . , 1981; White e/ ml., 1985; Nicolas e/ ( I / . , 1989; Sun rr d . , 1989; Cao el al., 1990; Schultcs and Slostak, 1990; Lichten and Goldman, 1995.)
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Wayne P. Wahls
exceptions: genes lacking polarity (Kitani and Olive, 1967) or having polarity gradients with the low frequency in the middle of the gene (Pees, 1966) have been described. The enhancement across yeast polarity gradients ranges from about 3-fold (at HIS2) to about 10-fold (at ARG4). In cases of a uniform polarity gradient, the high-frequency conversion end of the gene usually corresponds to the 5' end, but in the HIS2 gene the polarity gradient slopes up toward the 3' end. The shape of polarity gradients suggests that there are discrete sites at which recombination initiates. In this view, recombination events initiate preferentially near the high-frequency peak and propagate outward. The slope and extent of the gradient reflect the processivity of recombination events initiating at each hotspot. Double-strand DNA breaks have been found at these hotspots (Nicolas et a/.. 1989; Sun et a / . , 1989; Cao et al.. 1990), suggesting that the hotspots within polarity gradients are sites where recombination initiates. These initiating lesions are discussed in more detail subsequently (Section IV). On the other hand, one must consider the alternative explanation for the polarity, namely, that a repressor of recombination acts on the low-frequency end of the gradient and polarity results from a distance-dependent propagation of repression. For example, several trans-acting gene products repress recombination in the mat2-mat3 interval of the fission yeast, Schi,osnt.charorn?'ces pomhe (Klar and Bonaduce, 1991; Thon and Klar, 1993; Thon et nl., 1994), thereby making a recombination coldspot. However, this may reflect the special need to reduce recombination at the mat loci to achieve the appropriate regulation of mating-type switching. In some cases, polarity may be largely due to the extent of heteroduplex DNA that is formed to the right, left, or both sides of an initiating event. In other cases, the extent of the mismatch correction tract likely has a significant role in the shape of the polarity gradient. For a more comprehensive discussion of polarity ii recent review is available (Nicolas and Petes, 1994).
C. Recombination Hotspots of the Mouse MHC
The best examples of recombination hotspots in multicellular eukaryotes are those found in the mouse major histocompatibility complex (MHC). The MHC is a large genomic region that encodes multiple cell-surface proteins of the immune system involved in self/non-self recognition. Because of its importance in immune function, the MHC has been extensively studied. The MHC is highly polymorphic and different haplotypes (the particular combination of genes within the MHC) can be determined serologically, thus providing a large number of allelic markers for loci throughout the region. Those markers can be used to study recombination. Intriguingly, crosses between some mouse strains lead to an increase in recombination within the MHC relative to crosses between other strains (Shiroishi et N / . , 1982; Kobori et al., 1984). A strain-specific recombination enhancer (recombination hotspot) is inferred. The generation of contiguous DNA clones spanning large intcrvals of the
2. Recombination Hotspots and Minisatellite Change
45
MHC (e.g., Steinmetz et a/.,19x6) made possible a comparison of the physical and genetic maps. By using RFLP analysis and DNA sequencing, it is possible to narrow down the intervals within which [he reciprocal exchange events occurred. The data reveal that most recombination within the MHC is clustered within recombination hotspots. One hotspot is located in the EP second intron (Kobori et a/., 1984; Kobori e t a / . , 1986; Lafuse and David, 1986; Steinmetz et d.. 1986), one is within the Pb-Oh intergenic interval (Lafuse pt a/., 1990),another is found in the Int3-Tnx interval (Yoshino. Sagai, Lindahl, Toyoda, Shiroyashi. et a/., 1994) and so on. In each case. ;IS line physical mapping was done, it was discovered that most of the exchnnge events occurred within small physical intervals of a few thousand base pairs or less. These points of exchange may be even more tightly clustered; howevcr, resolution is limited by the proximity of the nearest heterologies flanking the points of exchange. Some of these MHC hotspots can be really hot: genetic crosses between Asian wild mice with the u w 7 haplotype and laboratory strains generate about a 2% recombinant frequency within a physical interval of h u t 1000 hp at the Pb-Oh hotspot (Shiroishi et al., 1993). This reflects a meiotic recombination rate that is more than 1000-fold higher than the genomic average. As with the mouse MHC, hotspots have been identified in the hurnan (van Endert rt ( I / . , 1992; McAdam et d . ,1994; Martin et a/., 1995), cow (Andersson cJt ul., I98X), and chicken (Hepkema et a / . , 1993) MHC regions, suggesting that hotspot\ i n the MHC are evolutionarily conserved. DNA sequence comparisons failed to reveal any major features common t o all the MHC hotspots. However, the EP and the Lmp2 hotspots do have three motifs in coininon. They each contain an isolated, ancestral retroviral long terminal repeat (LTR) element, a middlc repetitive clement of the M T family, and tandem repeats of a tetraineric sequence resembling hypervariable minisatellite DNA. Two of these three elements (UI'R and hypervariable minisatellite) are recombinogenic in plasmid-based or iri ilitro recombination assays (Edelmann et d . , 1989; Wahls rt d . , 1990a). Activity of the Lmp2 hotspot requires significant homology between the recombining chromosomes, with the number of copies of the minisatellite-like sequences being most important (Yoshino et d.,1995). Furthermore, the HLA-DQ hotspot in humans has similar DNA sequence elements: hypervariable minisatellite DNA and the repeating dinucleotide d(TG),,, which are hotspots in human cells (Wahls r/ d . , 1990a,b), are located close to (or at) the point of exchange. These repeated DNA elements may be partially responsible for hotspot activity. DNaw I hypersensitive sites have been found in the vicinity of these elements, suggusting that open chromatin may play a role in hotspot activation (Shenkar ct ( I / . . 1991; Mizuno (T a/.,1996). Roles for hypervariable minisatellite DNA and open chromatin are discussed later (in Sections VII and V. respectively). The control of hotspot function seeins to be complex. The presence of two MHC hotspots located about 100 khp apart does not have an additive effect on recombinant frequencies (Yoshino. Sagai. Lindahl, Toyoda, Shiroishi. ct ( I / . , 1994). [A similar phenomenon occurs lor two pairs of hotspots located near each
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Wayne P. Wahls
other in fission yeast (Zahn-Zabal et ul., 1995) and in budding yeast (Fan e t a / . , 1997)]. Presumably there is either competition for limiting recombination factors or direct regulation involved. Perhaps the most exciting findings have been for the Pb-Ob hotspot. Shiroishi and colleagues (1990) have shown that elements outside of the primary sequence in the hotspot region are required for activity. Two distinct components are inferred: an element centromere proximal to the hotspot is required for the hotspot to function at all, and an element that is distal to the hotspot represses hotspot activity in male meiosis (Shiroishi et al., 1991, 1995). (Trans-acting repression of hotspot activity also occurs at the cog hotspot of N . crassu (Catcheside and Angel, 1974; Catcheside, 1977)J. These findings reveal that hotspot activity is not simply a function of some local, cis-acting feature. At a minimum, two other factors or DNA sequences located away from the Pb-Ob hotspot must be required for function. It may be possible to identify the factors that control hotspot activity from a distance.
D. Mammalian Recombination Hotspots Revealed in Plasmid Recombination Assays
Genetic studies of hotspot activity in mammalian meioses are time-consuming, expensive, and restricted to well-characterized regions such as the MHC. In somatic cells, however, recombination between mutant DNA viruses (such as SV40) can be studied (Dubbs rt al., 1974). The development of shuttle vectors derived from these DNA viruses and carrying a dominant, selectable marker gene (e.g., neomycin phosphotransferase) permits analysis of recombination in many cell types (Southern and Berg, 1982; Subramani and Southern, 1983). The general approach is to measure recombination between two similar plasmids, each of which has a different mutation in the marker gene. Recombination between the two defective genes restores a functional wild-type gene that can be expressed in either bacterial or mammalian cells. Thus, it is possible to select directly for recombinants within mammalian cells that have been cotransfected with the two plasmids. Alternatively, one can select for recombinant plasmids within bacteria after incubating the plasmid recombination substrates in cell-free extracts of mammalian cells. The utility of this approach and a discussion of somatic recombination can be found in a review by Subramani and Seaton (1988). The general view is that plasmid-based recombination uses the same enzymes and pathways as chromosomal recombination. Thus, DNA sequences that are hotspots in the plasmid recombirlation system might also be active within chromosomes.
1. Z-DNA Certain DNA sequences, such as the repeating dinucleotides d(CG), and d(TG),, can undergo a structural transition from the familiar right-handed B-DNA form to a left-handed Z-DNA form. Extensive stretches of these repeats are not detect-
2. Recombination Hotspots and Minisatellite Change
47
able in the genomes of eubacteria. archaebacteria, or mitochondria (Gross and Garrard, 1986). On the other hand, there are ?los copies of these dinucleotide repeats scattered throughout the mammalian genome. The discovery that Z-DNA can form in supercoiled DNA molecules under physiological conditions (Klysik ef a/., 1981; Singleton et d., 1982) and evidence that Z-DNA might exist iiz vivo within chromosomes (Lancillotti P / nl., 1987) suggested a biological role for Z-DNA. Hotspot activity of the Z-DNA motif d(TG), was initially seen as an increase in intramolecular recombination between two tracts of d(TG),, in SV40 viruses replicating in monkey cells (Stringer, 1985). Recombination between tandem copies of the Z-DNA mot s about %fold higher than recombination between other tandem repeats. At about the same time it was shown that a single tract of the Z-DNA motif can enhance intramolecular recombination between adjacent tandem DNA sequences in replicating SV40 virus (Bullock et ul., 1986). Subsequently, the effect of the Z-DNA motif on intermolecular recombination between nonreplicating plasmids introduced into human cells in culture was tested (Wahls et ul., 1990b). A (dTG),,, sequence enhances recombination about 20-fold and exerts its effect at a distance. The hotspot promotes both reciprocal exchange and gene conversion to an equal extent, and during gene conversion the substrate containing (dTG),,, is preferentially converted to wild type. [Two models for hotspot activity can account for these observations (Wahls et ul., 1990b).] Furthermore, binding of SV40 T antigen adjacent to the (dTG)30 sequence abolishes hotspot activity, suggesting either that T antigen prevents the (dTG),,, sequence from acquiring a recombinogenic configuration (such as left-handed Z-DNA) or that it stearically blocks the interaction of recombination proteins with the hotspot (Wahls and Moore, 1990). A protein that may have a role in recombination, HPP- I , was partially purified by Z-DNA affinity chromatography from the extracts of mammalian cells (Fishel ct d., 1988; Moore and Fishel, 1990; Moore et ul., 1991). Whether HPP-I is a factor that binds to the d(TG), sequence to activate the hotspot remains to be secn. Other results also implicate the Z-DNA motif as being recombinogenic. Purified Ustilago inuydis recl protein, like RecA of E. coli, is a recombination enzyme that promotes synapsis and strand exchange between homologous DNA molecules (Kmiec and Holloman. 1983). Z-DNA forms during the initial synapsis of homologous DNA promoted by rec 1 (Kmiec and Holloman, 1984), and the recl protein can increase the pairing of two DNA molecules that contain Z-DNA (Kmiec and Holloman, 1986). Addition o f anti-Z-DNA antibodies or competitor Z-DNA, or altering the superhelical density of the plasmids, inhibits the pairing reaction. This suggests that Z-DNA may be required for rec 1 -mediated pairing of duplex DNA molecules during recombination, Similarly, the RecA protein of E. coli binds more stably to Z-DNA than to B-DNA (Blaho and Wells, 1987) again suggesting that recombination enzymes may have a preference for these recombinogenic sequences. Because the biochemistry of eukaryotic meiotic recombination is now being revealed i n yeast (reviewed by Petes et ( I [ . , 1991), it is
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worthwhile considering one more example of d(TG), hotspot activity. A short fragment of DNA from the human P-globin gene cluster, containing d(TG),,,, enhances meiotic recombination during meiosis in yeast (Treco and Arnheim, 1986). Thus, further study of how this hotspot is activated during yeast meiosis might be productive.
2. Hypervariable Minisatellite DNA These highly polymorphic DNA sequences were first identified in a random clone from human chromosome 14 (Wyman and White, 1980). Related sequences were discovered subsequently at a variety of loci (Higgs er al., 1981; Bell et a / . , 1982; Capon et id., 1983; Jeffreys et ul., 1985a). The common feature of hypervariable minisatellites is a short consensus core sequence that may or may not have variable-length flanking sequences and that is repeated in tandem head-to-tail arrays at 215,000 loci in the human genome (Melmer, 1994). Allelic variation is due to differences in the number of repeats at each locus, and typical loci have overall lengths from 0.1 to 50 kbp. Locus-specific and multilocus probes can be used to study multiple polymorphic loci by Southern blotting, giving rise to individual-specific DNA “fingerprints” (Jeffreys et a/., 1985b) that can be used to establish genetic relationships. The importance of DNA fingerprinting has fueled study of how the polymorphism arises and is maintained. The hypervariable repeat length at individual loci was initially thought to result from unequal reciprocal exchange between alleles, suggesting a high rate of meiotic recoinbination (Jeffreys et ul., 1985a). But analysis of markers flanking newly arising minisatellite alleles revealed a lack of expected reciprocal exchange (Wolff et d., 1989; Kasperczyk et nl., 1990; Kelly et a/., 1991; Buard and Vergnaud, 1994). This led many to conclude that minisatellite DNA variability cannot be related to recombination. In fact, it only shows that reciprocal exchange within or near minisatellite repeats seldom occurs when new length alleles are generated. The core sequences within individual repeats at any given locus and allele can vary slightly, which permitted further analysis of how new length variants arise. Surprisingly, alleles often exhibit polarized variability (Armour, Harris, et al., 1993; Armour, Monckton, et al., 1993; Buard and Vergnaud, 1994; Ellsworth er al., 1995). New variants have one parental type across most of the locus, and at a discrete point, usually within a few tens of repeats from one end of the array, they become mosaic for both parental types. This implies that minisatellite change begins or ends at a discrete position within each array and extends in a directional fashion toward one end. Furthermore, the mosaic pattern suggests either complex mutational events or, as proposed below (Section VII, part B), several short patches of mismatch correction within a length of heteroduplex DNA extending to a discrete point of exchange. Considerable circumstantial evidence suggests that minisatellites are recom-
7. Rccornbination Hotspots and Miiiiutellire Change
49
binogenic. New alleles can arise at a remarkably high rate. inore than 10% for some loci, during meiosis (Buarti and Vergnaud, 1994) and, very rarely, during mitotic growth (Kelly et a/., 1989). The pairing of homologous chromosomes and the high rate of induction o f recornbination during meiosis are thus good candidates for a meiotic mechanism of minisatellite change. The frequency of change at minisatellite loci can be markedly different in female and niale meioses (Buard and Vergnaud, 1994; Henke and Henke, 1995), again analogues t o sexspecific patterns of meiotic recombination. In addition, hypervariable minisatellite DNA has homology to the Chi recombination hotspot of E. coli (reviewed by Kowalczykowski ('f "/., 1993); however, that homology may simply be coincidental. As discussed previously (Section 111, part C), minisatellite DNA motifs are found within recombination hotspots of the MHC. I n addition, these sequences are sometimes found in thc vicinity of translocation breakpoints (Krowczynska r t u/., 1990; Jaeger et a/., I994), which suggests that they have a role in the formation of DNA breaks that occasionally lead to aberrant rcconibination. Similarly, independent insertion events in the hamster ciprt locus are accompanied by loss of a sequcnce resembling the minisatellite core (Meuth et id., 1987). A recent study identilied minisatellite DNA among DNA that was preferentially undergoing repair i n meiosis (Ramachandra and Rao, 1994), suggesting that these sites may enhance repair as well as recombination. On a larger scale, cytogenetic analyses have found hypervariable minisatellite DNA at chiasmata (Chandley and Mitchell, I 988), sites within chromosomes where recombination is thought to occur. Direct evidence for hotspot activity of minisatellite DNA was obtained with a plasmid-based assay. Six copies o f a hypervariable minisatellite DNA core sequence enhanced intermolecular recombination up to 20-fold between plasmids introduced into human cells (Wahls rt u/.. 1990a). The hotspot increases recombination within a distant test interval and exhibits three characteristics different from those of most other hotjpot4. First. there is parity during gene conversion: the hotspot-containing substrate functions cqually well as the donor or as the recipient of genetic information. I f the minisatellite DNA promoted dsDNA break formation, then disparity 01' conversion would be expected (Szostak Pt a/., 1983). Second, the effects of the hotspot and a distal dsDNA break are additive. If most recombination initiates at dsDNA breaks, then this provides further evidence that the minisatellite hotspot does not function in the initiation of recombination. Third, the hotspot enhances reciprocal exchange events much inore than gene conversion events. This provides compelling evidence that the minisatellite acts late in the pathway of recombination. Reciprocal exchange and gene conversion are alternative resolutions of an intermediate in the pathway of recombination, the Holliday junclion (Holliday, 1964; Meselson and Radding. 1975; Szostak et al., 1983). 1 propose that hypervariable minisatellite DNA functions as a hotspot for recombination by acting as a resolution point for Holliday junctions (Section VII, part B.)
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Wayne P. Wahls
A number of hypervariable minisatellite DNA-binding proteins have been detected (Wahls, 1989; Collick et ul., 1991; Shinder et a/., 1994) and purified (Wahls et al., 1991) from nuclear extracts of mammalian cells. However, little is known about the biochemistry of these factors, and no sequence data have been reported. Intriguingly, several of these proteins seem indistinguishable from transcription factors of the rel/NF-kappa B family (Trepicchio and Krontiris, 1992), and the minisatellite DNA repeat can influence transcription of reporter genes (Green and Krontiris, 1993). Binding of these proteins to minisatellite DNA also seems to be required for hotspot activation (W. P. Wahls and P. D. Moore, unpublished observations). However, until further mechanistic data are available, the link between these proteins and hotspot activity or transcription must remain anecdotal.
3. Mouse Retroviral LTR Element As mentioned earlier, an LTR eletnent is located near two hotspots in the MHC. In an in vitro assay, recombination between LTR elements is about 13-fold higher than between two control DNA sequences (Edelmann et al., 1989). This hotspot activity seems to be meiosis specific; enhancement occurs within extracts of mouse testes cells but not within extracts of ascites cells. Deletion analyses revealed that some feature within a 37-bp region is essential for hotspot activity and that two different nuclear proteins bind in a sequence-specific fashion. Intriguingly, one of the proteins was absent from extracts of testes cells, raising the possibility that it represses hotspot activity. The purification of two proteins that interact with the hotspot (Goller rr al., 1994) tnay facilitate further investigations.
IV. Double-Strand DNA Breaks and Open Chromatin A. Double-Strand DNA Breaks Activate Recombination Hotspots
Broken DNA can be deadly or recombinogenic. Based on observations with irradiated S. cerevisiue cells, Resnick and Martin (1976) concluded that a single unrepaired dsDNA break may be lethal. This led to a model in which recombination is used to repair dsDNA breaks (Resnick, 1976). Subsequent studies of yeast transformation showed that an artificially introduced dsDNA break can markedly increase the frequency of transformation by both nonreciprocal (gene conversion) and reciprocal recombination mechanisms (Orr-Weaver et d., 198 I , 1983), establishing the dsDNA break repair model of recornbination (Szostak et al., 1983). Meiotically induced dsDNA breaks were first detected within the ARC4 hotspot (Nicolas eta/., 1989; Sun eta/., 1989), and dsDNA breaks are present at other hotspots surveyed to date, as well as throughout the genome at sites not yet characterized genetically (Cao rt (11.. 1990; Game, 1992; Zenvirth et al.. 1992;
2. Recombination Hotspots and Minisatellite Change
51
Nag and Petes, 1993; Wu and Lichten, 1994; Fan et ul., 1995; Wu and Lichten, 1995). At any given locus the amount of cleaved DNA is roughly proportional to recombination rates. Furthermore. the frequency of dsDNA breaks varies coordinately with hotspot activity for ci.v- and 11-urwactingmutations affecting recombination (Leem and Ogawa, 1901; Fan et ( I / . , 1995; Rockmill et al., 1995; Wu and Lichten, 1995; Xu and Kleckner. 1995; Fan and Petes, 1996),except in some instances, such as when there is apparent competition between adjacent hotspots (Fan rt a/., 1997). These data suggest that most meiotic recombination i n yeast initiates at dsDNA breaks. In wild-type cells, meiotic &DNA breaks are transient and they are heterogeneous, resulting from resection of about 600 nucleotides of the 5' terminated strand on each side of the breakpoint (Sun et [ I / . , 1989; Cao e t a / . , 1990; Sun et ul., 199 1 ). At least nine genes involved in the formation or processing of dsDNA breaks have been identified. The most widely studied is a non-null mutation of RAD.50 (rad.50.~)(Alani et a/., 1990) that causes mutants to lack both the resection and subsequent repair of' dsDNA breaks. This mutant has been particularly useful for gauging the overall frequency of dsDNA breaks at various hotspots. Other genes whose products are required for the formation or processing of dsDNA breaks include SPOII (Cao et a/., 1990). ME14 (Menees et ( I / . , 1992; Scott Keeney, personal communication, 1997), M E K 2 (Rockmill et a/., 1995), M R E l l (A.jimura rt a/.. 1993; Johzuka and Ogawa. 1005). KEC102, RECI04, and RECl14 (Bullard et a/.. 1996), and XRS2 (Ivanov tit ( I / . . 1992). Intriguingly, in rcid.50~mutants the unprocessed dsDNA breaks accumulate with a protein covalently bound to the 5' ends of the DNA (de Massy et ( I / . , 1995; Keeney and Kleckner. 1995; Lui et NI., 1995). A brute force purification (Keeney et a/., 1997) and a genetic approach (Bergerat et al., 1997) reveled that Spol 1 is the bound protein. The Spol 1 protein has blocks of homology with type 11 topoisomerases (Bergerat etnl., 1997; Keeney et d., 1997). and mutation of a conserved tyrosine residue abolishes dsDNA break formation (Bergerat e t a / . , 1997).These data suggest that the biochemical function of Spol 1 is the same as that of topoisoinerase 11, at least for the strand scission step. Genes homologous to SPOI I have been identified in archeabacteria, fission yeast, and nematodes, suggesting conservation of activity across kingdoms. The fission yeast homolog. recl2, is a meiotically induced gene required for meiotic recombination (DeVeaux et ul., 1992; Lin and Smith, 1994). Although dsDNA breaks have not been reported for hotspots of any organism other than budding yeast, conservation of SPOI I suggests thal dsDNA break-promoted recombination may be evolutionarily conserved.
B. D N A Accessibility within Chromatin
One factor that dictates where Spol 1 protein introduces dsDNA breaks may be the accessibility of DNA. Increased accessibility of DNA within chromatin (as
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Wayne P. Wahls
gauged by increased sensitivity to enzymes such as micrococcal nuclease or DNase I) was first observed in the vicinity of the EP recombination hotspot of the mouse MHC (Shenkar et d., 1991). Chromatin with this increased sensitivity, called open chromatin, is found in the vicinity of all natural and created dsDNA break sites that have been examined (Ohta et id., 1994; Wu and Lichten, 1994; Liu et d . , 199.5: Wu and Lichten, 1995; Fan and Petes, 1996), as well as at recombination hotspots where dsDNA breaks have not yet been reported (Shenkar et a/., 1991; Mizuno et ( I / . , 1996. 1997). The association of recombination hotspot activity with open chromatin and dsDNA breaks in promoter regions is very strong (Lichten and Goldman, 1995). However, there are exceptions. Some hotspots are not in promoter regions (Malone e t a / . , 1992). and hotspot activation can be uncoupled from levels of transcription (Grimm et a/., 1991; Schultes and Szostak, 1991; White et ( I / . , 1992). The position of open chromatin does not always correlate precisely with the patterns of dsDNA break formation (Liu et d . , 199.5; Wu and Lichten, 1995; Fan and Petes, 1996). Furthermore, some regions of increased DNA accessibility within chromatin do not undergo highfrequency dsDNA break [ormation (Wu and Lichten, 1995; Fan and Pete>, 1996). It seems clear that accessibility of DNA within chromatin is not sufficient for hotspot activation. Intriguingly. the magnitude of hotspot activity can depend more on binding of certain transcription factors than on the relative abundance of open chromatin, Replacement of Bas I , Bas2, Gcn4. and Rap 1 transcription factor-binding sites in the HIS4 promoter region with the Rap1 binding site results in very strong dsDNA break formation and hotspot activity (White et ul., 1991, 1993; Fan ef a/., 1995) but not additional increase in DNA accessibility to nucleases. Three possible explanations were considered (Fan and Petes, 1996). First, open chromatin may be necessary and sufficient to confer hotspot activity, but the reagents used to a y DNA accessibility within chromatin do not accurately reveal the chromatin structure. Second, open chromatin structure might be necessary but not sufficient, and some other factor (such as a protein bound nearby) might be required. Third. hotspots might not require open chromatin for action but only a factor that is usually associated with open chromatin. Activation of the (1(le6-M26 hotspot by binding of Mts 1/Mts2 transcription factor supports the latter two possibilities (N. Kon et ( I / . , unpublished observations), particularly since hotspot-dependent, meiotically induced open chroinatin requires the presence of the MtsI/Mts2 heterodimer ( K . Ohta and W. P. Wahls, unpublished observations).
V. Roles of Protein-DNA Binding in Hotspot Activation Presumably, as for ck-acting transcriptional regulatory elements, there are proteins that interact with hotspots to mediate their biological activity. The paradigm is found in E.sc~hrrichiacoli: the RecBCD enzyme interacts with Chi sites to enhance recombination (reviewed by Kowalczykowski et o/., 1994). The cog
2. Recombination Hotspots and Miniwtellite Change
53
hotspot near the his3 locus in Neurosporci crassu is repressed by unlinked, dominant loci (Catchcside and Angel, 1974; Catcheside, 1977). suggesting that trans-acting factors can negatively regulate eukaryotic hotspots. Hotspots in the MHC require distal elements that positively and negatively regulate activity (Shiroishi et al., 1990, 1991, 1993), perhaps due to trans-acting factors that recognize yet-unidentified cis-acting sites. In addition, proteins that interact with the hypervariable minisatellite DNA hotspot have been purified (Wahls et al., 199 I ) , and circumstantial evidence suggests that protein binding is required for activity (W. P. Wahls and P. D. Moore, unpublished observations). An uncomplicated model would propose that discrete DNA-binding proteins are responsible for hotspot activation. Attempts to identify discrete DNA elements that activate hotspots in S. cerevisiar have been largely unsuccessful, which suggested that hotspot activity is not conferred by a single protein binding to a discrete DNA site (Rocco et al., 1992; de Massy and Nicolas. 1993: Coyon and Lichten, 1993). Similarly, comparison of DNA sequences in the vicinity of dsDNA breaks failed to reveal any clear consensus sequences at the site of the breaks (de Massy et ul., 199.5). The prevailing view is that the dsDNA breaks at recombination hotspots are dictated by some feature of the overall chromatin structure (reviewed by Lichten and Goldman, 1995). It is not clear why some regions of open chromatin contain hotspots and others do not, but the answer is likely to be found within the individual protein components of chromatin. For example, proteins binding near yeast hotspots can influence hotspot activity. Full activity of the HIS4 hotspot in S. cerevisiae requires binding of Bas 1 , Bas2, and Rap1 transcription factors to the promoter region, but none of the individual factors seems responsible for complete hotspot activation (White rt ( I / . , 1991. 1993). Deletion of all the binding sites for all these transcription factors abolishes hotspot activity and replacement with two Rapl-binding sites restores high hotspots activity, suggesting that Rapl-protein binding might be sufficient to activate the hotspot (White er id., 1993: Fan et ul., 199.5). The production of dsDNA breaks might be due to direct interaction of meiosis-specific nuclease with one or a few transcription factors that are bound within regions of open chromatin (Fan and Petes. 1996; N. Kon et ul., unpublished observations). Alternatively, the topoisomerase 11-like nuclease that makes the dsDNA break may have a loose consensus site that might be present within open chromatin for cleavage. Thus, other regions of open chromatin that lack the requisite subset ot transcription factors or consensus sequence for the nuclease would not be recombinogenic. The lack of clear consensus sequences at dsDNA break locatiorrs (de Massy rf al., 1995) favors that first possibility. One particularly well-studied eukaryotic recombination hotspot is the M26 hotspot of the fission yeast, S~~Iii,-o.viicc.litiroin~ces pomhe. M26 was first identified as an ude6 mutant that exhibited a high frequency of meiotic recombination within the ade6 gene (Gutz, 1971). The M26 mutation is a single base pair substitution (Ponticelli et nl.. 19x8: Szankaai et al., 1988) that increases meiotic
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Wayne P. Wahls
recombination within ~1de6about 15-fold relative to the nearby control allele, M375 (Gutz, 1971; Schuchert and Kohli, 1988). The hotspot functions during meiosis but not during mitosis (Gutz, 1971: Ponticelli et d., 1988; Schuchert and Kohli, 1988) and exerts its effect in the vicinity of udr6, as well as in nearby intervals (Grimm et a/., 1990), but not at other, more distant or unlinked loci (Ponticelli et id., 1988). M26 stimulates both gene conversion (Gutz, 1971) and reciprocal exchange events (Grimm et a/., 1994) to an approximately equal extent, but during gene conversion there is strong disparity, with the M26 allele being converted to wild type at a rate about 10-fold higher than vice versa [i.e., M26 acts as a recipient of information during gene conversion events (Gutz, 1971)l. Gene conversion events at M26 often coconvert alleles to the left. right, or both directions, and the average conversion tract length is about 700 bp, consistent with the notion that M26 is an initiator of recombination (GutL, 1971; Grimm et a/., 1994). Preferential transfer of the transcribed stand occurs (Schar and Kohli, 1994), suggesting that the mechanism of hotspot activation does not involve extensive resection of both DNA stands from a dsDNA break. These data are consistent with M26 being the site of a ssDNA or dsDNA break that initiates recombination. Other data suggest that recombination may not initiate precisely at ,4426. M26-enhanced recombination events do not always include conversion of the M26 site (Grimm et a/., 1990), and an M26-independent recombination initiation site near the 5’ end of the d e 6 gene has been inferred (Grimrn et a/., 1994). suggesting that M26 may enhance recombination that initiates near M26 but not necessarily within the site itself. Kohli and colleagues used in v i t r o mutagenesis to alter individual base pairs in the vicinity of the M26 site, they introduced the mutations into the ude6 gene, and they then tested the resulting mutants for hotspot activity in viw (Schuchert et a/., 1991). This revealed that a specific 7-bp DNA sequence, 5’-ATGACGT-3’,is absolutely required for activity of the M26 hotspot, suggesting that the M26 mutation might create a binding site for a protein involved in hotspot activation. (Such single base pair changes may be the best way to identify cis-acting sites involved in recombination at other hotspots because more extensive alterations, such as deletions, could perturb a local chromatin conformation that is necessary but not sufficient for hotspot activity. It may therefore be worthwhile using a similar point-mutation approach to search for cis-acting sites near other hotspots.) A heterodimeric protein that binds to the M26 site has been identitied and purified and the binding activity in v i t r o correlates with hotspot activity in vitw (Wahls and Smith, 1994). Binding of the heterodimer is essential for hotspot activation but not for basal recombination levels (N. Kon r t a/., unpublished observations). Intriguingly, this Mts 1 /Mts2 heterodimer is also a putative transcription factor that has roles in sexual development and stress response (Takeda et a/., 1995; Shiozaki and Russell, 1996; Watanabe and Yamamoto, 1996; Wilkinson et al., 1996). However, hotspot activity is not directly related to transcription because there is no detectable difference in the level of ade6 mRNA during
2. Recombination Hotspots and Minisatellite Change
55
meiosis in strains exhibiting normal versus hotspot recombination (Grimm er al., 1991). [Similarly, for hotspots i n S. cere\i.siae, some promoter mutations that alter gene expression do not alter hotspot activity (Schultes and Szostak. 1991; White et a/., 1992)]. Mtsl/Mts2 protein therefore has multiple biological roles: one in hotspot activation, one in regulating genes involved in sexual development, and one i n regulating genes during stress conditions. It is proposed that the Mts 1 /Mts2 heterodimer has evolved separate roles in transcriptional regulation and hotspot activation (N. Kon e/ ul., unpublished observations). In each case, the protein is constitutively present (Wahls and Smith, 1994) but apparently inactive during normal mitotic growth, permitting rapid induction of activity in times of crisis (such as nitrogen starvation, which leads to induction of meiotic differentiation). Activation of the protein involves signal transduction via MAP kinase and CAMP-dependent kinase pathways, and perhaps others (Takeda et a/., 1995; Shiozaki and Russell, 1996; Watanabe and Yamamoto, 1996; Wilkinson e/ d . , 1996; N. Kon and W. P. Wahls, unpublished observations). Although DNA breaks at the M26 hotspot have not been reported, M26 sitedependent, meiotically induced open chromatin is present at the M26 site and in the promoter region (Mizuno et (11.. 1997). A promoter deletion in cis to the M26 hotspot, ending 1 12 bp upstream of the M26 site, abolishes hotspot activity, but a promoter deletion in /runs to M2h has no effect (Zahn-Zabal e/ a/., 1995). Thus, in addition to MtsI/Mts2 binding to the M26 site, some other feature removed by the promoter deletion is required lor hotspot activation. Remodeling of meiotic chromatin at M26 requires MtsI/Mts2 protein (K. Ohta and W. P. Wahls, unpublished observations), suggesting that Mts l/Mts2 protein binding, possibly interacting with a factor bound i n the promoter region, is responsible for the open chromatin structure and hotspot activation. It remains to be seen whether Mts 1 /Mts2 recruits recombination enzymes via protein-protein interactions or whether recombination enzymes assemble at chromatin conformationally altered by Mtsl/Mts2 binding to M26 DNA. In either case, binding of MtsI/Mts2 to M26 sites increases recombination above basal levels, suggesting that the hotspot with its binding protein helps regulate where and when recombination occurs within the genome. The M26 heptamer is about fivefold underrepresented in the S. pornbe genome. If each naturally occurring M26 site is, on average, as active in promoting recombination as the M26 site in the udr6 gene (Gutz, 1971; Schuchert and Kohli, 1988), then about 50% of all meiotic recombination events could be due to Mtsl/Mts2 heterodimer binding those sites (Wahls and Smith, 1994). This proposal, although speculative, is testable. So far no study has reported whether or not any other naturally occurring M26 sites exhibit hotspot activity. However, intergenic recombination is reduced in wits mutants (N. Kon and W. P. Wahls, unpublished observations) and the M26 heptamer has hotspot activity when created at any of five locations in udeh and uru4 (M. E. Fox, J. B. Virgin, J . Metzger, and G. R. Smith, personal communication). These findings support the
56
Wayne P. Wahls
view that natural M26 \ites are active in meiojis and contribute to elevated meiotic recombination levels throughout the genome.
VI. Control of Recombination in cis and in trans, Near and Far The control of recombination likely involves a complex network of structural an enzymatic machinery. Hotspots act locally to enhance recombination events within a few tens of thousands of base pairs of their location. As discussed in the previous section, hotspots can be regulated positively and negatively by both ci.7- and trurzs-acting factors. On a wider scale, the timing and distribution of recombination are also regulated. Two examples of this control are provided here. On the chromosome-wide scale, the distribution of recombination events is nonrandom. It has long been recognized that chiasmata (the cytological structures thought to represent points of reciprocal exchange between chromosomes) are not randomly distributed (reviewed by Maguire, 1995). Individual chromosomes have some means of ensuring that at least one, but not many, chiasmata occur on each chromosome arm. That regulation is likely conferred (at least in part) by structural proteins of the synaptonemal complex (Sym and Roeder, 1994: Kleckner, 1996). The presence of chiasmata usually correlates with appropriate segregation of chromosomes. This correlation is supported by genetic observations in yeast where smaller chromosomes have higher rates of recombination than larger chromosomes, when adjusted for the physical size of the chromosomes (Kaback Pf al., 1992). Thus, recombination rates and distributions are controlled relative to individual chromosomes. In the fission yeast S. pomhe 18 complementation groups of mutants affecting meiotic recombination have been identified (Schmidt er NI., 1987; Ponticelli and Smith, 1989; DeVeaux et 01.. 1992; Tavassoli ef al., 1995 ). Three of the mutants, re&. rec.10, and r e c l l , profoundly reduce recombination over all tested intervals on chromosome 111, but the leave recombination essentially normal on the other two chromosomes (DeVeaux and Smith, 1994). Because these genes are recessive and are located on three different chromosomes, they must encode frcrn.sactivators of recombination that recognize specific cis-acting determinants of chromosome Ill. The gene products are likely involved either in the initial pairing of chromosome 111, which is required for meiotic recombination, or in chromosome 111-specific recombination. perhaps by recognizing DNA sequences such as recombination hotspots that are preferentially located on chromosome 111. Additional examples of genome-wide regulation of recombination may be found in a recent review article by Lichten and Goldinan (1995).
2. Recombination Hotspots and Minijdtellile Change
57
VII. Hotspots as Initiators or Resolvers of Recombination: Two Models A. Recombination Hotspots Enhancing an Early Step in Recombination: Convergence of Genetics and Biochemistry
The dsDNA break model of recombination (Kesnick, 1976: Szostak r t d . ,1983) can explain most recombination i n budding yeast, and this pathway is likely conserved in other eukaryotes (Hergerat et ul., 1997; Keeney Pt nl., 1997). The modified model in Fig. 3 incorporates resection of the 5' terminated strands at dsDNA breaks (Sun r t al., 1989: Cao r t d., 1990; Sun et d., 1991), a role for DNA accessibility within chromatin (Shenkar et d., 1991; Ohta et ( I / . , 1994; Wu and Lichten, 1994, 1995), a reciuirement for specific transcription factors but not for transcription (Fan and Pete\, 1996; N. Kon et ul., unpublished observations), and the recently discovered biochemical activity of Spol 1 protein as the nuclease that forms dsDNA breaks (Bergerat et ~ i / .1997: , Keeney r t d . , 1997). In step 1 in Fig. 3, chromowmes have associated DNA-binding proteins involved in gene expression and a characteristic mitotic chromatin configuration. For example, the Mtsl /Mts2 hotspot-activating protein of fission yeast is uniformly expressed and has equivalent DNA-binding activity in mitotic and meiotic cells (Wahls and Smith, 1994). presumably acting as a transcription factor (Takeda rf a/., 1995; Watanabe and Yamamoto, 1996), but the M 2 6 hotspot functions only during meiosis (Glitz, 1971; Schuchert and Kohli, 1988; Ponticelli and Smith, 1989). Similarly, multifunctional, constitutively expressed RAP1 protein of budding yeast activates a meiotic hotspot (White et d., 1991, 1993). These observations suggest that the hotspot-activating proteins are always interacting with their DNA sites, but during meiosis some change in these proteins (or their environment) triggers recombinogenic activity. During meiosis, as in step 2. posttranslational modification of the DNAbinding proteins occurs. It is proposed that the machinery of eukaryotic recombination has evolved to use some transcription factors for induction of meiotic recombination at hotspots (N. Kon et id., unpublished observations). Two transcription factors are known t o fully activate meiotic recombination hotspots (White et NI., 1993; Wahls and Smith, 1994; N. Kon et al., unpublished observations): however, recombination-enhancing and transcription-regulating activities are separable (Grimm et a/., 199l; Schultes and Szostak, 1991; White et d . , 1992). The protein changes involve signal transduction via MAP kinase and CAMP-dependent kinase pathways, and perhaps others (Shiozaki and Russell, 1996; Wilkinson et al., 1996). Alteration of the DNA-binding proteins either directly or indirectly results in a conformational change in the surrounding chromatin, making the DNA more accessible to nuclease digestion (Shenkar et al., 1991; Ohta et a/., 1994; Wu and Lichten, 1994, 1995; Mizuno et a/.,1997).
58
Wayne P. Wahls
3'
~
a &. t
(9 W
Without Reciprocal Exchange
t
..
OR
5'
( 6 L ~
3'
m W
With Reciprocal Exchange
Fig. 3 A dsDNA break repair model of recombination. 1. Mitotic chrotnosoniea with xsociatcd DNA binding protein5 (oval\). 2. During meiosis, the binding proteins are altered by posttranslational modification or by intcraction with meiotic facton to generate chromatin in which the DNA becomes more accessible. 3. Spol I protein (solid circle) assembles at the site, either via protein-protein interactions or by recognizing an altered chromatin conformation at the hotspot. 4. A topoi\ornerase
2. Recombination Hotspots and Minisatellite Change
59
Current evidence is consistent with open chromatin being either the cause or the effect of hotspot activation. In step 3, the meiosis-specific, topoisomerase I1 homolog Spol 1 arrives at the hotspot and introduces a dsDNA break (Bergerat et ul., 1997; Keeney et al., 1997). It remains to be seen whether Spol 1 is recruited directly via proteinprotein interactions with the transcription factors bound at the hotspot or whether Spol 1 assembles at chromatin that is conformationally altered by the hotspotbinding proteins. In either case, in step 4, two subunits of Spol1 cleave the DNA by transesterase reactions in which active site tyrosines make closely spaced ssDNA nicks and concomitantly form phosphodiester bonds with the 5’ termini of the broken DNA (Bergerat P t ul., 1997; Keeney et ul., 1997). Because the energy of strand scission is retained in the phosphotyrosine linkage, the dsDNA break could be religated during protein release. [Genetic evidence suggests this may occur: a high frequency of &DNA breaks created by insertion of a RAPlbinding site within HIS4 does not have genetic hotspot activity when cis-linked to a natural hotspot located nearly, presumably because of competition between the two sites (Fan et al., 1997)l. Alternatively, the covalent intermediate is further processed in the recombinant pathway. Release of the covalently bound protein likely occurs by one of two mechanisms: Hydrolysis of the phosphodiester bond (step 5 ) releases intact Spol 1 monomer and leaves behind a dsDNA break that is subsequently processed by exonucleolytic resection ofthe 5‘ rerminated strand (Sun et al., 1989; Cao et d., 1990; Sun et d., 1991). Alternatively, Spoll is released by endonucleolytic cleavage of one DNA strand, in (,isto the bound protein, that produces a variable length ssDNA fragment that remains covalently attached to the released Spol 1 monomer (step 5’). Genetic and biochemical studies suggest that these reactions are catalyzed by some combination of the Rad50, Mrel 1 , and Xrs2 protcins. A more detailed discussion of this possibility is available (Keeney et d . , 1997). The remaining steps ofthis model are as previously proposed (Szostak rt ul., 1983) and are only briefly outlined here. For the sake of clarity, only two of the
11-like, dsDNA cleavage reaction by SpoI 1 piutein produces covalent protein-DNA intei-mediates with phosphodiester linkages between acti\e site tyrosines and the 5’ terminal phosphate groups of the broken strands. 5. Release of protein i \ achieved by hydrolysis of the phosphotyrosine Iinlage or by nuclcolytic cleavage of the covalently hound strand (step S‘). Exonucleolytic resection (dotted line) generates free, 3’ asDNA tails that ciiii invade the homologous dsDNA to produce heteroduplex DNA. 6. The invading strand is extended by DNA synthesis (arrow) to produce a displacement loop. 7. Annealing of the remaining free 3’ strand to the displacement loop and further DNA synthesis fill\ the existing gaps. 8. Ligation of DNA strands results in chromosomes attached by two Holliday junctions. Branch migration of the junction\ may occur (not shown). The junctions are resolved by cleavage of two DNA strands. Depending on which strands are clcavcd, the resulting chroiiio\omes either lack reciprocal exchange of Hanking markers (step 9) or have reciprocal exchange (\tep 9’). Additional details and reference\ are provided in the text.
60
Wayne P. Wahls
four chromatids are shown. One of the free, 3’-terminated, ssDNA ends at the dsDNA break invades the homologous chromosome to form heteroduplex DNA (step 6). DNA synthesis extrudes a displacement loop that can serve as a template for DNA synthesis primed by the remaining 3’ ssDNA tail (step 7). Ligation of DNA nicks (step 8) and cleavage of the resulting Holliday junctions results in recombinant chromosomes without (9) or with (9’) reciprocal exchange of the flanking markers.
B. Recombination Hotspots Enhancing a late Step in Recombination: Hypervariable Minisatellite DNA as a Site of Resolution of Holliday junctions
Recombination hotspots enhance some rate-limiting step i n the biochemical pathway of recombination. The prevailing view, as exemplified for recombination via dsDNA break repair in S. cerevisiae, is that the rate-limiting step is early, presumably acting as the initiating step. However, as with other biochemical pathways, the rate-limiting step need not be early. I propose that some eukaryotic recombination hotspots act late, as resolvers of recombination intermediates. This model can be summarized by the following hypothesis: Hypervariable minisatellite DNA is a site of resolution of Holliday junctions. 1 initially proposed this model at the I 1th International Chromosome Conference (Edinburgh, 1992) to account for the observation of polarized changes at minisatellite DNA, presented at the meeting (Armour, Harris, et al., 1993), and the observation that minisatellites enhance recombination and have an additive influence when dsDNA breaks are introduced into the substrates (Wahls et al., 1990a). A number of findings since then further support the model shown in Fig. 4. New minisatellite alleles can arise at some loci in as many as 10%of meioses (Buard and Vergnaud, 1994) and alleles often exhibit polarized variability (Armour, Harris et al., 1993; Armour, Monckton, et al., 1993; Buard and Vergnaud, 1994; Ellsworth et al., 1995). The polarized variability is shown schematically in Fig. 4A. Since reciprocal exchange of markers flanking minisatellite loci is seldom observed (Wolff et al., 1989; Kasperczyk et al., 1990; Kelly et al., 1991; Buard and Vergnaud, 1994), most models for minisatellite change have discounted recombination in favor of other, more complex mechanisms. The model proposed in Fig. 4B can account for all of the change via meiotic recombination and associated heteroduplex DNA repair. In step 1, recombination initiates at some point distal to the minisatellire array but proximal to the nearest flanking marker, the “Y” locus. For the sake ofclarity, only two of the four chromatids are shown. Minisatellite enhanced recombination can have at least one point in a distant interval (Wahls et al., 1990a), and allelic changes at minisatellite arrays can be strongly influenced by cis-acting sites outside of the array (Monckton et id., 1994; Andreassen et al., 1996). The
2. Recombination Hotspots and Miniwtellite Change
61
initiating lesion may be promoted by minisatellite DNA-binding proteins (Wahls et al., 1991), or it may involve other cis- and tmns-acting factors such as those
inferred for hotspots in the mouse MHC (Shiroishi et al., 1991, 1995). Open chromatin has been found near the minisatellite DNA in the EP hotspot in the mouse MHC (Shenkar et d., l99l), suggesting that the initiating event is analogous to those at hotspots in yeast. Symmetrical ssDNA nicks are shown, as in the Holliday model of recombination (Holliday, 1964); however, other types of initiating events (Meselson and Radding, 1975; Szostak et d., 1983) will work equally well as long as they lead to formation of at least one Holliday junction betwcen the recombining chromosomes. In step 2, the recombining chrmnosoines have exchanged single strands to form a Holliday junction and branch migration has moved the junction halfway to the minisatellite array. On naked DNA a random walk can result in the junction moving in either direction. I n E. c d i the RuvAB heteromultimer catalyzes unidirectional migration of the junction (reviewed by Muller and West, 1994) and presumably eukaryotic counterparts carry out the same process. The extent of branch migration is dictated by the processivity of the enzyme. Branch migration might be reversed by dissociation and reassociation of the machinery in the opposite orientation, which would lead to “aborted” recombination events. Thus, in the absence of resolution. the Holliday junction is removed by reverse migration or it is pushed farther along the recombining chromosomes until it encounters a signal for resolution, the minisatellite DNA. In step 3, branch migration has generated heteroduplex DNA that extends from the flanking region into the minisatellite array. Some component within each minisatellite DNA repeat acts as a signal for resolution of the Holliday junction. The signal is quite strong but not absolute, because polarized variability can occur within a few tens of repeats from the end of the minisatellite array (Armour, Harris, et ul., 1993). I calculate that there is a probability of between 5 and 10% that a Holliday junction that is migrating past a minisatellite repeat unit will be resolved at that site. Thus, about 50% of resolutions will occur within the first 10 repeats, about 75% within the first 20 repeats, and so on, reflecting the distribution of polarized changes that has been described. This proposal is considerably strengthened by recent lindings of how the E. coli Holliday junctionresolving enzyme, RuvC, functions. RuvC preferentially resolves Holliday junctions at a consensus sequence in an orientation-dependent fashion, leading to preferential cleavage in one of two possible directions (Shah el al., 1994). That also supports the next step of the model, which is that resolution of the Holliday junction in the “patch” configuration (strand nicks at horizontal arrows) occurs with higher frequency than resolution in the “splice” configuration (strand nicks at vertical arrows). In step 4, the major pathway by which the junction is resolved, cleavage of two strands in the horizontal orientation allows separation of the recombining chromatids. There is no reciprocal exchange of the flanking markers distal to the
Wayne P. W a h l s
62 Parental 3 tl CI a a
A
II
tl
tl
ti
a
a
Parental New allele3
a
H ti i l tl Unchanged
, Polarized Variability I
X
Y
Fig. 4 A model for reconlbination hotspot activity and the fortnation of polariLed variuhility at hypervariable minisatellite DNA. ( A ) Schematic representation of polarized variability. Individual minisatellite repeats (boxes) can sometimes be distinguished by minor hequence difference.. within each repeat (black or white). Arising alleles are uniform acre\\ most ol the locus and then become mosaic toward one end of the locus. ( B ) Modcl lor recombination hotspot activity and generation of
2. Recombination Hotspots and Minisatellite Change
63
minisatellite locus. A “patch” of heteroduplex DNA extends from the point of recombination initiation to the point of resolution within the minisatellite array. When enhancing recombination between plasmids, minisatellite DNA preferentially increases recombination events accompanied by reciprocal exchange (Wahls et ul., 1990a). In the model, preferential increase of nonreciprocal resolution is proposed. The factors that dictate resolution in this orientation are unknown, but they may be contained within the minisatellite repeat itself or they may be conferred by the higher-order structure of the recombining chromosomes. In yeast, proteins that are central components of the synaptonemal complex, such as Zip 1, clearly influence thc outcome of recombination, but not necessarily its frequency (Sym et al., 1993). Yeast with the zip1 mutation have nearly normal recombination frequencies, but they no longer exhibit crossover interference (Sym and Roeder, 1994). A recent model proposes that the physical constraints of the synaptonemal complex, specifically the tension created by initial steps of recombination within the semirigid structure, lead to reciprocal exchanges that alleviate the built-up tension (Kleckner, 1996). Similar forces could be operating during recombination at the niiiiisatellite loci. Resolution of the Holliday junction, signaled by the minisatellite DNA and catalyzed by interacting proteins, might be influenced by constraints of the surrounding chromatin. In step 5 , the resulting heteroduplex DNA is corrected by mismatch correction. The best described mismatch repair systems in mammalian cells have very short (a few nucleotides) excision tracts, but there is evidence for long patch repair (reviewed by Friedberg et a/., 1995). Heteroduplex DNA that contains mismatches within the minisatellite loci is repaired by the short patch mismatch repair machinery. Each minisatellite repeat is corrected individually to excise mismatched nucleotides on the upper strand (upward-pointing arrows) or on the lower strand (downward-pointing arrows). This would generate the polarized variability. The observation that hypervariable minisatellite DNA undergoes a high rate of repair in mammalian meiosis (Ramachandra and Rao, 1994) supports the model. Furthermore, there i s genetic evidence for heteroduplex DNA at minisatellites during meiosis: the rate of meiotic change of minisatellites is high
polariied variability. I . Initiating cwiil leads t o strand exchange and generation of a Holliday junction. 2. Branch migration ofthe Holliday junctioii. 3. An element within each minisatellite DNA repeat provides the signal to resolve the Holliday junction. Recolution can occur by the major pathway (nick strands at horizontal arrow\) or by the minor pathway (nick strands at vertical arrows). 4. Rewlution by the major pathway yields recombinants with heteroduplex DNA and no reciprocal exchange of flanking markers. 5. Short patch mismatch repair (up and down arrows) act5 indepcndently on each repeat to generate polari/cd vai-iability. Loci with incomplete mismatch repair will exhibit postmeiotic segregation (not shown). 3 ’ . Resolution hy the minor pathway yields recombinants with heteroduplex DNA and reciprocal exchange of flanking markers. S ’ . Short patch mismatch repair of the minor product also generate\ polarized variability. Additional details and references are provided i n the text.
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Wayne P. Wahls
(Buard and Vergnaud, 1994), but the rate of change in mitosis is very low. Intriguingly, “somatic mutation during early development” has been reported (Kelly et d . ,1989; Gibbs et al., 1993). The H n - 2 locus experience a high rate of allelic change per meiosis and, surprisingly, about 20%)of mice with new vanants of the Hm-2 locus are mosaic. Based on the pattern of mosaicism. it was concluded that somatic mutant alleles arise within the first two cell divisions after fertilization (Kelly et ol., 1989; Gibbs et ( i l . , 1993). I suggest that the mosaicism is actually due to PMS of uncorrected heteroduplex DNA that is formed during meiotic recombination. Honiotypic variants arise from recombination events with complete mismatch correction of heteroduplex DNA. The remaining new variants undergo partial repair of heteroduplex DNA and produce PMS that, on fertilization, generates mosaic conceptuses, further supporting a role for short patch mismatch correction. In step 4’, the minor pathway by which the junction is resolved, cleavage of two strands in the vertical orientation allows separation of the recotnbining chromatids. This results i n a recombination event in which there is reciprocal exchange of the markers flanking the minisatellite locus. In step 5’. the processing of the heteroduplex DNA is exactly as for step 4. Tracts of short patch mismatch repair can independently correct each minisatellite repeat, with a random probability that either the top or the bottom strand will be excised and replaced at each repeat. As with the nonreciprocal recombinants, the reciprocal recombinants will have polarized variability. The model in Fig. 4 is drawn with an equal number of repeats in each allele for the sake of clarity. Individual alleles often have different numbers of repeats, so what is the consequence of recornbination between two alleles of different lengths‘? If the flanking sequences to the right of the minisatellite array are identical for both alleles (as in recombination between sister chromatids), then the Holliday junction would encounter minisatellite DNA simultaneously on both strands. Recombination would proceed as in Fig. 4 and the recombinant products would exhibit polarized variability for any internal allelic differences in the parental molecules, but no length change. These recombination events would be invisible to typical experimental approaches that score minisatellite change based on the appearance of new length alleles. If the allele lengths are different or the neighboring sequences are different (e.g., one allele extends farther into the adjacent DNA than the other), then strand exchange would likely bypass the heterology and branch migration would continue into the minisatellite array before encountering the signal to resolve the Holliday junction. The resulting heteroduplex DNA would be the same as shown in Fig. 4 except the right end would have additional minisatellite DNA repeats in one strand opposite a strand of nonminisatellite DNA (a loop opposite a shorter opposing strand could also form). Mismatch repair could replace either strand. Thus, individual DNA strands within the heteroduplex DNA at the minisatellite array may become longer, shorter, or stay the same length, depending on the initial minisatellite lengths and the direction of the mismatch correction. This
2. Recombination Hotspots and Minisatellite Change
65
mechanism can therefore generate new length alleles as well as account for polarized variability. However, it depends upon preexisting length polymorphism within the population. How is the length polymorphism generated? Most models of minisatellite length change invoke a coinbination of duplications and deletions involving both inter- and intrachromosomal interactions (Buard and Vergnaud, 1994) and additional independent mutations leading to polarized variability (Ellsworth r t ul.. 199.5). Length changes arc sometimes attributed to DNA polymerasc slippage on the template strand during DNA replication followed by failure of mismatch repair, as is the case for expansion and contraction of dinucleotide repeats (Strand et ml., 1993; Farber ef a/., 1994: Heale and Petes, 1995; Kolodner, 199.5). This seems unlikely for minisatellites because they are relatively stable niitotically and they undergo a high rate of length change during meiosis. There is no evidence for reduced fidelity of replicative DNA polymerases during meiosis, or any reason to believe that mismatch repair would be compromised. 1 propose that some length changes are due to DNA polymerase slippage on the template strand, but these events do not use the replicative DNA polymerases. Rather. the strand slippage probably occurs when repair-specific DNA polymerases help correct heteroduplex DNA present after resolution of recombination within minisatellite loci. The high rate of DNA repair at minisatellite DNA i n mammalian meiosis, which seeins to use DNA polymerase f3 (Ramachandra and Rao, 1994). supports this model. In conclusion, most of the complex changes that occur at hypervariable minisatellite DNA may be due to minisatellites acting as sites where Holliday junctions are resolved. The polarity oi' change, the variability of change, the changes previously attributed to somatic mutation during early development, and allele expansion and contraction can all be accounted for by a model invoking meiotic homologous recombination followed by heteroduplex DNA repair. This suggests that rate-limiting steps can occur late in the pathway of recombination as well as early. Although primarily a meiotic event, the process of minisatellite recombination might also occur between Yister chromatids or homologous chromosomes during mitotic growth. This would provide a mechanism for recombinational repair of DNA damage that preferentially uses gene conversion. If so, it would also provide a mechanism for some aberrant recombination events. For example, several independent translocation breakpoints at c - m y and Oc12 occurred precisely at minisatellite consensus sequences (Krowczynska et nl., 1990), strongly suggesting that the aberrant recombination was resolved (or initiated) at the minisatellite DNA.
VIII. Summary Recombination is increased up to 1000-fold during meiosis in order to promote accurate segregation of homologous chromosomes and to enhance genetic diversity. Part of this induction is due to recombination hotspots and their interacting
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Wayne P. Wahls
factors. For at least one eukaryotic hotspot a discrete DNA sequence and interacting protein are required for activity (Schuchert et Nl., 1991; N. Kon et al., unpublished observations), and for another hotspot a discrete DNA site (presuniably binding a specific protein) is required (White et al., 1993; Fan et al.. 1995). For other hotspots no single, discrete activating factors have been identified, but this could be due to redundancy of sites clustered near hotspots or limitations of the experimental approaches used. It remains to be seen whether hotspot activation is regulated specifically and independently, or whether it is a consequence of other biological processes such as transcription. Yeast are genetically parsimonious eukaryotes that have evolved to the point of having about a 50% gene density. Meiotic recombination initiates at dsDNA breaks that appear in regions of increased accessibility of DNA within chromatin. Although hotspots are usually found in the vicinity of transcriptional promoters, the emerging view is that recombination is not directly linked to transcription. Rather, it seems likely that some component of the transcription machinery, most likely one or a subset of transcription factors, has evolved dual roles in transcription and recombination (Fan and Petes, 1996; N. Kon et ul., unpublished observations). It remains to be seen whether hotspot-binding proteins directly recruit recombination enzymes via protein-protein interactions or whether recombination enzymes assemble at conformational changes in chromatin conferred by hotspot binding proteins. The recent discovery of' a topoisomerase 11-like nucleus that makes the dsDNA break (Bergerat et al., 1997; Keeney et ul., 1997), and conservation of this protein between distant species, suggests that the initiation of recombination via dsDNA breaks is evolutionarily conserved. Identification of the factors that direct this nuclease to recombination hotspots and elucidation of the biochemical steps following strand scission are reasonable near-term objectives. In the longer term, study of yeast hotspots will reveal other biochemical activities in the pathway of recombination and yield information about whether recombination throughout the genome is regulated predominantly by hotspots. Mammals, in contrast to experimental organisms such as yeast, have a very low gene density, an abundance of noncoding DNA, and a life cycle that makes genetic experimentation time-consuming and expensive. Despite the challenges of conducting such experiments, compelling evidence suggests cis- and trunsacting regulation of recombination at discrete sites in the mammalian genome, recombination hotspots. Furthermore, it seems that the fundamental mechanism of meiotic recombination in all metazoans is similar to that in yeast. Recornbination probably initiates at dsDNA breaks and the basic molecular mechanisms are likely conserved. It is proposed that one type of noncoding, repetitive DNA found in metazoans, hypervariable minisatellite DNA, acts late in the pathway of meiotic recombination as a signal to resolve Holliday junctions. It is tempting to speculate that other types of noncoding DNA in metazoan genomes have additional roles in regulating the pairing, recombination, and segregation of meiotic chromosomes.
2 Recombination Hotspots and Mini\,tteIlite Chdnge
67
Acknowledgments I thank Bernard de Massy, Scott Kccnc), Nancy Klechner, Mary Fox, Gerry Smith, and Toni Pctcs for sharing results prior to publication. 1 d \ o thank my readers and colleague\ for constructive comments, and Mary Ann Handel for hei- hard work and patience in putting together this volume. This work was supported by the Vanderbilt Llniversit) Department of Biochemistry and a grant from the National Institute\ of Health. W.P.W. I\ ii Leuhemin Society of America Special Fellow.
References Ajirnui-a. M., Lcern, S. H.. and Ogawa. I-I 11993). Identification of new gene.; required for meiotic recombination i n Sacc.ltoror,i?.c.r, rrwiticir.. G r w r i c s 133, 5 1-66. Alani, E., Padrnore, R., and Kleckner, N. ( 1990). Analysis o f wild-type and rod.750 mutant\ of yeast suggcsts an intimate relationship between meiotic chromosome synapsis and reconibination. C d l 61, 419-436. Andenson, L., Lundcn, A,, Sigurdardottii. S.. Davies, C. J . , and Rask, L. (19x8). Linkage relationships in the bovine MHC region: High reconibination frequency between regions. /rirrrnrr,ogrrrutic.s 27, 273-2x0 Andreaswi, R., Egeland. T.. and Olaiwii. B. ( 1996). Mutation rate in the hypervariable VNTR g3 (D7S22) i\ atfectcd by allele length and ii flanhing DNA sequence polymorphism near the repeat ar-ray. Am. J . Hirru. Gwet. 59, 360-367. Armour, J A,, Harris. P. C., and Jelftey\. A . J . (1993). Allelic divenity at minisatellite MS20.5 (D16S309): Evidence for polarized \ari:ihility. H i m M o l . G r ~ i r t2, . 1137-1 15.5. Armour, J. A. I-., Monckton, D. G., Neil. D. I... Ci-o\ier. M.. T m a k a , K., Maclcod, A,. and Jeffreys, A . J . ( 1993). Minisatellite iiiutatioii and recombination. /ti "Chromosomes Today" ( A . T. Summer and A . C. Chandle). Ld\.). pp. 337-3.50. Chapman & Hall. London. Bell, G . I., Sclby, M. J., and Rutter. W. J ( 19x2). The highly polymorphic region near the human insulin gene i s composed of \iniplc t,iiidenilq repeating sequences. Ncittrr-e 295, 3 1-35, Benson, E 1..Sta\iah. A,, and West. S . C. I 1994). Pui-ification and characteriiation of the hunian RadSl protein. an analoguc of E. c o l i RccA. E M H O J . 13, S764-5771 Bergem, A,, de Massy, B., Gadelle. 11.. Varoutas. P.-C., Nicolas, A,, and Forterre, P. (1997). An atypical topoisoincra\e I1 from archnc;i with implication for meiotic recombination. Nciritrr 386, 414-417. Bishop, D. K. ( 1994). RecA hornologs Dmc I and RadS I interact to form multiple nuclear c o n plewes prior to meiotic chrorncxmie \ynap\is. Cell 79, 108 1 - 1092. Bishop, D. K., Park, D.. Xu, L., and Kleckner. N . ( 1992). DMCI: A nieiosis-\pecific yeast homolog o f E. coli recA required for recoiiibinatiun, synaptoncmal complex formation. and cell cycle pi-ogrc\aion. Cell 69, 439-456. Blaho, J. A,. and Wells. R. D. (1987). Left-handed Z-DNA binding by the recA protein of Escherichia coli lpuhlished erratum appear\ i n .J. H i o l . C/wui. 1988, 263, I I O I S ] . J . Riol. Chrrn. 262, 6082-6088. Bryda. E. C., DePari, J . A,, Sant'Angelo. D. B.. Murphy, D. B., and Passmore, H . C. (1992). Multiple sites of crossing over w i t h i n the Ef3 recombinational hotspot in the mouse. M c itwm Gerforfle 2, 123- 129. Buard. J . . and Vergnaud, G. ( 1994). Coinplex recombination events at the hypermutable m i n satellite CEBl (D2S90). EMRO ./. 13, 3203-3210. Bullard, S. A,, Kim, S., Galbraith, A . M.. and Malone, R. E. (1996). Double strand breaks at the HIS2 recombination hotspot in S t r c , [ . h r i ~ ~ , r f i \ t . L . .c.rrn.ic.itrr. (. P roc. Null. A u l d . .%i. US4 93, 130.54- 130.59
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Shenkai-.R.. Shen, M . H., and Arnhcini. N . ( 1901 1. DNase I-hypcr\cnsitive \ite\ and tran\cription Pactor-binding motif\ within the tiiotiw t hcr;i meiotic recombination hot.;pot. M ol. Ccll. Rrol. 11, 1813-1819. Shindct-. G. A,. Manam. S., Ixdwith, 13. J . aiid Nichol\, W. W. (1994). Minisatellite DNA-hinding proteins i n mouse brain, liver, and kidney. E l / > . C'<,// Ras. 213, 107-1 12. Shinohara, A,. Ogawa, H., and Ogaw~i,'1. (IO92). KadSI protein involved in repair and recoiiibinntioii in S. c.rrc.iisitrc, i \ a RecA-lik p t o t c in [puhlished erratum appears in Cell 1992, 71, following p. I XOl. Cell 69, 457-470. Shioraki. K., and Kus\ell, P. (1996). Conlugation. meiosis, and the osmotic \II-CSS rcspon\c arc regulated by Spcl kinase through Attl trnn\criptioii factor i n fi\\ion yea\[. Gews /lei,. 10, 2276-2228. Shiroishi. T.. Haniawa, N., Sagai. T.. I\hiura. M., Go,iohori, T.. Steinmeti, M., and Moriivaki, K. ( 1990). Recornbinational hotspot spccilic to lenialc meiosis in the mouse major histoconpatihility complex. /i,i,,,ir/io,~e,irt,c..\31, 70-XX. Shiroi\hi. T., Koide, T., Yo\hino, M., S a p . T., :id Moriwnki, K . (1995). Hotspots of honiologous recombination i n mou\e iiieio\i\, A d i , . Bi(ip/ty\. 31, I 19- 132. Shiroi\hi. T., Sagai, T., Haniawa. N., Giitoh. 11.. and Moriwaki. K. (1991). Genetic control of sex-dependent meiotic recombination i n the niajor histocompatibility complex of the iiiouw. EMHO J . 10, 6 8 I -686. Shiroihhi, T., Sagai, T., and Moriwahi, K (IVX2). A new wild-derived H-2 haplotype enhancing K-IA recombination. Ntiriire 300, 370-372. -. ( 1993). Hotspots of- meiotic tecomhinatioii i n the niou\e majoi- histocornpatihilit) conplcu. Gerirtic,o 88, 187- 196. Singleton, C. K.. Kly\ik, J . . Stirdivatit, S. M . . and Wclls, R . D. (1982). Left-handed Z-DNA is induced by supercoiling in phy\iological ionic conditions. Nritirr-r 299, 3 12-2 16. Smith, G. R. ( I991). Conjugational reconihtii;ition in E. cvdi: Myth\ and mcchnnisin\. Cdl\ 64. 19-27. Super. L A,. Stein. M. H.. and P a s m o r c . H. C. (1988). The E beta hot\pot of recombination i n uild-deti\ed natural recomhinnnt hlH(' haplotype\. Cross-over site mapping and the identificat i o n o f a I .O-kh E beta deletion in the p and u I4 hoplotypes. J . I n w i i o i o l . 140, 984-990. Southern. P. J., and Berg. P. ( 1982). Tr~iii\t~)riii~ilioii 0 1 manimalian cells 10 mtibiotic resi\Liince with a bacterial gene under conti-cil 01 SV40 cai-ly I-egion promoter. J . M d . A p p l . Gericr. I ,
327-341. Steinmeti, M., Stephnn, D., and Fischei 12indahl. K. ( 1986). Gene organitation and rccomhinatioiial hotspot\ in the tiiui-iiie malor lii\tocoiirpntibility complex. Ccll 44, 895-903. Strand. M.. Prolla. T. A.. Liskay, R M.. 'ind Pete\, T. D. (1993). Destahiliiation of tracts of simple repetitive DNA in yea51 by n i i i t i i l i o i i \ allecting DNA mismatch repair [publi\hed erratum appears in N c i f i i l p 1994, 368, 5691. Nritrrr-c, 365, 274-276. Stringer, J . R. ( 1985). Rcconibination between poly[d(CT),d(CA)]sequences in 5imian vir~i\40infected cultured cell\. Mol. Cell. R,o/. 5. 1247- 12.59. Suhramani, S., and Seaton. B. I.. ( 19x8). Hoinoltigous recombination i n mitotically dividing trimiiiiili:in cell\. It7 "Genetic Recomhin;ilion" IR. Kucherlapati and C. R. Smith. Eds.). pp 549573. American Society l o r Microbiology, Wa\hington. D.C. Subrarnani. S.. and Southern. P. J. ( 19x31.An;ilyhi\ of gene expression using riiniiin \ i r u \ 40 vector\. A w l . Hioc.hn,r. 135, I - I S . Sun, H.. Treco, D., Schultes, N.P., and Smstah. J . W ( 1989). Douhlc-strand breaks at an initiation site for meiotic gene conversion M,rrrw 338, 87-90, Sun. ti.. Treco, D.. and Sm\tak, J. W ( IU9l ). I;xtcn\ivc 3'-overhanging. \ingle-\tranded DNA ;I\sociated uith the meio\is-specitic douhle-striiiid breaks at the ARC4 recoinbination initiation site. Cell 64, 1155-1 161. Sym. M.. Engehrecht, J . A,, and Roeder. C . S ( 1993). ZIP1 is a \ynaptonem;rl complex protein rcquii-ed tor meiotic chromosome \>n;ip\is Cell 72, 365-378.
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Sym, M., and Rceder. G. S. (1994). Crossover interference is abolished in the absence of a synaptonemal complex protein. Cell 79, 283-292. Szankasi. P., Heyer. W. D.. Schuchert. P., and Kohli. J. (1988). DNA sequence analysis of the ade6 gene of Schizosacchammyces pombe: Wild-type and mutant alleles including the recombination host spot allele ade6-M26. J. Mol. Biol. 204, 917-925. Szankasi, P., and Smith, G. R. (1992). A DNA exonuclease induced during meiosis of Schizosacchammyces pornbe. J . Biol. Chem. 267, 3014-3023. Szankasi. P.. and Smith, G. R. (1995). A role for exonuclease 1 from S. pombe in mutation avoidance and mismatch correction. Science 267, 1 166- 1 169. Szostak. J. W., Om-Weaver, T. L.. Rothstein. R. J., and Stahl. F. W. (1983). The double-strandbreak repair model for recombination. Cell 33, 25-35. Takeda. T., Toda, T.. Kominami, K., Kohnosu. A,, Yanagida. M.. and Jones, N. (1995). Schizosaccharornyces pombe arf! encodes a transcription factor required for sexual development and entry into stationary phase. EMBO J. 14, 6193-6208. Tavassoli. M., Shayeghi, M.. Nasim, A,, and Watts, F. Z. (1995). Cloning and characterisation of the Schizosaccharornyces pombe rad32 gene: A gene required for repair of double strand breaks and recombination. Nucleic Acids Res. 23, 383-388. Thon, G., Cohen. A,, and Klar. A. J. (1994). Three additional linkage groups that repress transcription and meiotic recombination in the mating-type region of Schizosaccharomyces pornbe. Generics 138, 29-38. Thon. G., and Klar, A. J. (1993). Directionality of fission yeast mating-type interconversion is controlled by the location of the donor loci. Genetics 134, 104-1054. Treco. D.. and Amheim. N. (1986). The evolutionarily conserved repetitive sequence d(TG.AC), promotes reciprocal exchange and generates unusual recombinant tetrads during yeast meiosis. Mol. Cell. Biol. 6, 3934-3947. Trepicchio, W. L., and Krontiris, T. G. (1992). Members of the rellNF-kappa B family of transcriptional regulatory proteins bind the HRAS 1 minisatellite DNA sequence. Nucleic Acids Res. 20,2427-2434. van Endert, P. M., Lopez, M. T., Patel. S. D., Monaco, J. J . . and McDevitt, H. 0. (1992). Genomic polymorphism. recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes. Pmc. Narl. Acad. Sci. USA 89, 1 1594I 1597. Wahls, W. P. (1989). "DNA sequences that stimulate homologous recombination in mammalian cells." p. 197. University of Illinois at Chicago, Chicago. Wahls, W. P.. and Moore. P. D. (1990). Homologous recombination enhancement conferred by the Z-DNA motif d(TG),, is abrogated by simian virus 40 T antigen binding to adjacent DNA sequences. Mol.Cell. Biol. 10, 794-800. Wahls. W. P., and Smith, G. R. (1994). A heteromeric protein that binds to a meiotic homologous recombination hotspot: Correlation of binding and hotspot activity. Genes Dev. 8, 1693- 1702. Wahls. W. P., Swenson, G., and Moore. P. D. (1991). Two hypervariable minisatellite DNA binding proteins. Nucleic Acids Res. 19, 32693274. Wahls. W. P., Wallace, L. J., and Moore, P. D. (1990a). Hypervariable minisatellite DNA is a hotspot for homologous recombination in human cells. Cell 60,95-103. Wahls. W. P., Wallace, L. J., and Moore, P. D. (1990b). The Z-DNA motif d(TG),, promotes reception of information during gene conversion events while stimulating homologous recombination in human cells in culture. Mol. Cell. Biol. 10, 785-793. Watanabe. Y.. and Yamamoto, M. ( I 996). Schizosaccharomyces pombe pcrl+ encodes a CREBlATF protein involved in regulation of gene expression for sexual development. Mol. Cell. Biol. 16, 704-7 I 1. +
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White. J. H., Lusnak, K., and Fogel, S . ( 1985). Misvnatch-specific post-meiotic segregation frequency in yeast suggests a heteroduplcx recornbination intermediate. Nafure 315, 350-352. White, M. A.. Detloff, P., Strand, M.. and Pete\, T. D. (1992). A promoter deletion reduces the rate of mitotic, but not meiotic, recombination at the HIS4 locus in yeast. Curr. Genet. 21, 109-1 16. Whire. M. A,. Dominska, M., and Petes, T. D. ( 1993). Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus in Soccharomyce.s cerevisicre. Proc. N u t / . Actrd. Sci. USA 90, 6621 -6625. White, M. A,. Wierdl, M., Metloff, P.. :tiid Pete\. T. D. (1991). DNA-binding protein RAP1 stimulates meiotic recombination at the HIS4 locur in yeast. Proc,. Not/. A c d . Sc.i. USA 88, 97559759. Wilkinson, M. G., Samuels, M., Takecia, T., Toone, W. M., Shieh, J.-C., Toda, T.. Millar, J. B. A,, and Jones, N. (1996). The Atfl transcription factor is a target for the Sty1 stress-activated M A P kinase pathway in fission yca\t. Cows Dnz. 10, 2289-2301. Wolff, R. K., Plaetke. R., Jeffreys, A. J., and White, R. (1989). Unequal crossingover between homologous chromosomes is n o t the iiiiilor mechanism involved in the generation of new alleles at VNTR loci. Genornics 5, 382-3x4. Wu. T. C., and Lichten, M. (1994). Meiohi\-induced double-strand break sites determined by yeast chromatin structure. Science 263, 5 I 5 I8 Wu, T. C., and Lichten, M. (1995). Factors that atfect the location and frequency of meiosisinduced double-strand breaks in SnL.c.litr,-orii\.(.c,.v cerevisiae. Genc,tic,s 140, 55-66. Wynian, A., and White, R. (1980). A highly polymorphic locus in human DNA. Proc. Nrrrl. Acud. Sci USA 77, 6754-6758. Xu, L.. and Kleckner, N. (1995). Sequence noii-specific double-strand breaks and interhomolog interactions prior to double-strand hrcak formation at a meiotic recombination hotspot in yeast. EMBO J . 14, 51 15-5128. Yoshino. M., Sagai. T., Lindahl. K. F., Toyoda, Y., Moriwaki, K., and Shiroishi, T. (1995). Alleledependent recombination frequency: tlomology requirement in meiotic recombination at the hotspot in the mouse major histocoinpatibility complex. Genornics 27, 298-305. Yoshino. M., Sagai, T., Lindahl, K. F.. Toyoda. Y., Shirayoshi, Y.. Matsumoto, K., Sugaya, K.. Ikemura, T., Moriwaki, K., and Shiroishi, T. (1994). Recombination in the class 111 region of f i c ~280-286. s the mouse major histocompatibility complex. I r ~ r i r t i ~ ~ g e n c ~40, Yoshino, M., Sagai, T., Lindahl, K. F., Toyoda, Y., Shiroishi, T., and Moriwaki, K. (1994). No dosage effect of recombinational hotspots in the mouse major histocompatibility complex. h mitriogenerics 39, 38 1-389. Zahn-Zabal, M., Lehmann. E., and Kohli. J . (1995). Hotspots of recombination in fission yeast: Inactivation of the M26 hotspot by deletion of the ode6 promoter and the novel hotspot um4uirn. Gerzetic.\ 140, 469-478. Zenvirth, D.. Arbel, T., Sherman, A,, Goldway, M., Klein, S., and Simchen, G. (1992). Multiple sites for double-strand breaks in whole meiotic chromosomes of Sncckarornyces cerei~isiau. EMBO J . 11, 344 1 -3447. Zimmerer, E. J., and Passmore, H. C. (1991). Structural and genetic properties of the Eb recombinational hotspot in the mouse. Ir,rniitrr~,gerietic.s33, 132- 140
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3 Pairing Sites and the Role of Chromosome Pairing in Meiosis and Spermatogenesis in Male Drosophh Bruce D. McKee Department of Biochemistry and Molecular Biology University of Tennessee Knoxville. Tennessee 37996
1. Introduction 11. Meiotic Pairing Sites in Chroinosome\ o f Ilrosophilo Males: Distribution, Molecular Composition. and Function A. Chromosomes and Sperinatogeiie\i\ iii Drowphiki
B. Distribution of Pairing Site\ u i t l i i i i Chromosomes C. Molecular Coinpo\ition of Pairing Site\ D. M e c h a n i m of Pairing Site Fiiiictioii E. What I s a Pairing Site'? 111. Ctiroino\oine Pail-ing and Spcrniiogeiie\is A . Sex Chromosome Rearrangement\ and Spermiogenic Disruption\ B. Mechanisms of Meiotic Drive ;ind Chi-oinosoinal Sterility
IV. S LI mmai-y and Iinpl icat ion \ A. Pairing Sites and the Mechani~iiiof Pail-ing B. Pairing and Spermiogenesis
References
Mechanistic and regulatory aspect\ o f meiotic chromosome pairing and segregation have received increasing attention i n I-L'CL'III gcai-s. Thi\ review I S concerned w i t h the role of chromo\onid \ite\ and chroinusomc org;ini/atioii i n pairing and \perm development iii Drocopliiln. Two major topics iire re\ i r w e d The lii-\t concerns the distribution and identification of meiotic pairing \ite\ in inidc />J.(twp/?i/u.Cytogenetic data hhow that pairing \ite\ are distributed widely in the eucht-oniiitiii of iitito\cmie\ but are absent from centroineric hetei-0chromatin. The rever\c distribution hold\ l o r the X. where the major pairing \ite i\ locatc.d i i i the cciitral region of the ceiitric lieterocliroiiiatiii. co-mapping v, ith the [ D N A Iucti\. Recent hui\geiiic \titdie\ have dernonstratcd that t h i h p i i r i n g site consists mainly of a ?IO-hp irepeoted sequence i n the intergenic sp;icer\ o f the rDNA repeat\. These spacer repeats contain RNA polymerase I promoter\, which must be functional for the repeat\ to have p i r i n g actii ity. \uggmting a mechani\tic c ~ o ~ i i ~ c c tbetween iot~ pairing and transcription. The general idea that pairing sites coincide with tr;in\crihcil ~cquencesis discussed. The second iiiajoitopic involves the elfect\ of sex chl-oiii~i~oiiie reari-nngemcnt\ on rpcrmiogcncsis. A iaricty of rean-angernent\ involving the \ c x c1iroiiiowincs. including heterochromatic deletion\ and translocations with autosomes, h a b c heeii \howti to lead either to meiotic drive or t o sterility. Recent e\ idcncc strongly iniplicatc\ thc X chi-oino\ome pairing site i n the etiology 01 there
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Bruce D. McKee effects. These findings are d i w u s w l in tcrrns 01' 21 novel model that interprets the sperrniogenic disruptions associated with \ex chromosome renrrangerncntr as resulting from disabling of spermatids due to triggering of a checkpoint concerned with monitoring chrornosomc alignment at meiotic rnetaphase. Copyright C 1998 by Academic Press.
1. Introduction Chromosome pairing is thought to play two important roles in meiosis. First, pairing brings homologs into register to allow reciprocal and nonreciprocal recombination events between alleles. Second, it enables homologs to interact as a unit with the meiotic metaphase spindle so that homologous kinetochores can orient reliably to opposite poles. One or the other of these functions may be absent in certain cases, such as in Dmsophila males and some other invertebrates that undergo intimate pairing without recombination. Nevertheless, homologous pairing is nearly universal in meiosis, and its apparent importance has made it the subject of considerable research interest. Cytological aspects of pairing have been thoroughly described in many organisms, and several excellent reviews have appeared (for example, von Wettstein ef LII., 1984; Jones, 1987; Loidl, 1990). Briefly, in the most common type of meiosis, in which recombination is a regular feature, three types of interhomolog connectors have been described: stringlike connectors, associated with the initial alignment phase (Loidl, 1990); synaptonemal complexes (SC), which develop soon after initial alignment and provide a platform for fully parallel intimate alignment (Loidl, 1990); and chiasmata, which stabilize bivalents during the period of chromosome condensation and spindle interactions (Jones, 1987). The latter two especially have been described in many diverse groups of organisms, but neither is universal. SC has been thought to be involved somehow in recombination, but its role is quite uncertain, in part because it is present in some nonrecombinational meioses and absent in some recombinational meioses. Chiasmata have been described wherever recombination is thought to be present and appear to be absent in all cases where recombination is known to be absent, reflecting their apparent origin in meiotic crossovers (Jones, 1987; Hawley, 1988). But achiasmatic bivalents achieve regular coorientation on the meiotic spindle, so clearly this function can be served by other structures. Much less is known about the genetic and molecular basis for chromosome pairing. Some SC proteins have recently been identified (Heyting, 1996), but their functions are poorly understood, and nothing is known about the molecular composition of the string connectors or chiasmata. Data from yeast studies indicate that SC formation and recombination are mechanistically connected and have identified gene functions required for both synapsis and recombination (Roeder, 1990; Padmore et al., 1991). However, it is not yet clear how universal this relationship is. A different approach to the pairing problem involves mapping and characteri-
3. Meiotic Pairing and Spermiogenesia in Drosophilri
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zation of meiotic pairing sites. The localization of pairing sites has been pursued in part because of the general interest in mapping chromosomal functions and in part because it has been hoped that identifying sites of pairing will provide clues to and further reagents for analysis of the mechanism of pairing. Although sites of alignment and of synaptic initiation have been visualized in several organisms, Drosophila is the only metazoan in which it has been possible systematically to apply both cytological and genetic mapping methods to pairing site localization. Recent years have witnessed considerable progress in characterization of the male Drosophila pairing system, including identification of a short repeated sequence that functions as the major pairing site for the X-Y bivalent and preliminary evidence concerning the role of transcription initiation in the function of this sequence. These findings, along with recent data concerning distribution of pairing sites on Drosophila autosomes, are summarized in the first part of this review. A unifying hypothesis relating pairing to transcription and pairing sites to expressed sequences is discussed. The second part of this review deals with the relationship between meiotic pairing and spermiogenesis in male Drosophila. A variety of rearrangements involving the sex chromosomes have been shown to disturb spermiogenesis, leading to meiotic drive and reduced fertility in some cases or, in others, to complete sterility. The genotypes that are associated with spermiogenic abnormalities include simple deletions of X heterochromatin, X-autosome or Y-autosome translocations, and particular combinations of rearrangements. Spermatid development is visibly abnormal in some but not all cases, and the variety of stages at which abnormalities have been described, combined with the variety of rearrangements that cause the phenotypes, have frustrated attempts to develop unifying models. Some of the rearrangements also cause disturbances i n meiotic pairing and disjunction, leading to suggestions that spermiogenesis depends in some direct way on pairing. Recent findings have clarified the role of meiotic pairing failure in these spermiogcnic disturbances. These data are reviewed and reinterpreted in terms of a novel meiotic regulatory model, the basic premise of which is that the various disturbances of spermiogenesis associated with sex chromosome rearrangements result from triggering of a metaphase checkpoint sensitive to chromosome misalignment.
II. Meiotic Pairing Sites in Chromosomes of Drosophih Males: Distribution, Molecular Composition, and Function A. Chromosomes and Spermatogenesis in Drosophila
Cytological aspects of Drosophiki male meiosis have been reviewed previously (Cooper, 1950; Lindsley and Tokuyasu, 1980; Kremer et al., 1986; Church and 1994) (see also Maines and Wasserman, this Lin, 1988; Fuller, 1993; Cenci r t d.,
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volume); they are summarized here briefly to set the stage for the cytogenetic analyses that form the main topic of this review. Drosophilu spermatocytes develop in cysts of 16 cells generated by four rounds of mitosis starting from a primary spermatogonium. Cyst-mates are connected by ring canals that allow sharing of diffusible substances and help ensure that development occurs synchronously. The period from the end of the last gonial mitosis until the first meiotic division lasts approximately 90 hrs and is characterized by extensive synthesis of both RNA and protein and by a 25-fold increase in volume (Lindsley and Tokuyasu, 1980). DNA replication is apparently completed very early in this period, within the first 3 hrs (Cenci et d., 1994). The chromatin at first forms a single clump in the center of the nucleus but quickly assorts into three major clumps that remain closely apposed to the nuclear membrane throughout the period of growth. These three clumps are thought to correspond to the X-Y bivalent, which is associated with the nucleolus, and the two major autosomal bivalents (Fig. I ) . The tiny fourth chromosome bivalent is not visibly resolved until shortly before meiosis. The chromatin clumps remain decondensed and transcriptionally active until the end of the growth phase, at which time they quickly condense to form rather compact spherical structures that are still arrayed around the inside of the nuclear membrane (Cooper, 1950; Cenci et ml., 1994). This stage, which has been referred t o as diakinesis, is excellent for scoring interbivalent conjunctions associated with chromosome rearrangements, because in wild-type cells the bivalents are both sharply resolved and clearly separate one from another (McKee et al., 1993). Thc fourth chromosome pair is often clearly visible at this stage; it too takes up a position separate from the other chromosome pairs. The condensed bivalents subsequently congress toward the inetaphase plate and form a tight cluster there equidistant from the poles. Metaphase is quite brief, and the chromosomes undergo a rapid and approximately synchronous anaphase. Electron microscopic reconstructions of sectioned prometaphase chromosomes have revealed little in the way of structural detail relevant to pairing mechanisms. Autosonial bivalents appear as a pair of overlapping oval-shaped chromatin clumps with no evident axes and no apparent connecting structures
ccn-4 Fig. 1 Karyotype of niale Dmsophi/o. Rectansle\ reprcwnt hcterochroniatin, lines repwsent euchromatin. circles represent centromeres. Cro55hatched region5 represent rDNA.
3. Meiotic Pairing and Spermiogcneais in Dr.o.c.opliila
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(Ault el ~ 1 . . 1982). The paired chromosomes overlap from end to end, but the actual surface of contact is probably quite limited because the chromosomes are highly condensed. The sex bivalent differs in that the region of overlap is limited to a fraction of the length of the chromosomes, presumed to correspond to the genetically defined pairing region, as discussed later. A fibrillar material different from chromatin is present in the pairing region of D. rnelanogastrr X-Y bivalents (Ault e t a / . , 1982), but it is lacking from the X-Y bivalents of D. .rinzu/ans, so it is doubtful that this material has anything to do with pairing (Ault and Rieder, 1994).
B. Distribution of Pairing Sites within Chromosomes
1. Autosomal Pairing Sites a. Autosomal Pairing Sites Are Restricted to Euchromatin. Two types of observations indicate that euchi-omatic and heterochromatic regions of autosomes differ substantially in pairing capacity. First, deficiencies of euchromatin and of heterochromatin of the large metacentric autosome 2 have very different effects on pairing patterns of autosomes. A minichromosome ( D p ( 2 , f ) I )that is deficient for most of the euchromatin (except the tip of 2 R ) but that retains most of the proximal heterochromatin (Fig. ? A ) is regularly found as a univalent (or as two univalents when present in two copies) at metaphase I when present with a pair of normal chromosomes 2 (Yamamoto, 1979). Conversely, large deletions of heterochromatin from either the left o r right arm of chromosome 2 have no effect on the ability of the resulting chromosomes to pair with fragments of chromosome 2 that share euchromatic homology (Yamamoto, 1979). Second, translocations that rearrange only heterochromatic regions of chromosome 2 typically have little eftect on pairing patterns, but otherwise similar rearrangements involving euchromatin often have dramatic effects. Thus, pairs of compound second chromosomes [chromosomes with two copies of one chromosome arm, such as C(2L) and C(2R)l that arose via breaks in the heterochromatin (Fig. 2Ba) disjoin randomly from each other despite sharing homology for substantial heterochromatic regions, whereas similar compound pairs that share homology lor proximal euchromatin as well as heterochromatin (Fig. 2Bb) disjoin regularly from each other (Hilliker c / ul., 1982). Moreover, transpositions of even rather small segments of euchromatin from chromosome 2 to the Y [known as 2-Y transpositions, Tp(2;Y)I cause detectable elevation in the frequency with which the Dp(2;Y) element (the Y carrying the inserted piece of chromosome 2) pairs with a normal 2 in late prophaae (Fig. 2C). These pairing events are detected as connected or fused bivalents (referred to as quadrivalents) that are present at late prophase when wild-type bivalents are still well separated. Genetic analysis shows that these quadrivalents are effective in orienting the Dp(2:Y) and
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Fig. 2 Genotypes for tests of pairing site distrihution in Chromosome 2. ( A ) Pairing of free duplication with normal chromosomes. Thich rectangles indicate heterochromatin, thin rectangles indicate euchromatin. Dotted lines indicate tested region. ( B ) Pairing ofC(2L) with 2(2R): (a) compounds that overlap in heterochroinatin only; (b) compounds that overlap in heterochromatin and euchromatin. (C) Pairing in 2-Y transposition heterozygotes. Shaded rectangles indicate sex chromosome heterochromatin.
normal 2 chromosomes to opposite poles, as these elements segregate from one another nonrandomly. Conversely, a transposition encompassing approximately two thirds of the heterochromatin of 2R is without effect on pairing patterns (McKee et al., 1993). Less extensive data suggest that the same distribution of pairing capacity holds for chromosome 3, the other large metacentric autosome, as well. When the heterochromatin and centromere of chromosome 3 are replaced with the corresponding region of chromosome 2 (T(2;3)lO8),the resulting hybrid pairs regularly with a normal chromosome 3 at metaphase and is not seen to associate with elements of chromosome 2 (Yamamoto and Miklos, 1977). Moreover, when the reciprocal hybrid, which has the euchromatic arms of chromosome 2 appended to the heterochromatin of chromosome 3, is also present along with two normal
3. Meiotic Pairing and Spermiogenesia in Dm.rophilu
83
homologs, two large autosomal bivalents are regularly present at metaphase. If the heterochromatic region of chromosome 3 had pairing capacity, quadrivalents should have been present at least some of the time. Lack of pairing in third chromosome heterochromatin is also indicated by the random segregation of C(3L)/C(3R) pairs (Holm and Chovnick. 1975). Limited data suggest that the pairing capacity of chromosome 4, a very small autosome, may also be restricted to the euchromatin. The 4pXl) (4-proximal, X-distal) element of an X-4 translocation (T(1;4)wnZ5), in which the fourth chromosome breakpoint is located i n the heterochromatin, fails to pair with a normal fourth chromosome at metaphase I, whereas the other translocation half (Xl’4r)), which contains all of the fourth chromosome euchromatin, often is seen to be associated with the normal fourth chromosome (Yamamoto and Miklos, 1977). This suggests that the pairing region is confined to the X’41)(euchromatic) half of the translocation. It has been shown that the pairing sites responsible for disjunction of the fourth chromosomes in female meiosis are confined to the 4f’X” (heterochromatic) half of this same translocation (Hawley et a / . , 1993). Thus the male and female pairing sites are separated by the fourth chromosome breakpoint of T ( ~ ; ~ ) M ?Although ” J - ~ . the position of thc breakpoint is not very precisely characterized, it clearly separates the euchromatin from the bulk of the heterochromatin and thus places the male meiotic pairing sites in a fragment that includes all of the euchromatin and lacks most of the heterochromatin.
b. Weak Pairing Sites Are Widely Distributed along Euchromatic Arms. As noted above, 2-Y transpositions that encompass portions of the euchromatin of chromosome 2 causc the resulting Dp(2;Y) chromosome to pair with a normal 2, leading to the formation of quadrivalents involving these two chromosomes plus the X and the deticient chromosome 2 (Fig. 2C). A comparative study of pairing capacity of a dozen transpositions that collectively encompass a region including the proximal one fifth of 2L and all of 2R was carried out. The transpositions ranged in size from several polytene bands to nearly an entire chromosome arm. It was found that the frequency of quadrivalent formation (presumed to reflect Dp(2;Y)-2 pairing) depended directly on the size of the transposed region. The smallest transpositions were associated with weak (but statistically significant) pairing frequencies, whereas transpositions encompassing most of a chromosome am1 resulted in nearly 100% pairing. The degree of preferential segregation of the Dp(2;Y) element from the normal chromosome 2 also depended directly on size of transposition. These data imply that weak pairing sites that interact additively are widely distributed within the euchrornatin of chromosome 2. It is not clear if this reflects general homology pairing or a widespread distribution of discrete pairing sites (McKee ef a/., 1993).
c. A Strong Pairing Site in Proximal 2L. One transposition, Tp(2;Y)G, which encompasses the basal one fifth of chromosome arm 2L, mediated both
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quadrivalent formation and preferential segregation of the Dp( 2;Y) element from the normal 2 at a frequency disproportionate to its size (Fig. 3). Two deletional derivatives of Dp(2;Y)C,one of which retained this strong pairing capacity and the other of which lacked it, narrowed the region of interest to two small intervals near the base of 2L (McKee et NI., 1993). The pairing site was further localized by determining the effects of replacing the normal 2 in Tp(2;YY)G/X;2males with each of six small deficiencies on the frequency of meiotic quadrivalents (Fig. 3). Three of the deficiencies retained the strong pairing site and two were deficient for it, delimiting the site to the 39DE interval. The site responsible for high levels of preferential Dp( 2;Y)-2 segregation mapped to the same small interval. implying that the pairing and segregation sites are the same, as expected. The proximal breakpoint of the sixth deficiency, Dj(2L)TW#4, is within the 39D3-El interval. This deficiency exhibits intermediate phenotypes with respect to both quadrivalent formation and segregation, suggesting that the 39DE pairing site is repetitive and that Df(2LjTW84 is partially deficient for the pairing sequences (N. Takano and B. D. McKee, unpublished observations).
366
37
40
39
38
P ~~
36E4-Fl ~~
~~~
~2 ~
38A6-7 37D2-El
~
1 37E2-F4 2
Tw84 TW65 TW161
TW50
his
38E6-9
’
his + 39D1-2
his -+
+ + +
S
-
ND
+ +
37F-38Al 37F-38Al 38A6-61
39E2-Fl
his-
4OA4-61 his-
-
-
-
Fig. 3 Mapping ii strong pairing sitc i n proxiinn1 2L. I’roxirnal region 0 1 7L is represented i i i the middle. Thin rectangles ahove ~hr(iniosoiiiercprc\eiit duplication\. line\ helow chromosome reprrsent deletions. P: pairing with normal 2 ( l o r I)p\) or with Dp(2:Y I(; (for Df\), a \ indicated hy sipnihcanl quadrivslent frequency. S: \ e g r c p t i o n o l chronio\oiiie froin iiornial 2 (for D p ) o r from D(2;Y)G ( f o r Df\).
3. Meiotic Pairing and Spermiogencais in f h s o p h i k r
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Localization of a strong pairing site to 39DE is consistent with evidence from analysis of C(2L)/C(2R) pairs (Fig. 2B). C(2R)cn contains proximal 2L euchromatin from the 39-40F region and segregates regularly from several C(2L) chromosomes (Gethmann, 1976; Lindsley and Zimm, 1992). C(2L)V12,SD72 and C(ZR)V43,SD72 share homology for both euchromatin and heterochrornatin from 39D to 42A and disjoin regularly from each other (Hilliker et al., 1982). As noted earlier, all other C(2L)/C(2R) pairs that lack euchromatic homology disjoin randomly from each other.
2. Sex Chromosome Pairing Sites a. The X Chromosome Pairing Sites Are Confined to Pericentromeric Heterochromatin. Superficially at least, meiotic pairing capacity on the X chromosome is distributed oppositely to that on the autosomes, being confined to heterochromatin. The evidence for this is that deletion of centric heterochromatin disrupts X-Y pairing but deletion of euchromatin is without effect. The effects of heterochromatic deletions (Fig. 4B) depend on size and location of the breakpoint, the smaller ones having no effect and the largest causing frequent X-Y pairing failure and random X-Y disjunction at anaphase I (Cooper, 1964; McKee and Lindsley, 1987). Two of the larger deletions merit special mention, as they have been the subject of numerous studies. Df(1 ).sc4L-sc8R (known as sc4-.~c8; Fig. 4Bb) is a product of recombination between the large inversions li?(l)sc4 and ln(I)sc8, which have a common euchromatic breakpoint within the scute locus near the tip and different heterochromatic breakpoints (Fig. 4A). It is deficient for a large central fragment of heterochroniatin that includes the rDNA, and has a block of distal heterochromatin relocated to near the tip of the X. Males carrying sc4-sc8 and a normal Y exhibit X-Y nondisjunction at a frequency that varies considerably, depending on genetic background and temperature (Gershenson, 1933; Sandler and Braver, 1954; Cooper, 1964; Peacock, 1965; Peacock et ul., 1975). D f ( 1 ) X - 1 (Fig. 4Ba) is a simple deletion recovered following X-ray mutagenesis and screening for simultaneous deletion of the rDNA and the .su(j') locus, the proximalmost identifecl gene in the euchromatin. It is indeed deficient for both of these loci and all of the intervening heterochromatin, as well as for some additional proximal euchroniatin (Lindsley and Zimm, 1992). Males carrying Qf(I ) X - 1 exhibit essentially random X-Y nondis,junction, generating X , Y, XY, and nullo-XY secondary spcnnatocytes at equal frequencies (McKee and Lindsley. 1987; McKee and Karpen, 1990). Conversely, even the largest tlelctions of euchromatin have n o evident effect on pairing capacity (Fig. 4Cf). The resulting chromosomes, referred to as free X duplications, disjoin regularly from an attached-XY in XY/Dp males and disrupt X-Y disjunction in X/Y/Dp males. as long as most of the heterochromatin is intact (Lindsley and Sandler. 1958; Park and Yamamoto, 1995). Free X duplications that are also deleted for most of the heterochromatin behave genetically as
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x-1
scv2 sc8 wm51 b sc4 wm4
sc4 wm4
scV2 wm5lb sc8
Disjunction Frequency
- - - -Df(1 - _)X-1 _-----
a
-000
-
-/ +
+ + Df(l)wrn4L-wm51bR 0
I
#----BlOU
+ + -
+ Fig. 4 Mapping the X chromosome pairing site. (A) Breakpoints of some inversions and deticiencies used to map the pairing site. Crosshatched region represents rDNA. ( B ) some deticiencies used to map pairing site. Dotted lines represent deleted region. X-Y disjunction: random ( - ) . regular (+). disrupted but not random ( - / + ) . (C) Representative free X duplications used to map pairing site.
univalents (Fig. 4Cg). A single copy segregates randomly from an attached-XY (Lindsley and Sandler, 1958) and fails to induce nondisjunction of an X and Y (Yamamoto and Miklos, 1977); when two copies are present, they segregate randomly from one another (Karpen et al., 1996). It is not clear whether the X euchromatin really lacks pairing capacity or merely appears to because it has nothing with which to pair. The X euchromatin and the Y actually do share some homology, though it may not be enough. The
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Stellate (Ste) locus at 12E contains a 1.2-kb repeated sequence, 900 bp of which are homologous with a portion o f a 2.8-kb repeated sequence in the Suppressor of Stellate (Su(Ste) ) locus on the Y. However, Ste and Su(Ste) are only 90% homologous over the common region, well below the average level of allelic homology, and it is possible that homology levels of 90% fall below the stringency cutoff, assuming such a cutoff exists (Hardy et al., 1984; Livak, 1984; Livak, 1990; Balakireva et al.. 1992). Also, although Sre repeats are present at high copy number on some X chromosomes, other X chromosomes contain only a few (Palumbo et a/., 1994), and the Ste copy numbers of heterochromatically deficient X’s that are pairing deficient have not been determined. Homology between the X euchromatin and the Y has also been created artificially via transposition of blocks of X-derived euchromatin to the Y. Transpositions as large as 300 kb do not promote X-Y segregation when the X heterochromatin is absent (McKee and Karpen, 1990), whereas transpositions of autosomal chromatin that appear to be of comparable size do have detectable pairing effects (McKee et al., 1993). However, this does not rule out the possibility that X euchromatin has a pairing capacity too weak to be detected in assays involving small fragments.
b. The Proximal-most Block of X Heterochromatin Lacks Pairing Capacity. The segment of Xh proximal to the breakpoint of In(l)sc8 (Fig. 4A) consists of at least 1 Mb of DNA (Le et a/., 199.5) and contains representatives of three different satellite DNA families-the 1.672 (AATAT), 1.705 (AAGAG and AAGAGAG), and 1.688 (a 3.59-bp repeat) sequences (Steffenson et a/., 1981; Hilliker and Appels, 1982; Lohe r t al., 1993; Le et a/., 1995)-as well as islands of complex DNA that likely consist of various transposable elements (Le et ul., 1995). Two lines of evidence indicate that this region lacks meiotic pairing capacity. First, the mini-X chromosome Dp(l;f)l187 (Fig. 4Cg), which was derived from In(l)sc8 and contains most of the region proximal to the sc8 breakpoint along with a small amount of heterochromatin from a more distal location (Le et ul., 1995), disjoins randomly from itself when present in two copies along with a normal X and Y (Karpen et al., 1996). Chromosome size is not a factor here since other minichromosomes only slightly larger than Dp1187 pair with perfect regularity (see next section). Second, pairing is never observed in the proximal heterochromatin of either the In(l)sc8 or sc4-sc8 chromosomes (Fig. 4Bb). In( I ) s d pairs regularly and sc4-sc8 pairs occasionally with the Y, but in both cases it is always the distal segment of heterochromatin, relocated to the tip of the X by the inversion, in which pairing occurs (Cooper, 1964). The failure of the 1.688 repeats to promote pairing is not surprising in light of the absence of homologous sequences on the Y. However, the other two repeat families are abundant on the Y (Lohe e f a l . ,1993), so their failure to promote pairing suggests a real lack of function. These lindings, combined with the inability of autosomal heterochromatin to contribute to the pairing capacity of those chromosomes,
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suggest that satellite sequences generally may be excluded from participation in male meiotic pairing. c. A Strong Pairing Site Maps near or within the Nucleolus Organizer of the X. The rDNA repeats of Drosophila are located in central Xh and near the base of the short arm of the Y, each block containing about 200-250 repeats. Most well-spread X-Y bivalents appear to be paired in the vicinity of the NOS, although occasional pairing in YL has also been reported (Cooper, 1964). X chromosomes that are completely deficient for the rDNA exhibit elevated frequencies of X-Y nondisjunction, which range from several to 50% (random disjunction) (Gershenson, 1933; Sandler and Braver, 1954; Cooper, 1964: Yamamoto and Miklos, 1977; McKee and Lindsley, 1987). Conversely, X chromosomes that retain at least some rDNA, even though they may be partly or mostly deficient for it, disjoin regularly from the Y (Appels and Hilliker, 1982; McKee and Lindsley, 1987). Included i n this latter class is one chromosome ( Q J ljiv"'4' W " ' - ~ " ' R : Fig. 4Bc) that has been estimated by in siru hybridization to retain only about 6-8 rDNA repeats (Appels and Hilliker, 1982). Thus, these results suggest that rDNA may compose the major X-Y pairing site but that normal pairing capacity requires far fewer repeats than are needed for viability. The pairing behavior of free X duplications also points to a major role of rDNA. All such chromosomes that have been shown on genetic or molecular grounds to be rDNA-positive also pair regularly with other pairing-competent sex chromosomes, whereas rDNA-deficient free X duplications disjoin randomly or at least highly irregularly from other sex chromosomes (Lindsey and Sandler, 1958; Yamanioto and Miklos, 1977: Park and Yamamoto, 1995: Karpen ef a/., 1996). A large set (27) of free duplications was recently generated by X-ray mutagenesis of a chromosome in which the distal half of the rDNA and the distal heterochromatin were inverted to the tip of the X. All 27 of the free duplications proved to retain pairing function and all that were tested proved also to retain at least some rDNA. Two of these chromosomes (one pictured in Fig. 4Ch) are thought, based on size and gene composition, to be deficient for all of the X heterochromatin except for the material proximal to the h ( l ) s c # breakpoint (which we have seen to lack pairing capacity) and a small segment of rDNA (Park and Yamamoto, 1995). The regular disjunction of these chromosomes from an attached-XY argues strongly for a major role of the rDNA in sex chromosome pairing. However. the resolution of all of these cytogenetic analyses is limited, and i t is impossible from them to rule out the alternative favored by Cooper (1964), namely, that the major pairing sites are distinct from but immediately flanking the NO. The molecular data discussed in Section C confirm the interpretation that the rDNA repeats themselves mediate pairing.
d. Weak Pairing Capacity Maps to the Distal Region of Xh. Although all rDNA-deficient X chromosomes exhibit significant levels of X-Y nondisjunc-
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tion. these levels vary over a considerable range. Two observations indicate that the sequences responsible for this partial pairing are in distal Xh. First, as noted earlier, the sc4-scB chromosome (Fig. 4Bb), which retains a block of distal Xh relocated to the tip and a block of proximal Xh in its normal location around the centromere, pairs occasionally with a Y, and in those cases it is always the tip of the X that associates with the Y (Cooper, 1964). Second, X chromosomes selected for simultaneous deletion of the rDNA and of the proximal euchromatic sucf') locus following X-ray treatment display very high rates of X-Y nondisjunction, whereas X chromosomes selected only for loss of the rDNA exhibit highly variable levels of X-Y nondisjunction (Yamamoto and Miklos, 1977; McKee and Lindsley, 1987). Presumably some of the latter class, but none of the former, retain sequences from distal Xh that have weak pairing capacity. Nothing is known at present about the composition of this pairing material. It is interesting that fragments ofrDNA (both gene region and spacer) have recently been documented within complex repeats cloned from the distal Xh region (Nurminsky rt ol., 1994). Another plausible candidate would be the Stdlate repeats, a block of which are located in the distal heterochromatin (Shevelyov, 1992). The apparent absence of pairing capacity in the euchromatin would argue against participation of Ste, since copies are located at 12E in addition to distal Xh. However, the euchromatic Ste locus is highly variable in copy number, ranging from a few copies up to 300 (Palumbo et d., 1994), and no comparisons of Ste copy number have been made among heterochromatically deleted X chromosomes with different pairing capacities.
C. Molecular Composition of Pairing Sites 1. X Chromosome Pairing Site a. rDNA Insertions Restore Pairing Capacity to Heterochromatically Deficient X Chromosomes. Decisive evidence for a role of rDNA in X-Y meiotic pairing emerged from transgenic studies involving cloned rDNA repeats and fragments. Insertions of a P element containing a single complete rDNA gene flanked by spacers were able to partially restore pairing capacity to an X chromosome (Df(1)X-1) deficient for the native pairing site (Fig. 5Ba). X-Y disjunction improved from 50% (random) in the absence of an insertion to 64% in the presence of a single insertion to 75% in the presence of two insertions (McKee and Karpen, 1990). X chromosomes with rDNA insertions near the tip formed terminal associations with the Y, indicating that the rDNA insertions were functioning as chromosomal pairing sites. All of the insertions were in the euchromatin, so it is apparent that there is no general repression of pairing activity of the X euchromatin, at least not in X chromosomes that are deleted for the heterochromatin. Insertions at several different X euchromatic sites had similar
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x-Y
B
Disjunction
~-
.64*(1),.7$(2) [riblo] c
&44+44[7$
d
a--++.[c711
e
a-++++++~vA
.72*
f
I++++++d [rib7](1A)/ 211
67*
g
b+++W/A [rib-/l(lA)A7B
62*
.51(1),.64*(2)
[ribl0~3] [riblOA3]
h i
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.55*(1), 73*(2)
[rib71(1A)AUt
b*&7A
[ribA(lA)n49A 1A)AZlO [rib121
I m
I~KKYICX~+WK.(~LI - & - [SPMx25 mwlg
[SPMxlO]
.59*
.56* .51 .52 .52(1),.52(2) .53 .51
Fig. 5 Mapping the rDNA pairing \ite using transgenic rDNA insertions. ( A ) An rDNA repeat. Rectangle represents rDNA transcription unit, arrowheads represent spacer promoter\. IGS: intergenic spacer. ETS: external transcribed spacer, ITS: intcrnal transcribed spacer (includes S.8S and 2s xquences). (I31 Constructs used to map pairing site. Open rectangle\ represent eye color marker where , it is w ' ); solid rectangles represent P element sequences. X-Y (rosy ' in all cases except line !i disjunction: frequency from cytological assay (frequency of opposite pole disjunctions) averaged across all X insertions for each con\truct; asterisk indicates \igiiifciintly diiferent froni random disjunction; number in parentheses is copy number of insertion.
effects on X-Y disjunction, so pairing capacity of rDNA repeats does not appear to be particularly sensitive to chromosomal position. These results indicate that single rDNA repeats function autonomously as pairing sites.
b. Pairing Capacity Maps to the Intergenic Spacer (IGS) of rDNA Repeats. The capacity of rDNA repeats to mediate pairing does not depend on an intact rRNA transcription unit. Deletions of part or all of the transcription unit were generated irz \tifro and X chromosome insertions obtained by P element
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microinjection (Merrill er a/., 1992). Three different constructs containing intact IGS regions and varying amounts of the 5’ end of the transcription unit were tested and found to mediate pairing in a dose-dependent manner, and at levels at least roughly comparable to that of complete rDNA repeats (Figs. 5Bb-e). No clear-cut effects attributable to amount of rDNA transcription unit were found. Deletions also were induced within an inserted complete rDNA repeat on the X by remobilization of the P element (McKee et al., 1992). Several of the resulting chromosomes retained most or all of the IGS region (Figs. 5Bf-j) but lacked part or all of the rDNA transcription unit. Absence of the transcription unit had little effect on pairing capacity, which seemed to depend more on the size of the remaining IGS region. Several of the pairing-positive chromosomes contained no rDNA other than a 240-bp sequence normally present in 6-12 tandem copies immediately upstream of the rDNA promoter. Pairing capacity detectable in the X-Y disjunction assay required a minimum of 6 copies of these sequences; at copy numbers above 6, pairing strength was proportional to 240-bp repeat copy number. The idea that X-Y pairing is mediated primarily by IGS repeats, rather than by rDNA genes as a whole, is supported by observations on the structure and pairing behavior of the Y chromosome of D. sirnulam, a close relative of D. melanogast r r . The Y chromosome of this species lacks an NO altogether, the only rDNA being on the X . However, a large (megabase-sized) block of 240-bp IGS repeats not associated with rDNA genes is located near the tip of the long arm of the Y (Lohe and Roberts, 1990). Moreover, the Y chromosome of this species associates with the X at the tip of YL, very unlike the proximal connections seen in D. melanogaster (in which the 240-bp repeats are near the Y centromere on the short arm) (Ault and Rieder, 1994). Thus it seems likely that the 240-bp repeats also serve as X-Y pairing sites even in D. sirnulam, where a Y-chromosomal NO is lacking.
c. rDNA Transcription Unit without Promoter Lacks Pairing Capacity. A fragment that encompasses most of the transcription unit but lacks a promoter or IGS sequences was also tested and found to lack pairing capacity. This fragment includes most of the 18s and 28s sequences and all of the ITS and 5.8s sequences but lacks the IGS and ETS (Fig. 5Bk). Two single insertions of this element on the X chromosome were obtained by P element microinjection. Four lines containing double insertions were obtained by P element remobilization using the dose-dependent mini-white eye-color marker in the construct to select copy number increases. Both single insertions and all four double insertions proved completely incapable of stimulating disjunction of Df(1)X-1from a Y-disjunction values in all cases were very close to 50%, the same as for D f ( 1 ) X - l with no insertion (Ren rt d., 1997). The rDNA fragment in this case encompasses approximately 5 kb, so double insertions contain as much as 6 times as much rDNA as the smallest IGS fragments that have proved to mediate
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pairing. Thus, the failure of the transcription unit insertions to mediate pairing is not likely due to insufficient DNA. Rather, these results indicate that there is something special about the IGS repeats with respect to meiotic pairing capacity.
2. The Strong Pairing Site at 39DE of Chromosome 2 There have been no transgenic analyses of autosomal pairing function, so no definitive statements can be made about molecular composition. However, the strong pairing site at 39DE (Fig. 3) coincides with the location of the his repeats, 4.8/5.0-kb fragments containing the structural genes for the five major histones, which are distributed across the same interval (Lifton e f al., 1977; Matsuo and Yamazaki, 1989). Moreover, the Df(2LJTw84chromosome that exhibited intermediate pairing and segregation properties is reported to be a partial his deletion (Lindsley and Zimm, 1992). If the reported breakpoint can be confirmed, these findings will strongly implicate the his repeats in the 39DE pairing site, and will provide justification for transgenic tests of pairing function of his repeats.
D. Mechanism of Pairing Site Function
1. Transcription and Pairing
a. Transcription of Spacer Promoters Is Required for Pairing of 240-bp Repeats. Each of the 240-bp IGS repeats from the rDNA contains a functional RNA polymerase I promoter embedded in a 52-bp sequence that is a nearly perfect copy of the sequences flanking the rDNA transcription initiation site (Coen and Dover, 1982; Kohorn and Rae, 1982b; Miller er al., 1983; Simeone et al., 1985). Transcripts are initiated at these “spacer promoters,” some terminating precisely at the next spacer promoter and others reading through into downstream sequences (Kohorn and Rae, 1982b; Miller et a/., 1983; Murtif and Rae, 1985; Tautz and Dover, 1986). Arrays of spacer promotes function as directional enhancers of transcription initiation from the rDNA promoter (Grimaldi and Di Nocera, 1988; Grimaldi et al., 1990). To determine whether this transcriptive function is related to the pairing capacity of IGS repeats, the spacer promoter of a cloned 240-bp repeat was disabled by site-directed mutagenesis and an array of these mutated 240-bp repeats was introduced into the Drosophila germ line. The mutation involved substitution of four conserved sites in the promoter including the - 1 and + I positions with the respect to the transcription initiation site. X-linked arrays of these repeats have now been analyzed for transcriptional activity and ability to mediate X-Y pairing (Figs. 5B1,m) and found to be completely null with respect to both phenotypes (W. T. Sun, D. Chakaravarti, L. Habera, and B. D. McKee, unpublished observations). This is a remarkable result in that similarly sized or smaller arrays of wild-
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type repeats initiate transcription from the spacer promoters and function as very effective pairing sites. Thus, altering only a few bases in the promoter sequence suffices to completely disable the pairing function of 240-bp repeats. This strongl y suggests a fundamental link between the mechanisms of transcription and meiotic pairing. b. The Relationship between Transcription and Pairing. How might pairing and transcription be linked'? The fact that the introduced mutations affect the transcription initiation site and disable transcription point to a direct link. One possibility is that transcription is needed to unwind the DNA, rendering it accessible to pairing enzymes and/or to homologous base-pairing interactions. A restriction on models of this type i s that apparently both participating molecules must be activated by transcription, since the promoter mutations affected only the X chromosomal array. This observation argues against models involving one active and one passive partner, such as standard RecA-type reactions. RecA requires single-stranded DNA t o initiate formation of the DNA-protein filaments that function as homology search complexes, but there is no requirement for ssDNA in the other member of the paired complex (Radding, 1988). Another possibility is that the nascent transcript is an essential component of the pairing reaction. The 5' ends of nascent transcripts could base pair with complementary regions of a homologous template strand, thus forming a double heteroduplex RNA bridge. An alternative is a triplex structure involving a nascent transcript and DNA strands from two homologous molecules. It has been shown that transcribing a linear duplex prior to interaction with a RecA-ssDNA complex stimulates formation of a novel and remarkably stable product that involves the RNA, the ssDNA, and the template strand of the duplex (which is the same polarity as the ssDNA) (Kotani and Kmiec, 1994). An RNA bridge might provide a way of linking chromosomes homologously without requiring DNA breaks; moreover, because a linkage of this type could be dissolved by removing the RNA, there is no reason to think it would lead to crossovers. Thus the RNA bridge idea is especially attractive with respect to achiasmatic chromosome pairing where homology is clearly a critical factor but where crossovers are not normally found among the products. However, RNA-mediated interactions could also play a role in the early stages of homologous alignment even in organisms with recombination, as i t might be of value to align chromosomes prior to committing to recombination, to avoid the risk of ectopic recombination. Even in yeast, where dispersed repetitive DNA is far less common than in more complex genomes, there is evidence for homologous interactions prior to and independent of the double-strand-break dependent recombination pathway (Weiner and Kleckner, 1994). It is also possible that the relationship between pairing and transcription is somewhat less direct. Although the mutations introduced into the spacer promoter interfered with transcription. they might also have altered promoter structure
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in some way, for example, by interfering with proper binding of transcription factors or by altering chromatin conformation. Hotspots for initiation of recombination in yeast have also proved to map within gene promoters, but in the two most thoroughly studied cases, the ARC4 and HIS4 genes, the promotion of recombination is related to the relative accessibility of promoter chromatin (Lichten and Goldman, 1995) and/or to the presence of certain transcription factors (White er al., 1993) rather than to the act of transcription per se.
2. Relationship between Pairing and Nucleolus Formation Another possible type of indirect connection, one that is more specific to the rDNA, is that pairing is fostered in some way by the formation of nucleoli. Perhaps one or more nucleolar proteins double as pairing proteins, or perhaps the presence of homologous rDNA loci in the confines of a nucleolus provides a kinetic advantage in a homology search. This model would explain the connection between transcription and pairing by invoking the likely dependence of nucleolus formation on the ability to initiate RNA polymerase I transcription. It has been shown that isolated euchromatic rRNA genes are capable of nucleating formation of mininucleoli in polytene chromosomes (Karpen er al., 1988). However, many rDNA fragments that retain pairing ability do not form mininucleoli. These include not only naked IGS arrays but also rDNA repeats deficient only for a major part of the 28s gene (McKee rt al., 1992). Thus the requirements for nucleolus formation are more stringent than those for pairing. This argues against a role of nucleolus formation in pairing but does not rule out the possibility that transcriptionally active rDNA fragments are incorporated into nucleoli organized by intact repeats and that pairing interactions take place in the context of those nucleoli.
E.
What I s a Pairing Site?
1. Pairing Sites in Male Meiosis The foregoing data suggest that pairing sites in Drosophila males may correspond to expressed sequences (McKee, 1996). This idea would account economically for the widespread distribution of pairing capacity in euchromatin and its absence from most of the heterochromatin, as this distribution parallels that of active genes. It also provides an explanation for the fact that the two strong pairing sites that have been identified thus far coincide with tandemly repeated clusters of highly expressed genes, the rDNA and histones. Finally, it is consistent with the molecular analysis of the rDNA pairing site, which shows not only that pairing capacity is concentrated in the spacer region where the tandemly repeated promoters are located, but that it depends on the function of spacer
3. Meiotic Pairing and Spermiogenejij in Dm.sophi/tr
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promoters. The data obtained thus far do not allow us to discriminate between promoter pairing versus transcription-activated pairing, although the association between lack of transcription and pairing incompetence in the mutant spacer promoter array leads us tentatively to favor the latter idea. Sequence repetition may also be important for pairing strength, given the fact that both the rDNA and his loci arc moderately repetitive. Sequence repetition could promote pairing in a kinetic fashion by enhancing the probability of homologous sequences finding each other, or in a structural fashion by forming a repeating chromatin conformation that promotes stable interhomolog bonding. However, while sequence repetition may contribute to pairing capacity, it clearly does not suffice to guarantee pairing participation. As noted earlier, satellite sequences in the proximal X hetcrochrotnatin and Ste repeats in the euchromatin do not promote X-Y pairing despite the presence of homologous sequences on the Y. Satellite sequences also compose much of the heterochromatin of chromosome 2, another region that has been shown to lack pairing capacity. The lack of pairing function of all of these repeats could reflect transcriptional inactivity, although in the case of Str thew arc other possible explanations, such as insufficient homology (Ste and Su(Sto) repeats are only about 90% homologous) or insufficient copy number. The data at present do not allow us to choose between the interpretation that the exceptional potency of the rDNA and h i s loci with respect to pairing results from a combination of transcriptional activity and sequence repetition or, alternatively, that it results from an unusually high density of very active transcription units. repetition being irrelevant.
2. Comparison of Male and Female Pairing Sites There are some important differcnccs between male and female meiosis in D m sophila with respect to the rules for participation in pairing. There are two distinct types of pairing in female meiosis: recombinational pairing (pairing that leads to recombination), which is limited to the euchrotnatic arms of the three large chromosomes (X, 2 , and 3). and distributive pairing (pairing that promotes segregation of achiasmatic homologs), which seems to be primarily heterochromatic (Grell, 1976; Hawley ct d., 1993, Dernburg, Sedat, et ml., 1996).The heterochromatin of both the X and fourth chromosomes has been shown to contain “distributive” pairing sites (Haley et NI., 1993; Dernburg, Sedat, et d., 1996). The involvement of autosomal heterochromatin in achiaamatic pairing is a clear difference between the male and female systems. Although the X heterochromatin is involved in achiastnatic pairing in both sexes, there are important differences here as well. In particular, the satellite-rich region near the X centromere has no pairing activity in males but is highly active in females (Karpen rt al., 1996), whereas the rDNA contains the major pairing site in males but appears to be inactive in females (Hawley, 1988). The most significant similarity between the pairing systems in the two sexes
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would seem to be the reliance on euchromatic homology for pairing of the major autosomes. In both sexes, euchromatic pairing can occur over short regions of homology (Craymer, 1981; McKee el d.,1993), and there is at least some relationship between pairing efficacy and length of homology. It is unclear, however, how similar the rules for participation are. It seems unlikely that the apparent participation of tandemly repeated euchromatic sequences, such as the histones, in male meiotic pairing will prove to be common to the female system as well, though definitive evidence on this point is lacking. There is also no evidence concerning possible roles of promoter sequences or transcription in meiotic pairing in females, except that distributive pairing seems to involve sequences that have no transcriptional capacity of their own.
I 11. Chromosome Pairing and Spermiogenesis A. Sex Chromosome Rearrangements and Spermiogenic Disruptions
1. Meiotic Drive
a. Sex Chromosome Rearrangements and Distorted Sperm Recovery Ratios. Deletions of Xh that disrupt X-Y pairing and disjunction also cause distortion of recovery ratios of reciprocal sex chromosome classes (Fig. 6A). From Xh-/Y males, progeny that inherit the paternal X are recovered in substantial excess of progeny that inherit the paternal Y, and progeny derived from nullo-XY sperm are recovered in great excess (as much as 100-fold)over progeny from XY sperm (Gershenson, 1933; Sandler and Braver, 1954; Peacock, 1965; McKee, 1984; McKee and Lindsley, 1987). These non-Mendelian recovery ratios are referred to as meiotic drive. Sex chromosome meiotic drive has been most extensively studied in males carrying the sc4-sc8 chromosome, but it is present to one degree or another during spermatogenesis in males carrying any of the rDNA-deficient X chromosomes (McKee and Lindsley, 1987). The patterns are consistent with drive acting both against the X and Y in these genotypes, although drive against the normal Y chromosome is clearly greater than that against the heterochromatically deficient (and shorter) X chromosome. Addition of a heterochromatic free X duplication, such as D p ( l ; f ) 3 (Fig. 6B), to an Xh-/Y genotype alters the pairing patterns but not the drive patterns. The Dp outcompetes the Xh- chromosome for Y pairing sites and segregates regularly from the Y, but substantial drive against both the Y and the univalent Xh- is still observed (McKee, 1984; McKee and Lindsley, 1987). Certain other sex chromosome rearrangements cause similarly distorted recovery ratios. In males heterozygous for an X-4 translocation T(1;4)B,’ with breakpoints in the proximal X euchromatin and near the tip of 4R, the two halves of the translocation disjoin regularly and independently from the Y and normal chromosome 4 (the XP4” element from the Y and the 4pX” element from the normal 4),
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A
B
D
Y
Y s X . Y ,' In(1)EN
k & y I
1
Some genotypes associated v,ith meiotic drive. ( A ) Xh-/Y. (B) X h b / Y D p . (C) T( I :4). ( D ) Attached-XYiDp. Crosshatching reprehenis rDNA.
Fig. 6
but both bivalents exhibit substantial distortion of recovery ratios (Fig. 6C). As in Xh--induced meiotic drive, it is the larger member of each pair that exhibits the reduced recovery (Novitski and Sandler, 1957). Other X-4 translocations with breakpoints in the proximal third of the euchromatin exhibit similar distortion (McKee, 1987). Males heterozygous lor the attached-XY chromosome YSX.YL,ln(l)ENand some (but not all) free X duplications induced by X-ray deletion of the X euchromatin exhibit strong meiotic drive, which again acts against the larger member of the pair, the attached-XY (Fig. 6D) (Lindsley and Sandler, 1958). Meiotic drive has also been documented in males carrying the compound-2-entire (C(2)EN)chromosome, which contains two copies of 2L and two copies of 2R, all attached to a single centromere in the order 2R2L.2L2R (Novitski et al., 1981). C(2)EN is transmitted at frequencies as low as 1-2% in sperm from C(2)EN/O males, but maternal transmission rates are normal (Novitski et d . , 1981; Dernburg, Daily, e f d., 1996). Although C(2)EN is not a sex chromosome, it contains Y-derived heterochromatin at the junctions between 2L
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and 2R on each arm, and, as developed below, there are reasons to think that the sex chromosome material is responsible for the distortion.
b. Meiotic Drive Results from Spermatid Elimination and/or Sperm Dysfunction. In Xh-/Y males, meiosis itself is not distorted, since reciprocal secondary spermatocyte classes are present in equal ratios (Peacock and Miklos, 1973; McKee and Lindsley, 1987; McKee and Karpen, 1990). Spermiogenesis appears relatively normal in sc4-sc8 males until the individualization stage, at which time the waste bag, which normally accumulates cellular debris from the individualization of previously syncytial spermatids, accumulates substantial numbers of abnormal-looking spermatid tails; in addition, many bundles of postindividualization spermatids contain syncytial spermatids, presumably destined for elimination. The frequency of such abnormal spermatids is positively correlated with the severity of meiotic drive among a sample of males that vary for that factor, indicating that the spermatid lethality is a likely cause of distortion (Peacock et al., 1975). A plausible alternative is that distortion results not from spermatid lethality but from preferential function of the favored sperm following insemination. This idea is supported by evidence that the number of progeny produced by females mated to .rc4-sc8males raised at 25°C (at which temperature nondisjunction and distortion levels are both high) is substantially lower than the number produced by females mated to sc4-sc8 males raised at 18°C (low nondisjunction and distortion rates). Importantly, the numbers of stored sperm present in the ventral receptacles and spermathecae of females recently mated to the same males are very similar (Peacock et af., 1975). Thus, perhaps the various classes of sperm are transferred and stored at equal frequency but differ in efficiency of fertilization. It has recently been shown that distortion in C(2)EN/O males results from differential sperm storage in the female reproductive tract; the frequencies of C(2)EN and nullo-2 sperm are only slightly different in testes and among recently transferred sperm but are present at highly unequal frequencies, comparable to the progeny ratios, in the female storage organs (Demburg, Daily, et al., 1996). Thus, sperm selection can apparently occur at various stages before and after transfer to the female. c. Lack of a Specific Target in Sex Chromosome Meiotic Drive. A remarkable feature of the cases of meiotic drive reviewed above is the apparent lack of a specific “target.” In other well-studied examples of meiotic drive, such as those associated with the Drosophilu Sd locus or the murine t complex, sensitivity to meiotic drive is a property associated with a particular allele or complex of alleles ( R s p and t + , respectively) that have distinct genetic locations (Lyttle, 1993). However, in sex chromosome meiotic drive, there is no evidence for such a target. Instead sensitivity seems to be a widespread or universal property, so that the probability of recovery of a sperm class from such males is
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inversely proportional to its chromatin content. In Xh-/Y males, sperm viabilities vary in the order: Xh-Y < Y < Xh- < 0 (Sandler and Braver, 1954; Peacock, 1965; McKee and Lindsley, 1987). In T(1;4) males, the smaller member of each of the two bivalents (the Xl’4” element from the XP4”:Y bivalent, the and normal fourth chromosome from the 4”X”:4 bivalent) is recovered in excess of the larger member (Novitski and Sandler, 1957). In the cases of meiotic drive associated with compound chromosomes (attached-XY/Dp or C(2)EN/O),it is the sperm carrying the large compounds that are recovered poorly (Lindsley and Sandler, 1958; Dernburg, Daily, r t NI., 1996). The relationship between chromatin content and sperm recovery was addressed directly by manipulating the genotype of sc4-sc8/Y males to generate sperm with different amounts of sex or autosomal chromatin. When the Y chromosome was replaced with pairs of Y chromosome fragments, the recovery of those fragments was inversely proportional to their size. When an asymmetric translocation between chromosomes 2 and 3 (T(2;3)bwV4),which generates sperm with three, four, or five maior autosome arms, was introduced into the sc4sc8/Y genotypes, sperm recovcrics proved to be inversely proportional to the number of autosome arms. This was true for all sex chromosome classes, even the nullo-XY, proving that autosomal as well as sex chromatin can act as a “target” in this type of meiotic drive (McKee, 1984). It is important to note that this sort of dependency of sperm function on karyotype is not the usual case in Drosophilu spermatogenesis. Sperm with no chromosomes at all except for the tiny fourth chromosome are functional, and chromatin-rich sperm classes suffer no unusual recovery deficit as long as thcy are derived from males with normal sex chromosomes (Lindsley and Grell, 1969; McKee, 1984). Thus, the inverse relationship between sperm function and chromatin content in Xh-/Y males is related in some way to the absence of X heterochromatin.
d. Role of X-Y Pairing Sites in Meiotic Drive. Since all of the Xh deticiencies that are associated with meiotic drive are quite large, the fact that they also cause nondisjunction need not imply that a single locus controls both phenotypes; there could be several distinct loci with spermatogenic phenotypes clustered in Xh. However, three lines of evidence strongly support a single-locus model for control of these phenotypes. One is that among the rather large number of Xh deficiencies that have been described over the past 60 years (none of which has been selected for spermatogenic phenotypes), not one has been found to induce X-Y nondisjunction without also inducing meiotic drive (McKee and Lindsley, 1987). Second, there is a strong correlation between the frequency of nondisjunction and the severity of meiotic drive. Males that carry the deficiency .sc4-.sc8 vary quite dramatically in both nondisjunction frequency and drive level, depending on genetic background and on temperature during meiosis (Peacock, 1965; Peacock and Miklos, 1973; Peacock ef ul., 1975), and the two variables are very strongly (and positively) corrclated. The correlation between nondisjunction
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and drive also holds across a sample of Xh deficiencies that exhibit a broad range of X-Y nondisjunction frequencies (McKee and Lindsley, 1987). Finally, the partial restoration of pairing ability associated with X-linked insertions of complete rDNA genes on Xh- chromosomes is accompanied by a dramatic amelioration of drive in Xh-/Y males; recovery of the Y chromosome improves from less than 10% to nearly 50% in Df(I)X-I/BSYvi males carrying a single inserted rDNA gene, and to 75% when two copies are present (McKee and Karpen. 1990). Moreover, the same rDNA fragments that have been shown to function as pairing sites-arrays of 240-bp IGS repeats-also mediate the amelioration of distortion; the degree of drive is as strongly (inversely) correlated with the number of 240-bp repeats as is the frequency of nondisjunction. Conversely, rDNA fragments that lack pairing activity, such as the large transcription unit fragment (Ren et al., 1997) and the array of promoter-mutant 240-bp IGS repeats discussed earlier, have no effect on drive (B. D. McKee, W. I. Sun. and C. Merrill, unpublished observations). These data provide strong evidence that a single cis-acting Xh locus that consists of the 240-bp IGS repeats from the rDNA genes is responsible for pairing and for preventing meiotic drive. In light of these findings, it seems likely that the presence of Y-derived pairing sites in the C(2)EN chromosome is responsible for the meiotic drive seen in C(2)EN/Omales. C(2)EN tests positive for the presence of rDNA, disjoins regularly from an attached-XY when that is the only sex chromosome present in the genome, and often disjoins from both sex chromosomes when together with a free X and Y (Falk, 1983). Recent data suggest an association between the frequency of C(2)-XY disjunction (leading to a male-biased sex ratio among C(2)-bearing progeny) and the degree of meiotic drive among a group of three C(2)EN variants with different levels of drive (Dernburg, Daily, et al., 1996). The structural basis for the differences among C(2)EN variants is not known, but the correlation between X-Y pairing capacity and severity of drive among the variants is clear.
2. Chromosomal Sterility a. X-Autosome Translocations. Many rearrangements that involve sex chromosomes cause male-specific sterility. The most extensively studied examples are X-autosome translocations, which are known to cause male sterility in flies, mice, and humans. Complete male sterility is associated with at least 75% of reciprocal translocations between the X and one of the major autosomes in Drosophila (Fig. 7A) (Lifschytz and Lindsley, 1972) and 100% of such translocations that have been identified in mice and men (Handel, 1987).In Drosophila, the fertile exceptions fall into two classes: those with relatively terminal breakpoints on both the autosome and the X and those with breakpoints within or proximal to the NO in Xh (Lifschytz and Lindsley, 1972). X-A translocations are associated with unique cytological phenotypes. In Drosophila, spermiogenesis is
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Y
YPAD
X
YPAD
X
APXD
A APYD
A
APYD
A
D
X
I y + ma1 ~
+
Fig. 7 Some genotypes aswciated with chromosomal sterility. ( A ) X-2 or X-3 translocation. ( B ) Y-2 or Y-3 translocation. absolutc \terility. (C) Y-2 or Y-3 translocation, synthetic Ykrility. (D) Xh- I?’. Ynicil’ .
arrested at the sperm head elongation stage, producing the so-called pin-head phenotype (Shoup, 1967); both the normal elongation of the nucleus and the accompanying replacement of lysine-rich histones with arginine-rich histones are lacking. In mammals, all known X-A translocations cause arrest of meiosis during prophase or metaphase of the first division in males (Handel, 1987).
b. Y-Autosome Translocations. Y-autosome translocations also cause sterility in Drosophila and mammals. In Drosophila, there are two classes of “dominant’’ T(Y;A) sterility, one class consisting of translocations that are sterile only when the X is deficient for a large fraction of the heterochromatin (synthetic sterility, Fig. 7C) (Besmertnaia, 1934; Lindsley et a/., 1979; Lindsley and Tokuyasu, 1980) and the second class consisting of translocations that are sterile irrespective of X karyotype (absolute sterility, Fig. 7B) (Kennison, 1983). Both classes are dominant with respect to the Y because the presence of an extra Y does not restore fertility. They are thus distinguished from cases of recessive
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sterility associated with interruption of a Y fertility gene (Hardy et al., 1981; Gatti and Pimpinelli, 1983). There are also some Y-A translocations that do not cause either type of dominant sterility, but they are a small fraction consisting mostly of translocations with very distal autosomal breakpoints (Lindsley et al., 1979). The absolute class of T(Y;A) sterility has been estimated at 55% of an unselected sample (Kennison, 1983) but otherwise has not been studied. The synthetic type of sterility is interesting because the Xh deficiencies that cause it are the same ones that in an otherwise normal genotype cause distortion (Linds ley et ul., 1979), namely, deletions that encompass the rDNA (see later discussion). There is also a report that combining T(1;4)BS, a strongly distorting X-4 translocation, with a Y-autosome translocation results in sterility (Stone, 1984). It is not known what feature(s) distinguishes the absolute from the synthetic class of Y-A translocations. However, the distribution of Y chromosomal breakpoints among the synthetic class is strongly biased toward terminal and near-terminal sites (Gatti and Pimpinelli, 1983), suggesting that those with more internal breakpoints may be overrepresented among the absolutely sterile class. Although cytological data have not been published, it is reported that Xh-/T(Y,A) testes exhibit the same defect in sperm head elongation seen in testes of T(X;A) males (Lindsley and Tokuyasu, 1980). This strongly suggests that the mechanisms of X-A and Y-A translocation sterility are closely related.
c. The y+YmaZ+ Chromosome. One other example of synthetic chromosomal sterility in Drosopliila has been described. Males carrying the X-Y transposition y + Ymal+,in which a substantial fragment of proximal X encompassing both euchromatin and heterochromatin is inserted into the Y, are fertile in the presence of a normal X but sterile in the presence of Xh deficiencies (Fig. 7D). The sterilizing effects of y + Ymd+ can be partly reversed by removal of some of the inserted X chromatin (Rahman and Lindsley, 1981). Again, the same Xh deficiencies that induce sterility in the presence of y + Ymu/+also cause distortion in otherwise normal k q o t y p e s (Rahman and Lindsley, 1981; McKee and Lindsley, 1987). Moreover, the same X-4 translocations that cause distortion have also been reported to interact with y+Ymul+ to cause sterility (Stone, 1984). These observations suggest that y + Ymal+-induced sterility is closely related to T(Y;A) sterility. Otherwise, however, it is unclear how this genotype relates to the sex chromosome-autosome translocation genotypes. d. Role of Sex Chromosome Pairing Sites in Chromosomal Sterility. Two of the sterile phenotypes discussed earlier involve Xh deletions, and thus it is natural to ask whether the X pairing site is the deleted factor responsible for causing sterility in these genotypes. The Xh deletions that cause both Xh-/T(Y;A) sterility (Lindsley et al., 1979) and Xhk/y+ Ymal+ sterility (Rahman and Lindsley, 1981; McKee and Lindsley, 1987) are all rDNA deficient, and are the same deficiencies that cause X-Y pairing failure and meiotic drive in
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Xhk/Y males, which argues strongly that the pairing sites are the important factors. Compelling evidence for this view comes from phenotypic analysis of transgenic rDNA insertions. Insertions of complete rDNA genes on 1)X-l rescued fertility in combination with J Y n d + (McKee, 1991) and in combination with eight out of nine Y-autosome translocations that are sterile in the absence of a ribosomal insertion. Moreover, insertions of rDNA fragments containing 240-bp IGS repeats on Dfl1)X-lalso rescued fertility in combination with a Y-autosome translocation (McKee et LII., 1997). Thus, synthetic chromosomal sterility in Drosophila is caused in part by X-Y pairing failure.
of(
+
e. Relationship of Chromosomal Sterility to Meiotic Drive. The overlap in the types of rearrangements that cause chromosomal sterility and meiotic drive, combined with the evidence that X-Y pairing failure is involved in the etiology of both effects, has led us to suggest that they should be viewed as different levels of a common sperm dysfunction syndrome rather than as qualitatively distinct phenotypes. In addition to the genetic data cited above, this view is supported by the fact that the level of meiotic drive is correlated (inversely) with fertility. The most severely driving Xh deficiencies, such as D f l I ) X - l , reduce offspring number quite markedly; for example, under our standard experimental conditions, which involve mating to two females and counting the progeny that emerge over a period of approximately 20 days, Df(l)X-l/B.yYy+ males produce an average of 14 progeny, compared to almost 100 for wild-type males. Insertions of single rDNA repeats improve fertility to an average of 42 progeny per male (B. D. McKee, K. Wilhelm, C. Merrill, and X.-J. Ren, unpublished observations). Drive levels arc also inversely correlated with fertility in sc4-sc8 males (Peacock et al., 1975). These similarities suggest that the two phenomena are closely related in mechanism.
B.
Mechanisms of Meiotic Drive and Chromosomal Sterility
1. Pairing Site Saturation How is pairing failure related to meiotic drive? One suggestion has been that “unsaturated” pairing sites on the sex chromosomes of Xh-/Y males act as “undefused bombs” that kill spermatids unlucky enough to inherit them (Baker and Carpenter, 1972; Peacock and Miklos, 1973). Pairing is supposed to result in a change in epigenetic state of the pairing site sequences that is essential to prevent them from functioning as spermatid lethals. This idea accounts for the especially poor recovery of XY sperm relative to the nullo-XY class, as all the bombs would end up at the XY pole, and for the excess of X over Y sperm, since the Y would be expected to have more unsaturated sites than a heterochromatically deficient X. The idea also accounts for the correlation between nondisjunction and distortion in .sc4-.sc8males raised at different temperatures or
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carrying different genetic backgrounds and in males carrying Xh deletions of different sizes, because these features have been shown to affect the frequency of X-Y pairing. The pairing site saturation model has been generalized to account for associations in a variety of organisms between chromosome rearrangements that disrupt pairing and partial or complete sterility (Miklos, 1974). The model has also been invoked recently to account for distortion in C(2)EN/O males in Drosophila, the argument being that the C(2) chromosome, which is known to carry Y-derived pairing sites, may often have those sites unsaturated (Demburg, Daily, rt d., 1996). However, several observations argue both against this specific model and against the more general idea that meiotic drive is a direct consequence of X-Y pairing failure. First, complete deletions of the X pairing site, such as in the Dj(1)X-/ chromosome, do not prevent drive against the X, despite the fact that such complete deletions should produce a bomb-free chromosome. Instead, drive against such X chromosomes is actually more severe than is drive against X chromosomes carrying partial pairing site deletions, such as sc4-,~c8(McKee and Lindsley, 1987). Second, distortion is not substantially ameliorated by addition of a free X duplication ( D p ( / , f ) 3 carrying ) all of Xh to an Xh-/Y genotype (Fig. 6B). The Dp is recovered substantially in excess of the Y, from which it disjoins regularly, and the univalent X is recovered in well under half of the progeny. Thus, despite providing a pairing partner for the Y and presumably “saturating” its pairing sites, addition of a free Dp fails to eliminate drive. Third, drive is present in other genotypes in which X pairing sites are present and there is n o evidence of pairing failure. As noted above, some X-4 translocations and several attached-XY/Dp combinations exhibit meiotic drive very comparable to that in Xh-/Y males. In some of these cases, X-Y disjunction has been shown to be regular (Novitski and Sandler, 1957; Lindsley and Sandier, 1958). These observations indicate that pairing failure is not a prerequisite for meiotic drive and instead suggest that it is only one of several conditions that can trigger it. The difficulties mount when the idea of pairing site saturation is applied to chromosomal sterility. X-Y pairing failure is certainly part of the explanation for sterility of Xh-/T(Y;A) or Xh-/?+ Y m r l i - males, but the pairing site saturation model does not fit the data very well. One difficulty is that Xh deletions alone do not suffice to cause sterility, even when, based on disjunctional behavior. the X pairing sites are completely deleted, and the Y pairing sites should thus be completely unsaturated. Another difficulty is that addition of a full dose of X pairing sites in trans in the form of the free X duplication D p ( / ; f ) 3 does not restore fertility of Xh-/T(Y;A) males (Besmertnaia, 1934). Finally, it is unclear how the model applies to the absolute sterility associated with many X-A and Y-A translocations. In these cases, sex chromosome pairing sites are present and intact, and at least some X-A translocations have been reported to form the expected quadrivalents (Stone, 1984), although it must be admitted that very little cytological analysis of meiotic behavior of these rearrangements has been carried
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out. Nevertheless, there is nothing in the pairing site saturation model that would lead us to expect either X-A or Y-A translocations to cause sterility. Given these observations, it seeins likely that the relationship between X-Y pairing failure and spermiogenic disruption is not as direct as postulated in the pairing site saturation model. Instead, meiotic drive and sterility are more likely consequences of some defect downstream of pairing failure, such as misalignment of univalents or certain types of multivalents at inetaphase. This idea would allow for other types of rearrangements that also might disrupt meiotic behavior without causing actual pairing failure to generate phenotypes similar to those of Xh deficiencies. This idea is developed in more detail in Section 3.
2. X-Inactivation Some X-autosome translocations with breakpoints in proximal Xh are fertile, whereas all other X-autosome translocations, except those with quite distal breaks on both chromosomes, are sterile. This observation stimulated the idea of a cis-acting site in Xh that functions to inactivate the X euchromatin during meiosis; separation of this site from the euchromatin was postulated to be responsible for the sterility associated with X-autosome translocations (Lifschytz and Lindsley, 1972). This hypothesis has the major virtue of being based on a wellestablished fact, namely, that i n ;I wide variety of heterogametic males, the X chromosome is rendered heterochromatic and transcriptionally inactive in spermatocytes (McKee and Handel, 1993). The function of this intriguing phenomenon has been mysterious and controversial. A recent review concluded that there is no basis for believing that X-inactivation serves a gene regulatory function, and that a more plausible interpretation is that i t functions to repress pairing/recombination activity of X- and Y-linked sequences that lack appropriate pairing partners. Such repression would serve to prevent ectopic pairing and recombination of such genes with repeated sequences elsewhere in the genome and prevent the appearance of possibly irreparable double-strand breaks (McKee and Handel, 1993). Dmsophilri males lack recombination and, therefore presumably, double-strand breaks, but still face the problem of ectopic pairing because the sex chromosomes are largely heteromorphic. There are two difficulties with applying these ideas to Drosophih. The first is that it is not clear whether X-inactivation occurs in Drosophila spermatocytes. Unlike in most heterogametic males, the sex chromosomes and autosomes are not obviously allocyclic. As discussed earlier in this chapter, all chromosomes are decondensed and, apparently, transcriptionally active, throughout most of the spermatocyte growth period. They then condense, rapidly and approximately synchronously, shortly before entering into prometaphase (Kremer ef d., 1986; Cenci ef a/., 1994). Although little is known about specific expression patterns of X-linked genes during this period, Y genes are known to be heavily expressed throughout much of primary spermatocyte development. Indeed, primary sper-
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matocytes are the only cells in which substantial fractions of the Y chromosome are transcriptionally active. It has been suggested that there may be a subtle difference in the timing of inactivation of the sex chromosomes and autosomes (Lifschytz and Lindsley, 1972). This is certainly possible, but is not easy to test. The second difficulty is that attempts to map the putative cis-inactivation site have been unsuccessful. There is no site in Xh for which removal causes absolute sterility, since neither Xh deficiencies nor translocations between the X and the Y or fourth chromosomes are absolutely sterile. However, the data do point to a cisacting site in Xh that plays some role in spermiogenesis. Both Xh deficiencies and X-4 translocations cause distortion and contribute to synthetic sterility. Thus, if we think of the distortion and sterility phenotypes as a complex of spermiogenic abnormalities arrayed along a continuum of severity, as suggested earlier, the data in support of a cis-acting site in Xh are quite consistent. If there is a cis-acting site in Xh required for normal spermiogenesis, what is it and what does it do? Evidence outlined earlier provides strong reasons to believe that the cis-acting site involved in both meiotic drive and synthetic sterility is the pairing site. Could the pairing site also be the site involved in T(X;A) sterility'? There are two reasons to think that it may be. First, the only translocations involving the X that do not disrupt spermiogenesis are X-Y translocations, which are also the only translocations that leave the X euchromatin in cis with an rDNA pairing site. Second, preliminary data indicate that the fertile X-A translocations with Xh breaks have their breaks proximal to those that are sterile, probably within or proximal to the rDNA (Lindsley and Tokuyasu, 1980). If so, then the fertile translocations could be those that do not separate the X euchromatin from the X pairing site. Two plausible explanations can be offered for how separation of the bulk of the X euchromatin from the pairing sites might disrupt spermiogenesis. The first and simpler explanation is that the X pairing sites do nothing but pair, and that separation of the euchromatin from the pairing sites is disruptive because it interferes with chromosome alignment at metaphase. This idea is developed in more detail in the next section. An interesting but more complex alternative is that very strong pairing sites such as the IGS repeats might inhibit pairing activity of sequences in cis, a phenomenon similar, perhaps, to the inhibiting effect of crossovers at one site on a chromosome on other crossovers in cis (interference). In this case, the &-repression would serve, presumably, to prevent ectopic pairing of sequences that lack natural pairing partners. This conjecture predicts that release of pairing inhibition through deletion of the pairing site or separation by translocation should lead to involvement of the X in heterologous associations. 3. A Metaphase Checkpoint Model
An alternative that has not been considered is that the various types of rearrangements discussed in the preceding paragraphs all disturb metaphase alignment and trigger a checkpoint that acts via arrest of spermiogenesis or generation ofdycfunc-
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tional sperm. Data from vertebrates and insects show a metaphase checkpoint sensitive to chromosome misalignment that operates both in mitosis and in meiosis. In grasshopper and mantid spermatocytes and in mammalian mitotic cells, failure of bipolar attachment of even one chromosome or pair leads to metaphase arrest by a mechanism thought to involve a signal transduction system that monitors kinetochore tension (Li and Nicklaa. 1995; Nicklas et ul., 199.5; Rieder et ul., 199.5). Tension results from bipolar attachment and leads to quenching of a kinetochore phosphoepitope thought to be a component of a signaling pathway that prevents entry into anaphase until all chromosomes have achieved metaphase alignment. Lack of bipolar attachment does not cause metaphase arrest in Drosophila. Univalents such as the unpaired X and Y in Xhk/Y spermatocytes oscillate unstably between the poles and usually fail to achieve stable bipolar attachment (Church and Lin, 1988), but anaphase proceeds despite these difficulties. Nevertheless, there is some evidence for a tension-sensing mechanism in male meiosis. The ZWlO protein, which is required for proper anaphase segregation in both mitosis and meiosis, is redistributed from the kinetochores of prometaphase chromosomes to the spindle niicrotubulcs at metaphase when normal chromosomes achieve bipolar orientation. However, ZW 10 protein remains attached to the kinetochores of univalent chromosomes at both metaphase 1 and 11, suggesting that the redistribution in normal chromosomes is a response to the state of tension resulting from bipolar orientation. Consistent with this view, ZW 10 redistributes back to the kinetochores a1 the onset of anaphase when tension is released (Williams et al., 1996). In light of these observations, we have proposed that Drosophilci spermatocytes have a metaphase checkpoint that, when triggered, results in a general disabling of the spermatids that result from affected spermatocytes (McKee et al., 1997). This checkpoint would be sensitive to the same types of meiotic errors, such as univalents and other types of configurations that fail to achieve bipolar orientation, that the metaphase checkpoint in grasshopper and mantid spermatocytes detects, and thus would guard against transmission of aneuploid gametes. However, instead of being used t o trigger a “wait anaphase” response, the signal associated with untense kinetochores would trigger a pathway leading to disability of the resulting spermatids. Depending on the degree of disability, the consequence could be arrest 0 1 spcrmiogenesis as early as head elongation or as late as individualization, or it could be mature sperm that have reduced ability to function after insemination. The checkpoint idea accounts well for the two distinctive features of sex chromosome meiotic drive reviewed earlier; the lack of a specific target and the causal relationship between X-Y pairing failure and meiotic drive. The nonspecific target suggests a general disability that affects all sperm classes (although unequally), rather than a poison produced by the drive-inducer that binds to a specific target site on sensitive chromosomes. Such a general disability, in turn, is more consistent with a checkpoint response to a meiotic error than with the cheating behavior of a selfish genetic element. The connection with pairing
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failure can be explained by the prevalence of univalents in genotypes such as Xh-/Y. The univalent sex chromosomes in such genotypes have been shown to oscillate unstably between the poles, never achieving bipolar orientation (Ault and Lin, 1984; Lin e t a / . , 1984; Church and Lin, 1988). Addition of pairing sites to the Xh- chromosome enhances the bivalent frequency and thus is expected to increase the frequency of sex chromosome bipolarity. The checkpoint model also accounts for the failure of pairing sites added in trans (in the form of a heterochromatic free X duplication) to an Xh-/Y genotype to suppress meiotic drive, because the Xh- chromosome in the resulting Xh-/Y/Dp genotypes behaves as an obligate univalent (McKee, 1984; McKee and Lindsley, 1987). The checkpoint model might also account for some of the features of the chromosomal sterility data, although here the explanations are somewhat more tenuous. For example, the ability of pairing site insertions on Xh- to rescue sterility in Xh-/T(Y;A) males (B. D. McKee rt af.,unpublished observations) could result from a “normalization” of the pairing patterns, i.e., substitution of two bivalents for the 3 + 1 (trivalent plus univalent) pattern likely to prevail in the absence of inserted pairing sites. Fertility rescue has been possible only for translocations with quite distal autosomal breakpoints. The trivalent in these translocations is held together only by the small pairing region between the YPAD element and the unrearranged autosome. It is possible that the addition of pairing sites to the Xh- chromosome would allow it to compete with the rearranged autosome for pairing with the YI’A” element, and sometimes to resolve the 3 + 1 configuration into two bivalents. I t seems likely that two bivalents would be more likely to achieve bipolarity than would a trivalent. A similar explanation might account for the fertility of some X-autosome translocations that have relatively distal autosomal breakpoints (Lindsley and Tokuyasu, 1980). Again, the multivalents (quadrivalents in this case) depend on the short pairing region between the XI’AI)element and the unrearranged autosome, and might sometimes resolve into two bivalents. This explanation is consistent with the lack of sterility associated with X-4 translocations, which typically segregate as two bivalents rather than as a quadrivalent (Novitski and Sandler, 1957). The behavior of multivalents during meiotic prophase and metaphase has not been examined in Drosophila spermatocytes. Although trivalents and quadrivalents have been shown in some systems to achieve bipolarity (Rickards, 1983), the situation in Drosoplzilu spermatocytes might be different. Even wild-type bivalents have considerable difficulty achieving bipolar orientation: they move chaotically and make and break connections to both poles numerous times before settling down at the metaphase plate (Church and Lin, 1985). Given this instability, along with the lack of “wait anaphase” response, it is plausible that some kinds of rearrangements might exacerbate the instability sufficiently to cause the multivalents to still be in flux at anaphase and thus trigger the checkpoint. This type of problem has been identified in sc4-s1WY males, in whom bivalents form at variable frequencies. These bivalents often fail to achieve bipolar orientation by the end of metaphase: homologous kinetochores sometimes face the same
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pole and sometimes face neither pole (Ault and Lin, 1984). Thus, pairing is no guarantee of achieving bipolarity. and it would not be surprising if more complex rearrangements also often failed to achieve bipolarity despite being paired. The checkpoint hypothesis raises some questions that cannot be definitively answered at this time. One is why sex chromosome-autosome translocations often trigger the checkpoint but autosome-only translocations rarely do. It seems likely that the explanation lies in differences between the behavior of the types of multivalents that form in the two cases. but the basis for such differences is not clear. Such differences perhaps could result from effects of the large unpaired regions necessarily present in multivalents involving the sex chromosomes, or from the unusually tight linkage between pairing sites and centromeres in the sex chromosomes, which may inhibit the reorientational behavior needed to achieve bipolarity. Careful cytological comparisons of chromosome behavior in various fertile and sterile rearrangements might provide clues to the underlying mechanism. Another question concerns the nature of the disability imposed on the spermatids derived from defective meioses. In light of the variety of developmental stages at which selection has been shown to occur, the disability must be rather general in nature. At low to moderate levels of disability, selection acts at stages likely to involve intergamete competition: during individualization, when all sperm are invested in membranes essentially simultaneously and any developmental kggards can be eliminated. or after insemination, when sperm function becomes subject to direct selection. In meiotic drive phenotypes, the favoring of sperm classes with reduced chromatin may result from differences in rate of completion of nuclear reshaping. with the inore chromatin-rich classes tending to lag, or from differences i n the vigor of transferred sperm, the more chromatinrich sperm being more sluggish than their lighter cyst-mates. Whatever the nature of the advantage held by nullo-XY sperm over the other products from abnormal meioses, such sperm are presumably at a disadvantage relative to sperm from normal meioses. This interpretution predicts that chromosomally normal males should exhibit an efficient and relatively unbiased mechanism for eliminating sperm from abnormal meioses. a mechanism that could be evident from a cornparison o f nondisjunction frequencies measured cytologically during meiosis versus from progeny counts. It also predicts that the competitive advantage of nullo-XY sperm over more chromatin-rich classes should decrease as the ratio of abnormal t o normal meioses falls.
IV. Summary and Implications A. Pairing Sites and the Mechanism of Pairing
The last few years have seen considerable progress in the localization and characterization of meiotic pairing sites in Drowphi/u males. Cytogenctic data indicate that autosomal pairing sites are distributed broadly within the euchroniatin but
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are absent from the heterochromatin, and that a strong pairing site coincides with the histones locus. From a combination of cytogenetic analysis and transgenic methodology. it has been learned that the major X-Y pairing site, long known to be in the heterochromatin. consists of ;I repeated sequence found i n the intergenic spacers between each rDNA transcription unit. The finding that the R N A polymerase I promoters present in each spacer repeat must be functional for the repeats to have pairing activity provides strong evidence for a mechanistic relationship between transcription and meiotic pairing in Dt-osoplzil~i.The nature of this link has not yet been determined. but the findings raise the intriguing possibility that nascent transcripts play a direct role in homologous interactions in meiosis. Taken together. these findings suggest that pairing sites may generally consist of transcribed sequences. arid that pairing site strength may be a function of the density o r activity ( o r both) o f transcription units.
B. Pairing and Spermiogenesis Chromosoine rearrangements involving the sex chromosomes have been found to interfere with spermatogenesis in ways that are often quite mysterious. In L~rwsophiltithe phenotypic effects o f such rearrangements range from complete sterility accompanied by arrest of spermiogcnesis at the head elongation stage to distorted sperm recovery ratios. Some of these effects are caused wholly or in part by deletion of X hcterochroniatin. We have shown that in three cases of spcrmiogenic disruptions involving deticiency of X heterochroinatindistortion in Xh / Y males and synthetic sterility i n Xh /T(Y;A) o r X h / is the absence of X pairing sites that is responsible for J Yniril' males-it the effect o f the Xh deticiency. This tinding indicates that X-Y pairing failure is one cause o f both distortion and chromosomal sterility. However. other genotypes in which X-Y pairing occurs normally, such as X-autosome and Y-autosome translocations, can experience the same spertniogenic disruptions. Thus. it seems unlikely that the relationship between X-Y pairing failure and distortion or sterility is a direct one. as postulated in the pairing site saturation model. Instead. we have suggested that the conimon feature of the diverse sex chromosome rearrangements that cause distortion and sterility is a failure to achieve stable alignment at metaphase. and that the consequence o f such failure is triggering of a checkpoint that leads to arrest o r partial arrest o f spermiogenesis. We have proposed that this checkpoint may utilize information concerning the state of tension on kinetochores that has been shown to trigger a "wait anaphase" response in other type of cells. This hypothesis generates a number of predictions mentioned in the chapter; hopefully it will stimulate renewed interest in cytogenctic analysis of the mysterious effects of this class of rearrangements. In light of the taxonomically widespread evidence for effects of sex chromosome rearrangements on spermatogenesis and of the current interest
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in cell-cycle regulation and checkpoints, this hypothesis clearly has significance well beyond the realm of Dro.sopliilo meiosis.
Acknowledgments Thanks go to W. Bradherry, C.-S. Hong. S. Das. and M. A. Handel for helpful di\cuaaions. and to M. A. Handel and J. Schimenti for rcvicws of the nianumipt. Work from the author's laboratory was supported by NIH grant No. GM40480.
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Peacock, W. J . . Miklm. G . L. G., antl Gootlchiltl, 1). J. (1975). Sex chromosonir ineiotic &ice systems in Drosophila melanoga\tcl. I . Ahiiornial \permatid development in nialer with ii hcterochromatin deficient X chromo\oine (rc4rcX ). C;riretic~,\79, 6 13-634. Radding. C. M. ( 1988). Homologous p;iu ing nncl strand exchange promoted by Ercherichia coli recA protein. Iii "Genetic Recombination" ( R . Kucherlapati and G. R. Smith. Ed\. ), p p I c L 229. American Society for Microhiology. Wa\hiiigton. D.C. Rahman. R., and Lindslcy. D. L. (198 I I . Male-rtcriliring interactions between duplication\ and deficiencies for proximal X-chroniowiic inatel-in1 i i i Drosophila nielanogaster. Gcwc~trc\99, 49-64. Rcn. X.-J., Eisenhour. L.. Hong, C.-S.. Lee. Y., and McKee, B. D. (1997). Roles of rDNA \pacer and transcription unit sequences i n X-Y iiiciotic pairing in Di-o.sophilo ~ J J ~ , / f i i J t ) , ~ f /C'hn~iuo.\f~,r, . Y O f J l ( l 106. 29-36. Rickurds. G. K. ( 1983). Orientation hehaclot- o f chroniosoine multiples of interchange (reciprocal translocation) heteroiygote\. Arviil. Rci.. C ; o w r . 17, 343-498. Ricdcr, C . L., Cole. R. W., Khodjakov. A.. iitid Sluder. G. (1995). The checkpoint delaying anaphase in rerponse to chroinosoiiie iii(iii(i0ricntation is mediated by an inhibitory signal produced by unattached kinetochores. ./. ('rll Brol. 130. 941 -948. Koeder. G. S. ( 1990). Chromosome h y n a p s i h antl genetic recombination: Their role\ in meiotic chromosome segregation. Trerid.s C;erit,r. 6, 385-389. Sandler, L., and Braver, G. ( 1954). The iiiciotic lo\\ of unpaired chromosomca in DJ-o.\o/I/J~/~I rnclnirogtrsrer. G'eJleric.r 39, 365-377. Shevelyov, Y. Y. ( 1992). Copies of a Stellcite gene varinnt arc located i n the X heterochroiiiatin of Drowphiln mekrriogti\rer and are piohahly expi-csscd. Grrirric.c 132, 1033- 1037. Shoup. J. R. (1967). Spermiogenesis in wild-type and ii male \lerility mutant of Drosophiln melanogaster. J. Cell B i d . 32, 663-675. Simeone, A.. La Volpe. A,. and Boncinelli. I:. (19x5). Nucleottde sequence of a complete rib()\()ma1 spacer of D. me/tiriogrister. N i ~ l ( ~ A(,itic rc R c t . 13, 1089- I 101. Stcffenson, D. M., Appels, R.. and PcacticL. W. J . ( 19x1 ). The distribution of two highly repeated DNA requences within Urosophilu r ~ i c , l o i i ~ ~ g t i tchroino\omer. /~'r Chroimmruo 82, 525-54 1. Stone. J. C. ( 1984). Observations o n chroino\omal male sterility in Drosophiln rnel;inoga\tci-. Ctirr. .I. Gerrrt. Cjiol. 26, 67-77. Triutz. D., and Dover, G. A. ( 1986). Traii\cription of the tandem array of ribowma1 DNA i n Drosop/?ilti riieltrnogci.\fer does not terminale at any fixed point. EMBO J . 5, 11-67- 11-73. von Weitstein. D.. Rasmussen. S. W.. a n d Holm. P, B. (1984). The synaptonemal complex i n genetic \egrcgation. A J I J I LRn,. I . G e ~ f 18, . 33 1-4 14. Weiner, B. M., and Kleckner. N. (1994). Chromosome pairing via multiple interstitial iiiteractioiis before and during meiosis in yeart ( b l l 77, 977-09 I . White, M. A., Dominska, M., and Pete\. 7'. D. f 1993). Transcription factors are required I o i - the meiotic recombination hotspot at the HIS4 Iocii\ in Sncc1zcirornyce.s cereiisinc. Pro(c M i t l . Actid. Sci. USA 90, 6621-662s. Williams. B. C.. Gatti, M., and Goldbcrg. M . L. ( 1996). Bipolar spindle attachments affect i-rdistributions of ZW 10, a Drosophila centroiiiere/hinetochore component required for accut-iitc chroniorome segregation. J. C e / / Hrol. 134, I 177- I 140. Yainamoto, M. ( 1979). Cytological sttidie\ o f hetei-ochromatin function in Dro\o/,/rilo i w l c i r w g t w tcr males: Autosomal meiotic pairing. ~ / i ) - [ ) f J i ~ ) , \ t ) f 72, r i ~ ~ 293-328. Yainainoto. M.. and Miklos, G. L. G. ( 1077). Genetic dissection of heterochi-oiiiatin i n Drosophila: The role of basal X heterochroinatiii in inciotic sex chromosome behavior. Chromo\ot)i(i 60, 283-296.
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4 Functions of DNA Repair Genes during Meiosis W. lason Cummings and Miriam E. Zolan Department of Biology Indiana University Bloomington, Indiana 47405
1. DNA Repair and Organisinal Phy\iology 11. Pathways of DSB Repair 111. Genetics of DSB Repair I v. Relative Abundance of Homology-Ra\ed DSH Repair Events V. A Coprinus cirrerws Epistasis Group for DNA Repair and Meiosis
V1. Conclusions and Perspective References
One of the most basic functions in a n y organi\m is DNA repair. In addition, pi-ogrammcd DNA "damage," i n the form of DNA Jouble-strand breaks (DSBs). is a regular part ol the phy\iology 0 1 inwt organisms. There 'ire three main types of DSB repair: homolofous recombination; single-strand annealing: and nonhomologous end joining. The gene product\ known to be required for these repair prncesse\ are conserved in evolution. but the relativc dependence o n different pathway\ lor 1)SB repair is different when sy
e
1. DNA Repair and Organismal Physiology One of the most basic functions in any organism is DNA repair. In eukaryotes, unrepaired DNA damage can lead to mutations, chromosomal rearangenients, cell cycle arrest, and cellular death (Bennett r t al., 1993). The complex layering of these effects provides a challenge for the experimenter i n determining the precise function of genes that have been implicated in any aspect of D N A repair. 117
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Furthermore, mutant alleles of these genes can generate complicated and unpredicted secondary phenotypes, as directed DNA “damage” is u t i h e d as a means to complete various physiological tasks during the life cycles of many organisms. For example, the creation of mature immunoglobulin genes involves the programmed formation of a double-strand break (DSB) in chromosomal DNA to initiate V(D)J recombination (Gellert, 1992). The mechanism of matingtype switching in Sacchcrmmyces c‘erevisiae involves a DSB-induced gene conversion event between an HMR or HML donor sequence and the MAT locus (Herskowitz et a/., 1992). Immediately after conjugation in the ciliate Tetrahynena thermophi/a, development of the macronucleus from the newly formed micronucleus occurs by the cleavage and elimination of DNA from approximately 6000 chromosomal sites (Prescott. 1994). Meiotic gene conversion events at the ARC4 recombination hotspot in S. cerevisiae have been shown to initiate from the scheduled formation and processing of DSBs at the 5’ end of this locus (Sun et a/., 1989, 199 1 ). In addition, DSBs, either programmed or incidental, are likely a normal part of the early stages of mammalian development; evidence for this is the embryonic lethality of mice homozygous for a disruption of the DNA repair gene Rad.51 (Tsuzuki et a/., 1996). Based on these observations, mutations that cause deficiencies in the repair of DSBs would be predicted to have deleterious effects on the mammalian immune response, mating-type switching and meiotic recombination in yeast, and maturation of the macronucleus in ciliates. Secondary phenotypes not only demonstrate the diverse physiological roles that DSBs can play, they can also provide insight into DSB repair itself. One of the most striking connections made between pathways of DSB repair and other physiological processes was observed in the severe combined immunodeficiency (scid) mouse. The scid mouse was initially reported as inimunodefective (Bosma et al., 1983), and it was later shown to be sensitive to ionizing radiation (Biedermann et a/., 1991) and defective in the repair of DSBs in DNA (Hendrickson et al., 1991; Chang et al., 1993). It is now known that the immunodeficiency of these mice is associated with an inability to process a DNA hairpin intermediate (formed after DSB formation) during V(D)J recombination (Roth rt d . , 1992; see also reviews by Gellert, 1992; Roth et a/., 1995; Weaver, 1995). An intense area of study has been the formation and processing of DSBs during meiosis in the yeast S. cerevisiae. The pattern of meiotic DSBs in yeast correlates with the temporal and positional requirements of recombination, at least at recombination hotspots (Padmore et al., 1991; Game, 1992; Zenvirth et al., 1992; Wu and Lichten, 1994), and it has been suggested that the majority (if not all) of meiotic gene conversion events that occur in S. cerevisiae are initiated by the formation of fully processed DSBs (Kleckner et a/., 1991). Thus, much of the recombination between homologous chromosomes in yeast can be viewed as a means to “repair” meiotic DSBs. Indeed, meiosis has been proposed to have
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evolved, in part, from the recruitment of vegetative DNA repair functions (Kleckner rt a/., 1991).
II. Pathways of DSB Repair There are three basic types of DSB repair pathways. In the first type, DSB repair is based completely on a mechanism for homologous recombination that is similar or identical to meiotic recombination between homologs; this is a conservative mechanism in that no new DNA sequences are created (although one allele may be converted to another). In the second type, homology is utilized but the mechanism of repair is different, and repair can result in large deletions. The third type of DSB recombination involves nonhomologous joining of the ends of DNA and can result in either insertions or deletions. Observations of how budding yeast cells repair DSBs in plasmid DNA (Szostak et a / . , 1983) have contributed significantly to the reigning model of homologous recombination. This model (Fig. l), referred to as the double-strand break repair model, involves the transfer of genetic information from an uncleaved donor molecule to a cleaved recipient. This mechanism initiates with the formation of a DSB to generate strand ends that are resected in a 5' to 3' direction. A single-stranded 3' tail from the cleaved molecule invades the uncleaved double-stranded donor. A strand from the donor molecule is displaced and serves as a template for repair of the estranged broken strand. One of the distinguishing characteristics of this model is that it predicts a double-Holliday structure intermediate. Depending on how these junctions are resolved, a gene conversion or a crossover event will result. By using two-dimensional gel electrophoresis, Schwacha and Kleckner ( 1995) were able to demonstrate the physical presence of a double-Holliday intermediate during prophase 1 in meiotic yeast cells. This discovery provided strong support for critical aspects of the DSB repair model as a mechanism of meiotic recombination in yeast (see Stahl, 1996, for a review). The DSB repair model presents a series of steps that can give rise to gene conversion events that are either associated or unassociated with crossing over. Although this model (Fig. 1) predicts roughly a 1:l correspondence between crossover and noncrossover resolution events, some studies in yeast and other organisms have revealed that homologous recombination events involved in the repair of DSBs may resolve as crossovers less than half the time. Plessis and Dujon (1993) investigated plasmid repair in yeast and showed that when the amount of transforming DNA is decreased, the proportion of gene conversion events associated with crossing over decreases below the 50% level originally demonstrated by Orr-Weaver and Szostak ( 1983). A reevaluation of recombination events at the urn locus in Neiirosporu crussa demonstrated that more than
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E.
*
F.
B
Fig. 1 The double-strand break (DSBj repair inodcl for recombination (see Stahl, 1996, for a review). A DSB ( A ) is resected 10 form single-stranded 3’ tail\ (Bj. A single-stranded 3’ tail from the cleaved inolecule invades the uncleaved double-stranded donor (C). A \trand from the donor molecule i’i displaced and \crves as ii template tor repair of the estranged broken strand (C, D). Ligation and branch migration result in the formation of two Holliday junctions (D) that can be resolwd to yield either noncrossovcr ( E ) or crossover (I;)products. The vertical arrow represents a DSB site: hori7ontal arrowheads represent the 3’ cnds 0 1 DNA strand\.
90% of the conversion events occur in the absence of crossing over (Bowring and Catcheside, 1996, and references therein). In addition, studies of plasmid gap repair in Ustilago muydis revealed that repair events involving reciprocal crossovers are rare (Ferguson and Holloman, 1996). These authors also determined that the two ends of the gap are processed differently so that the gap is asymnietrically enlarged. To explain these observations, the authors proposed a model that invokes the formation of a D-loop on the donor molecule that migrates along with DNA synthesis, primed by an invading 3‘ end of the cleaved molecule. Additionally, Gloor et al. ( 1991) found that when gaps left by P-element excision are repaired by homologous recombination in Drosopliila melnnopstrr, conver-
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sion events are not associated with crossing over. To explain this observation (and others from the article) the authors proposed that, as in the DSB repair model, a double-Holliday junction intermediate is formed. However, the authors suggested that resolution of the junctions could occur independently of endonucleolytic cleavage by a model originally proposed by Hastings (1988), in which the two Holliday junctions are pushed together so that they cancel one another. Experiments in yeast (Fishtnan-Lobell rt u/., 1992), involving the repair of a DSB on a plasmid that contains repeats of the lacZ gene, have revealed a mechanism of correction that is similar to one originally proposed by F. L. Lin et a/. (1984) yet quite different froin the model proposed by Szostak et a/. (1983). Like the original DSB repair model, this mechanism, referred to as single-strand annealing (SSA; Fig. 2 ) , involves the formation of a DSB and 5' to 3' singlestrand resection. However, the strands of the lacZ gene are re-annealed such that
A.
-
B.
C.
D.
E. Fig. 2
A single-strand annealing pathway for the repair of DSBs (Fishman-Lobell et u/., 1991: Liii e t a / . , 1984). A DSB is generated (A) and i \ Ie\ectcd t o lorrn single-stranded 3' tails ( B ) . Hornologou\ single-stranded regions anneal (C), tail\ (D) are degraded, and ligation result\ in the formation of intact DNA. In the example shown, hecpences in between two repeated DNA sequences have been deleted by this process. The vertical arrow represents a DSB site; horizontal arrowhead5 represent the 3' ends of DNA strands.
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the two copies are reduced to one. When this process is investigated on plasmids, the SSA mechanism can be distinguished from conventional gene conversion events (which are accompanied by a crossover) by the absence of the predicted reciprocal circular product (containing the sequence that originally separated the two copies; Fishman-Lobell et d., 1992). Disappearance of the product is believed to result from the enzymatic removal of nonduplex DNA. Unlike cases of gene conversion in the DSB repair model, these events can occur independently of RAD52 function (Fishman-Lobell et d., 1992). The mechanism of SSA resembles events involving the end-to-end joining of “nonhomologous” DNA that has been transformed into cultured mammalian cells (Roth and Wilson, 1986) or X r w o p u . ~/ w \ i . s oocytes (Goedecke e t a / . , 1992). As in SSA, degradation of the single strands occurs in a 5’ to 3‘ direction. However, subsequent processing is dependent on annealing at short stretches of DNA identity (1-5 bp; Kramer et d., 1994). This process, referred to as nonhomologous end joining (NHEJ; Fig. 3). has also been revealed in S. c.eraisiur
YFig. 3 DSB rcpair by nonhomologous end joining (Roth and Wilson. 1986).A DSB (A,€ 3 ) leads to resection (C) as for homologou5 recombination (Fig. I ) and single-strand annealing (Fig. 2 ) . Singlestranded regions anneal at regions of microhomology (D; a ?-bp overlap is shown in the hatched rectangle). Degradation of single-stranded tails. polymerization, and ligation result in the formation of intact DNA, which may contain small insertions (if the initial DSB is staggered: J. K . Moore and Haber, 1996), or small or large deletions. The vertical arrow represents a DSB site: horizontal arrowheads represent the 3’ ends of D N A strands.
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by the characterization of DSB survivors from Rad- strains. Specifically, the process has been observed in r~1clS2deletion cells that lack the ability to repair using conservative mechanisms of homologous recombination.
111. Genetics of DSB Repair A genetic correlation between D N A repair and meiosis has long been established (Baker et id., 1976). As cautioned by Game (1993), i t may be naive to assume a direct parallel between mitotic DNA DSB repair and meiotic recombination. However, at least some of the genes known to be required for both DNA repair and meiosis in yeast have recently been shown directly to be necessary for one or more pathways of DSB repair. These genes are from the RAD.52 epistasis group, which consists of RADSO-RADSY, M R E I I , and XRS2 (reviews of most of the members in this collection of genes are found in Game, 1993; Friedberg rr L I / . , 1995; and Shinohara and Ogawa, 1995). The Rad5O protein is a member of the SMC family of condensation proteins (Hirano e t a / . , 1995) and has been shown, by two-hybrid studies, to form a complex with Mrel 1 (Johzuka and Ogawa, 199.5). Johzuka and Ogawa (1995) also demonstrated that Mrel 1 interacts with itself and with Xrs2. The human homologs of RadSO and Mre1 1 have been shown to associate in a stable protein complex, which may contain three other proteins (Dolganov el al., 1996). RadS 1. Rad55, and RadS7 are RccA homologs (reviewed in Yeager Stassen et al., 1997), and have been shown to form a complex with Rad52 (Hays et d., 1995; Johnson and Symington. 1995). RadS3 is necessary for checkpoint control of mitosis (Allen et d . , 1994; Wcincrt r t ( I / . , 1994), Rad54 contains DNA helicase motifs (Emery et N / . , 1991), and Rad56 is a relatively uncharacterized protein (reviewed in Game, 1993). RadS8 has recently been found to be necessary both for survival after gamnia irradiation and for meiosis (Chepurnaya et id., 1995), and RadS9 is a recently discovered Rad52 homolog that can function in Rad5 1-independent homologous recornbination between inverted repeats (Bai and Symington, 1996). Except for RADS8, RAD.59, M R E I I , cind XRS2. these genes were initially identified within a larger collection of “RAD” mutants that exhibit increased sensitivity to ioniiing radiation (reviewed in Game, 1993). RadS2 function is essential f o r homologous recombination events in yeast. Because RadS1 has been shown iri t i t r o to catalyze strand exchange between homologous DNA molecules (Sung, 1994) and because the protein is homologous to the prokaryotic protein RecA (reviewed in Yeager Stassen rt al., 1997). it is tempting to speculate that it also plays an essential role in homologous recombination. In order to dissect the separate functions of RAD.51 and RADS2 i n conservative intrachromosomal homologous recombination independently of nonconservative events (such as SSA), Rattray and Symington ( 1994) developed an assay to analyze recombination between inverted repeats. They determined
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that the overall rate of recombination was reduced approximately 2900-fold in a rad.52 deletion strain. However, the relative distribution of simple gene conversions, reciprocal crossovers, and crossovers associated with gene conversion did not differ from that seen in wild-type (Rad+) cells. A strain carrying a null allele of RADSI demonstrated only about a 4-fold reduction in recombination frequency. In this strain, however, gene conversion events unassociated with a crossover were reduced about 18-fold (compared to wild-type), whereas gene conversion events associated with crossing over decreased only about 2.5-fold. The authors interpreted these findings to mean that RADSI is not essential for intrachromosoma1 reciprocal crossover events but is important in gene conversion, whereas RAD52 is essential for both of these events (Rattray and Symington, 1994). Among the genes of the RAD.52 epistasis group, RADSO, MRE11, and XRS2 are noteworthy in that they function in both homology-based repair and in NHEJ (Moore and Haber, 1996). During meiosis, mutations at the RAD50 (Alani e t a / . , 1990), MREII (Ajimura et al., 1992; Johzuka and Ogawa, 1995), and XRSZ (Ivanov et a/., 1992, 1994) loci behave like s p o l l mutations (Klapholz et a/., 1985) in that their conferred meiotic deficiencies are suppressed by the mutation spol3-1 (Klapholz and Esposito, 1980). Cells homozygous for spol3-1 undergo only a single division during meiosis to form dyads. In some instances, chromosomes segregate equationally rather than reductionally (Hugerat and Simchen, 1993). It has been proposed that cells with mutations causing defects in initial events that precede meiotic recombination or the earliest events of recombination can be bypassed by proceeding directly to an equational division (Malone. 1983). Additionally, it is believed that spol3-1 fails to rescue mutations in genes involved in “later” stages of recombination (Malone, 1983; Malone et ul., 1991). Because rad50, rnrell, and xrs2 strains are rescued by the spol3 mutation, and because strains carrying deletions in these genes fail to form meiosis-specific DSBs, these genes likely function in “early” recombination events (Malone, 1983). Sharples and Leach (1995) have identified the existence of sequence similarities between the bacterial proteins SbcC and SbcD and the S. cerevisiae Rad5O and Mrel 1 proteins, respectively. In Escherichiu coli, the SbcCD complex is believed to function as an exonuclease and has been specifically associated with a role in the replication of palindromic DNA (Leach, 1994). Analysis of crystal structures of other bacterial phosphoesterases suggests that residues that are conserved between Mre 1 1 and SbcD compose, in part, the active site for the recognition and cleavage of phosphate bonds (Griffith et al., 1995; Goldberg ef al., 1995). This information suggests that Rad5O and Mrel 1 may have exonucleolytic activity that could function in the repair of DSBs (Sharples and Leach, 1995). However, Moore and Haber (1996) have pointed out that RADSO (and probably MRElI and XRSZ) likely contributes more to DSB repair than a 5’ to 3‘ exonuclease function. These authors examined the repair of an HO
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endonuclease-induced DSB at the MAT locus in cells deleted for H M L and HMR, the homologous donors for the repair of this DSB. The HO endonuclease makes a DSB with staggered ends, and one type of repair event involves rejoining of' the ends such that small insertions are produced. In order for these insertion products to be formed, essentially no exonucleolytic degradation can occur. When rad50 deletion strains are compared to wild-type yeast strains, the mutant cells exhibit a decrease in the frequency of formation of the insertion products and an increase in the level of deletion repair products (Moore and Haber, 1996). If the primary role of RadSO were to function as an exonuclease, then the loss of exonuclease activity (expected in rud50 deletion strains) would lead to an increase in the formation of insertion products (where no nucleolytic processing of the DNA ends has occurred). Based on this observation and other data, Moore and Haber ( 1996) proposed that RadSO, Mre 1 1, and Xrs2 function in an interaction between sister chromatids. They have suggested that this interaction can serve to protect DNA ends from exonucleolytic degradation following DSB formation. Another conserved group of proteins involved in NHEJ mechanisms of DSB repair was initially identified in studies o f rodent cell lines exhibiting increased sensitivity to ionizing radiation. These cells are phenotypically similar to cells from the scid mouse in that they are defective in the formation of V(D)J recombination products (Weaver, 1995, and references therein). With the use of cell fusion techniques, these mutanrs have been divided into at least nine complementation groups (Weaver, 1995; Wood, 1996). The deficiencies that some mutations confer are known to result from defects in a pathway of DSB repair involving the abundant nuclear protein Ku and a DNA-dependent serinekhreonine protein kinase (DNA-PK; this is the gene thought to be defective in the scid mouse; see Wood, 1996, for review). The K u protein is a heterodimer composed of subunits that are approximately 70 kDa and 80 kDa (Wood, 1996). Gel mobility shift experiments demonstrated that the heterodimer binds strongly to double-stranded ends of DNA (Weaver, 1995). K u is thought to be the predominant end-binding complex in eukaryotic cells and has been identified in budding yeast (Feldmann and Winnacker, 1993; Milne et ( I / . , 1996), fruit flies (Beall and Rio, 1996), and mammals. The Ku heterodimer has been shown to interact with DNA-PK (Weaver, 1995) and is believed to servc as the DNA-binding component of the larger kinase complex. Thus, Ku is essential lor DNA-PK activity. The specific function of this complex in DNA repair is still not well understood, but some of the many substrates that can be phosphorylated ir7 vitro include p53, replication factor A (RPA), many transcription factors, and the Ku70 and Ku80 subunits themselves. Recent work in mammalian cells (Liang el ul., 1996) has demonstrated that Ku functions in an NHEJ mechanism of repair of an inducible DSB, but has essentially no effect on correction by homologous recombination. This conclusion is supported by work in yeast (Milne et a/., 1996; Siede et al., 1996).
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IV. Relative Abundance of Homology-Based DSB Repair Events The data so far suggest that NHEJ is the preferred mechanism of DSB repair in mitotic vertebrate and plant cells, whereas yeast principally uses honiologydriven mechanisms (reviewed i n Milne et ~ l . 1996; , Puchta and Hohn, 1996). The dependence of S. cerevisine on homologous recombination for repair of DSBs was demonstrated directly by experiments i n which the HML and HMR cassettes were deleted from haploid cells (Moore and Haber, 1996). These sequences function as donors of genetic information during mating type conversion. The switching of mating type is initiated by cleavage at the MAT locus by the HO endonuclease (Herskowitz rf ( I / . , 1992). Expression of the endonuclease in cells that lack both HML and H M K results in greater than 95% lethality (Moore and Haber, 1996). Thus, in the absence of a donor molecule, yeast cells cannot repair a DSB. Additionally, cells that have intact copies of thc donor sequences but carry a deleted version o f RAD.52 exhibit similar levels of DSB-dependent lethality. Gaining insight into the predominant mechanism of DSB repair in multicellular eukaryotes is confounded by the fact that the necessary experiments are often difficult to perform in systems less tractable than unicellular models. Experiments in which the repair of an inducible DSB is monitored focus primarily on mechanisms of homologous recombination. In these assays the repair event generates a functional copy of a selectable or identifiable marker developed for the organisin i n question. Constructs carrying a synthetic gene encoding the I-Scel endonuclease along with DNA substrates that when recombined can produce a functional copy of the gene encoding p-glucuronidase (GUS) were cotransformed into protoplasts made from plant cells (Nicvticiiza p/urithaginoj~~~/ia; Puchta et a/., 1993). Recombination within these extrachroinosomal constructs was assayed by measuring transient levels of GUS activity. The authors suggest that the repair products generated in this assay are best explained by the SSA model. In a subsequent publication (Puchta et a/., 1996), the authors demonstrated that when single-stranded extrachroinosomal DNA (introduced by Agmhcrcteriuin transformation) is used to repair an inducible chromosomal DSB, two types of events are observed. Principally, the DSB is repaired by purely homology-based reconibination in a manner that the authors interpret as being similar to the DSB repair model. However, the authors also observed products that must have employed NHEJ as part of the repair process. Much of what is known about DSB repair in multicellular organisms is derived from studies involving the integration of exogenous DNA after its transformation into cells. Targeted events are indicative of homologous mechanisms of DSB repair, whereas random integration events indicate illegitimate recombination processes (Roth and Wilson, 1988). Milne rt al. (1996) have speculated that the genomic complexity and gene structure of an organism may be the basis for the
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evolution of mechanistic preferences for DSB repair. They suggest that an organism with densely packed genes and relatively few introns might be constrained to repair DSBs by precise, homology-based mechanisms. In contrast, for organisms with large genomes and large amounts of dispersed repetitive DNA, homologybased DSB repair might increase the probability of ectopic recombination and hence genetic translocations. In these organisms, which also have a high percentage of noncoding DNA, nonhomologous mechanisms such as NHEJ would be tolerated and in fact preferred. However, there is no absolute correlation between genome size, amount of repetitive DNA, and biases in DSB repair (Roth and Wilson, 1988). It is known that systems such as animal cells, plants, and many fungi primarily use mechanisms of DNA end joining (an illegitimate mechanism) for repair of DSBs (Milne et ( I / . , 1996; Puchta and Hohn, 1996). However, despite the physiological bias toward repair by illegitimate mechanisms in multicellular systems, all pathways (homologous and nonhomologous) appear to be present in every organism in which DSB repair has been studied. That is to say, mechanistic preferences probably do not stem from the loss of other mechanisms of repair. Despite a preference for NHEJ in mammalian cells, the induction of the site-specific endonuclease I-Scel in mouse cells has shown that gene-targeting events (by homologous recombination) are increased by more than two orders of magnitude when a regulated break is made at the target locus (Ronet c’t ~ 1 . 1994). . The use of an inducible DSB to promote homologous targeting has also been employed in cells from tobacco (Puchta ct NI., 1996). Similarly, although the pritnary mechanism of DSB repair in S. c~or\~i.vicie involves homologous recombination, pathways of NHEJ have been observed to occur in this organism. Cotratisfortnation of linear DNA and restriction enLymes leads to integration into chromosomal sites by illegitimate recombination (Schiestl and Petes, 1991). The occurrence of these events is dependent on RAD.50 function but is essentially independent of RADS2 (as well as RADSI and RAD.57; Schiestl et al., 1994). In a related study, Moore and Haber (1996) examined the role of members of the RAD-52 epistasis group on NHEJ events at the MAT locus. These authors lound that mutants carrying single deletions of RADSO, M R E I I , or XRS2 denionstrared the greatest defects in NHEJ, each reducing the overall frequency of these events by approximately 70-fold. Levels of NHEJ were virtually unaffected by deletions in RAD52 and the other members of the RADSO epistasis group. Interestingly, on closer examination of the joint molecules generated by repair, it was found that mutations in RADSU, M R E I I , and XRS2 caused a severe reduction in the observed frequencies of a two-base insertion product. This product was the predominant outcome of repair in all phases of the cell cycle except for G 1. The fact that relative levels of this repair product decreased in the mutants while the levels of other deletion events remained unaffected led the authors to propose that there are multiple pathways of NHEJ, and that only one had specifically been affected by the three mutations. The notion of the evolutionary conservation of DSB repair pathways is sup-
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ported by the discovery of HDFI (Feldmann and Winnaker, 1993) and KU80 (Milne et ul., 1996), the S. crrevisiur homologs of the mammalian genes that encode the 70-kDa and 80-kDa subunits, respectively, of the Ku heterodimer. This protein heterodimer was shown, as in mammals, to exhibit strong binding affinity for double-strand DNA ends (Milne rt a/.,1996). Milne et d.(1996) reported that haploid strains carrying single and double deletion mutations in these two genes exhibit a slight sensitivity to the radiomimetic agent methyl methanesulfonate (MMS). In a separate study, Siede et trl. ( 1996) used survival curves to demonstrate that a homozygous diploid h 4 1 deletion strain is no less deficient in its ability to survive treatment with MMS and gamma radiation than wild-type diploid strains. However, when the ability to repair damage by homologous recombination was prevented by utilizing hdfl cells that also carry a deleted version of the RAD52 gene, an increased sensitivity to ionizing radiation and MMS was observed. The need to remove homologous mechanisms of repair in order to analyze Ku function in yeast was further demonstrated by testing haploid mutant and haploid wild-type cultures in stationary phase (Siede er ul., 1996). These cultures consist primarily of cells in the G , phase of the mitotic cell cycle. Because these cells lack the ability to use a sister chromatid for repair, the prediction is that DSBs would be processed by an end-joining mechanism. Cells in which HDFI is deleted do show a small increase in sensitivity to gamma radiation. Interestingly, a RADSO deletion was shown to be epistatic to KCIKO and H F D l deletions with respect to MMS sensitivity and the efficiency of repair of a linearized plasmid in vivo (Milne et a/.,1996). These results suggest that in S. cerevisiae, Ku may function with RadSO (and possibly Mrel 1 and Xrs2) in an NHEJ pathway to repair DNA DSBs. In both of these studies the authors did not detect any effect on sporulation efficiencies in strains homozygous for the deleted versions of either HDFI (Milne et al., 1996; Siede eta/., 1996) or KUXO (Milne et d., 1996).However, a meiotic function for the Ku heterodimer in other organisms cannot be ruled out because of the possibility that the dependence of S. cerevisiae on homologous recombination for DSB repair would effectively mask any contribution of the end-joining pathway to meiotic DNA metabolism.
V. A Coprinus cinereus Epistasis Group for DNA Repair and Meiosis An attractive system for the analysis of pathways of DSB repair and their relative prominence during meiosis would be one that is amenable to genetic analysis, has an accessible meiotic process, and relies more heavily on nonhomologous mechanisms of DSB repair than does S. cerevisiae. Coprirzus cinereus is a basidiomycete fungus in which the meiotic process is naturally synchronous (Raju and Lu, 1970). C. cinereus can grow vegetatively as a haploid mycelium or as a
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stable dikaryon in which each cell contains two nuclei of different mating types. The dikaryon can be photoinduced to form mushroorns, which develop to contain 10 million meiotic cells. The starting point of meiosis is karyogamy, the fusion of the two nuclei of the dikaryon, and this fusion is followed by rapid chromosome condensation, alignment, pairing, and synapsis (Seitz et ul., 1996, and references therein). The timing of meiotic initiation can be controlled experimentally, by manipulation of light conditions, and the stages of meiosis are tightly coupled to fruiting body development. Thus, this organism provides an excellent population of meiotic cells in which to analyze chromosome behavior and gene expression. The fact that C. cinereus can grow vegetatively as a haploid also facilitates experiments involving mutagenesis and DNA-mediated transformation (Binninger rt d . , 1987). Using screens similar to those described for other organisms (Baker et al., 1976), we identified four complementation groups of C. cinereus mutants that are sensitive to gamma radiation and also exhibit defects in meiosis (Zolan et ul., 1988; Ramesh and Zolan, 1995; Valentine et ul., 1995; Zolan et al., 1995). Epistasis analysis has demonstrated that these mutants, called rud3, rud9. run1 I , and radl2, make up a single gamma radiation survival pathway (Valentine et ul., 1995). Additionally, all of the mutants exhibit distinct abnormalities in basidiospore formation (Valentine et d., 1995). Thus far, all four rud genes have been physically mapped to electrophoretic karyotypes (Zolan et ul., 1993), and rud9 has been cloned by complementation with a chromosome-specific library (Zolan et al., 1992). The rud9 gene encodes a 6.8-kb transcript that has been shown to be induced following exposure to gamma radiation and during meiosis (Seitz et id., 1996). Homologs o f rudY have been found by genomic sequencing in S. cerevisiae and Schizoscic~chnrot,i\.c.espombe (Seitz et cil., 1996), but the importance of the homologs to DNA repair and meiosis in these organisms has not been determined and is currently under investigation. Mutations in rud3, rud9, rticlll, or rudl2 cause abnormalities in meiotic chromosome condensation and synapsis (Pukkila et al., 1992; Ramesh and Zolan, 1995; Seitz et ul., 1996; Zolan et al., 1988; E. Gerecke and M. E. Z., unpublished results). In wild-type meiosis, chromosomes undergo two distinct phases of chromatin condensation (Fig. 4). The first condensation culminates in pachytene synapsis of homologs and is accompanied by formation of the synaptonemal complex (Figs. 4A,4B). Full synapsis is followed by a relaxation of the condensation and entry into diffuse diplotene, as has been observed in animals (Wilson, 1 9 2 3 S. cerevisiue (Dresser and Giroux, 1988), and Sordurin 171acro.sporu (Zickler, 1977). Although chromosomes remain visibly paired and do not completely decondense (Figs. 4C.D) a significant increase in compaction occurs between diffuse diplotene and metaphase 1 (Figs. 4E,4F). We have found that some of the C. cinereus rud mutants uncouple the two phases of chromatin condensation in meiotic cells. For example, rudY-1 cells do not reach full pachytene; chromosome do not fully condense (Fig. 5C). nor do
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Fig. 4 Two stages of chromatin condensation during meiosis. Chrornosomcs were spi-ead as described in Pukkila c t u / . ,( 1992).stained with either silver ( A , C ) or acridine orange (B, D-F), and photographed using electron (A, C ) or fluorescence ( B , D-F) microscopy. A and B show nuclei at pachytene. 4 ( A ) or 6 (B) hrs after karyogamy. C and D show nuclei at diffuse diplotene, 8 hrs after karyogamy. E and F show chromosomes entering metaphase I ; the images were made 9 hours after karyogamy. The arrow\ in A and B denote nucleoli. Scale bars = 2 pm. ( A , B, and D-F reproduced with permission from Seitz etcil., 1996.)
they achieve significant synapsis (Seitz et a/., 1996). However, 55% of the meiotic cells in a md9-1 mushroom undergo a metaphase I-type of chromosome condensation (Fig. 5D; Seitz et al., 1996). Because no rud9-/ basidia ever exhibit full pachytene-stage chromosome condensation but more than half show
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Fig. 5 Chromatin defects in rru/Y-I and r . ~ r ~ / / _ 7 - - nititiints. I A and B arc from B wild-type strain that i \ congenic with the rrrd mutant strain\. C', D, and E are lrom a rtrdY-I mutant, and F and G are from a rtrrll2-4 mutant. For all panels, chroniosonie\ were spread as described by Pukkila C/ ( I / . (1992), stained with acridine orange, and photogi;iplicd uvng Iluoresccnce microscopy. A. C, and F show nuclei at 6 hrs after karyoganiy, when the wild-type \train is i n pachytene. B shows a spread made 9 hr after karyogamy. D, E, and G shou \pread\ at 10 hr\ post karyogamy. The arrows in A, C. D, and F denote nucleoli. Note that the nucleolu\ is eliniin;ited by metaphase I: a greatly diminished nucleolu\ is seen i n D. All panels are to thc same \c;iIc. ScAc har = 2 kin. (A-E reproduced with permi\\ion from Seitz o f ( I / . , 1996; F and G reproducctl with permission from Ramesh and Zolan. 1995.)
at least some compaction at metaphase I, metaphase I condensation is not dependent on successful pachytene condensation. Therefore, these two processes are distinct, differently regulated events. The reason why only 55% of the radY-1 meiotic cells progress into metaphasc I is not known. It is possible that the rud9I mutation is leaky and that ;I complete absence of the Radc) protein during
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meiosis would lead to complete arrest prior to metaphase I. Alternatively, the absence of functional Rad9 in these meiotic cells may result in a stochastic success or failure to enter metaphase I. The meiotic chromosomes of rndI2 mutants undergo significant chromosome compaction (Fig. 5F) but arrest at diffuse diplotene (Fig. 5G). This has been observed for three different alleles of rudl2 (Ramesh and Zolan, 1995); in each case, 5% or less of the meiotic cells progress into metaphase I. There are three possible explanations for these observations. First, the basidial cells could be dead. We think that this is unlikely, because the fruiting body continues to develop; the stipe elongates and the cap opens and undergoes apparently normal deliquescence (Moore and Pukkila, 1985). Further, if cells were dying, they might be expected to show apparent arrest at various stages. Second, the Radl2 protein might be required as part of metaphase I condensation or may regulate this process. However, defects in rudl2 mutants are manifest early in meiosis (Fig. 5F; Ramesh and Zolan, 1995), and we think it likely that the biochemical need for Rad12 is during prophase I. We cannot, though, rule out the possibility that Radl2 is needed twice, once for chromosome synapsis and once for entry into metaphase I. A third explanation for the behavior of r(id12 mutants is that they are sensitive to a meiotic checkpoint that prevents them from entering into metaphase I. Diffuse diplotene is the point of arrest of mammalian oocytes at birth (Albertini, 1992). The arrest of the rad12 mutants may provide evidence for a fungal checkpoint at this stage. By this logic, the md9-I mutant does not trigger the checkpoint, perhaps because its prophase I development arrests before a checkpoint arrest signal is generated.
VI. Conclusions and Perspective In S. cerevisiae, the dual phenotypes of gamma radiation sensitivity and meiotic disfunction are strong indicators of defects in DSB repair. By incorporating a search for meiosis-defective mutants into a radiation screen, it is possible that we have isolated mutants of C. cinereus with defects in the repair of DSBs. However, it is worth noting that (to our knowledge) naturally occurring meiotic DSBs have not been identified in any organism other than S. cereiisiae. Therefore, it is possible that the C. cinereus rud genes function in some other process that is common to gamma radiation survival and meiosis. The ability to analyze yeast chromosomes by pulsed field gel electrophoresis, the high frequency of meiotic recombination in that organism, and the availability of the rud5OS mutation (Alani et nl., 1990), which forms meiotic DSBs but fails to process them further, have allowed yeast meiotic DSBs to be mapped with high precision (for example, Liu et a/., 1995, and references therein; de Massey e f ul., 1995). However, this has not been possible in other organisms. A
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recent article (Klein et al., 1996) analyzed yeast artificial chromosomes (YACs) containing human DNA from the XY pseudo-autosomal region and from chromosome 21. The authors examined DSB formation within these YACs during yeast meiosis, using rad50S strains. For the XY YACs, DNA from a meiotic “hotspot” demonstrated approximately three times the number of DSBs as YAC clones containing a nearby “cold spot.” A similar use of YACs carrying human DNA from chromosome 21 showed that one of three clones from a putative hotspot region was enriched in meiotic DSB formation relative to other clones from that region and relative to a putative chromosome 21 cold spot. Whether this observation represents an accurate, more detailed mapping of a real meiotic recombination hotspot in humans is not clear, however. In addition, the DSBenriched YAC was not shown to contain the actual human meiotic hotspot, nor were these regions shown .to receive DSBs during meiosis in humans. Keeney et al. (1997) demonstrated that the Spol 1 protein likely catalyzes the formation of DSBs during meiosis in yeast. Putative Spol 1 homologs have been identified by peptide sequence comparisons in an evolutionarily diverse collection of organisms. Pairwise alignments between the S. cerevisiae sequence and the Rec 12 protein of Schizosacc-hcirom?res pornbe, an uncharacterized putative peptide from Cuenorhabditis elegans, and an uncharacterized putative peptide from Methunococcus jannaschii show 22-28% sequence identities (Keeney et al., 1997). Interestingly, the reel2 gene of S. pornbe has been demonstrated to function in intragenic and intergenic recombination during meiosis (Lin and Smith, 1994). Bergerat et al. ( 1997) have identified the genes that encode the A and B subunits of a type I1 topoisomerase from the archaebacterium Sojdobus shibatue. The gene encoding the A subunit appears to represent another member of the Spol I subfamily of topoisomerase 11-like proteins. Together, the two protein subunits have been demonstrated to function as a decatenase and can unwind positive and negative superturns (Bergerat et al., 1994). These functions are consistent with the known activities of Spol 1. However, the eukaryotic Spol 1 homologs (from S. pornbe and C. elegans) have not yet been shown to function in meiotic DSB formation. Therefore, the issue of whether DSBs initiate meiotic recombination in other organisms has not yet been resolved. If rad genes in C. cinereus and other complex organisms do function in DSB repair, then it is important to determine the types of mechanisms that are used for correction, and the organism’s relative dependence on different types of DSB repair. Is DSB repair largely a homology-based mechanism similar to that presented by the DSB repair model, or is NHEJ, which does not require an uncleaved donor molecule, more prevalent? The integration of transforming DNA in C. cinereus occurs at nonhomologous sites at least 95% of the time (Binninger et al., 1987, 1991). The prediction, therefore, is that C. cinereus, like plants and animals, relies extensively on NHEJ for DSB repair in vegetative cells. If this is true, are these pathways also active in meiotic cells? DSB repair in
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vegetative S. cerevisiae cells is predominantly by homologous recombination; in order to explore mechanisms of repair by NHEJ in yeast, it is necessary to genetically remove homology-based events. Because S. cerevisiup uses homology-based mechanisms for repair in mitotic cells, the organism does not require a change in mechanism for the processing of programmed DSBs during prophase 1 of meiosis. However, if DSBs are universal initiators of meiotic recombination, as has been suggested (Klein et al., 1996; Keeney er al., 1997), how are DSBs processed in organisms that rely extensively on NHEJ in vegetative cells? Is at least some DSB repair constrained, in meiotic cells, to the homology-based events that result in gene conversion and crossing over? It is possible that programmed DSBs might form frequently along the length of the chromosomes, with the majority of the breaks repaired by mechanisms like NHEJ. On occasion, however, a break would be repaired using the homologous chromosome as a template, and Holliday junctions could then be resolved to form a crossover. Such a mechanism of repair would be predicted to generate fewer crossover events per chromosomal length in mammals relative to their frequency in a system like S. cerevisiue. This prediction is consistent with observations that crossing over occurs about once per chromosome arm in mammals (Hawley, 1988) and about three to eight times per chromosome in S. cc~rrvisiur (Olson, 1991). An alternative mechanism for ensuring the formation of crossovers would be either to functionally increase the activity of repair by homologous recombination or to decrease the activity of end-joining pathways. How could this be achieved? It is possible that features of meiotic chromosome structure or meiotic homolog association limit DSB repair to homology-based processes, or ensure that at least the programmed breaks formed during meiosis are usually repaired i n a homology-dependent manner. A different explanation is that recombination does not initiate with DSBs in all organisms. However, there is evolutionary conservation of the genetic link between DNA repair and meiosis. For example, the Rad5 1 protein is highly conserved and found ubiquitously in reconibinogenic and meiotic tissue (reviewed in Yeager Stassen er ul., 1997). As pathways for DSB repair are investigated in more systems also amenable to the genetic analysi5 of meiosis. it should be possible to determine whether DSB repair is a unifying theme in meiotic recombination, or whether conserved gene products have other essential functions that tie together DNA repair and meiosis. Furthermore, because of the preferential utilization of conserved DNA repair pathways in different organisms, the same screens should lead to the discovery of genes in C. cinereus and other appropriate systems that are different from those identified initially in S. cerevisiae (for example, the rad9 gene of C. cinereus; Seitz et ul., 1996). In addition, the study of orthologous genes in multiple organisms will reveal whether meiotic recombination and DNA repair consist of the same conserved pathways or instead involve different arrangements of modular components of conserved mechanisms.
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Acknowledgments Our review of pathways and mechani\ni\ lor I X B repair was meant to be illustrative, not exhaustive; we apologiLe to investigators whose work wii\ omitted. We thank S. Acharya, M. Cclerin, E. Gerecke. and G. Faurote for comments on the inantiscript, and L. Li and K. Jepson-Innes for preparing Figs. 4 and 5. The work on DNA repair and nicio\i\ in Zolan's laboratory is supported by grant GM4.7930 from thc National In\titute of Gcnei-al Mctlical Sciences.
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Hirano. T., Mitchison, T. J . , and Swedlow,. J. R. 1995). Thc SMC family: From chromosonic condensation to dosage compcnwtioii. ( ~ ' i i r i(:) p i n . Cell B i d . 7, 329-336. Hugerat, Y., and Simchcn. G. (1993). Mixcd \egregation and recombination of chromosomes and YACs during single-division meio\i\ i n vpo/.j strains of Siicdiuror~iyc~e.v c~erevisine.Geuc,tic.v 135. 297-308. Ivanov, E. L., Korolev, V. C., and Fabre. I.. (1992). XRS?, a DNA repair gene of Sac~c~htiroffr!.cf~.c cerei,isriie, is needed for meiotic recombinntion. Geriefic,.v132, 65 1-664. Ivanov, E. L, Sugawara, N., White, C. 1.. tnhrc, W. F., and Haber, J. E., (1994). Mutatations in XRS2 and RADSU delay but do not pi-c\cnl mating typc switching in Sucdiurortiyr.v w r e vi,\i(ie. Mol. Cell. Biol. 14, 3414-3425. Johnwn. R . D.. and Symington, L. S . (1995). Functional differences and interactions among the putative RccA homologs RadSI, Rad55. and Rad57. M o l . Cell. B i d . 15, 4843-4850, Johmka, K., and Ogawa, H. (1995).Interaction 01' MreI I and Rad50: Two proteins required for DNA repair and meiosis-spccific doublc-\trancl break formation in Smchuromyces cereiiyiur. Griirtic.s 139. 1521-1523. Keeney. S., Giroux. C. N.. and Kleckner, N. ( 1997). Meiosis-specific DNA double-strand hrcahs are catalyzed by Spol I , a nienihcr ol :I widely conserved protein family. Cell 88, 375-384.
Klaphol/, S., and Esposito, R. E. (1980). I\olatioii of vpu/2-/ and s p o l 3 - / from a natural variant of yeast that undergoes a single meiotic division. G'erletics 96, 567-588. Klapholz. S., Waddell, C. S., and Espo\ito. R. E (1985).The role of the .spo/l gene in meiotic recoinbination in yeast. Geiirtic.s 110, I X7-2 16. Kleckner, N., Padmore, N. R., and B i \ t i q . D. K. ( 1991). Meiotic chromosome metabolisni One view. Cold Spring Hurhor Sytiip. Qiitirr/ 8ioI. 56, 729-743. Klcin, S., Zenvirth. D., Sherman, A,. Ried. K., Rappold, G., and Simchen, G. ( 1996). Doublestrand breaks on YACs during yeast mriosi\ niay rellcct ineioitic rccombinaLion in the hunian genome. Nriturc. Genet. 13, 48 I-4x4. Kramcr. K. M.. Brock J . A,. Bloom, K. , Moore, J K.. and Haber, J . E. ( 1994). Two different type\ of double-strand breaks in Sri(.f./rof-oiri\(.(,.s crreiisitre are repaired by similar KAU.52independent. nonhomologous reconibiiiiitioii events. M u / . Cell. Biol. 14. 1293- 1301. Leach, D. R. F. ( 1994). Long DNA palindrome\. ci-uciforin structures, genetic instability and secondary structure repair. BioE.s.ciiv.s 16, X93-900 Liang. F., Romanienko, P. J., Weaver, D. 'l., Jeggo. P. A,, and Jasin, M. (1996). Chrornoaomal double-\trand break repair in Ku8O-dclicicnt cells. Proc. Nut/. Accitl. Sci. USA 93, 8929-8933. i n . F. L.. Sperle, K., and Sternbcrg, N. ( 19x4). Model for homologous recombination during transfer of DNA into mouse L cells: Role lor DNA ends in the rccombination process. Mol. C ~ l l . Biol. 4. 1020- 1034. Lin, Y., and Smith, G. R. (1994). Traii\ient. ineio\i\-induccd expression of the rec6 and r w / 2 gene\ of' Sc~ii~o.stit~c~hiirorii\cr.v powihe. G i ~ r i ~ i e136, ~ s 769-779. Liu, J., Wu, T. C., and Lichten, M. ( 1995). The location and structure of double-strand DNA breaks induced during yeast iiiciwi\: F.1 itlcncc for a covalently linked DNA-protein intcrmediate. EMBO J . 14, 4599-4608. Malone, R. E. ( 1983). Multiple mutant annly\i\ 0 1 recoinbination in ycast. Mol. Gen. Geiicr. 189, 405-412.
Malone, R. E., Bullard, S., Hermiston. M., Kieger. R., Cool, M., and Calhraith. A. (1991). Isolation of mutants dcfcctivc in early step\ 0 1 niciotic recombination in the yeast S ~ ~ c c ~ l i o r n i i i ~ c ~ i ~ . ~ cerei~iricir.Grrwrics 128, 79-88. Milnc, G. T., Jin, S., Shannon, K. B., and Wcaver. D. T. (1996). Mutations in two Ku homologs ;or. M ol. Cell. BfOl. 16. dclins a DNA end-joining rcpair pathway in Siii,chrir~,i?iye.('.fcer 41 89-4198. Moore, D., and Pukkila, P. J . (19x5). Copriiiii.\ c,itiererr.\: An ideal organism for studies 0 1 genetics and devclopmcntal biology. J . Riol. Ed. 19. 3 1-10.
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139
4. DNA Repair and Meiosis
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-
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Gene Expression during Mammalian Meiosis E. M. Eddy Gamete Biology Section Laboratory of Reproductive and Developmental Toxicology National Institute of Environmental Health Sciences National Institutes of Health Research Triangle Park. North Carolina 27709
Deborah A. O'Brien Laboratories for Reproductive Biology Departments of Pediatrics and Cell Biology and Anatomy University of North Carolina at Chapel Hill Chapel Hill. North Carolina 27.599
1. Introduction 11. RNA Synthesis during Meiosi\ 111. Genes Expressed during Meiosi\ A. Structural Proteins of the Nucleus
B. DNA Repair and Recombinntion C. Transcriptional Regulation D. RNA Processing E. Cell Cycle E Intercellular Communication G. Signal Transduction H . Eniymcs of Enei-gy Metabolisiii I . Other Components I v. Conclusion References
The expression of a wide variety 01' genes is developmentally regulated during mainnialiaii meiosis. Drawing mainly on studies in \permatogenesis, this review shows that some of thew genes are transcribed exclusively iii germ cells, while others are also tran\cribed in mnatic cell\. Some of the genes e x p r e w d cuclu\ivcly in spermatogenic cells are unlike an) exprcs\ed in somatic cells, while other\ ;IIC isologou\ to genes expressed in somatic cell\ and are in the wine gene family. Sane oS ihe tlevclopmentally regulated gcnes also expressed in somatic cells produce \permatogenic cell-\pecitic transcripts, while others produce transcripts that are apparently the same in somatic and pci-in cells. Possible answers to why so many genes have atypical patterns of exprc\\ion during meiosis are that: ( I ) all cell types expre\\ certain genes that deline their cell type and lineage. ( 2 ) spermatogenesis i \ a developmental proces\ that progresses according to a penctic pi-ograin directing the sequential and coordinate expre\sion o f specific genes. (3) sonic penes iirc expressed that encode proteins requircd for meio\i\. (41some genes are expre\\ed that encode proteins not required until after meio\i\. ( 5 ) some genes are expre\aed to conipcn\aic for other genes that become inactivated with X
141
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E. M. Eddy and Deborah A. O'Brien
chromosome condensation. and (6) it ha\ hccn sugge\ted that regulation of gene expiession becomes leaky during spermatogenesis due to change\ in DNA organization, leading to production of irrelevant transcripts. However, it is largely unknown how extrinsic cue\ from the endocrine system and surrounding somatic c e l l \ interact with intrinsic mechani\ms of germ cells to activate signal transduction pt-occsse\ regulating transcription during mammalian meiohis. Copyright 0 1998 by Academic Press.
1. Introduction This review focuses on gene that are transcribed in spermatogenic cells undergoing meiosis in mammals. Less is known about genes cxpressed in oocytes because the critical events in the female happen only once, they occur in the fetus and are relatively inaccessible to experimental manipulation, and the amount of mat. 'a1 available for analysis is small. On the other hand, spermatocytes are readily available because meiosis in males is an ongoing process throughout life, and the unique organization of the seminiferous epithelium allows precise determination of when expression of a gene occurs during the developmental process of spermatogenesis. It is well established that the expression of many gcnes is developmentally regulated during spermatogenesis. Some of these genes are expressed exclusively in germ cells, while others are also expressed in somatic cells (Table I). There are two categories of genes expressed exclusively in spermatogenic cells. Some are unique and have nucleotide sequences with little similarity to those of genes expressed in somatic cells, while others are isologous to genes expressed in somatic cells, being members of gene families with common sequence features. Furthermore, there are two categories of genes that are upregulated during a particular phase of spermatogenic cell development but are also expressed in somatic cells. Some produce transcripts that apparently are the same in both spermatogenic and somatic cells, while others produce spermatogenic cellspecific transcripts. The regulatory mechanisms responsible for developmental changes in gene expression during spermatogenesis are largely unknown, but they presumably operate in response to signal transduction processes that selectively activate transcription in response to internal and external cues. These cues are also likely to regulate the production of spermatogenic cell-specific transcripts via transcription initiation or tennination processes, transcript-splicing mechanisms, and alternative exon utilization. Genes that are regulated developmentally and are either expressed exclusively during spermatogenesis or produce spermatogenic cell-specific transcripts have been called chauvinist genes because male germ cells favor their expression with strong prejudice (Eddy, 1995). Spermatogenesis is a highly ordered process that occurs in mitotic, meiotic, and postmeiotic phases. Although the focus of this review is on gene expression during meiosis, other genes crucial for the process of spermatogenesis are ex-
143
5. Gene Expression during Manitiialian Meio5is Table I
Gene\ Expressed During Mcio\i\
Genes
Expre\\ion pattern
Structural Protein.; of the Nucleu\ Lamins lamin C2 S&G:d t r m w 1131 variant’’ lainin B, S&G, traiici-ip viii-iaii~ Histones THZB G,
\2
S&G
Ptirp DNA polymerase-P LINE- 1 Atm
S&G S&G S&G S&G S&G
Pill
Arr
Transcriptional Regulation Transcription Factors Crrh I Crrm HSfZ Sprm I
S&G, tTiin\cript \lariiints S&G, traii\cript \ariant\ S&G G , uniquc genc S&G, t raii\cri pt variant S&G, tran\cript v;Lri;int
Waeber et d.( I99 1 ), Ruppcr! o r (1992) Foulkes et t i / . (1992) Sarge rr n/. ( 1994) Anderscn e f d.( 1993) Kim and Criswold ( 1990) Hirose r t td. ( 1994)
S&G, tramcript
Schmidt and Schihlcr ( I W S ) , Perwngiev e f c i / . (1996) xu et (11. (1994)
RAR-cu TAK- I Transcriptional Machinery S&G TBP SII-TI RNA Processing PL I 0
Kim r f a/. ( 1987) Huh rt ( I / . (1991) Cole et ( I / . ( I 987), Kremer and Ki\tler (1991) Grime\ er al. ( 1987) Welch Y / ( I / . (1990)
Morita rt u / . (1993), Shinohara t’f t i / ( 1993) Kovalenko rt a/. (1996) Habu et nl. ( 1996) Chen et el/. (1995) Walter et crl. ( 1996) Tan et d.( 1966) Baker er d.( 1995) Edelmann et cil. (1966). Baker (’I t i / . ( 1996) Alcivar et ( I / . ( 1992) Alcivar et ( I / . ( 1992) Branciforte and Martin (1994) Barlow et ul. ( 1996) Keegan rt t i / . ( 1996)
S&G
Mlll I
Furukawa P I ciI. ( 1994) Furukawa and Hotta ( 1993)
Meuwissen et a / . (1992) Dobson rt ( I / . ( 1994) Chen rr a/. (1992) Calenda rt ( I / . ( 1994)
S&G G, unique gene
S&G S&G S&G
Reference
G, gene
kiiiiily
vilriiiiit
Leroy
PI
crl. ( 1989)
(I/.
144
E. M. Eddy and Deborah A . O’Brien
Table 1 CnffIfrfrrcd Genes P68 l’trbt 2 P53Ip56 MSY 1 refff-
Prl1p Spfff-
Cell Cycle Cyclin/CDCZ Cmh I CCIlhZ C t ~ f fI t l
Ctlc25C MUk C1k3
Mogl Ncd I Tumor Supprcs\oi ps3 KhI Brc,rr I Heat-Shoch H.sp70-2
Exprehsion pattern
Reference
G, gene iamily G , gene iainily G. gene family S&C, gene family G , gene fmiily S&G, gene family S&G. gene family
Lamaire and Heinlein (1993) Kleene et ti/. ( 1994). Gu r / ( I / . ( I995 j Kwon et ol (1993) Tafuri er t i / . (1995) Schumacher ct d.(199Sb) Lee et ( I / . ( 1996) Schumacher et a / . (1995aj
S&G
S&G G, gene family S&G. transcript variant G , gene family S&G. gene faniilq G. gcne iamily S&G
Choprnan and Wolgemuth ( 1992) Chapinan and Wolgeinuth (1993) Sweeney ct a/. (1996) Wu and Wolgemuth (1995) Mat\ushime et ( I / . (1990) Becher ct ti/. 1 1996) Don and Wolgemuth ( 1992) Letwin ol. ( 1992)
S&G S&G S&C
Schwart7 (’1 rrl. (1993) Bernards cI rrl. (19x9) Zabludoli rt (11. ( 1996)
G , ,gene family
Zakeri P / r i l . ( 19x8). Kosario ( 1992)
S&G. Iransci-ipt Lat-iant
Ayer-LeLicvrc
ct (I/
Intercellular Communication Grcwth Factor\ N<SfB
trl.
of trl. (
I9XX), Parvinen e /
(1992)
hFGF
S&G
Mayerhofer c/ t i / . ( I99 I 1. Lahr ct ( I / . (1997) Watrin or t i / . ( I99 1 ) Zhatr and Hogan ( 1996) Zhao t’? t i / . ( 1996) Han\\oii or ol. ( 1989). J . Baker e/ trl. ( 19961 B o d y ef i f / . 11994). T\uruta and
Acrogranin
S&G, gene lamily
Baha c r rrl. ( 1993)
S&G S&G
Foo trl. (1994) Kilpalrich (’I t i / . (19x5). Kilpati-ick and Millctte (1986) Per\son c’f ( I / . ( 19x9)
O‘Ri-ien (19951
Neuropcptides
VP Proenkeplial i n CCK Receptor?, Prolactin Type 111 TGF-P
S&G, transcript ~ariaiit S&G S&G, trnn\ci-ipt vai-iaiit\
(21
145
5 , Gene Expression during Marnriiali:un Meiosis
ActiLiii
S&G
IGI~-II/CI-M6P
S&G S&G
BRS-3 DTMT A ndcnoainc Signal Tran\ductioii Kinahe\ PKA RI,r
S&G, getie l i l m l 1 ~ S&G. gene lsllllly
@ycn i't ol. ( 1990)
MAS T2 5
Walden and Cowari ( 1993). Walderi and
Millettc ( I 996) Wolic\ ('I (I/. (1989). Wadewitr ('1 r i / . (1993) Muttei- and Wolpernuth (1987) Toshiina c't t i / . (1995) Ke\her c't o/. (1990). Fishmnn ef ( I / ( 1990) Mean\ et d . ( I991 )
Mi1 \
TESK I Fri-t2 CAMKIV Pho\phodiesterasc Type IV CAMP PDE Regulatory Proteins G,,u. G,,(Y. G,,n, G,,u K ,H .N-rtr,\ 01 2 -C'h iiiieri ti Cal modu I i t i
Dc Winter PI trl. ( 19921, Kaipiii c'f ti/. (1992) O'Brien et ol. (1994) Fathi et ti/. ( 1993) Parmentier Ct d.(1992) Meycrhof o r t i / . (1991), Rivkeeh ( 1994)
Welch ('t t i / . ( 1992). Morena r / ol (1995)
S&G. gene l a i i i i l y Sorrentino et d.( 1988) Hall c'/ t i / . (1993) Sano c'f trl. ( 1987). Slaughter and Means (1989) Watanabc (If ( I / . (1994, l995), Ohsaho ('/ (I/. ( 1994)
Eri/yiiie\ o l Energy Metabolism P,yl.? G , gene
talllll)
Other Component\ Cyto\kelclal Proteins KKP2
I'~ln1ll)
G, gene
McCarrey and Thomas ( 1987). Boer ( I / . ( 1987) Mori C/ (I/. (1993. 1996)
<J/
146 Table I
E. M. Eddy and Deborah A. O'Brien Coritinrrrd ~~
Genes
Expresion pattern
MCSISCMP
G. unique gene
Reference Kleene er
ti/. (
1990). Cataldo
('I
rrl.
( 1996)
p
S&G, transcript variant
Lin and Morrison-Bogorad ( I 99 1 )
Pc'4
G , gene family
fertilin u ftrtilin p cyritestin
S&G, gene family G , gene family G. gene tarnily
proacrosin
G, gene family
Kakayaina r'r ti/. ( 1992), Scidah cf ti/. ( 1992) Wolfsberg cr ol. (1993, 1995) Wolfsberg P I ol. (1993, 1995) Heinlein F I ( I / . ( I994), Wolfsberg et a/. (1995) Klemm (11 t i / . ( 1990), Kashiwabard ef d.( 1990)
thymosin Proteam
Other Enryme\ dnd Protein\ PKPS3 G. gene family mCS7M.5 G, gene family flrltllf S&G. transcript variant ODC S&G ASA
ChAT CCK GAD 1,ipophilic tran\port piotein sp17 CFTR
S&G S&G S&G, tran\cript variant S&G G
G S&G, splice variant
Taka r / crl. ( 1990) Fulcher ei a/. (1995) Benoit and Trasler ( 1993) Alcivar r'/ t i / . (1989). Kaipia er d. ( 1990) Kreysing er d.( 1994) I b a W C I ti/. ( I99 I ) Persson r'f t i / . ( 1989) Persson t't rr/. ( I O Y O ) Schmitt PI u/. (1994)
Kong c't trl. (1995) Trezise. Buchwald, C I trl. (1993). Trezise. I.inder. P I trl. (1993). Delaney ('I r r / . ( 1994)
<'S&G: gene expressed in somatic and germ cells. "Transcript variant: alternative transcripts produced in sperniatogenic cell\ by unusual transcription initiation or termination processe\. transcript-splicing mechanisms. or exon utiliration. G: expressed only in germ cells. ./Gene family: i\ologous gene(s) expressed i n other ti\\ue\. ',Unique gene: n o isologoua gene(s) have been identified.
pressed developmentally in the mitotic and postmeiotic phases as well (reviewed by Willison and Ashworth, 1987; Eddy rt u[., 1991, 1993; Wolgemuth and Watrin, 1991; Winer and Wolgemuth, 1993; Hecht, 1993, 1995; Eddy, 1995; L6pez-Fernandez and del Mazo, 1966; Kleene, 1996). Gene expression during meiosis can be viewed as part of a larger developmental program for the germ line, stretching from the origin of primordial germ cells in the early embryo to the production of gametes in the adult and the joining of these cells at fertilization to begin the next generation.
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II. RNA Synthesis during Meiosis Early autoradiographic studies o f KNA synthesis during spermatogenesis i n the mouse observed maximal levels of [ '1-1 luridine incorporation during meiosis (Monesi 1964, 196%). In these studies. RNA synthesis was low during the perleptotene stage and undetectable in leptotene, zygotene, and early pachytene spermatocytes. The incorporation of [ 'H luridine increased rapidly to a transient peak in mid to late pachytene spermatocytes and then declined during the remaining prophase stages to reach undetectable levels during the meiotic divisions. High levels of RNA synthesis have been detected in pachytene spermatocyles isolated from mouse (Geremia c't ( I / , , 1978; Handel et ul., 1995), rat (Soderstriim, 1976), hamster (Utakoji et nl., 1968), and human (Saussine et NI., 1994) testes. Electron microscopic autoradiography confirmed high levels of RNA synthesis in pachytene spermatocytes, and also demonstrated [ 3H]uridine incorporation during earlier stages of meiotic prophase (Kierszenbaum and Tres, 1974b). Nucleolar RNA synthesis reached a peak during the zygotene stage in both mouse and human spermatocytes, providing evidence that ribosomal RNAs are synthesized during meiosis (Kierszenbaum and Tres, 1974b, 1978; Tres, 1975). RNA precursors were also incorporated into the nucleoli o f rat primary spermatocytes (Stefanini et ul., 1974). During the pachytene stage 01' meiosis in both mouse and human spermatocytes, RNA synthesis detected by high-resolution autoradiography was primarily localized adjacent to the autosomes (Kierszenbaum and Tres, l974b; Tres, 1975). This perichromosomal localization of ['Hluridine suggests that RNA synthesized during meiosis is predominantly heterogeneous nuclear RNA (hnRNA), the precursor of cytoplasniic mRNAs. Subsequent biochemical studies have shown that isolated pachytene spermatocytes synthesize both ribosomal RNA and hnRNA, as identified by polyacrylamide gel electrophoresis (Grootegoed et ul., 1977) or sucrose gradient centrifugation (Geremia Pt ul., 1978). Furthermore, approximately one third of the hnRNA (Soderstrom. 1976) and one third of the cytoplasmic KNA in pachytene spermatocytes was polyadenylated (D'Agostino et ul., 1978; Germia er d., 1978), and some of the polyadenylated RNAs were bound to polysomes (D'Agostino et af., 1978; Gold et al., 1983). These studies provided initial evidence that substantial levels of mRNA are transcribed during meiosis, particularly during the pachytene stage, and that these transcripts are used for protein synthesis. In contrast to the autosomes, the condensed X and Y chromosomes in mouse and human pachytene spermatocytes incorporated little or no RNA precursors (Monesi, 1965a; Kierszenbaum and Tres, 1974a,b; Tres, 1975). Subsequent studies have provided further evidence for transcriptional inactivation of the sex chromosomes during meiosis, including the loss of mRNAs transcribed from several X-linked genes (McCarrey, Dilworth, et ul., 1992; Shannon and Handel, 1993) and the absence of RNA polymerase I1 and other components of the RNA
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splicing machinery from the condensed XY chromosome pair (Richler et a / . , 1994). Early studies monitoring [3H]uridine incorporation during meiosis indicated that much of the RNA synthesized during the pachytene stage accumulated in the nucleus and was then released into the cytoplasm during the division stages (Monesi, 1964. 196%). A significant portion of this RNA persisted throughout spermatid development, and was ultimately discarded in the residual bodies (Monesi, 1964, 196%; Geremia et ul.. 1977). The results suggested that longlived mRNAs synthesized during meiosis may be stored for translation during spermiogenesis. Definitive evidence for translation of meiotic transcripts in spermatids is difficult to obtain, since many genes are transcribed both during meiosis and in the early stages of haploid differentiation. The accumulation of polyadenylated RNA in the nucleus of pachytene spermatocytes has been confirmed in the rat, particularly during stages IX-XI of the seminiferous cycle (Morales and Hecht, 1994). However, there is no conclusive evidence for sequestration and storage of mRNA in specific cytoplasmic structure in spermatocytes (Morales et al., 1993; Morales and Hecht, 1994).
I 11. Genes Expressed during Meiosis It has been estimated that at least 100 genes are associated with meiosis in Sacchurornyces cerevisicie (Burns el a / . , 1994), and it would not be surprising if
even more genes are associated with meiosis in mammals. Although many genes are developmentally expressed during the meiotic phase of spermatogenesis in mammals, relatively few have been characterized thoroughly and shown to encode proteins with specific roles in meiosis. Furthermore, reports of spermatogenic cell-specific transcripts frequently have been based on Northern blot analysis of a limited number of tissues from adult animals. These studies seldom have established rigorously that spermatogenic cell-specific transcripts are products of genes expressed exclusively in spermatogenic cells or are alternative transcripts of genes also expressed in other cell types. Genes expressed during meiosis can be grouped into categories based on the location or presumed function of the proteins that they encode (Table 1). These include genes for proteins involved in nuclear structure, DNA repair and recombination. transcriptional regulation, RNA processing, cell cycle, intercellular communication, signal transduction, enzymes of energy metabolism, and other components. However, this organization is artificial because some proteins may have multiple roles, and the roles of others may be incorrectly defined or unknown. In addition, some transcripts produced during meiosis encode proteins with postmeiotic roles. This review focuses on genes transcribed during meiosis, and most reports based only on protein data from biochemical or immunochemical studies are not included.
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A. Structural Proteins of the Nucleus
The structural proteins of the nucleus include the lamins, histones, and synaptonemal complex (SC) proteins (Table I ) . The nuclear envelope is supported by an internal scaffolding that contains the lamins and to which other components of the nucleus are tethered. The histones are structural proteins of the chromosomes that both form the nucleosomes around which the DNA is wound and associate with the intranucleosomal DNA. Although these structures are ubiquitous, there are lamins and histones unique to spermatogenic cells. However, the SC is found exclusively in the meiotic cell nucleus. It provides a structural substrate for pairing and other chromosomal events that are the hallmarks of meiosis. In addition to the proteins that make up its structure, the SC contains proteins that are involved in DNA repair and meiotic recombination.
1. Lamins The nuclear lamins are a family of karyoskeletal proteins that maintain nuclear envelope integrity and nuclear structure. cDNAs have been cloned that hybridize with transcripts in pachytene spermatocytes encoding nuclear lamins unique to spermatogenic cells. The mRNA for an A-type lamin, named lamin C 2 , was detected in mouse pachytene spermatocytes, but not in juvenile testis or other tissues from adult animals (Furukawa et ul., 1994). The nucleotide sequence was identical to that for mouse A-type lamin, except that the N-terminal 119 amino acids of lamin A were replaced by a 7-amino acid sequence in lamin C2. This suggests that the mRNA encoding lamin C2 is generated by differential splicing from the A-type lamin gene during spermatogenesis (Furukawa et NI., 1994). A cDNA was also cloned for ;I B-type lamin unique to the testis, and the mRNA and protein were present in isolated pachytene spermatocytes of the mouse (Furukawa and Hotta, 1993). The protein was named lamin B, and the mRNA was determined to be an alternative transcript of the lamin B2 gene, produced by differential splicing and use of a different polyadenylation signal. However, the period of expression of lamin B, during spermatogenesis remains to be described.
2. Histones Marked transitions in basic chromosomal proteins occur during spermatogenesis, including the replacement of somatic histones by testis-specific histone variants. Genes encoding three of these variants, TH2B (Y.-J. Kim et nl., 1987), TH2A (Huh et NI.. 1991 ), and HI t (Cole et ( I / . , 1986), are expressed during meiosis (Table I). Unlike somatic histones, the synthesis of these testis-specific variants is not dependent on DNA replication (Brock et d . , 1980). A distinct histone H4 gene ( H 4 t ) that is within 1.5 k b of the H l t gene is also expressed in pachytene spermatocytes (Grimes eta/., 10x7: Wolfe Pt nl., 1989; Wolfe and Grimes. 1991).
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The predicted amino acid sequences of H4t and the somatic H4 histones are identical, even though the nucleotide sequences and regulation of these H4 genes are substantially different. Genes encoding the testis-specific histone variants TH2B and TH2A i n the rat have the same structural organization as the somatic H2B and H2A genes. The TH2B and TH2A genes are transcribed in opposite directions and share approximately 300 bp of 5’ flanking sequence (Y.-J. Kim et ul., 1987; Huh et al., 1991 ). This shared promoter region contains a bidirectional transcriptional regulatory element that is typically associated with S-phase-specific expression (Hwang et ul., 1990; Huh et a/., l99l), even though these genes are expressed in prophase. Additional studies suggest that expression of the TH2B and TH2A genes is correlated with DNA hypomethylation, because CpG sites in the promoter region are methylated in somatic cells but are unmethylated during spermatogenesis (Choi and Chae, 1991, 1993). TH2B and TH2A mRNAs (Y.-J. Kim et ul., 1987; Huh er al.. 1991) and proteins (Meistrich et al., 1985) have been detected during meiotic prophase in the rat. Both or these variants participate in nucleosome formation (Rao et ul., 1982; Chiu and Irvin, 1986). The amino acid sequence of histone TH2B, which replaces most of the somatic H2B histone in pachytene spermatocytes (Meistrich et al., 1985), is most divergent in the N-terminal third of the molecule that binds to DNA (Y.-J. Kim et al., 1987). I t has been proposed that replacement of somatic H2B with TH2B is responsible for a relaxed chromatin structure in pachytene spermatocytes that may be important for recombination (Rao et al.. 1983; Rao and Rao, 1987). The H 1 t histone protein is a member of the family of H 1 histones that bind to linker DNA between nucleosornes, influence chromatin structure, and may act as general repressors of transcription (reviewed by Wolfe and Grimes, 1993). The H l t histone gene is expressed exclusively in mid to late pachytene spermatocytes in rat (Meistrich et ul., 1985; Kremer and Kistler, 1991; Grimes e t a / . , 199221)and mouse (Drabent et a/., 1993, 1996). The H l t protein was not detected by immunohistochemistry at 9 days after birth in mouse but was present in pachytene spermatocytes at 20 days, the next age examined, and was later seen in the nuclei of round spermatids (Drabent et d . , 1996). In surface-spread preparations of mouse pachytene spermatocytes, HI t was first detected on meiotic prophase chromosomes after synapsis and SC assembly (Moens, 1995). Compared to other H1 histones, H l t has fewer DNA-binding motifs and is a poor condenser of chromatin (Khadake and Rao, 1995).These studies suggest that H1 t has a role in spermatogenic cell chromatin structure from late pachytene spermatocyte development until chromatin condensation begins in spermatids during the postmeiotic phase. Another nuclear protcin has been identified that is unique to spermatogenic cells and is an apparent histone-binding protein. This is NASP (nuclear autoantigenic sperm protein), and the mRNA and protein were first found in pachytene
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spermatocytes in the rabbit (Welch and O'Rand, 1990; Welch et ul., 1990). The deduced protein contains two histone-binding motifs and has regions of similarity to the Xenopus N1 /N2 histone-binding protein (Kleinschmidt et ul., 1986). The NASP protein was detected by immunostaining in small patches in the nucleus of leptotene and zygotene spermatocytes. It was present in larger patches in pachytene spermatocytes, dispersed into the cytoplasm during the meiotic divisions, and was again localized in patches in the spermatid nucleus (Welch et ul., 1990). NASP may be involved in the histone switching events that occur during meiotic and postmeiotic chromatin remodeling.
3. Synaptonemal Complex Components The SC is a unique feature of spermatocyte and oocyte nuclei during meiosis. Pairing of the condensed chromosomes during meiotic prophase occurs concurrently with assembly of the SC. The two longitudinal elements of the SC are connected by transverse filaments and the SC forms the structural substrate upon which crossing over and recombination occur (see Chap. 7, by P. B. Moens. R . E. Pearlman, W. Traut, and H. H. Q. Heng, this volume). The SCPl protein is a major component of the transverse filaments of SCs in rat and mouse spermatocytes (Schmekel et al., 1996; Liu et ul., 1996). I t also contains potential DNA-binding motifs and target sites for CDC2 protein kinase (Meuwissen et ul., 1992). cDNAs have been identified for Scpl homologs in rat (Meuwissen et ul., 1992), mouse (Kerr c't ul., 1994, 1996; Hoog, 1995), and hamster ( S y n l ; Dobson et LII., 1994).Transcription of Scpl occurred in zygoteneto diplotene-stage spermatocytes in the rat (Meuwissen et ul., 1992). The CORl protein is another SC component that is encoded by a gene expressed only during meiosis. It was detected by Western blotting in cells in the leptotene-zygotene stages of meiotic prophase. It localized to unpaired chromosomal cores before synapsis, to the lateral domains of the SC after synapsis, to the chromosomal cores after disjunction, and is then axial to the metaphase I chromosomes and in association with pairs of sister centromeres (Dobson er a/., 1994). cDNAs for the Corl gene have been isolated for the hamster (Dobson et d . , 1994) and rat homologs (Scp3; Lammers et al., 1994), and the mRNA was detected by Northern analysis i n rat testis. but not in liver, kidney, brain, thymus, or spleen (Lammers et al., 1994). A clone encoding a third SC protein (SC65) was isolated from a rat testis cDNA expression library. The SC6S protein localized to the inner side of the lateral element of the SC (Chen et nl., 19Y2).Although the SC65 cDNA sequence showed no similarity to any previously reported sequences, Northern blot analysis on RNA from four tissues indicated that major 2-kb and minor 2.4-kh transcripts for SC65 were readily detected in testis, brain, and heart and were present at low levels in liver. It apparently was not determined when the SC6S mRNA or
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the 65-kDa protein appears during spermatogenesis, or where the protein is localized in brain and heart cells. A protein associated with the condensed X Y chromosome pair in pachytene spermatocytes appears to have features in common with SC proteins. I t is encoded by the Xrnr gene, a member of the Xlr (X-chromosome linked, lymphocyte regulated) gene family (Calenda et ( I / . , 1994). The transcript was detected by Northern blot analysis only in mouse testis, where it was present in low amount at 2 weeks and more abundant at 3 weeks of age. The 25-kDa XMR protein was first detected in the nucleus of preleptotene cells, increased in pachytene spermatocytes, was reduced in diplotene spermocytes, and was not deleted by metaphase I. The protein was present initially throughout the nucleus, but by zygotene it was localized to the sex body, where the greatest concentration of protein was associated with the axes of the X and Y chromosomes (Calenda et al., 1904). The Xmr- gene sequence has similarities to the MER2 gene from Succhuromycrs cerevisiae that is essential for meiosis and appears to be involved in gene conversion or formation of SCs (Engebrecht et a/., 1990).
B. DNA Repair and Recombination
Studies in yeast have established that DNA repair proteins also participate in meiotic recombination. The mammalian homologs of some of the yeast genes involved in these processes have been identified and the proteins that they encode have been localized to the SC (see Chap. 4, by W. J . Cummings and M. E. Zolan, and Chap. 6, by T. Ashley and A. Plug. this volume). Many of these genes are expressed ubiquitously, but, as is noted below, several have been found to be developmentally regulated during spermatogenesis. The elevation of transcript and protein levels during pachytene spermatocyte development strongly suggests that these components have a significant role in meiosis (Table I). The Esclzrrichiti coli RecA enzyme has an essential role in initiation of pairing and strand exchange, homologous recombination, and DNA repair. The RLiti-71 gene of the mouse is the homolog of RecA and is transcribed at high levels in thymus, spleen, testis and ovary, those organs where genetic recombination events associated with lymphocyte differentiation and meiosis normally occur 1993). Rod51 mRNA was present in (Morita et ul., 1993; Shinohara et d., spermatogonia and pachytene spermatocytes (Morita et d., 1993) and the protein was highly enriched in the SC of pachytene spermatocyles (Haaf et 01.. 1995). RAD5 I was also associated with SCs of mouse oocytes, and the axial localization of RAD5 1 occurred earlier in oocytes than in spermatocytes (Ashley et a/., 1995). A study using the yeast two-hybrid system to identify proteins that interact with RAD51 identified HSCJBCY, the human homolog of the S. cerivisitre gene for the Ubc9 ubiquitin-coiijugating enzyme (Kovalenko et LII., 1996). The
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mouse MinUbcY gene was expressed ubiquitously, but at elevated levels in testis and thymus. The protein was found to co-localize with RADSI on the SC in pachytene spermatocytes of the mouse and was suggested to play a regulatory role in meiosis (Kovalenko et u / . . 1996). The Dinc.1 gene of mammals is the homolog of the yeast S. cerevisiw “disrupted meiotic cDNA” gene that functions specifically in meiotic recombination (Bishop et u/., 1992). The yeast I1riic.l and Rad.51 genes are structurally and evolutionarily related, and their proteins co-localize on zygotene chromosomes. Dmc1 transcripts were detected i n mouse only in adult testis by Northern analysis and in adult testis and fetal ovary by RT-PCR (Habu et NI., 1996). High accumulation of Di77cl transcripts was seen in spermatocytes by irz situ hybridization. Sequence analysis of cDNAs indicated that alternative splicing produces two Lli?zcl transcripts, and DMC 1 proteins o f 3 1 kDa and 37 kDa were detected in testis homogenates by Western blotting (Habu et nl., 1996). The DNA ligase I11 enzyme functions in DNA single-strand break repair, homologous recombination, and sister chromatid exchange in mammals. Its mRNA was present at 10-fold higher levels in the testis than in other mouse tissues, and was most abundant in pachytene spermatocytes (Chen et al., 1995). DNA ligase I11 copurified by affinity chromatography with XRCC 1 (Caldecott et NI., 1994), previously identified by its ability to restore normal phenotype when transfected into repair-deficient cells (Thompson et nl., 1990). I t appears that XRCCl and DNA ligase 111 exist as a complex i n mammalian cells and that XRCCl is required for normal DNA ligase 111 activity. Xrcc.1 mRNA was abundant in the testis of rat, mouse. and baboon (Yo0 et d., 1992; Walter et d., 1994; Zhou and Walter, 1995), and Xi-ccl transcript levels were low in leptoteneLygotene spermatocytes and high in pachytene spermatocytes and round spermatids of mouse (Walter et d . , 1996). This suggests that XRCCI-dependent DNA ligase 111 activity is importiuit in DNA strand-break repair during meiosis (Walter et al., 1996). APEX nuclease (apurinic/apyrimidinic IAP] endonuclease) is another mammalian DNA repair enzyme whose mRNA is expressed at relatively high levels in the testis. It is a multifunctional DNA repair enzyme thought to be involved in DNA repair of AP sites and 3’ blocked single-strand breaks, and to function as a redox factor that stimulates the DNA binding activity of Fos-Jun heterodimers (Sancar and Sancar, 1988; Xanthoudakis 1992). APEX transcript levels increased substantially between days 21 and 28 i n the rat testis (Tan et nl., 1996). This corresponds to the first wave of spermatogenesis in the juvenile rat testis, when pachytene spermatocytes appear and increase substantially in number, strongly suggesting that APEX nucleaae mRNA expression increases during meiosis and that the enzyme has a role in this process. The MutH, MutS, and MutL proteins are essential components of the bacterial postreplication DNA-mismatch repair machinery. Null mutation of the Pins2
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gene, a MutL homolog, caused infertility in male but not female mice (Baker et al., 1995). Epididymal sperm were abnormal in appearance, vacuoles were present in pachytene spermatocytes, and the number of spermatids was reduced. Surface spreads of spennatocyte nuclei from PMS2-deficient mice indicated that synapsis and SC organization were disrupted, suggesting that PMS2 is expressed during meiosis and has an important role in synapsis and genetic recombination. Null mutation of the Mlhl gene, another MutL homolog, caused infertility in both male and female mice (Edelmann et a/., 1996; S. M. Baker et a / . , 1996). The MLH 1 protein was localized on synapsed portions of SCs in oocytes earlier than in spermatocytes in wild-type mice. MLH 1-deficient spermatocytes showed high levels of prematurely separated chromosomes and arrest in late pachytene or metaphase. MLH 1 deficient males did not produce sperm, but females produced oocytes that failed to develop (S. M. Baker et id., 1996; Edelmann et id., 1996). However, null mutation of the Msh2 gene, a MiitS homolog, had no effect on the fertility of male or female mice (Reitmair rt u/., 1995). (See also Chap. 6, this volume, for further information on these proteins.) The ATM gene encodes a putative protein or lipid kinase, and mutation in this gene is responsible for an autosomal recessive hereditary disorder resulting in increased cancer risk and inefficient G I/S-phase cell cycle checkpoint function. The ATM protein is found along synapsed chromosomal axes, and targeted mutation of the mouse A/rn gene resulted in male and female infertility, with meiosis being arrested at the zygotene/pachytene stage (Barlow et d., 1996; Elson e f a/., 1996; Xu et ( I / . , 1996). Protein and mRNA for the related A f r gene (for ataxia-telangiectasia- and rad3-related) are at highest levels i n pachytene spermatocytes in the mouse, and the protein is found at sites along unpaired or asynapsed chromosomal axes (Keegan et uI., 1996). It has been suggested that these proteins have a direct role in recognizing and responding to DNA strand interruptions that occur during meiotic recombination (Keegan et a/., 1 996). Poly(ADP)ribose polymerase (PARP) is a chromatin-associated protein that catalyzes covalent attachment of ADP-ribose to numerous nuclear proteins. Activity of the enzyme increases greatly after DNA damage, suggesting that it is involved in DNA repair (Althaus and Richter, 1987; de Murcia and MCnihsier-de Murcia, 1994). PARP is also a component of the DNA replication complex (Simbulan-Rosenthal et ul., 1996), and its cleavage by a highly specific protease is a key step in apoptosis (Nicholson et d., 1995). High levels of Par/> mRNA were seen in the testis of mouse and rat (Ogura et a/., 1990; Menegazzi er uI., 1991). It was found using isolated spermatogenic cells that the two Ptrrp transcripts reached their highest levels in pachytene spermatocytes and were greatly reduced in spermatids (Alcivar et d . , 1992). This finding correlated with other studies indicating that Parp enzyme levels were highest in 25- to 30-day-old rats and that the enzyme was localized most strongly on the condensed chromosomes of pachytene spermatocytes (Concha et u / . , 1989). However, mice with a knock-
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out of the P a y gene were healthy and fertile, indicating that the enzyme is not required for normal chromatin function (Wang et a / . , 1995). DNA polymerase-6 (Pol-6) also is involved in excision mismatch repair and genetic recombination. Pol-6 mKNA levels increased during testicular development in juveniles and were high i n the testis of adult rat and mouse (Nowak et a/., 1989, 1990; Alcivar et a / . , 1992). The Pol-6 mRNA levels were low in leptotene-zygotene spermatocytes and increased markedly in pachytene sperniatocytes and round spermatids isolated from mouse testis (Alcivar et al., 1992). These results correlated well with earlier studies indicating that high levels of POL-6 enzyme activity were present in meiotic and postmeiotic cells from rat and mouse testes (Hecht et d., 1976. 1979; Grippo et a/., 1978; Hecht and Parvinen, 1981). Another possible participant i n recombination processes during meiosis is LINE-I, an interspersed repeated DNA present in high copy numbers in the mammalian genome. Although transcripts containing partial LINE- 1 sequences can be found in all tissues, the full-length mRNA is abundant in leptotene and zygotene spermatocytes in the mouse (Branciforte and Martin, 1994). This may be signilkant because the full-length LINE-I mRNA encodes two proteins, one of which is a reverse transcriptase. It has been suggested that LINE-I may function as a molecular glue to repair breaks in chromosomal DNA (Voliva et d., 1984), and it might have a role in the generation of retroposon genes (e.g.. Pgk2, Pclhci2) during meiosis.
C . Transcriptional Regulation The control of transcription is key to the regulation of gene expression. Distinctive DNA promoter elements of the gene and transactivating proteins that bind specifically to these elements arc responsible for modulating the transcription process. Some sequence-specilk transcription factors are ubiquitous and bind to DNA elements of' genes expressed in many cells, whereas others appear to be expressed only in specific cell types and to regulate the expression of specific genes in those cells. Activation of gene expression during meiosis presumably requires interaction between DNA-binding regions of particular transcription factors and DNA sequences specilic to the promoters of certain genes. Although many of the ubiquitously expressed transcription factors have been characterized, few of the unique transcription factors expressed in pachytene spermatocytes have been identified.
1. Transcription Factors Common DNA-binding motifs of transcription factors include zinc finger, homeobox, helix-loop-helix, leucine Lipper. POU-domain, and high-mobility-group
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(HMG) box sequences. A previous review identified several transcription factors bearing these motifs that are expressed during meiosis in males (Eddy et a/., 1993). These include: ( I ) zinc finger proteins ZFY-I, ZFY-2 (Nagamine et a/., 1990), ZFP-29 (Denny and Ashworth, 1991), ZFP-35 (Cunliffe et ul., 1990), REX-I (Rogers et a/., 1991), TSGA (Hiiog et u/., 1991), and RAR-a (Kim and Griswold, 1990); (2) the homeobox-containing proteins HOX 1.4 (renamed HOXA-I; Rubin et a/., 1986; Wolgemuth et a/., 1987; Viviano et d., 1993), and Gtx (Komuro et al., 1993); and (3) leucine zipper proteins J U N D (Alcivar. Hake, et a/., 1991), CREB (Waeber et al., 1991; Ruppert et d., 1992), and CREM (Foulkes el a/., 1992; Delmas and Sassone-Corsi, 1994). Although many of these transcription factors are expressed during other stages of spermatogenesis and in other tissues as well (Eddy ef id., 1993; Winer and Wolgemuth, 1993), some appear to have a significant role during meiosis in spermatogenic cells. Certain transcription factors expressed during spermatogenesis have been shown to regulate spermatogenic cell-specific gene expression. Transcription factors that appear to be involved in regulating gene expression during meiosis are described in the following discussion. Unfortunately, the target genes for most transcription factors expressed in spermatogenic cells are still unknown. The cyclic AMP/protein kinase A signaling pathway activates the CAMPresponsive element binding protein (CREB) transcription factor. Transcripts for truncated CREB isoforms, with alternatively spliced exons responsible for premature termination of translation, were detected at high levels in pachytene spermatocytes in rat and mouse (Wacber et d., 1991; Ruppert et al., 1992).These CREB isoforms lacked the bZIP domain and nuclear localization signal and were unable to act as transcription activators. Another isoform was identified recently in late pachytene spermatocytes that is produced by a splicing event that results in synthesis of an inhibitor CREB (I-CREB) (Walker et a/., 1996). This isoform apparently downregulates CAMP-activated gene expression by inhibiting CREB binding to CAMP response elements, and probably is responsible for cell- and stage-specific repression of CAMP-regulated genes in spermatocytes. Knockout of the Crebl gene had no effect on male fertility (Hummler et a/., 1994). The closely related CAMP-responsive element modulator (Crem) is another transcription factor that binds to the same promoter element as CREB. lsoforms of CREM that are expressed in pachytene spermatocytes were found to be repressors of transcription, whereas the isoform expressed in spermatids is a transcription activator (Foulkes eta/., 1992). Knockout of the Crem gene resulted in infertility in male mice due to disruption of spermatid development (Blendy et [ I / . , 1966, Nantel et a/., 1996). There are two heat-shock transcription factors, HSFl and HSF2. HSFl is responsible for mediating the cellular stress response, and HSF2 regulates heatshock gene expression under nonstress conditions, including processes of differentiation and development (Morimoto et al., 1992). HSf2 mRNA is more abundant in the testis than in heart, brain. liver, spleen, or kidney of the mouse. Testis
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Hsj2 mRNA levels increase between Days 14 and 21 in juvenile mice and are most abundant in pachytene spermatocytes and round spermatids (Sarge et ul., 1994). Two HSF2 protein isoforms have been identified, a larger HSF2-a isoform that predominates in the testis and a smaller HSF2-P isoform that predominates in heart and brain (Goodson c’t ( I / . , 1995). Hsj2 is a single copy gene in the mouse (Sarge et al., 1991), and its protein isoforms appear to be the result of alternative transcript splicing. The HSF2-(r isoform seems to be a more potent transcriptional activator than the HSF2-P isoform (Goodson et al., 1995). Several members of the HSP70 and HSP9O gene families are expressed in spermatogenic cells (Allen et al., 1988; Zakeri c/ d., 1988; Matsumoto and Fujimoto, 1990; Gruppi et u/., 1991, 1993), and HSF2 was reported to interact with the promoter sequence of Hsp70-2 (Section 111, part E, 1) that is expressed specifically during meiosis (Sarge et al., 1994). However, the HSP70-2 protein is abundant prior to the increase in Hsf2 mRNA levels in pachytene spermatocytes (Rosario et al., 1992: Dix, Allen, et al., 1996; Dix, Rosario-Herrle, et al., 1996), suggesting that HSF2 does not regulate Hsp70-2 expression. The mRNA for a POU-domain transcription factor, named Sprrnl, was detected by RNase protection assay in the testis but not in several other adult and embryonic tissues. In situ hybridization was used to determine that Sprrnl is expressed during a 36- to 48-hour period immediately preceding the first meiotic division in the rat testis. It is a single copy gene that encodes a protein that binds to a variant of the typical octamcr DNA response element (Andersen et d., 1993). It was suggested that SPKM- I protein may exert a regulatory function in meiotic events required for the subsequent terminal differentiation of male germ cells. The expression of certain members of the steroid receptor gene superfamily, which encode ligand-activated transcription factors, is developmentally regulated during spermatogensis. Two spermatogenic cell-specific retinoic acid receptor-a (RAR-a) mRNAs are present i n pxhytene spermatocytes and spermatids of the rat, in addition to the transcript found elsewhere (Kim and Griswold, 1990). Two RARy transcripts, including a smaller germ cell-specific mRNA, are abundant in rat pachytene spermatocytes but present at very low levels in spermatids (Huang et ul., 1994). A novel orphan receptor, named TAK-1, for which the ligand is unknown, has a ubiquitously expressed 9.4-kb transcript and a 2.8-kb transcript that is largely restricted to the testis in the human, rat, and mouse. In situ hybridization studies indicated that it is most abundantly expressed in pachytene spermatocytes in mouse and rat (Hirose et al., 1994).
2. Promoter-Binding Factors Promoter analysis studies have provided indirect evidence that proteins present in crude nuclear extracts regulate expression of certain genes during the meiotic phase of spermatogenesis or repress expression of such genes in somatic cells or
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other phases of spermatogenesis. These studies typically use DNase I footprinting assays, in vitro protein-DNA-binding assays (referred to as electrophoretic mobility shift, gel retardation, or band-shift assays), or cell-free transcription assays to identify sequences in a promoter region that bind nuclear proteins. Because mobility shift patterns are often specific for extracts of nuclei from a particular cell type, the proteins that bind to DNA or effect cell-free transcription are presumed to be transcription factors that are expressed in these cells. Further information about the size of the proteins can be determined using Southwestern blotting assays, in which radiolabeled DNA fragments are used to probe blots of nuclear proteins separated by gel electrophoresis. However, such assays do not allow precise identification of the proteins in the nuclear extracts that are used as the source of putative transcription factors. Studies using these approaches have shown that two proteins present in extracts of nuclei from adult rat testes bind to a region in the promoter of the histone H l r gene (see Section 111, part A, 2), referred to as the Hlt/TE element. These proteins were not detected in extracts of nuclei from prepubertal rat testes. Because H I t is expressed exclusively in pachytene sperinatocytes, these proteins appear to be spermatogenic cell-specific transcription factors regulating H l t expression (Grimes rt a/., 1992a,b). Thc HI t/TE promoter element is conserved in other species (vanWert rt a/., 1996), and recent studies have identified TEI and TE2 regions within the H 1 t/TE element in the mouse that bind different testisspecific proteins (vanWert rt a/., 1996). The Pgk2 gene is expressed exclusively in spermatogenic cells and encodes the phosphoglycerate kinase 2 enzyme that is essential for glycolysis (Section 111, part G). Nuclear extracts from adult testis, prepuberal testis, and HeLa cells were used to examine the regulation of expression of the Pgk2 gene. Either one or two proteins from adult testis bound to sequences within a 40-bp region 5’ to the 190bp core promoter of Pgk2, while a third protein in the other nuclear extracts also bound to this region. The proteins from adult testis were suggested to be associated with Pgk2 gene activation during meiosis, while the other protein may suppress expression (Gebara and McCarrey, 1992). Another study reported that a protein named TAP-I was a positive regulator of the Pgk2 gene. The TAP-I protein bound to a different regulatory element 82-64 bp 5’ of the transcription initiation site and stimulated cell-free transcription of Pgk2 in testis and liver extracts (Goto rt ul., 1993). The transcription in the liver extract was suggcsted to be due to the absence of a negative regulatory element in the Pgk2 promoter construct. Other studies have indicted that a protein named TIN-1 binds to a silencer-like element of mouse Pgk2 that is located 882-796 bp 5’ of the transcription initiation site. This region contained two sequences that bound proteins present in nuclear extracts of mouse B8/3 cells and rat liver. The protein also was present at low levels in rat testis nuclear extracts, presumably from somatic cells and
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spermatogonia in which Pgk2 expression is repressed (Goto et al.. 1991; Nishiyama et al., 1994). Ldh3 is another gene expressed exclusively in spermatogenic cells that encodes an enzyme important in energy metabolism (Section 111, part H). Analysis of the promoter region of the Ldh3 gene i n a transcription assay system indicated that a nuclear extract from adult mouse testis contained factors that caused promoter activation, while factors in a liver nuclear extract repressed LDH-C activity (Zhou et al., 1994). Mobility shift and Southwestern assays indicated that a 103-kDa protein in the testis nuclear extract bound to a 60-bp palindromic region in the core promoter containing the TATA box and the transcription initiation site. Liver nuclear extracts contained a 65-kDa protein that also bound to this promoter region. These results suggest that the 103-kDa protein in the testis nuclear extracts is a transcription activator in germ cells, and that the 6.5-kDa protein in the liver nuclear extracts is a repressor of Lti123 transcription in somatic cells. Footprint analysis of the 5’ flanking rcgion of the rat proacrosin gene (Section 111, part H, 2) identified two binding sites that interacted with nuclear extracts from testis (TS2 and TS3), three sites that bound nuclear extracts from testis and brain (F7, F1, and F3), and eight sites that bound nuclear extracts from brain (Kremling et al., 1995). Mobility shift assays indicated that footprint TS2 bound a sequence-specific testis nuclear protein complex, although footprint TS3 did not appear to be generated by a sequence-specific DNA-binding protein. The mobility shift patterns of testis and brain nuclear extracts were different with the F7, F 1, and F3 footprint sequences, with testis-specific protein complexes being associated with footprints FI and F7. A repressor protein was reported to be responsible for the low level of expression of histone TH2B (Section 111, part A, 2) in spermatogonia and somatic cells of rats. This protein bound to a region referred to as site E in the TH2B promoter and was present at higher levels in nuclear extracts from adult liver and 7-day-old testis than in extracts from adult testis. It bound to a site between the TATA element and the transcription initiation site of TH2B and repressed in Litm transcription (Lim and Chae, 1992). The decrease in the level of this protein in the testes with postnatal development correlated inversely with the expression of TH2B mRNA in pachytene spermatocytes. A protein that bound to a negative regulatory element (NRE) of the M o s gene promoter was present in nuclear extracts of somatic cells but not in nuclear extracts mainly from pachytene spermatocytes and spermatids. Because M o s is expressed during meiosis, when the protcin appears to be absent, this protein was suggested to be a repressor of M o s gene expression in other cells. Sequences nearly identical to the NRE are present in the promoter regions of other genes expressed in spermatogenic cell5 (Pr-m2, Pgk2, C y t , Hst70). and i t was suggested that this sequence may also be involved in repressing the expression of these genes in somatic tissues ( X u and Cooper, 199.5).
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Such findings suggest that specific transcription factors regulate spermatogenic cell-specific gene expression. However, because these proteins and their genes have not been identified, their expression patterns cannot be determined.
3. Transcriptional Machinery The TATA-binding protein (TBP) is an important component of the general transcription machinery and functions in promoter recognition and transcriptional initiation by all three RNA polymerases of eukaryotes (reviewed by Hernandez, 1993). TBP mRNA levels increased substantially in meiotic and postmeiotic spermatogenic cells in rat and mouse, and were more abundant in these cells than i n somatic cells. However, TBP protein levels appeared to be higher in postmeiotic cells than in meiotic cells (Schmidt and Schibler, 1995; Persengiev r t al., 1996). Protein levels for two other components of the RNA polymerase 11 complex. TFIIB and RNA polymerase 11, were also present at high levels in round spermatids (Schmidt and Schibler, 1995). These changes in protein levels suggest that TBP expression in pachytene spermatocytes may not relate directly to meiosis but rather to mRNA processing events that occur in postmeiotic cells, including the delayed translation of many mRNAs (reviewed by Kleene, 1996). Rat and mouse cDNAs have been characterized recently for the testis-specific transcription elongation factor (SII-TI) (Xu et LII., 1994; It0 rt d., 1996). S-I1 is a ubiquitous nuclear protein whose role is to enable RNA polymerase I to read through transcription pausing sites that are present in genes (Reines el 01.. 1992). SII-TI mRNA was not detected in spermatogonia or spermatids and appeared to be expressed exclusively in spermatocytes in the mouse (Ito er al., 1996). The deduced rat S-I1 and SII-T1 protein sequences are highly similar, except for a unique intervening sequence of 46 residues in the SII-TI protein (Xu et NI., 1994), suggesting that these proteins are products of alternatively spliced transcripts from the same gene. D. RNA Processing
Transcripts for several RNA-binding proteins that are products of developmentally regulated genes have been identified in spermatogenic cells (Table I). These proteins either have structural roles modulating the conformation and organization of DNA or RNA, or are associated with mechanisms and machinery of transcription, RNA processing, message stability, and translation. These proteins include ATP-dependent RNA helicases, poly(A)-binding protein, Y-box-binding proteins, and RNA-binding proteins. Some of these may participate in RNA processing events leading to the formation of unique transcripts, or may be involved in effecting translational delay (Kleene, 1996). The cDNAs for two putative ATP-dependent RNA helicases have been reported to hybridize with mRNAs that are expressed initially in pachytene sper-
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niatocytes during mouse spermatogenesis. They show high homology to mouse translation initiation factor e l F-4A and other members of the so-called DEAD box family of proteins that are believed to be involved in many aspects of RNA metabolism, including splicing, translation, and ribosome assembly. One helicase, referred to as PLIO, is encoded by two transcripts detected only in spermatogenic cells, at high levels in pachytene spermatocytes and lower levels in spermatids, and by a larger transcript found at low levels in the liver (Leroy et d., 1989). Southern analysis indicated that the PLlO transcripts come from a single copy gene, suggesting that spermatogenic cells and liver contain alternative transcripts. Another cDNA encoding a mouse P68 RNA helicase hybridized with an mKNA restricted to late pachytene and diplotene spermatocytes and spermatids (Leniaire and Heinlein, 1993). Northern analysis with a probe from the central part of the cDNA hybridized only with mRNA from testis, while a probe from the 3‘ end hybridized with an mRNA of about the same size as well as with a larger mRNA in the eight tissues examined. It was not determined if these were alternative products of the same gene or were transcripts from related genes. Another protein implicated in the regulation of mRNA stability and translation is the poly(A)-binding protein (PABP), which binds to the 3’ poly(A) tail of mRNAs. Two PABP cDNAs were isolated from mouse testis cDNA libraries; one (PABPI) was nearly identical in sequence to a PABP cDNA from human liver, while the other (PABP2) was only 80% similar (Kleene et a/., 1994). Although PABP mRNA i s present in all cells, the levels in testis are 5- to 10-fold higher than in somatic tissues. There are multiple sizes of PABP transcripts in testis, but a 5’ untranslated region (UTR) probe for PABP2 hybridized only with a 2.7-kb transcript (Kleene et ( I / . , IY94). The level of PABP mRNA increased in early pachytene spermatocytes, was highest in round spermatids and became low in elongating spermatids (Kleenc et a/., 1994; Gu et ul., 1995). Western blot and immunocytochemistry analyses indicated that PABP protein levels closely follow the mRNA levels in pachytene spermatocytes and round spermatids and diminish as spermatids undergo nuclear elongation and condensation (Gu et a/., 1995). Southern blot analysis indicated that three or more genes for PABP exist in the mouse genome (Kleene eta/., 1994).The differences in the nucleotide sequences of the PABPI and PABP2 cDNAs suggest that they represent mRNAs transcribed from different genes. There is a family of dual-function proteins that bind to RNA in a sequenceindependent manner and also bind to the DNA Y-box sequence. The mouse homologues of the Xenopus ~ 5 4 1 ~ 5Y6box proteins were present in pachytene spermatocytes and reached their highest levels in round spermatids. They bound to nonpolysomal RNA (Kwon et N / . , 1993) and also interacted with the Y-box element in the Prml promoter (Nikolajczyk et al., 1995). A cDNA encoding another mouse Y-box protein, MSY 1, hybridized with mRNA initially present in pachytene spermatocytes (Tafuri et a/., 1993). The protein was suggested to function in regulating storage and translation of spermatogenic cells RNAs.
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Because many mRNAs expressed i n the testis contain Y elements (Han et al., 199S), Y-box-binding proteins may have a key role in the storage of messages that are produced in late pachytene spermatocytes and round spermatids and transcribed later. Three cDNAs that appear to encode mRNA-binding proteins were isolated in a screen designed to select proteins that bind to the 3‘ UTR of mouse protamine 1 ( P r m l ) mRNA and effect the translational repression or activation of this message. One cDNA encoded a nuclear RNA-binding protein named TENR, for testis nuclear RNA-binding protein, that is transcribed exclusively in the testis (Schumacher et ul., 199Sa). Although Tenr mRNA was present in pachytene spermatocytes, the protein was first detected in a lattice-like network in the nuclei of round spermatids in the mouse. A second cDNA encoded PRBP, a protamine mRNA-binding protein that is present in the cytoplasm of late-stage spermatocytes and round spermatids and apparently is restricted to the testis (Lee rt d . , 1996). PRBP contains two domains that bind to double-stranded RNA and is a member of a known family of RNA-binding proteins. It is not completely specific for PrmI mRNA and may act as a general suppressor of translation (Lee et ul., 1996). The third cDNA isolated in this screen encoded a protein named SPNR, for spermatid perinuclear RNA-binding protein (Schumacher et ul., 199Sb). The deduced SPNR protein sequence contains two RNA-binding domains. and in v i m assays confirmed that it binds to RNA, including the 3’ UTR of Prml mRNA. The highest level of Spnr mRNA was in the testis, but lesser amounts were also found in the thymus, kidney, liver and spleen. The SPNR protein was detected only in testis by Western blotting, and was found by immunohistochemistry only in spermatogenic cells, being first detectable in step 9 spermatids (Schumacher rt ul., 199Sb). Because the Spnr cDNA was recovered from both pachytene spermatocyte and round spermatid libraries, transcription probably begins during meiosis.
E. Cell Cycle
Pachytene spermatocytes and oocytes are in a prolonged G,-phase of the cell cycle and pass through the G,/M-phase transition of meiosis I to become secondary spermatocytes and oocytes. Cyclin-dependent CDC2 protein kinase regulates the G,/M-phase transition during mitosis and has a similar role in meiosis. Proteins encoded by some tumor-suppressor genes also are involved in regulating cell cycle events in mitosis, and their presence during meiosis suggests that they have a role in this process as well. In addition, a unique member of the HSP70 heat-shock protein family expressed during meiosis in the male has been shown recently to have an essential role in determining CDC2 activity in pachytene spermatocytes (Dix, Allen, et ul., 1996).
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1. Cyclin/CDC2 The cyclin proteins undergo cyclic synthesis and destruction during the cell cycle and are critical regulators of cell cycle events. The association of cyclins with CDC2 (also referred to as p34cdcz),a cyclin-dependent serine/threonine protein kinase (cdk), initiates changes in phosphorylation that result in CDC2 activation and passage through cell cycle boundaries (reviewed by Pines, 1993; see also Chap. 10, this volume). Both CDC2 and cyclins are members of gene families. Developmentally regulated gene expression during spermatogenesis has been reported for certain members of the cyclin family. Cyclin B1 ( C y c B I ) transcript and protein levels were elevated in pachytene spermatocytes and early round spermatids (Chapman and Wolgemuth, 1992, 1993). However, cyclin B I dependent CDC2 kinase activity was found in pachytene spermatocytes, but not in spermatids (Chapman and Wolgemuth, 1994). Cyclin B2 (CycB2) expression also was developmentally regulated during spermatogenesis, with transcript levels being highest in late pachytene and diplotene spermatocytes (Chapman and Wolgemuth, 1993). A cDNA was cloned recently from a mouse testis library for a distinct cyclin A1 (Ccrial).The mRNA was present in mouse testis and ovary, as well as in the inner cell mass and trophectoderm of blastocysts, but was not detected in the brain, thymus, kidney, ovary, or heart. The level of Ccnal mRNA rose dramatically in late pachytene spermatocytes, but was undetectable soon after completion of the meiotic divisions. Cy(.Al was present in metaphase 1 and I1 oocytes, where a proportion co-localized with the spindle, suggesting a functional interaction between the CycAl protein and a component of the spindle apparatus (Sweeney et ml., 1996). CDC25 proteins are products of the Ccic2.5 gene family for threonineltyrosine phosphatases that effect some of the changes in phosphorylation of CDC2 that are necessary for its activation. The transcripts of Cdc25c are present at high levels in the testis of mouse (Wu and Wolgemuth, 1995). Although the 2.1-kb transcript present in other tissues was detected at low levels in mouse testis, a unique 1.9-kb transcript was present at high levels in late pachytene and diplotene spermatocytes and was still present in round spermatids. The cDNA for the shorter transcript was cloned from a mouse testis cDNA library and the comparison of its sequence with that of a previously published Cdc25c suggested that tissue-specific splicing occurs to produce the spermatogenic cell transcript (Wu and Wolgemuth, 1995). In addition to these well-known cell cycle-regulatory components, genes for other proteins expressed in germ cells may be involved in this process. Mak (male germ cell-associated kinase) may be a unique member of the CDC2 family and is present in rat, human, and mouse testis (Matsushime et al., 1990; Koji et al., 1992). Mak has homology to the S . pornbe cdc2 and is expressed in late pachytene spermatocytes in the mouse (Koji rr d., 1992). A 3.4-kb transcript is
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present in pachytene sperinatocytes and an additional 2.6-kb transcript is present in spermatids in the rat (Wang and Kim, 1993). A cDNA clone of another protein kinase (Clk3)which was high similarity to the Clk (Cdc2-like kinases) subfamily was isolated from a rat brain library. By Northern blot analysis, Clk3 mRNA was abundant in rat testis, and faint or no signals were seen in other tissues (Becker er a/., 1996). It was not reported what cell type contained Clk3 transcripts or if expression was developmentally regulated. Another cDNA was isolated from a mouse testis library for M e g l (meiosis expressed gene) that hybridized with an abundant transcript in pachytene spermatocytes that encoded a lysine-rich protein (Don and Wolgemuth, 1992). Although it was suggested that this gene may be involved in meiotic processes, this relationship has not been demonstrated. Nekl is a novel seridthreonine kinase gene (Letwin et ul., 1992) that contains an N-terminal domain similar to thc catalytic domain of NIMA, a protein kinase that controls initiation of mitosis in Aspergil/us nidulans. Although ubiquitously expressed in mouse tissues, N e k l mRNA is present at substantially higher levels than elsewhere in ovaries of fetuses at Day 15.5 of development and in spermatocytes and immediate postmeiotic spermatids of adult males. These findings led to the suggestion that NEKl may be involved in the regulation of meiosis (Letwin et a/., 1992).
2. Tumor-Suppressor Proteins Some tumor-suppressor genes are involved in cell cycle regulation and are expressed at elevated levels during the meiotic phase of spermatogenesis. The p53 protein is involved in DNA repair, acts as a transcription factor, is a cell cycle checkpoint component, and has a role in induction of apoptosis. Studies using transgcnic mice (Almon et NI., 1993) and irz .ritu hybridization (Schwartz et al., 1993) found that p53 was expressed at relatively high levels in the testis, with transcription occurring mostly in pachytene spermatocytes. In addition, spermatocytes in p53 gene knockout mice occasionally appeared to be unable to complete meiosis and formed multinucleated giant cells, although these mice were fertile (Rotter et ul., 1993). The retinoblastoma (Rb) susceptibility gene product is another regulator of cell growth. The Rbl gene is expressed at high levels in the testis, with a shorter transcript appearing near the end of meiosis in the mouse (Bernards rt ul., 1989). The Rbccrl gene also is expressed at high levels in pachytene spermatocytes and round spermatids of the mouse (Zabludoff et al., 1996). This gene is linked to breast and ovarian cancer susceptibility, but rather than being expressed in early progenitor cells in breast epithelium, spermatogenesis, or other tissues, as expected, it appears to be associated most often with final rounds of cell division or terminal differentiation in tissues. The BRCA 1 protein is associated with the
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developing SC, being present on the axial elements of meiotic chromosomes prior to synapsis (Scully et a/., 1997).
3. HSP70-2 Protein Members of the HSP70 heat-shock family are molecular chaperones that mediate protein folding, translocation and assembly of other proteins. Spermatogenic cells of mice contain at least three HSP70 proteins in common with somatic cells, and two other HSP70 proteins encoded by genes exclusively expressed in spermatogenic cells. One of these ( H s p 7 0 - 2 ) is expressed during meiosis; the other (Hsc70t) is expressed postmeiotically. Hsp70-2 gene transcription begins in leptotene-zygotene cells and occurs at a high level in pachytene spermatocytes (Zakeri et al., 1988; Rosario et id., 1992; Dix, Rosario-Herrle, et a/., 1996). Although abundant in the cytoplasm, the HSP70-2 protein also was found to be associated with the lateral elements of the SC in pachytene spermatocytes (Allen et al., 1996). The gene knockout approach was used to disrupt the Hsp70-2 gene to determine if the protein has a critical role in meiosis. It was found that male mice homozygous for the mutant allele (H.sp70-2-’-) were infertile because spermatocytes arrest in development and undergo apoptosis at the transition from G, to M-phase of the first meiotic division (Dix, Allen, et al., 1996). The HSP70-2 protein is not present in oocytes, and the fertility of female Hsp70-2-/- mice was unaffected. The failure to complete the GJM-phase transition suggested that HSP70-2 was a molecular chaperone for the CDK that regulates this aspect of the meiotic cell cycle. Co-immunoprecipitation and in vitm binding experiments demonstrated that HSP70-2 directly interacts with CDC2 in the mouse testis, is a molecular chaperone for CDC2. and is required for CDC2/cyclin B I complex formation (Zhu et ul., 1997). Furthermore, CDC2 kinase activity was nearly absent from extracts of testes from Hsp70-2-/- mice. Since most CDC2 kinase activity is present in pachytene spermatocytes (Chapman and Wolgemuth. 1994), it appeared that disruption of CDC2/cyclin B1 complex assembly and the absence of testicular CDC2 kinase activity was responsible, at least in part, for the meiotic arrest in Hsp70-2-I- mice. In addition, the failure of the other constitutively expressed HSP70-2 proteins to compensate for the deficiency of HSP70-2 indicated that the chaperone role for CDC2 is specific for this member of the HSP70 family (Zhu et al., 1997).
F. Intercellular Communication
Although there is substantial evidence that paracrine interactions in the seminiferous epithelium are important regulators of germ cell differentiation (Jegou,
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1993), specific pathways that mediate communication between spermatogenic and Sertoli cells have not been adequately defined. However, it has been demonstrated that several growth factors and neuropeptides are expressed in spermatogenic cells undergoing meiosis. In addition, a few surface receptors have been identified in spermatocytes.
1. Growth Factors Both mRNA and protein for several growth factors have been localized in spermatocytes, suggesting that these cells synthesize and secrete paracrine factors which interact with neighboring Sertoli cells or germ cells, or with other cells in the testis. Spermatocytes and early spermatids in the mouse and rat express nerve growth factor-@ (NGF) (Ayer-LeLievre et a/., 1988; Parvinen et a!., 1992). In both species, the predominant NGF transcripts in the testis were larger (21.5 kb) than those detected in somatic tissues (1.3 kb). NGF protein levels rose during the midpachytene stage of meiotic prophase in the rat and remained high during the meiotic divisions and steps 1-8 of spermiogenesis (Parvinen et a/., 1992). Furthermore, NGF stimulated dose-dependent increases in DNA synthesis in rat seminiferous tubule segments containing preleptotene spermatocytes (Parvinen et u/., 1992). NGF effects on spermatogenesis appear to be mediated by Sertoli cells, which express receptors for this growth factor (Persson, Ayer-LeLievre, et a/., 1990; Parvinen et a/., 1992; Djakiew et a/., 1994). Basic fibroblast growth factor (bFGF) has been isolated from bovine and 1988). Subsequent studies in human testes (Ueno et d.,1987; Story et d., rodents indicated that both bFGF mRNA and protein are expressed predominantly in pachytene spermatocytes in the adult testis (Mayerhofer et u/., 1991; Lahr et a/., 1992). On Western blots, the bFGFs from rodent germ cells were larger (24-39 kDa) than those isolated from bovine and human testes, suggesting that these proteins are precursors of the more typical 18-kDa forms (Lahr et a/., 1992; Han et a/., 1993). This growth factor has also been isolated from germ cell-conditioned medium, providing further evidence that bFGF is released from spermatocytes (Han et a/., 1993). It has been proposed that bFGF may exert paracrine effects on Sertoli cells and spermatids, since bFGF receptors have been identified in both cell types (Han et d . , 1993; Le Magueress-Battistoni et al., 1994). During testicular development in rat and boar, members of the transforming growth factor-@(TGF-@)family are expressed primarily by Sertoli, peritubular, and Leydig cells (Mullaney and Skinner, 1993; Avalett et [I/., 1994; Gautier et a/., 1994). In adult rodents, a shorter 1.8kb Tgfbl transcript (Watrin et u/., 1991) and the TGF-@I protein (Teerds and Dorrington, 1993) have been detected in pachytene spermatocytes and spermatids. Further studies have shown that additional
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members of the TGF-P superfarnily, bone morphogenetic proteins BMP8a and BMPXb, are expressed in spermatogenic cells in neonatal and adult mouse testes (Zhao and Hogan, 1996). Most male mice homozygous for a mutation of the Bmp8h gene were infertile and exhibited defect in spermatogonial proliferation and increased apoptosis in primary spermatocytes (Zhao et al., 1996). The insulin-like growth factors, IGF-I and IGF-11, appear to be expressed by both somatic and germ cells i n the testis. Species differences in the expression profiles of IGF-I in the testis have been reported for both juvenile and adult animals. l g f l mRNA was detected priiiiarily in the interstitium of the testis of prepubertal mice (J. Baker et ( I / . , 1996),although IGF-I protein has been detected in Sei-toli, Leydig, and germ cells in the neonatal rat (Hansson er al., 1989). IGF-I niRNAs were found predominantly in spermatids in the adult mouse (J. Baker et ul., 1996), while IGF-I immunoreactivity was highest in primary spermatocytes in the adult rat (Hansson et al., 1989). Igf2 transcripts have been detected in the seminiferous epithelium of the adult rat (Bondy et al., 1994) and in Sertoli cells and spermatogenic cells isolated from the mouse (Tsuruta and O’Brien. 1995). cDNAs encoding acrogranin, named because of its homology to epithelin/granulin peptides with growth-modulating properties, were cloned from guinea pig and mouse testis libraries (Baba et al., 1993). Acrogranin mRNA was found in all tissues examined, but in the testis the protein was present in pachytene spermatocytes and in the acrosomes of spermatids and sperm.
2. Neuropeptides Several neuropeptides have been identitied in spermatogenic cells. Their roles in spermatogenic cells are unknown, but they could be involved in cell-cell communication in the testis as in thc nervous system. They may have indirect effects on meiosis by being involved i n I’cedback loops in the testis that coordinate the processes of spermatogenesis. The neuropeptide vasopressin ( V P j is best known as a hormone that regulates water reabsorption in tubular cclls of the kidney. Indirect evidence indicated that mRNA for the preprohormone lorm of this 9-amino acid peptide was expressed in pachytene spermatocytes in the rat (Foo et al., 1994). The mRNA for the opioid precursor proenkephalin was detected in rat, mouse, hamster, and bovine testis (Kilpatrick et al., 1985; Kilpatrick and Millette, 1986; Yoshikawa and Aizawa, 1988) and was localized to pachytene spermatocytes and round spermatids in the mouse (Kilpatrick and Millette, 1986). The transcripts present in rat and mouse spermatogenic cells were larger than those in somatic cells due to use of alternative transcription start sites and an alternative acceptor splice site in spermatogenic cells (Garrett rt d., 1989; Kilpatrick et al., 1990). Cholycystokinin (CCK) is another neuroendocrine peptide for which a unique transcript was detected in spermatocytes o f rat, mouse, and monkey (Persson et ( i / . , 1989).
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3. Receptors Limited information is available on the expression of genes for specific growth factor receptors and hormone receptors during meiosis. However, some receptors have been identified on spermatogenic cells and may be involved i n triggering development and cell division processes. Prolactin receptor mRNAs were localized by in situ hybridization in rat interstitial cells and in spermatogonia and spermatocytes, but not in spermatids (Hondo et ul., 1995). Prolactin binding was detected on the surfaces of spermatogonia and spermatocytes, as well as on late-stage spermatids, suggesting that this hormone may act directly on spermatogenic cells (Hondo et al., 1995). Transcripts for the type 111 TGF-p receptor, a membrane-anchored proteoglycan that does not transduce TGF-P signals, have been detected in pachytene spermatocytes and round spermatids (Le Magueresse-Battistoni et ul., 1995). Unlike testicular somatic cells, which expressed a single 6-kb transcript, pachytene spermatocytes expressed three transcripts for this receptor. Activin is a member of the TGF-P superfamily, and type I1 activin receptors are also expressed in rat germ cells, predominantly in pachytene spermatocytes and round spermatids (de Winter et ul., 1992: Kaipia et ul., 1992; Cameron e t a / . , 1994). A single transcript of 4 kb was detected in rat germ cells, whereas transcripts of 4 kb and 6 kb were detected in immature Sertoli cells (de Winter et ul., 1992). IGF-Wcation-independent mannose 6-phosphate receptor mRNA and protein are expressed throughout spermatogenesis, and are particularly abundant on spermatogonia and early spermatocytes (O’Brien et NI., 1989; O’Brien, Gabel, rt ul., 1993; O’Brien et al., 1994). Studies indicate that ligands for this receptor are secreted by Sertoli cells and stimulate dose-dependent increases in c-fos mRNA and 18s ribosomal RNA levels in isolated spermatogenic cells (0’Brien, Gabel, et al., 1993; Tsuruta and O’Brien, 1995). Three G protein-coupled receptors that are expressed in spermatocytes have been cloned recently. BRS-3, a novel bombesin receptor, is expressed in human lung carcinoma cells and in rat testis, but not in rat somatic tissues (Fathi et ul., 1993). In situ hybridization suggests that transcripts for this receptor are localized in spermatocytes and/or spermatids (Fathi et al., 1993). Transcripts for a member (DTMT) of the olfactory receptor gene family have been detected in dog testis and not other tissues, with high levels of expression in an isolated germ cell fraction enriched in pachytene spermatocytes and round spermatids (Parmentier et ul., 1992). Peptide antisera raised against deduced amino acid sequences of this receptor detected the DTMT protein in dog spermatids and mature spermatozoa (Vanderhaeghen et ul., 1993). Another novel G protein-coupled receptor cloned from a rat testis cDNA library and expressed at high levels in spermatocytes and spermatids (Mayerhof et nl., 199 1) was subsequently identified as the A, adenosine receptor that inhibits adenylyl cyclase (Rivkees, 1994).
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G . Signal Transduction Components of signal transduction pathways are beginning to be identified in spermatogenic cells. The expression of several of these constituents, including kinases, phosphodiesterases, and regulatory proteins, is developmentally regulated during meiosis in the male. Many of the proteins involved in cell cycle regulation (Section 111, part E) can also be considered signal transduction components.
1. Kinases Early studies of CAMP-dependent protein kinase (PKA) activity in the mouse indicated that type I PKA, the predominant form in pachytene spermatocytes, decreased during spermatid development, while type I1 PKA became the major form in elongating spermatids (Conti et d . ,1983). Subsequent studies in the rat have generally confirmed this transition and have detected mRNAs for all four regulatory subunits (RIcx,RI,, RIIcx,and RII,) in spermatogenic cells (@yen et a/., 1987, 1990). Rat pachytene spcrinatocytes expressed an unusual 1.7-kb RI,, mRNA, as well as transcripts l o r RIB and the C, catalytic domain (0yen et a/., 1987, 1990). The mRNAs for Ri,,, Ri,, and C, increased substantially in the testis between 2.5 and 30 days of age, suggesting elevated expression of these subunits during meiosis (@yen el a/., 1990). Although RI,, transcripts persisted from midpachytene through the round spermatid stages, RI, niRNAs abruptly disappeared before diakinesis and the meiotic division (Lonnerberg et NI., 1992). This differential pattern of e x p r e s h i suggests that the PKA subunits may have distinct roles in spermatogenic cells. MAST205, a novel testis-specific serine/threonine kinase with a catalytic domain related to those of PKA ( C C yand ) protein kinase C, was cloned from a mouse testis cDNA library (Waldcn and Cowan, 1993). Although MAST205 mRNA levels were similar in pachytene spermatocytes and round spermatids, the MAST205 protein was expressed only in spermatids and was associated with manchette microtubules (Walden and Cowan, 1993; Walden and Millette, 1996). It has been proposed that this protein may be involved in signaling during the organization of the manchette and sperm head shaping. Two proto-oncogene families of serindthreonine kinases are expressed during meiosis in the mouse. In the r-~!fl'amily,Rafl mRNA was expressed at highest levels in pachytene spermatocytes ( Wolfes et a/., 1989). Testis-specific Rafl transcripts of 4.0 and 2.6 kb were detected at low levels in pachytene spermatocytes but were more abundant in spermatids (Wadewitz el a/., 1993). M o s transcripts also are present in both pachytene spermatocytes and round spermatids in the mouse (Mutter and Wolgemuth. 1987). However, the 43-kDa tcsticular MOS protein was detected only in pachytene spermatocytes in the rat (Van
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der Hoom et a/., 1991). Recent studies of Mos-deficient knockout mice suggest that MOS is required for meiotic arrest in oocytes but is not essential for spermatogenesis (Colledge et a/., 1994; Hashimoto et al., 1994). However, overexpression of Mos in spermatocytes leads to increased germ cell proliferation (Higgy et a/., 1995). TESKl, a testis-specific protein kinase with an unusual structure, appears to be expressed late in meiosis, with higher levels of mRNA detected during the early stages of spermiogenesis (Toshima er a/., 1995). The N-terminal kinase domain of this protein is most closely related to the LIM kinases and exhibits serinehhreonine kinase activity when TESKl is expressed in COS cells (Toshima et al., 1995). A specific function for TESKl has not been determined. Fert2, a unique transcript from the mouse Fert2 tyrosine kinase gene, is expressed exclusively in pachytene spermatocytes as a result of alternative splicing and the probable use of a testis-specific promoter (Fischman et a/.. 1990; Keshet et a/., 1990). This transcript encodes a truncated 51-kDa protein (Fischman et a/., 1990) that is detected in the nucleus during diakinesis and in meiotically dividing spermatocytes (Hazen et a/., 1993). Ca2+/calmodulin-dependent protein kinase 1V (CaMKIV) is a serine/ threonine kinase expressed in rat testis, brain, thymus, and spleen (Means et a/., 1991; Matthews et a/., 1994). In the testis, CaMKlV transcripts were most abundant in early spermatocytes (Means et a/., 1991). The same gene encodes a and p forms of CaMKIV, as well as calspermin, a calmodulin-binding protein expressed only in spermatids (Means el a/., 1991; Sun et al., 1995). Recent transfection studies suggest that CaMKIV may enter the nucleus and mediate calcium-dependent activation of transcription (Matthews et a / . , 1994).
2. Phosphodiesterases Members of two phosphodiesterase (PDE) families, the Ca2’-/calmodulin type I and the CAMP-specific type IV enzymes are most abundant in the rat testis (Morena et a/., 1995). Four genes of the type 1V CAMP-specific PDE family are expressed in testicular cells (Swinnen et a/., 1989; Morena et a/., 1995). These phosphodiesterases degrade CAMP, thereby regulating intracellular CAMP levels and attenuating CAMP-mediated responses. One member of this gene family (PDE1/IVc) is expressed predominantly in pachytene spermatocytes (Welch et a/., 1992), particularly during stages VIII-XI11 of the cycle of the rat seminiferous epithelium (Morena et a/., 1995).
3. Regulatory Proteins The expression of several other constituents of the signal transduction machinery is developmentally regulated during meiosis. These include guanine-nucleotide binding proteins, a GTPase activating protein, and calcium-binding proteins.
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A key regulatory protein in the CAMP pathway, the stimulatory guanine nucleotide-binding protein (G,) responsible for activation of adenylyl cyclase, has not been identified in spermatogenic cells (Paulssen et al., 1991; Kamik et al., 1992; Lamsam-Casalotti et al., 1993). In contrast, a subunits of several inhibitory guanine nucleotide-binding proteins (GiI , Gi2, Gi,, and Go) are expressed in pachytene spermatocytes and round spermatids in both mouse and rat (Paulssen et al., 1991: Karnik et al., 1992; Lamsam-Casalotti et al., 1993). Unlike the Gi a genes, which produce single transcripts in spermatogenic cells, four G,, (Y transcripts were expressed in pachytene spermatocytes, with a 6.9-kb mRNA being the most predominant (Paulssen c’t al., 1991). By immunocytochemistry. G,, c1 subunits were detected throughout the cytoplasm of mouse pachytene spermatocytes, while Gi a subunits were concentrated in the proacrosomal granules (Karnik et al., 1992). Other GTP-binding proteins are expressed during meiosis in the male. Transcripts for three member of the I-us proto-oncogene family (K-rus, H-rus, and N-ras) were detected in mouse pachytene spermatocytes (Sorrentino et u/., 1988). a2-chimerin, a GTPase-activating protein that regulates members of the ras superfamily, is selectively expressed in the rat brain and testis (Hall er al., 1993). Transcripts for a2-chimerin are derived by alternative splicing of the n-chimerin gene and are expressed in early pachytene spermatocytes in rat testis (Hall et al., 1993). Calmodulin, a ubiquitous calcium-binding protein that participates in multiple signaling pathways, is present at low levels in spermatogonia and accumulates during meiosis in mouse and human testes (Sano et al., 1987; Tsuji et al., 1992; Moriya et ul., 1993). High levels of calmodulin were detected by immunohistochemistry in both pachytene spermatocytes and round spermatids (Sano et al., 1987; Tsuji et al., 1992). Further analysis of the multigene family that encodes calmodulin has shown that at least three calmodulin genes are expressed in rat spermatogenic cells (Slaughter and Means, 1989). Transcripts from the calmodulin I and calmodulin I l l genes increase in leptotene-zygotene spermatocytes and remain constant throughout meiosis, while transcripts from the calmodulin I1 gene increase transiently in early to midpachytene spermatocytes (Slaughter and Means, 1989). This differential expression and accumulation of calmodulin during meiotic prophase suggests that calcium-calmodulin-regulated pathways play important roles during this period of spermatogenesis. Calmegin, a testis-specific calcium-binding protein, is expressed only in pachytene spermatocytes and in round and early elongated spermatids (Watanabe et a/., 1992). cDNA and genomic calmegin clones isolated from the mouse encode a protein with significant homology to calnexin and calreticulin, two molecular chaperones that preferentially bind to nascent glycoproteins in the endoplasmic reticulum (Watanabe et al., 1994, 1995). Calnexin-t, another cDNA cloned from a mouse testis expression library (Ohsako et al., 1994), has a nucleotide sequence that is 98.7%’identical to that of calmegin and may cncode
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the same protein. The monoclonal antibody used to isolate the calnexin-t clone recognizes a similar protein in mouse, rat, and hamster testis that has been localized by immunoelectron microscopy to the endoplasmic reticulum and nuclear envelope of hamster pachytene spermatocytes and spermatids (Ohsako et a/., 1991). Although the function of calmegidcalnexin-t has not been defined, its calcium-binding activity has been confirmed (Ohsako et a/., 1994; Watanabe et al., 1994).
H. Enzymes of Energy Metabolism
Energy production is a highly conserved process that in most cells requires metabolism of glucose to pyruvate by the enzymes of the glycolytic pathway. However, several enzymes in the pathway appear to have spermatogenic cellspecific isozymes, based on functional or electrophoretic characteristics (reviewed by Eddy rt al., 1994). For most of' these, i t has not been determined whether they are different isozymes that result from spermatogenic cell-specific gene expression or if they have been modified posttranslationally. Phosphoglycerate kinase (PGK) was the first glycolytic enzyme shown to be encoded by a separate gene expressed specifically in spermatogenic cells. Earlier studies had indicated that two isozymes of PGK were present in the testis but that only one was present in other tissues (VandeBerg et al., 1976; Kramer and Erickson, 198 1). I t was subsequently learned that the testis-specific PGK isozyme was encoded by the autosomal Pgk2 gene, which apparently arose as a functional retroposon from the ubiquitously expressed P g k l gene on the X chromosome (Boer et a/., 1987; McCarrey and Thomas, 1987). Transcription of the Pgk2 gene first occurs coincident with the onset of meiosis and continues to increase in later spermatocytes and in round spermatids. In contrast, expression of the Pgkl gene declines in pachytene spermatocytes (Goto et d . ,1990: SingerSam et al., 1990; McCarrey, Berg, et a/., 1992; Kumari et N I . , 1996). These changes in gene expression parallel the changes reported earlier for the protein levels of the two PGK isozymes (VandeBerg rt d . , 1976; Kramer and Erickson, 1981). It has been hypothesized that the Pgk2 gene evolved to provide the PGK enzyme required for glycolysis following loss of P g k l gene function that occurs with X-chromosome inactivation during meiotic prophase (reviewed by McCarrey, 1994). Hexokinase is the first enzyme in the pathway of glycolysis, and earlier studies suggested a sperm-type hexokinase activity (Katzen et a/., 1968; Sosa et ml., 1972). When clones from a mouse testis cDNA library were isolated and sequenced, they were found to represent three different hexokinase mRNAs ( H k l sa, Hkl-sh, H k l - s c ) (Mori et a/., 1993). These mRNAs were similar to the somatic H k l cDNA sequence throughout most of their coding regions. but differed from it at the 5' end. H k l - s a (and probably Hkl-sc) transcripts were present
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during meiosis, whereas H k l - . t h transcripts were expressed after meiosis (Mori et a / . , 1993). Similar tindings have been reported recently for the human (Mori et a/., 1996). Furthermore, it has been determined that the H k l - s u , Hkl-sh, and H k l - s c , rnRNAs in spermatogenic cells are transcribed from the same gene as the ubiquitously expressed H k l mRNA. The unique sequences are acquired through the use of alternative exons and splicing events that occur during expression of the H k l gene in spermatogenic cells of the mouse (C. Mori et id., unpublished observations). Phosphoglycerate inutase (PGAM) is another enzyme in the glycolytic pathway that is differentially expressed during meiosis. An earlier study using immunohistochemistry and electrophoretic separation of isozymes reported that the muscle-specific PGAM-B subunit was detected in pachytene sperrnatocytes and spermatids, but not in earlier stages of spermatogenesis in the mouse (Fundele et d., 1987). A subsequent study demonstrated that the Pgarn2 gene encoding the PGAM-B isozyme begins to be expressed at Day 22 in the rat testis, when germ cells start to enter meiosis (Broccfio c’t a/., 199.5). There was no evidence of coordinate reduction in mRNA levels for the constitutively expressed Pgarnl gene. I t was concluded that a special isoform for PGAM-B is not present in the rat testis, because Northern blot analysis detected identical-size transcript in testis and other tissues (Broceiio ~t u / . , 199.5). However, until rat testis cDNAs for Pguri72 are cloned and sequenced, it cannot be ruled out that an alternatively spliced Pgurr12 inRNA is present that encodes a spermatogenic cell-specific PGAM-B, as has been found for type 1 hexokinase. In addition to genes for glycolytic enr.ymes, other genes are expressed exclusively in spermatogenic cells for cnr.ymes important in different aspects of energy metabolism. Lactate dehydrogcnase (LDH) interconverts lactate and pyruvate, and pyruvate is used in the citric acid cycle. One member of the Ldh gene family, Ldh3, encoding LDH-C, is expressed exclusively in spermatogenic cells. L r M mRNA and protein were first present i n preleptotene and leptotene-zygotene sperniatocytes, and mRNA and en7yme activity levels were highest i n round spermatids (Li et a/., 1989; Thomas et 01.. 1990; Alcivar, Trasler, et a/.. 1991). The mRNA for WhI (encoding LDH-A), abundant in muscle, and the enzyme activity for LDH-A and LDH-B were also present during and after meiosis (Li et ( I / . , 1989; Alcivar. Trasler, rt u / . . 1991). However, the LDH-A and LDH-B activities declined throughout meiosis, while LDH-C activity increased (Li et d., 1989). Two other genes expressed exclusively in spermatogenic cells encode proteins essential for energy production. a component of the pyruvate dehydrogenase complex and a cytochrome c. Thc pyruvate dehydrogenase (PDH) enzyme complex consists of at least five subunits and converts pyruvate to acetyl-CoA within the mitochondria1 matrix. an essential step i n aerobic glucose oxidation. The E l a subunit contains the cofactor binding site and the phosphorylation site through which PDH activity is regulated (reviewed by Reed and Yeaman, 1987). Testis-
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specific cDNA clones for the PDH E l a subunit have been isolated for human (Dahl et al., 1990) and mouse (Pdhu2; Takakubo and Dahl, 1992; Fitzgerald ef ul., 1994). Paclha2 mRNA was first present in leptotene-zygotene spermatocytes and the protein was abundant in pachytene spermatocytes (Takakubo and Dahl, 1992; Fitzgerald er a/., 1994). The Pdhal isologue is present on the X chromosome which undergoes condensation in spermatocytes, causing expression of 1994). The Pdh02 gene most X-linked genes to cease (reviewed by Handel et d., is on an autosome and appears to take over for the Pdhal gene when it is inactivated during spermatogenesis (Dahl et al., 1990). Cytochrome c (Cyc)functions in the transport of electrons in the mitochondria1 intramembrane space. The Cycs gene is expressed in all somatic cells. but the Cycr gene is expressed exclusively in spermatogenic cells (Virbasius and Scarpulla, 1988; Hake et al., 1990). The expression of both Cycs and Cycf mRNAs are regulated during spermatogenesis. The Cyct transcript was first detected in zygotene spermatocytes and reached maximal levels in round spermatids, and the protein increased in amount in mitochondria during the zygotene to pachytene period (Hess er a/., 1993; Morales er al., 1993). A unique transcript of Cycs appeared in late meiotic prophase and reached its highest levels in round sper1993). The spermatogenic cell-specific Cycs message matids (Morales et d., arises from the utilization of an alternative transcription initiation site upstream of that for four shorter Cycs mRNAs (Hake and Hecht, 1993).
I. Other Components
The expression of a variety of other genes is regulated developmentally during spermatogenesis. Although the relationship of these genes to meiosis is uncertain, their expression is a result of the genetic program responsible for this process. At the very least, their expression during this period indicates that they have the appropriate promoter elements to respond to the specific complement of transcription factors present during the meiotic phase of spermatogenesis. However, several of these are expressed exclusively in spermatogenic cells and are members of well-characterized gene families, suggesting that their expression is advantageous to spermatogenic cells. Included are genes for cytoskeletal proteins, proteases, and other enzymes and proteins.
1. Cytoskeletal Proteins The kinesins are motor proteins that are associated with a variety of motile events that are often related to cell division. They typically have a force-generating head domain and a tail domain that links to a target cargo protein. Transcripts for five kinesin-related proteins (KRP) identified by RT-PCR were expressed primarily in the testis (Sperry and Zhao, 1996). Northern analysis indicated that one of these
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(KRP2) was expressed only in the testis, while the other transcripts were also expressed at low levels in the ovary, or in ovary and brain. In situ hybridization showed that transcripts for KRP2 and the three KRPs expressed also in the ovary were localized to regions of seminiferous tubules containing mainly pachytene spermatocytes. Because KRP2 shows sequence homology to hamster meiosis centromere-associated kinesin (MCAK; Wordeman and Mitcheson, 1995), it was suggested that KRP2 is associated with the centromere in dividing spermatocytes (Sperry and Zhao, 1996). The cDNA for mitochondrial capsule selenoprotein (MCS) of mouse sperm was shown to encode a protein rich in cysteine and proline (Kleene et ul., 1990). It appears to be a single copy gene that is expressed exclusively in the testis, encoding a protein that forms a capsule around the closely packed mitochondrial helix in the sperm midpiece. Northern analysis indicated that MCS mRNA was first transcribed in late meiotic cells and that the levels increase in round spermatids, but the translation was delayed until the elongating spermatid stage. Subsequent studies confirmed that the protein is not detected until elongating spermatid formation (Cataldo et d., 1996). However, these studies also indicated that translation probably originates downstream of the potential selenocysteine codons and that the protein does not bind selenium. The protein was renamed SMCP, for sperm mitochondrial associated cysteine-rich protein (Cataldo et d., 1996). A larger transcript was found i n rat testis than in other tissues for thyinosin plo, a member of the thymosin actin-sequestering protein family. These proteins are believed to complex with G-actin and to regulate the equilibrium between monomeric and filamentous actin (Stossel, 1989). When thymosin p10 cDNA clones isolated from a rat testis cDNA library were sequenced, the unique transcript was found to arise by a combination of differential promoter utilization and alternative splicing. The unique transcript was present in pachytene spermatocytes, but immunoblot analysis indicated that the protein was detected only in spermatids (Lin and Morrison-Bogorad, 199 1).
2. Proteases The expression of a variety of proteases is regulated developmentally during meiosis. These include endoproteases, plasma membrane-associated proteases, and serine proteases. Some of these have been implicated in sperm-egg interactions. The conversion of a precursor protein to an active form often involves cleavage at a specific site by an endoprotease. The cDNAs for a proprotein convertase, named PC4, were cloned for mouse and rat, and Northern analysis indicated that PC4 mRNA was present only in the testis in both species (Nakayama e t a / . , 1992; Seidah et al., 1992). Although PC4 mRNA was detected by Northern analysis and in situ hybridization only in round spermatids in the mouse (Nakayama et ul.,
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1992; Torii et al., 1993). it was found in pachytene spermatocytes and a t higher levels in round spermatids by these same methods in the rat (Seidah et al., 1992). Alternative transcripts of PC4 are present in testis and are produced by utilization of alternative exonic regions that result in transcripts with unique 5' and 3' ends (Seidah et a/., 1992; Mbikay et d . , 1994). The substrate(s) for this enzyme in spermatogenic cells has not been identified. At least six members of a gene family encoding membrane proteins with a disintegrin and a metalloprotease domain (ADAM) are expressed in the testis (Wolfsberg et nl., 199.5). The first ADAMs described, fertilin ci and fertilin p (ADAM 1 and ADAM 2 ) , exist as a heterodinier in the sperm plasma membrane and are involved in sperm-egg binding (Primakoff et al., 1987). They are encoded by single copy genes (Cho et d., 1996). Although guinea pig fertilin ci and fertilin p mRNAs were detected only in testis by Northern analysis (Wolfsberg et al., l993), fertilin CY mRNA was detected by RT-PCR at low levels in all eight mouse somatic tissues examined (Wolfsberg et ul., 1995). On in situ hybridization in the mouse, fertilin ci (Ftnu) transcripts were detected from late pachytene spermatocyte until elongated spermatid development, and fertilin p (Ftnh)transcripts were found from midpachytene until early round spermatid development (Wolfsberg et ul., 199.5). The fertilin p protein was first detected in the endoplasmic reticulum of pachytene spermatocytes and on the surface of elongating spermatids in the guinea pig (Carroll et d . , 199.5). Cyritestin (ADAM 3) mRNA was present only in testis by Northern analysis and RT-PCR of mRNA from mouse tissues, and was detected in pachytene spermatocytes and round spermatids by in situ hybridization (Heinlein et ul., 1994; Wolfsberg et a/., 199.5). Cyritestin is encoded by a single copy genc, CnizI (Lemaire et a/., 1994). Cyritestin inRNA was detected in leptotene-zygotene spermatocytes by RT-PCR, but the protein was not detected by Western blotting until pachytene spermatocytes were present in the mouse (Linder et nl., 199.5). Cyritestin was shown to be an integral transmeinbrane protein localized to the acrosome region of mouse sperm (Linder et cil., 199.5). Proacrosin, the precursor for the serine protease acrosin, is one of several hydrolytic enzymes present in the sperm acrosome (reviewed by Eddy and O'Brien, 1994). It has been shown to be a single copy gene (Acrj in mouse (Kremling et a/., 1991; Watanabe et ~ i l . ,1991) and several other mammalian species (see Adham rt a/., 1996). Although initial studies using Northern analysis and in situ hybridization reported that proacrosin transcription occurred only in spermatids in boar (Adham et u /., 1989) and mouse (Klemm et (11.. 1990; Kashiwabara, Baba, et d., I 990), subsequent studies indicated that proacrosin mRNA was present in pachytene spermatocytes of mouse (Kashiwabara, Anai, et 1994). Proacrosin id., 1990; K r e m h g et d., 1991) and rat (Naycrnia et d., mRNA was associated with polysomcs in mouse pachytene spermatocytes. suggesting that it was being translated (Kashiwabara, Arai, et d . , 1990,. Other studies using immunohistochemistry detected proacrosin protein only in spermatids in boar (Bozzola P / (11.. 1991j and guinea pig (Anakwe et ul.. 1991).
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Proacrosin was believed previously to be essential for sperm penetration through the zona pellucida. However, male mice with a knockout for the proacrosin gene were fertile, indicating that the enlyme i \ not essential for fertilization (Baba et ol., 1994).
3. Other Enzymes and Proteins The expression of genes for a variety of other enzymes has been shown to be developmentally regulated during spermatogenesis. These genes are involved in nucleotide synthesis, detoxifying activities, lysosomal function, DNA methylation, polyamine biosynthesis, general metabolic pathways, and neuropeptide synthesis. Additional genes are e x p r e w d that d o not lit into the arbitrary categories used in this review. A single copy autosomal gene Ioi- phosphoribosylpyrophosphate synthetase (PRPS3) is expressed in human. I - ~ I , and mouse testis, and the mRNA appears coincident with pachytene spermatocyte development in the rat (Taira ct d., 1990). This enzyme catalyzes a crucial step in the utilization of ribose 5-phosphate from the pentose phosphate pathway to form purine, pyrimidine, and pyridine nucleotides. The PKPSI and I’KPS2 genes are on the X chromosome and presumably are inactivated during spermatogenesis, when the PRPS3 genes become activated. Developmentally regulated expresion has been reported for genes encoding enzymes involved in neutralizing or eliminating other proteins. The transcripts for a unique k-class glutathione S-trainsferase (rizCSTM5)was found by Northern analysis to be expressed only i n the testis and to first appear during meiotic phase o f mouse spermatogenesis (Fulchcr c’t (/I.. 1995). The G S T enzymes are multifunctional proteins having the capacity to inactive cytotoxic substances via conjugation with glutathione. The met~illotliioneingenes ( M t l , M t 2 ) encode proteins involved in the homeostatic control of‘ metals, including toxic metals such a s cadmium. The transcripts for these enLymes also increase during pachytene spermatocyte development in m o u s e ( D e et d., I99 1 ). Another gene developmentally regulated during spermatogenesis encodes DNA methyltransferase (DNA Mtaw), an enzyme that is involved in geiic regulation, genomic imprinting, and X inactivation. This is a single copy gene, and mice with a Driri7t gene knockout die at midgestation (Li et 01.. 1992). In addition to the ubiquitously expressed 5.3-kb tr?ui\cript also present in pachytene spermatocytes and spermatids of mouse. pachytene spermatocytes contained ;I ~iiiiclue 6.5-kb transcript (Benoit and Ti-a\ler, 1994). However, less D N A Mtase protein was detected by Western blotting in pachytene sperniatocytes than in spermatids, suggesting that the larger transcript is not translated. Ornithine decarboxylase (OD<:) is the rate-limiting enzyme in the polyamine biosynthetic pathway. The mRNA Icvcls o f Odc increase substantially during pachytene spermatocyte development in mouse and rat (Alcivai- et (/I.. 1989: Kaipia et d..1990). A~-ylsulI‘ata~e A (ASA) is a lysosomal enzyme that is
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involved in the degradation of sulfated glycolipids and is considered to be a housekeeping gene. The level of ASA mRNA was found to increase 20-fold in pachytene spermatocytes compared to other tissues in the mouse (Kreysing et a/., 1994). The mRNA for glutamic acid decarboxylase (GAD), a key enzyme in the synthesis of the inhibitory neurotransmitter y-aminobutyric acid, was found in human brain and testis. The mRNA was present in pachytene spermatocytes and spermatids in the rat, and the protein was detected predominantly in spermatids and sperm (Person, Pelto-Huikka, et a/., 1990). In addition, the mRNA for choline acetyltransferase (ChAT), an enzyme involved in the synthesis of the neurotransmitter acethycholine, was also expressed in human and rat testis. In situ hybridization indicated that the transcripts were present mainly in pachytene spermatocytes and spermatids, and the protein was detected by immunohistochemistry on the postacrosomal region of human sperm (Ibaiiez et al.. 1991). A novel member of the cellular lipophilic transport protein superfamily has been cloned from a rat testis cDNA library (Schmitt et ol., 1994). This cDNA encodes a testis-specific 1 -kb mRNA and a 15-kDa protein that is first detected in midpachytene speramtocytes, is present throughout spermiogenesis, and is retained in epididynial sperm. Although the ligand for the transport protein has not been identified, sequence features of the transport protein suggest that the ligand is an acidic retinoid or other lipid with a specific role in sperm development (Schmitt ef a/., 1994). Sp17 is another protein that has been suggested to be involved in sperm-egg interaction at the level of the egg zona pellucida (Kong et al., 1995). The Sp17 protein is encoded by a single copy gene and is present initially in the cytoplasm of pachytene spermatocytes and subsequently on the equatorial segment of live, acrosome-reacted sperm of the mouse. It contains regions of similarity to human testis CAMP-dependent protein kinase type I I a and a putative calmodulin-binding site (Kong et al., 1995). Men with cystic fibrosis are usually infertile due to blockage or absence of the distal epididymis and vas deferens. A splice variant of the cystic fibrosis transmembrane regulator (C’tr) gene was identified in the mouse testis and found to be expressed in pachytene spermatocytes and round spermatids (Trezise, Linder, et ul., 1993; Delaney et a/., 1994). The alternative splicing event introduces an additional exon that results in a protein truncated in the first nucleotide binding fold and unlikely to act as a channel protein (Trezise, Buchwald, et al., 1993; Delaney et ul., 1994).
IV. Conclusion What is known about gene expression during mammalian meiosis is that a wide variety of genes are upregulated in spermatocytes, that some of these genes are
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transcribed exclusively in spermatogenic cells. and that others produce transcripts unique to spermatocytes. However, it is not known why so many genes have atypical patterns of expression during meiosis or how most of these genes are regulated. The list of genes expressed during mammalian meiosis continues to grow (Table I), largely by a random proccss and one gene at a time. In addition, human and mouse testis cDNA libraries have been randomly sequenced, and several hundred ESTs (expressed sequence lags) have been reported that correspond to genes expressed by all of the somatic cells in the testis and by germ cells at all stages of development (Affara e/ u/., 1994; Kerr et d.,1994; Hoog, 1995; Pawlak et a/., 1995; Lopez-Fernandez and del Mazo, 1996). Unfortunately, these approaches are not likely to lead to the rapid identification of many of the genes involved in meiosis. There has not been a focused effort to identify genes regulated during meiosis in mammals, as has occurred in yeast (Bums et al., 1994). However, there are large-scale sequencing efforts underway to develop EST databases that catalog essentially all of the genes expressed in each tissue. If an EST database of genes expressed i n isolated meiotic cells is produced, it will be possible to use computers to compare the meiotic cell EST database with those of other tissues and of germ cells at other stages of development to identify genes regulated during meiosis. Furthcr studies would be needed to characterize the genes and to determine the role o f thcir products in meiosis, but the methods are readily available for carrying out such studies once candidate genes are identified. There are several possible answers to the question of why so many genes have atypical patterns of expression during meiosis: 1. All cells are likely to express some unique genes and/or transcripts that define them as cell types with distinctive functions. For germ cells this might include genes that specify the unique developmental potential of this cell lineage, as well as other structural or functional features of this cell type. This may account for some of the genes expressed during meiosis, and may include unique genes and transcripts that are already identified but whose purposes are currently unknown. 2. Gametogenesis is a developmental process that progresses according to an intrinsic genetic program, and various genes probably are expressed during meiosis that direct germ cell development. However, remarkably little is known about how germ cell development is regulated in mammals and how extrinsic cues from the endocrine system and surrounding cells of the gonad influence this process. Because rat spermatogonial stem cells transplanted to the mouse testis can produce normal-appearing rat spermatozoa (Clouthier Pt al., 1996). this strongly suggests that the intrinsic genetic program of germ cells defines the developmental outcome. 3. Genes are expressed during meiosis that encode proteins required for specific events that occur during this process. This is known to be true for some
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genes transcribed during meiosis, such as those encoding the SC proteins and components of the DNA repair and recombination machinery. Although the pace of discovery of such genes is increasing, the number identified that are required for mammalian meiosis is still low. It is interesting that the genes for the SC proteins have little homology with genes in lower phyla, whereas the genes for DNA repair and recombination processes are conserved from bacteria and yeast to mammals. Because mammalian meiosis apparently uses both conserved and unique genes, searches for additional genes based either on homology with known genes or on computer screens of EST databases seem well justified. 4. Genes are expressed during meiosis that encode proteins not required until after meiosis. This apparently is the case for certain genes, particularly those expressed during oocyte development and for some expressed late in pachytene spermatocyte development and not translated until spermatid development (e.g., Tenr, Spizr, MAST205, SMCP, thymosin p,,,). This is understandable for oocytes because transcription is delayed until the two-cell stage of embryogenesis. Until then, protein synthesis depends on the use of stored transcripts. However, transcription is active during the haploid phase of spermatogenesis, and it is not apparent why transcription is initiated earlier for certain genes in these cells. 5. Some genes are expressed during meiois because of changes in expression of reciprocal genes. This is known to occur for certain genes on autosomal chromosomes ( P g k 2 , Pdlzu2; Section 111, part H) that are activated during spermatogenesis, as isologous genes are inactivated during condensation of the X chromosome. However, there does not appear to be any evidence that inactivation of a gene on the X chromosome or elsewhere in the genome directly influences activation of a gene o n another chromosome. Reciprocal gene expression is probably the result of long-term evolutionary processes involving gene duplication and leading to families of genes with different promoters but encoding similar proteins (see McCarrey, 1994: Handel et ul., 1994). 6. Genes might be expressed during meiosis because transcription becomes leaky. It has been proposed that transcripts are produced in spermatogenic cells that are unexpected or inay have no functional relevance because the DNA is undergoing major structural changes (Ivell, 1992). However, just because it is not obvious why some genes are expressed in spermatogenic cells, such as those for neuropeptides, this does not indicate their irrelevance to germ cell development. It is particularly significant that transcripts for Crein and Crehl that were thought to produce nonfunctional proteins during meiosis have been found instead to yield factors that repress gene expression (Foulkes et id., 1992; Deltnas and 1996). Such findings suggest that other Sassone-Corci, 1994; Walker rt d., unusual transcripts in germ cells probably are not the result of leaky transcription but rather are produced to generate proteins with unique roles adapted to the regulation and function of germ cells. More than 30 genes have been reported that are developmentally regulated, are expressed during meiosis. and are transcribed only in spermatogenic cells (Table
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I). In addition, over 20 genes are reported to produce unique transcripts during meiosis, and more than 40 additional genes that are developmentally regulated during this process are known (Table I). I t is clear for only a few of these genes that they are required during meiosis, and much more remains to be learned about the roles of most genes expressed during mammalian meiosis. The final question, how is gene expression regulated during meiosis, remains largely unanswered. Promoter analysis studies have proved to be difficult for germ cells. There are no germ cell lines available to allow the usual irz vitro transient transfection studies of promoter function, and it is quite laborious to purify germ cells i n sufficient quantities for isolation of transcription factors. Because of these difficulties, there have been relatively few studies of the regulation of genes expressed during meiosis, and even fewer efforts to identify spermatogenic cell-specific transcription factors. However, novel factors appear to regulate the genes that have been studied (Section 111, part C, 2). These factors need to be identified so that their roles can be defined and their regulation in turn can be determined. In addition, we need to know how other meiosis-related genes are regulated to determine if there are common factors involved in the expression of multiple genes. Insofar as isoforms of CREM (Foulkes et al., 1992) and CREB (Walker el al., 1996) suppress expression of several genes during meiosis (Section 111, part C, 1 ), other transcription factors may coordinately activate meiosisrelated genes. Remarkably little is known about how cues from the endocrine system influence meiosis and other processes of gametogenesis in mammals. This is probably an indirect process in most cases, with steroid and peptide hormones binding to receptors in surrounding somatic cells to modify their communication with the germ cells. The germ cells presumably process this information through their signal transduction pathways to modulate the synthesis, modification, and binding of transcription factors to promoter elements and to effect the regulation of gene expression. Some components of the intercellular communication mechanisms and signal transduction pathways that may participate in these processes have bcen identified (Section 111, parts F and G). However, much more needs to be learned about the components and mechanisms involved before the regulation of gene expression during marnmalian meiosis can be understood. Although identifying the genes expressed during meiosis is only the beginning of understanding how this process is regulated, the pieces of the puzzle need to be found before they can be assembled.
Acknowledgments Thi\ work h a s wpported in part by grant\ HI126485 (D. A . O.), P30-HDI8968 (The I.abor;itorit.\ f o r Reproductive Biology). and CA 16086 (llNC Linebetper Compi-ehenhive Cancer) l'roni the N;rlional Institutes of Health. The author5 thatih Mai-v Ann Hiindel and Michael Shelby for thcii- hclpful c o m m e n t on the manuscript.
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acterization of TH3, a germ cell-specific variant of histone 3 in rat testis. J . Biol. Churn. 259, 8769-8776. Tsuji, Y., Sano, M., Nagahama, M.. Tsutsui, Y., and Miyake, K. (1992). Immunohistochemical localization of calmodulin. / r r / . J . Aridrol. 15, 448-454. Tsuruta, J. T., and O’Brien, D. A. (1995). Sertoli cell-sperniatogenic cell interaction: The insulinlike growth factor-Il/cation-independentinannose 6-phosphate receptor mediates changes in sperniatogenic cell gene expression in mice. B i d . Reprod. 53, 1454- 1464. Ueno, N., Baird, A,, Esch, F., Ling, N.. and Guillemin, R. (1987). Isolation and partial characterization of basic tibroblast growth factor from bovine testis. Mol. Cdl. Eridocrinol. 49, 189- 193. Utakoji, T., Muramatsu, M., and Sugano, H. (1968). Isolation of pachytene nuclei from the Syrian hamster testis. Exp. Cell R r s . 53, 447-458. VandeBerg, J . L., Cooper. D. W., and Close, P. J . ( 1976). Testis specitic phosphoglyceratr kinase B in mouse. J . E.rp. Z o o / . 198, 231-239. Vanderhaeghen, P., Schurmans, S., Vassart, G., and Parmentier, M. (1993). Olfactory receptors are displayed on dog mature sperm cells. J . Cell Hiol. 123, 1441-1452. Van der Hoorn, F. A,, Spiegel, J . E., Maylie-Pfenningcr, M.-F., and Nordeen, S . K. ( 1991 ), A 43 k D c-rr~o.~ protein is only expressed before meiosis during rat spermatogenesis. Oircogme 6, 929-932. vanWert, J. M., Wolfe, S. A., and Grimes. S . R. (1996). Binding of nuclear proteins to a conserved histone HI t proinoter element suggests an important role in testis-specific transcription. J . Cell. Biochem. 60, 348-362. Vastrik, I., Kaipainen, A,, Penttila, T.-L.. ILymboussakis. A., Alitalo, R., Parvinen, M., and Alitalo, K. (1995). Expression of the r w d gene during ccll differentiation i n vivo and its inhibition of cell growth in vitro. J . Cell Riol. 128, 1197-1208. Virbasius. J. V., and Scarpulla, R. C. (19881. Structure and expression of rodent genes encoding the testic-specific cytochrome c: Differences in gene structure and evolution between somatic and testicular variants. J . B i d . Chrm. 263, 6791 -6796. Viviano, C. M., Galliot, B., and Wolgernuth, D. J . (1993). Multiple levels of regulation cxist for expreasion of the H0.m-4 (H0.x-1.4) gene in the mouse testis. Cell. Mol. Biol. Rrs. 39, 383495. Voliva, C. F., Martin, S. A., Hutchinson, C. A., 111, Edgell. M. H. (19x4). Dispersal proce\s associated with the LI family of interspersed repetitive DNA sequence\. J . Mol. B i d . 178, 795813. Wadewitz, A. G.. Winer. M. A., and Wolgemuth, D. J. (1993). Developmental and cell lineage specificity of rcrf family gene expression i n the mouse testis. Oricogerw 8, 1055-1062. Waeber, G., Meyer, T. E., LeSieur. M., Hermann. H . L.. GCrard, N., and Habener, J. F. ( 1991). Developmental stage-specific expression o f cyclic adenosine 3’.5‘-inonophosphate response element-binding protein CRER during spermatogenesis involves alternative exon splicing. Mol. Eirdocrinol. 5, 1418- 1430. Walden. P. D., and Cowan, N. J. ( 1993). A novcl 205-kilodalton testis-specific sermdthreonine protein kinase associated with microtubules of the spermatid manchette. Mol. Cell. B i d 13, 7625-7635. Walden, P. D., and Millette, C. F. (1996). Increased activity associated with the MAST205 protein kinase complex during mammalian spermatogenesis. H i o l . Reprod. 55, 1039- 1044. Walker, W. H., Girardet, C., and Habener, J . F. ( 1966). Alternative exon splicing control\ a translational switch from activator t o repressor isoforms of transcription factor CREB during spermatogenesis. J . B i d . Chrm. 271, 20145-201 50. Walter, C. A., Lu, J . , Bhakta, M., Zhou, Z.-Q.. Thompson, L. H., and McCarrey, J . (1994).Testis and somatic Xrcc-I DNA repair gene expression. S o i r i c i t i c . Cell M ol. Genet. 348, I 1 1 - 1 16. Walter, C. A., Trolian, D. A., McFarland, M. B., Street, K. A,, Gurran, G. R., and McCxrey, J . R. (1996).Xrcc-l expression during male meiosis in the mouse. B i d . Rcp-od. 55, 630-635. Wan&. Z., and Kim, K. H. (1993). Vitamin A-deficient testis germ cells are arrested at the end of
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tin, G. R. (1987). Differential expression of the mouse homeobox-containing gene Hox- 1.4 during male germ cell differentiation and embryonic development. Proc. Ntrtl. Actrd. S r i . USA 84,5813-5817. Wolgemuth. D. J., and Watrin, F. (1991). List of cloned mouse genes with unique expre\$ion patterns during spermatogenesis. Mtrmm. Grriorw I, 283-288. Wordeman, L., and Mitchison, T. J. ( 1995). Identitication and partial characterization of mitotic centromere-a\sociated kinesin, a kinesin-related protein that associates with centromcres during mitosis. J . Cell B i d . 128, 95-105. Wu, S., and Wolgemuth, D. J. ( 1995). The distinct and developmentally regulated pattcrris of expression of members of the mouse Cdc2S gene family wggest differential function during gametogenesis. flw B i d . 170, 195-206. Xanthoudakis, S., Miao, G., Wang, F., Pan. Y. C. E., and Curran. T. (1992). Redox actiLation of Fos-Jun DNA binding activity is mediated by il DNA repair enLyme. EMBO J . 11, 3323-
3335. Xu, Q., Nakanishi, T.. Sekimizu, K.. and Natori, S . ( 1994). Cloning and identification of testisspecilic transcription elongation factor S-11. ./. B i d . (%em. 269, 3 100-3 103. Xu, W., and Cooper. G. M. (1995). Identification of a candidate c-tnos repressor that rmtricts transcription of germ cell-specific genes. M o l . Cell. Hiol. 15, 5369-5375, Xu, Y., Ashley, T., Brainerd, E. E., Bronson. R. T.. Meyn. M. S., and Baltimore, D. (1996). Targeted disruption of ATM leads to growth retardation, chromosomal fragmentation during meiosis, immune defects, and thymic lymphoma. Grrie.s L ) r i . . 10, 141 1-2422. Yoo, H.. Li, L., Sacks, P. G., Thompson, L. H.. Becker. F, F., and Chan, Y.-H. (1992). Alterations in expression and structure of the DNA repair gene XRCCI. Biochern. Bioph\s. Krs. L'otnmu~i. 186, 900-910. Yoshikawa, K., and Aizawa. T. (1988). Enkephalin precursor gene expression in postmeiotic germ cells. B i ~ c h e m Biophys. . Rrs. Conmwi. 151, 664-67 I, Zabludoff, S . D., Wright. W. W., Harshman, K., and Wold, B. J. (1996). BRCAI mRNA i \ expressed highly during meiosis and hpermiogenesij but not during mitosis in male germ cells. Oncogew 13, 649-653. Zakeri. Z. F., Wolgemuth, D. J., and Hunt, C. R. ( 1988). Identitication and sequence analysis of a new member of the niouse HSP70 gene family and characterization of its unique cellular and developmental pattern of expression in the male germ line. Mol. Cell. R i d . 8, 2925-2932. Zhao, G.-Q., Deng, K . , Labosky, P. A,. Liaw, L.. and Hogan, B. L. M. (1996). The gene encoding bone morphogenetie protein 8B is required for the initiation and maintenance of spermatogenesis in the mouse. Genes Dei'. 10, 1657-1669. Zhao, G.-Q., and Hogan, B. L. M. (1996). Evidence that mouse BnipHa (Op2) and Bin/iHh are duplicated genes that play a role in spermatogenesis and placental development. Mech. Dev. 57, 159- 168. Zhou, W., Xu, J., and Goldberg, E. (1994). A 60-bp promoter sequence of murine lactate dehydrogenase C is sufficient to direct testis-specific transcription in vitro. B i d . Reprod. 51, 425432. Zhou, Z.-Q., and Walter, C. A. ( 1995). Expression of the DNA repair gene XRCCl in baboon tissues. Mutat. Res. Lett. 348, I I 1 - I 16. Zhu, D., Dix, D. J., and Eddy, E. M. (1997). HSP70-2 is required for CDC2 kinase activity in meiosis I of niouse spermatocytes. Development (in press).
6 Caught in the Act: Deducing Meiotic Function from Protein Immunolocalization Terry Ashley and Annemieke Plug Department of Genetics Yale University School of Medicine New Haven, Connecticut 065 10
1. The Plot 11. Setting the Stage: Meiosis Plain ;ind Siiiiple Ill. Surbcillance Methods A. The Old Method B. The New Method\
IV. Reconstructing the Scene V. Verifying an Alibi (Temporal and Sp.iti;il Resolution) Vl. Developing a List of Suspects A. Presence at the Scene of the Criinc B. Suspicious Professional Acti\ C. Family Connections D. The Body in the Parlor
VII. Setting U p ii Sting Operation V 111. Prel I minary Conclusions A. Parallel\ between Case\ (Yea\t and Manrnials) B. Differences between Ca\e\
IX. Uncol\~edCaw5 Reference\
Meiotic divi\ion comprise\ a complex st'ricb of cvcnts, many of which are unique in the I ~ i e cycle of the organism. The proces\ utilize\ both proteins that participate in norniiil mitotic cell cycle progre\\ion and DNA damage repinr and proteins cxpre\sed o n l y during meiosis. L.iitil I-ecently, fe\* meiotic protein particip;int\ had been identifed and characterited. but irecent developments have changed thi\ \ i t u i i l i o n . Proteins can he selected for study bared on their cDNA seyuencc and similarity to hnoa II pi-oteins with "siispiciou\" repair/reconhination or cell cycle actiLity and antiboilic\ ;ig;~inslthese proteins applied to meiotic nuclei 10 test for activity. With the developincnt ol gene \equcnce data base\ from many organi\ins. \imilnrity to a known protein need not be ba\ed on the same or even a closely related .;pec~e\. Potenti:il interactions between two or morc protein\ can be identified and involvement in ;I common process inferred based on antibody coloc;ilization. The gene sequence can be di\ruptcd and the effect o n meiotic progi-cs\ioii dii-ectly examined. Previously identilied \truetiires. the \ynaptonemal complex (SC) and both e;ii-ly and late recombination nodule\ ( K N \ ) . provide structural and temporal landm;irh\ that as\i.;t in interring meiotic activity o f the protein heing studied. Mainmalian nicio\i\ I\ especially attractive for thew hind\ of studie\ \ince \permatocyte and oocyte nuclei ;IIK large with distinct nuclear organelles and \ince
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meiosis is highly protracted, occurring over a period of several days. In this chapter, an approach to the study of mammalian meiosis based on use of specific antibodies is outlined and methods of coupling this approach to other techniques, such as targeted gene disruption or chromosome aberrations, are described. Some of the proteins already identified as participants in meiotic prophase are reviewed and their presumed functions discussed. Copyright 0 1998 by Academic Press.
1. The Plot Like all good mysteries, this chapter has a dual story line. The obvious story recounts recent discoveries in the field of meiosis. However, an underlying theme may well be the resurrection of cytogenetic studies as a major contributor to our increased understanding of meiotic events. Although “resurrection” may appear to be an overly dramatic pronouncement, it seems fitting in light of a minireview published in Cell in 1993 entitled “Yeast Genetics and the Fall of the Classical View of Meiosis” (Hawley and Arbel, 1993). Although Scott Hawley has since recanted his proclamation “that studies in Succharomyces cerevisiae have challenged the central paradigm of meiosis,’’ this chapter is not about the discovery that the previously presumed corpse is still breathing. It is about the return to health of a rowdy and robust adolescent determined to make its presence felt. In a mystery, it is hard to have even a presumptive corpse without an antagonist that poses a threat. The threat to the extinction of mammalian meiotic studies arose both from an apparently insurmountable weakness in mammalian meiotic studies and an immense strength in the newly emerging field of yeast meiotic studies. The weakness of the mammalian investigations and the strength of the yeast studies were the same-mutants. For years, the search was on for meiotic mutations in mammals. But this search met with considerable obstacles. Disruption of meiosis has an obvious consequence: sterility that cannot be circumvented in most sexually reproducing organisms, as it can be in yeast. Discovery of a sterile male in the human population, even when the sterility could be traced to meiotic abnormalities, was an obvious dead end from a genetic perspective. Although recessive mutants in mice might seem to provide a means of avoiding this problem, the search for mouse genes acting in meiosis was hardly more productive than the human studies. In 1988 Handel summarized the list of then known sterility mutants in mouse and found that most of these had pleiotropic effects that complicated interpretation of the primary defect. Moreover, few of these mutants seemed to disrupt the course of meiosis itself. A classic mouse mutant hunt for meiotic defects is further hampered by the fact that mutagenizing the mouse genome does not allow for conditional mutations such as temperature-sensitive mutants, and therefore there is no way to recover aberrant gametes for analysis. These constraints do not exist in yeast. It is easy to screen for temperaturesensitive mutants, and the spol3 mutant, which bypasses the first meiotic division, rescues many mutations with defects in meiotic prophase. Dozens of meio-
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tic mutants have been recovered, and the phenotype of many of these has been characterized (Prakash ef ul., 1993; Mitchell, 1994). Methods for in virro manipulation of these genes and targeted replacement followed by in vivo analysis (Struhl, 1993) have yielded a rich lode of information. In 1988 Dresser and Giroux introduced a technique for surface-spreading yeast meiotic nuclei. This procedure, an adaptation of the Counce-Meyer (1973) technique widely used in multicellular eukaryotes, allowed visualization of the synaptonemal complexes (SCs) that hold homologous chromosomes together during meiotic prophase and provided yeast geneticists with a means o f correlating their biochemical observations on mutants with cytological events within meiotic cells. Things in the world of yeast meiosis indeed looked rosy. However, as will be discussed in more detail in Section V, a yeast nucleus is small and the cytology far from ideal. In contrast, the cytology of mammalian spermatocytes and oocytes is superb. However, until recently it was difficult to link the cytological scene to specific proteins and biochemical processes. If only there were mouse meiotic mutants! But wait-new techniques have recently become available that both provide mutants and abrogate the need for them. This chapter discusses these new developments for examining maminalian meiotic prophase. Look out, yeast! Here come the mice!
II. Setting the Stage: Meiosis Plain and Simple The setting is the meiotic nucleus ilscll, and the scene, as it unfolds, is actionpacked. The focus in this chapter is on mammals in general and mice in particular. The function of meiosis is to deliver a complete haploid set of chromosomes to each gamete, be it an egg or a sperm. Consequently, all meiotic activities are focused on ensuring successful segregation of homologous chromosomes. (Production of a functional gamete involves additional activities and pathways and will n o t be discussed here.) There is a long list of events generally considered essential for meiotic progression. Because there is little, if any, evidence of premeiotic association of homologs in most multicellular eukaryotes (Scherthan ef a/., 1996), one of the first requirements is that homologous chromosomes must find one another and at least roughly align, or possibly pair via paranemic “sideby-side’’ interactions (Kleckner c f d., I99 1 ; Kleckner and Weiner, 1993). This alignment is postulated to involve progressively more sequences and to result in progressively more stable homologous pairing, and is a separate process occurring prior to synapsis (Kleckner et d., 1991; Kleckner and Weiner, 1993). Once this is accomplished, synaptic initiation is thought to require a further molecular check for homology involving a subset of the total genomic sequences, possibly via a gene conversion event (Carpenter, 1984, 1987; Smithies and Powers, 1986; Kleckner et ul., 1991; Kleckner and Weiner, 1993). This check of sequences is believed to continue as synapsis proceeds, but in the case of some chromosome
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aberrations, it may not adhere to the same stringency of checking as seems to characterize the synaptic initiation step (Ashley, 1988). Synapsis is thought to be followed by reciprocal recombination, which is generally considered essential for chiasma formation (Maguire, 1978; Engebrecht et a[., 1990). Sister chromatid cohesion distal to the site of recombination also appears to be needed to hold homologs together at chiasma sites to ensure maintenance of the bivalent as a cohesive structure until anaphase 1. Next, bivalents must move to the metaphase I plate and orient homologous kinetochores to opposite poles in order for homologs to segregate at anaphase I. Sister chromatids must then reorient their kinetochores to the newly formed anaphase I1 poles in preparation for separation. Superimposed on all these events are likely to be meiotic cell cycle checkpoint controls, monitoring each of these steps to ensure their successful completion before allowing initiation of the next step in the sequence. Although these are the activities that receive the most attention. there i s also an often ignored event: premeiotic S-phase. Neglecting it may be a big mistake. Meiosis may be analogous to a magic act, distracting us with a lot of hand waving while the stage is being set and the trick set up behind the scenes in premeiotic S. One of the peculiarities of the meiotic cell cycle is a protracted S-phase. In the mouse, for example, premeiotic S-phase requires about 14 hrs, versus the usual 8 required for mitosis (Monesi, 1962). However, as is the case in the mitotic cell cycle, premeiotic S-phase nuclei have no unique subnuclear structures to facilitate their study. But let us return to the defined primary events. Each of the steps discussed in the preceding paragraphs can be expected to require multiple genes and proteins in several different pathways. Dissecting any one of these pathways may prove a formidable task. However, before raising the curtain on this fast-paced drama, let us acquaint ourselves with the general scene and the main props for the show. They are described only briefly here. To fill in the scene, the reader is referred to other chapters in this volume (Chap. 7, by Peter B. Moens and colleagues; Chap. 8, by Daniel P. Moore and Terry L. Orr-Weaver; Chap. 10, by M. A. Handel and J . J. Eppig; and Chap. 11, by Patricia A. Hunt and Renee LeMaire-Adkins). Over the years, cytogeneticists, through detailed analysis of electron micrographs (EMS)taken at different stages of meiotic prophase, have become adept at recognizing changes in meiotic-specific structures as the nuclei progress through meiotic prophase. The most uniquely obvious and first described of these is the meiotic organelle known as the synaptonemal complex (Fawcett, 1956; Moses, 1956). The SC is an electron-dense tripartite structure consisting of two axial/lateral elements and a central element (Moses, 1968; von Wettstein et d., 1984). Before homologs synapse, an axial element forms between the sister chromatids of each homolog. The chromatin itself is organized into loops, with only the chromatin at the base of the loops attached to the axial elements. This means that only a small fraction of chromatin is physically in contact with the axial elements (Weith and Traut, 19x0). As synapsis progresses, homologous
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sister chromatids
Fig. 1
Diapram of organi/ation of hoinologous chrornomrnes as they synapse
portions of homologous chrotnosomes i n the vicinity of the axes come into progressively closer alignment. When homologous axial elements approach one another at about a distance of 100 nm, the central element forms between them. Once synapsed, the axial elements are called lateral elements. In addition to the central element, transverse filaments also extend across the SC between homologous axes. A simplitied version o i the arrangement of the chromatin and associated meiotic structures is depicted in Fig. 1. Although the basic structure is well described, the exact function of the SC remains a sub.ject of debate. The SC is not the only meiotic-specific structure, however. In 1075, Carpenter observed small electron-dense spherical or ovoid structures nonrandomly distributed along the length of the SCs i n miclpachytene nuclei of Dnuophilrr oocytes
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(Carpenter, 1975). Their size suggested they might be multiprotein complexes. Because pachynema is the stage at which recombination is thought to occur, Carpenter suggested that the structures, which she termed recombination nodules, might be involved in crossing over. In the intervening years, considerable evidence has accumulated substantiating this hypothesis (see Carpenter, 1984, for this review). Subsequently, however, another type of small electron-dense structure was observed both on the unsynapsed axial elements at zygonema as homologs begin to synapse and briefly on the fully synapsed SCs in early pachynema (Albini and Jones, 1987; Anderson and Stack, 1988). The position of these structures at corresponding sites on the unsynapsed axes of the two homologs, or sometimes at points of contact between homologous axes, led Albini and Jones (1987) and Anderson and Stack (1988) to suggest that they might be involved in a check for homology accompanying synaptic initiation. Carpenter ( 1984, 1987) further developed this theme and proposed that these electron-dense bodies might be involved in a gene conversion event associated with a check for homology prior to or accompanying synapsis. These structures have been termed zygotene or early recombination nodules (early RNs), and the earlier described structures are now called late RNs. Albini and Jones ( 1987) have suggested that early R N s are converted into late RNs, presumably through a turnover of some, but not all, of the protein components. Insofar as early R N s are identified based on their association with axial elements, it has not been possible to ascertain whether or not they are present before axial element formation. Mammalian spermatocyte nuclei have two additional uniquely meiotic structures: the sex body (sometimes erroneously called the sex vesicle), and an electron-dense body or “double-dense body.” The sex body (reviewed by Solari, 1974) appears to be the structural and functional domain of the sex chromosomes. Its location and appearance change during pachynema and can be used to substage meiotic prophase (Solari, 1974). During early pachynema, the density of the chromatin around the partially synapsed axes of the X and Y chromosomes is similar to that of the rest of the nucleus and the XY axes themselves are often centrally located. However, as the XY axes begin their precocious desynapsis relative to the autosomes, density of the chromatin surrounding the XY axes increases and the entire sex body (axes plus chromatin and protein components) moves to the periphery of the nucleus. In addition, a “dense body” or “doubledense body” (whether single or double depends on the species) is usually observed in the vicinity of the sex chromosomes and reacts to the same stains as do nucleoli (Dresser and Moses, 1980). Although not meiotic specific, nucleoli are also prominent nuclear components in both spermatocytes and oocytes. They undergo characteristic changes in morphology during mammalian meiosis, making them another useful marker for substaging meiotic prophase events (Moses, 1980). Despite superb morphological descriptions of this cast of meiotic organelles, little is known about their protein components. Moreover, their modus operandi
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remains a mystery of epic proportions. To crack this mystery will require a team of detectives and an arsenal of techniques. Let’s check out what is available.
111. Surveillance Methods A. The Old Methods
Prior t o 1973, analysis of meiotic prophase nuclei involved three-dimensional reconstruction of EMS from serially sectioned plastic-embedded nuclei. Because examination of a single nucleus required sectioning, examining, and reconstructing 60-100 serial sections, the technical skill, the daunting effort, and the sheer expenditure of time ensured that the size of the population of cells examined would be small and therefore might not include critical stages or substages. The light microscopic (LM) technique i n nearly universal use prior to 1973 was the “air-dried” method (Evans et a/., 1964). Although a much larger population of cells could be examined, this technique retains only the chromatin, particularly the DNA, but not the SCs. In 1973 Counce and Meyer provided a quantum leap in methodology with the introduction of a “microspreading” or “surfacespreading” technique that proved to work equally well for LM or EM. Unlike the air-dried method, the Counce-Meyer technique (and its myriad variations) preserves the SCs while flattening the entire nuclear contents into a two-dimensional plane. Although these preparations are generally stained to enhance visualization of the protein components, the chromatin is also well preserved. The SCs o f an entire nucleus can be visualized in a single EM, and dozens of nuclei can be analyzed more easily than a single serially sectioned nucleus. Consequently a more complete series of nuclei from zygonema through pachynema or diplonema can be assembled and sequenced than was previously possible. When trying to evaluate the time of action of a protein. this temporal sequencing becomes especially important. But the advantages of the Counce-Meyer technique don’t end with EM. Although the SC lies at the limit of resolution of LM, Moses (1977) found that the SC could be visualized by low-magnification phase microscopy. (Under higher magnification, with an oil immersion lens, refraction is lost and the SCs are no longer visible.) Therefore, by changing the substrate (glass for LM, plastic for EM) on which the meiocytes are spread, cells may be examined by either EM or phase contrast LM. Shortly thereafter Fletcher (1979) and Dresser and Moses (1980) independently showed that SCs could be stained with silver nitrate and visualized with either EM or conventional LM. This advance made meiotic prophase analysis available to anyone with access to a light microscope. However, there is no contest when the degree of resolution of silver-stained preparations examined by classic LM methods is compared to the resolution afforded by EM. The details seen o n EM are clearly superior to those observed on
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LM. Nonetheless, for gross analysis of synapsis and chromosome association, such as determining whether or not synapsis of a bivalent is complete or whether an unpaired autosome is associated with the sex chromosomes, LM analysis is adequate, especially when it is used for quantifying types of synaptic configurations to supplement the finer detail but lower number of nuclei analyzed by EM.
B. The New Methods
Technology has again advanced, and a further comparison of the relative merits of LM versus EM techniques is in order. The basic preparative technique remains relatively unchanged, but new reagents permit replacement of heavy-metal staining of chromatin and meiotic proteins with fluorescent detection of selected DNA probes or antibodies against specific proteins. Now what is required is epifluorescence optics, a C-CCD (cooled-charge coupled device) camera, and megastorage for digital images. As always, the choice of technique involves some trade-offs, and selection of the method depends on the intent of the analysis. Whereas a heavy-metal stain, such as phosphotungstic acid (PTA) or silver nitrate, provides generalized staining for visualization of total proteins i n SCs or RNs, antibody technology permits detection of individual protein components of these structures. Moreover, since the fluorescent dye DAPI (4’,6’-diarnidino-2phenylindole) preferentially stains AT-rich regions, its use as a counterstain on surface-spread nuclei provides a clearer differentiation of AT-rich heterochromatin versus euchroinatin than does PTA or silver nitrate in EM studies. The position of individual DNA sequences relative to the synaptonemal complex can also be easily evaluated by fluorescent in sitir hybridization techniques and combined with either antibody detection techniques (Moens and Pearlman, 1990; Spyropoulos and Moens, 1994) or silver staining techniques (Solari and Dresser, 1995). The primary focus of this chapter will be on antibody localization. Because the purpose of this chapter is not to provide an antibody how-to guide, the reader is referred to a manual on the subject, such as the one by Harlow and Lane (1988). However. a few words on the specifics of detection with regard to meiotic preparations are appropriate. As is always the case with cytological procedures, specimen preparation is crucial. The procedure of Peters et ( I / . (1997) yields superb rneiocyte preparations from either sperrnatocytes or oocytes. Antibody detection works best when carried out on fresh specimens. The reader is referred to Spyropoulos and Moens ( 1994) for a protocol developed specifically for meiotic surface-spread preparations. If the study involves more than one antibody, the siniplest and safest way to differentiate between the two is to use antibodies raised in two different species (for example, rabbit and mouse) to ensure differential detection by the secondary antibodies. Theoretically, the number of antibodies that can be employed at once is limited only by the number of different species employed for antibody produc-
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tion and the number of fluorochromes that can be detected with the available filter sets. I n actual practice, however, monoclonal antibodies are almost always made from inouse and polyclonals are most frequently raised in rabbits. When detection is accomplished through seconclary antibodies, this cornbination limits co-localization to two antibodies. The species problem can be circumvented by either of two methods, each of which raises additional d culties. The first of these methods is direct labeling of' the primary antibody, the second is sequential labeling. Again. the reader is encouraged to consult a manual for details. The success of antibody detection with fluorescent labels is enhanced by advances in imaging systems. For starters, the sire of a fluorescent image on a 17-in. (43-cin) coinputer monitor is actually larger than a standard 8 X 10-in. (20 X 25-cm) EM, and a fluorescent iniage of this size can be achieved without loss of detail. Because the images are collected as gray-scale images, they can be printed on a laser printer at minimuin cost. (Photographs are needed only for publication prints.) In contrast, exnmining, photographing, and printing a similar number of nuclei observed by EM would require days of effort at a far greater financial cost. For LM work, the CCD camera provides advantages over standard photographic techniques: ( 1 ) For two-color co-localization, standard photographic techniques require either a dual band-pass filter or double exposure, either of which can result in loss of image quality and information. The CCD camera allows capture of each fluor as a separate image. Different fluor images from the same cell can then be digitally merged with ii computer program. Such a pi-ogram provides an option of turning the two images on and off (merging and separating the images) to more easily verity whcther individual Joci co-localize. ( 3 ) Although signals for most antibodies are strong, the CCD camera can be used to focus and image a signal too weak to be visualized by eye, an impossibility with a standard camera system. (3) The size of the working image is larger. inore images can be captured for a more detailed analysis, and digital storage requires less space than do negatives and prints. When an antibody such as CORI or SCP3 (Section I V ) is used to immunostain the axial/lateral elements, the Iluorescent images and the EM images provide similar information. It is instructive to compare silver-stained EMS of iiiouse meiotic prophase oocytes to SCP.3 immunostained epifluorescent images o f the same cell types and stages acquired as digitized images. Figure 2 provides a side-by-side coinparison of nuclei at 4iiiiilar stages of meiotic prophase. In rygonema it is equally easy to distinguish between synapsed and asynapsed segments in the EM and fluorescent images (compare Figs. 2A and B), in p x h y nema the fully synapsed bivalent5 are equally evident (Figs. 2C,D), while i n diplonema desynapsis and sites where the bivalents are held together ut chiasmata are also correspondingly clciir in the two image types (Figs. 2E,F). These images demonstrate that the use of antibodies t o a component of axial/lateral elements of the SC detected with fluorescent secondary antibodies and viwalired
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Fig. 2 A compariwii ol'electron micrographs arid epifluorescent micrograph\ from CCD-generated images of inciotic stage\ i n the niwse. I n thc electron micrographs (left, the SC axes arc stained with silvcr nitrate; in the light micrographs (right) they arc initnunostained with SCP.3, an antibody against the axial/lateral element\, and are shown in white. ( A . B) Zygnneina i n ooyctes, (C, D) pachynema in oocytes, (El diplonema in a n oocytc, (F) diplonciiia i n a spermatocyte.
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by C-CCD (or equivalent) image capture and computer analysis can provide the same information with a similar degree o f resolution as EMS. Because the number of nuclei that can be examined by fluorescent microscopy is markedly greater than the number that can be visualized by EM with the same expenditure of time, the end result is the possibility of a more complete meiotic picture.
IV. Reconstructing the Scene The plot has now been outlined, and the scene of the action has been described, but before moving on to the list of suspects, it is time to consider some of the tools (antibodies) available for reconstructing the scene. One of the advantages of classic EM studies is that nonspecific staining of the proteins of the SC and R N provides both a structural and temporal framework for interpreting meiotic events throughout meiotic prophase. An ideal antibody arsenal would include marker antibodies to ( I ) the axial/lateral elements of the SC, (2) the central element of the SC, (3) an identified component of early RNs, and (4) an identified component of late RNs. Of these, an axial/lateral element antibody is the most beneficial. Labeling of the axial element provides landmarks from the time it begins to form as partial axes in leptonema. In zygonema, such an antibody stains both unsynapsed and synapsed axes: in pachynema, it reveals both the fully synapsed bivalents and the partially synapsed X and Y; and in diplonenia,
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it reveals the axes of desynapsed bivalents and the sites of cohesion between homologs (chiasmata). Two mammalian antibodies of this nature have been described: one to COR 1 (Dobson er d., 1994) and another SCP3 (Lammers P I NI., 1994). The SCP3 protein was isolated from rat (Lammers et d., 1994), the COR 1 from Syrian hamster (Dobson ef ( i l . , 1994). CORl has been shown to be a hoinolog o f SCP3 (Dobson et (11.. 1994). The value of antibodies to these components of the axial/lateral element is readily apparent in Fig. 3, in which an antibody t o SCP3 has been used to follow meiotic prophase from early leptonema through pachynema in spermatocytes. Two mammalian antibodies react with protein components of the central region of the SC: SCPl (Meuwissen et d., 1992) and SYNl (Dobson et c i l . , 1994). These proteins are also homologous and were isolated from rat and Syrian hamster. respectively. Zip 1 is a component of the central region in S L ~ ~ ~ I L I roniycw ccre\isiae (Sym cf NI., 1993), and although the cytology in yeast is not as good as in mammals, antibodies against Zip1 can provide similar data: defnitive information on the synaptic status of the chromosomes. Antibodies against protein components of the RNs have also been tentatively identified. I t appears likely that RADSI and DMCl, discussed later under the list of suspects (Section VI), are components of early RNs and that at least one mismatch repair protein (MLHI) is a component of late RNs. As additional proteins are localized via their antibodies, this list of' potential markers can be expected to grow rapidly. There is another source of potential marker antibodies: sera from certain human patients with autoimmune diseases and preimmune sera from animals being injected for production of antibodies. Sera from auloimmune patients with CREST (cdcinosis, Raynaud syndrome, esophageal dismobility, sclerodactyly, and relangiectasia) syndrome is the best-known example of the first category. All CREST sera react with a component of kinetochores, but some also react with SC components (Dresser, 1987; Haaf et al., 1989). If available, this serum is especially valuable, since it is derived from humans (a third species) and can therefore be used for co-localization studies with antibodies from rabbit, mousc, o r yet a fourth species. Rabbits have a high level of autoimmune sensitivity, and therefore preimmune serum from some rabbits may cross-react with SC proteins.
V. Verifying an Alibi (Temporal and Spatial Resolution) To clarify the roles of individual proteins in the meiotic process, it is necessary to localize them within meiotic nuclei both temporally and spatially. Although studies in yeast have provided a plethora of important information about meiosis (see Roeder, 1990, 1995; Kleckner et ul., 1991; Kleckner, 1996), yeast suffers from two shortcomings for antibody localization studies: a short meiotic cell cycle and a small nucleus with relatively poor cytology. The duration of the
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A comparison of the duration 01 m e i o t i c htages i n yeast vs. mouse sperinatocytes. Tiiiiiiig for progression in yeast i s lroiii Klecltiei ( 1906). and for mouse from G o e ~ ('I t i / . ( 1985).
meiotic cell cycle of yeast and niotisc is compared in Fig. 4. Because deducing meiotic function may hinge on precise determination of the time or time interval within meiotic prophase during which an event occurs or a meiotic component appears or disappears relative to another, the ability to separate events temporally cannot be overemphasized. For exnmple. in mammals, several lines of evidence suggest that recombination occurs within the early to inidpachytenc interval (Moses r t ctl., 1982a,b; Moses and Poorinan. 1984). In mouse, not only are all of the main stages o f prophase easily identilied, but pachynema can be divided into a series of substages based on estnblished criteria developed for silver-stained preparations. These criteria vary only slightly from species to species: human (Solari, 1980). tnouse (Moses, I980), Chinese hamster (Moses r't d., 1977), or the deer mouse, Pero/~~.rcus (Grcenhaum ('I ( i / . , 1986). Because most of the came subnuclear structures employed l.or substaging with silver staining are visible with the appropriate antibodies, these criteria can be readily adapted for tluorescence studies. However, in addition to the original criteria, localization of COR I o r RADS 1 (Ashley rt NI., 1995; Plug P I o/., 1996) provides additional teinporal markers that further improve the accuracy o f substaging. A second factor important i n the temporal pinpointing of an event also comes into play. The larger sample s i x obtained with fluorescence microscopy conies closer t o approaching a continuum and increases the possibility o f catching a protein "in the act." Because moclern scientific investigation often centers on quantitation of data, investigators tend to assume that quantitation of signals in meiotic prophase is essential. That is not iiecessarily the case. Descriptive interpretation of a large population o f meiotic prophase cells that have been accurately sequenced is probably much inore informative. As an analogy. consider the classic photographic series by Edward Muybridge, made in the late 1870s188Os, depicting the gaits o f camels, horses. elephants, and the like. Counting the number of feet an animal has on oI off the ground could never tell you as much about an animal's gait as a sequential series of images achieved by having an animal run through a series of trip wires. Similarly, a reasonably complete sequence of photographs catching the precise substage of co-localiration o f two
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antibodies, or their respective times of arrival or departure relative to a specific meiotic event, is far more informative than the average number of foci in a pooled group of zygotene or pachytene nuclei. This analogy continues to hold true when zygotene or pachytene stages are broken into substages. Spatial resolution is equally as important and easier in mammalian cells than in yeast. The S. c.erc.visiae genome has only 12.5 Mb of DNA distributed between 16 pairs of chromosomes. The largest of these, chromosome XII, has 1095 kb of DNA plus a variable amount of rDNA: the smallest, chromosome I, has only 240 kb. Silver-stained meiotic prophase spreads from yeast show parallel lateral elements between the honiologs of each bivalent, as well as nucleoli and spindle pole bodies (Dresser and Giroux, 1988). R N s (either early or late) have been reported only in sections (Byers and Goetsch. 1975). With the exception of SC XII, the nucleolus organizer chromosome, individual bivalents cannot be identified (Dresser and Giroux, 1988). In contrast, the mamnialian genome i s large, around 3000 Mb; a single midsized chromosome is as large as the entire yeast genome. The central element is frequently observed, and RNs have likewise been reported both in sectioned (Holm and Rasmussen, 1983; Glamann. 1986) and spread material (Solari, 1980).It is important to note, however, that visualization of niarnmalian R N s frequently requires either a modification of the silver staining techniques (Sherman ef N I . , 1992) or use of a different combination of heavymetal staining.
VI. Developing a List of Suspects Although the more complex genome of mammals can be expected to require more genes to carry out meiosis, one look at the list of meiotic mutants already identified in yeast (Hoekstra et d., 1991; Mitchell. 1994) suggests there is no shortage o f “meiotic suspects.” Despite the existence of many logical suspects, there may be others that are equal or more important players, though less obvious. Therefore it is important to devise a plan for developing a list of potential protein suspects. This section deals with strategies for selecting candidate proteins.
A. Presence at the Scene of the Crime
Obviously, any protein that shows testicular or ovarian expression on Wcstern or Northern blots is a candidate for meiotic involvement. However, expression in these tissues could be associated with sperm or oocyte differentiation rather than meiosis. If the antibody is availablc, cytological localization may be worth the effort. However, an antibody that gives good results on a Western blot may prove to be less effective on cytological preparations.
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In a similar vein, any cDNA sequence trom a testis or ovary library is ;I viable candidate to encode a protein with meiotic activity. However, pulling a random sequence out of a library and making a protein and antibody is still too much a shot in the dark. If the sequence is already in a data bank and one or morc of its motifs suggests a possible meiotic function, prospects improve, and the production of an antibody for localization studies may be merited.
B. Suspicious Professional Activities
Meiosis is an extremely complex process and can hardly be expected to have arisen cle /zo\'ojust as eukaryotcs wcre evolving from prokaryotes. Thcrefore it is logical to presume that the meiotic process recruited proteins from prokaryotic cells as well as the mitotic cyclc ot ancestral eukaryotes. Prime candidates in this category of proteins include those involved in repair of DNA damage and recoinbination. However, likely candidates are not restricted to this pool of suspects. They may also include proteins involvecl in DNA synthesis and cell cycle control. Protein complexes involved in both repair and transcription have been isolated, making known transcription factors also highly suspect. This activitiesoriented approach has already lead to the indictment of several protein characters, described in the following paragraphs.
1. RAD51 Perhaps the most successful example of this approach to date has been the quest for homologs of the E. coli protein RecA. As a repair enzyme, the RecA protein has been extensively studied and well characterized (reviewed by Radding. 199 I : KowalcLykowski et d . , 1994). RecA is known to polymerize on the 3' singlestranded DNA tail and to form a nucleoprotein filament following the formation of double-strand breaks and resection of the 5' end. The RecA protein enhances the ability of the single-stranded DNA (ssDNA) to find homologous doublestranded DNA (dsDNA), and once i t is found, the nucleoprotein complex participates in heteroduplex formation and strand exchange involved in both DNA repair and recombination. These properties are exactly those expected of a protein involved in a search for homology via a gene conversion event in meiotic prophase and led to the prediction that a eukaryotic homolog of RecA would be found to be an early player in meiotic prophase (Carpenter. 1984, 1987; Smithies and Powers, 1986). Several eukaryotic RecA homologs have now been identified. They include RADSI (H. Ogawa, 1993; T. Ogawa et a/., 1993) and two RecA homologs with meiosis-specific expression: Dmc I , first described in yeast (Bishop e f al., 1992), and LIM1.5, first described in lily (Terasawa et d., 1995). These latter two proteins are homologous to each other (Terasawa et cil., 1995).Antibody IOL.a I'iza-
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tion studies have confirmed the presence of Dtncl (Bishop et a / . , 1992; Bishop, 1994: Rockmill et a / . , 1995). RADSI (Bishop, 1994; Ashley e f d., 1995; Rockinill et d., 1995; Terasawa r t [//., 1995; Plug ct [ I / . , 19961, and LIM 15 (Terasawa et d . , 1995) in meiotic nuclei of a range of taxonomically diverse species. These studies have revealed interesting differences in the meiotic behavior o f RADS 1 in different taxa, especially between yeast and mice. Bishop ( 1994) and Rockinill et a / . found that the time of appearance of yeast KadSI foci is approximately concurrent with the previously reported time of occurrence of double-strand breaks and SC precursors (Padmore et 01.. 1991). Both Bishop (1994) and Rockinill f t d. also found that the number of RADSI foci they observed was similar to the number of “association sites” previously observed between unsynapsed axes in yeast 5 / 9 1 mutants (Sym rt ( I / . , 1993). Because association sites are the location of initial contact between hotnolog sequences, these sites are thought to be places where homology is checked prior to synapsis. This is an interesting correlation, for it suggests that there is only onc RadSI focus lor each association site between the two homologs. Therefore, in yeast, both the appearance of RadS 1 concomitant with double-strand breaks and the location of RadS 1 at sites previously postulated to be sites of a homology check are consistent with the gene conversion model of synaptic initiation proposed by Carpenter ( 1984, 1987) and others. However, although RadS 1 -promoted strand exchange has been demonstrated iri \,it,v (Sung. 1994: Sung and Robberson, 1995: Baumann (it ( I / . , 1996), the kinetics of this reaction are not as favorable as those for RecA, suggesting that RadS 1 may he playing a different biochemical role than its RccA ancestry niight lead one to believe. In mouse spertnatocytes and oocytes, RADS 1, as detected by antibody localization, is evident in premeiotic S-phase nuclei (Plug et ( I / . , 1996). It is already present on axial element components when they begin to assemble i n leptonema (Fig. SA). Moreover, RADS I foci are located at similar sites on the two homologous axes. As homologs synapse, the axes themselves often alternately converge and diverge. RADS I foci on the two homologous axes are situated at constrictions on the axes but riot in the region between the axes, although the latter location is predicted if RADSI is the primary player responsible for finding and checking homology (Plug et a/., 1996). In all respects these points of convergence are similar to the “association sites” tirst described in EM studies and postulated to be involved in the search and check for homology (Albini and Jones, 1987; Anderson and Stack, 1988). Although it is currently not feasible to map the location of RADSI foci dong the axes during zygoncma (Fig. SB), RADSI foci remain on fully synapsed SCs into early pachynenia (Fig. SC). During this substage the RADSI focal pattern roughly corresponds to the interchromotnere pattern observed on pachytene bivalents i n air-dried preparations (Evans, 1989) or R-band pattern of mitotic inetaphase chromosomes of mouse (Plug et ( I / . , 1996). RADSI then disappears from the synapsed axes during the transition from early to inidpachynetna (Fig. SD).
Figure 5 Progression of meiotic prophase followed with CORl and RADS I . (A) Leptonema. Note the small amount of white (CORI), indicating that the axial elements are only beginning to form. In contrast. there are numerous linear arrays of RADSI foci (green) in the nucleus. (B)Late zygonema. The bivalents are almost entirely synapsed. Note the forked appearance of some bivalents that are still not completely synapsed. Also note the reduction in number of foci. although there are still many RADSI foci on each synapsed axis. (C) Early pachynema. Note the slight decrease in number of RADSI foci on some bivalents. (D) Mid pachynema. Most RADSI foci have disappeared. (The slight bluish cast is a color shift in the white CORI axes.)
Fig. 5C and D
Comparison of number and distribution of MLHl foci (red) in (A) spermatocytes versus (B) oocytes on the mouse. Note the number of MLHl foci near the ends of bivalents in spermatocytes and the absence of foci at these locations in oocytes. The axes are immunostained with CORl (white).
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RADS 1 has also been detected by antibody localization during meiotic prophase in lily microsporocytes (Terasawa ('/ ( I / . , 1995).As in mammals, RADS 1 is present in zygonema but drops nipidly i l l pachynema. However, in contrast to the case in mammals, most RADS 1 foci in zygonema of lilies do not appear at corresponding sites on the two honiolog\, but on only one of the two homologs iTerasawa et ol., 1995). These studies all suggest that RADS 1 is a component of early RNs, although none has provided direct proof. pos\ible only with i m m u n o g o l d - ~ ~ n t i b ~local)d~ ization by EM. The most intriguing aspect of these studies is that the results from study of each species are consistent with what is known about synaptic initiation in that particular species. In yeast. RadSI appears at the time of double-strand breaks and the advent of SC precursors (Sun et ( I / . , 1989; Cao et a / . , 1990: Padmore et d., 1991; Sun, 1991 ), supporting the suggestion that synaptic initiation in yeast is triggered by a gene conversion event in which RadS l or a ximilar molecule is an active participant. Assuming RADS 1 is indeed a component of early RNs, the RADS 1 data froin plants i'rerasawa r t a / . , 1995) are consistent with the observations of' Albini and Jones ( 1987) and Anderson and Stack ( 19x8) of an early RN, often but not always located on one axis of a pair of homologs, but not at a corresponding site on the other. The correspondencc between the RADS I focal pattern and the interchromomere pattern (pachynenia) or R-bands (mitotic inetaphase chromosomes) in mammals is consistent with the suggestions of Chandley ( 1986) and Ashley ( 1988) that a subset of sequences within R-bands are sites where homology is recognired aiid synapsis is initiated. The association of RADS 1 with chromatin during premeiotic S-phase in mammals might be related to the evidence from plants suggesting that a subset of the genome (0.1 0.3), termed zygotene DNA, or /ygDNA, is not replicated during premeiotic S-phase but is delayed i n replication until iygonema (Hotta Pt ( I / . , 1966: Hotta and Stem, 1971; Stern and HoIta, 1985). Replication of this DNA during ~ y gonema is essential; blockage 01' its synthesis also blocks synapsis and halts the progression of meiosis (Roth and Ito, 1967). Although the evidence for L ~ ~ D N A is more tenuous in mammals than in plants, small single-stranded regions can be predicted to occur at the transition from replicated to unreplicated re,'"ions at replication torks. These are potential RADS 1 -binding sites, and this concept provides a link between the observations on RADSI localization and the preselection model of meiotic synapsis (Plug et (11.. 1996).
2. RPA Replication protein A (RPA) is another ssDNA-binding protein that exhibits suspicious professional activities. RPA is ;I trimeric complex composed of a 70-, a 32-, and a 14-kDa subunit (Fairman and Stillman, 1988; Wold and Kelly. 1988). Documented roles for a RPA-ssDNA nucleoprotein filament include DNA replication and nucleotide excision iepair and recombination (Wobbe r t L I I . .
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1987; Fairrnan and Stillman, 1988; Wold and Kelly, 1988; Heyer ri < I / . , 1990; Moore ei LII., 199 I ; Coverley et LII., 1991). In E. cdi,single-strand binding protein ( S S B )can stimulate RecA-promoted strand exchange and recombinationrelated activities (McEntee rt 01.. 1980; Radding (Jt ( I / . , 1982; Dixon and Kowalczykowski, 199 I ) . Similarly, RPA in i V t w has been shown to enhance strand exchange i n the presence of RADSI (Baumann ct ul., 1996). Antibody localization studies using both a polyclonal antibody raised against the entire trimeric coniplex and a monoclonal antibody against the 70-kDa subunit show that RPA is associated with meiotic chromatin distal to synaptic forks in zygotene nuclei (Plug ct ~ 1 . .submitted). Although i t co-localizes with RADS 1 at these sites, it is not detectable on unsynapsed axes i n advance of these synaptic forks. However, if synapsis is delayed. RPA is found bound to sequences hribt.rcn the synapsing axes, whereas RADS I binds to sequences directly associated with the axes. These results suggest that RPA does not necessarily bind to the same DNA sequences as RADS 1 but may bind to adjacent sequences as an active search for homology is initiated. The variation in size and shape of the RPA signals on synapsing bivalents. especially when synapsis is delayed, provides an interesting clue to previous EM observations. In a variety of organisms. the unpaired axes have been described as alternately diverging and converging at multiple association sites (Rasmussen and Holm, 1978; Hasenkampf. 1984: Albini and Jones, 1987; Anderson and Stack. 1988; Syrn ri (11.. 1993). Electron-dense nodular material has often been observed at these association sites on the unpaired axes and along the synapsed SC into early pachynema. The shape of these structures has been variably described within the same plant nucleus as spherical to elliptical to barlike, and their size has been reported to vary in diameter between 100 and 170 nm (Anderson and Stack. 1988). Their reported location on axes is equally variable: single sites on asynapsed axes, matching sites on hotnologous asynapsed axes, and positions bridging the distance between converging asynapsed axes, as well as sites on newly synapsed axes (Albini and Jones, 1987).I t has been suggested that despite these variations in size, shape, and location, all of these structures are sites of homolog recognition (Albini and Jones, 1 987), although no explanation had been offered to explain the morphological differences and positions. The combined localizations of RADS I and RPA provide the likely explanation. Because both proteins bind to ssDNA, their respective signals can be expected to reflect the amount of protein bound to varying quantities of ssDNA at each site. Thus the variation in si7e and shape of the electron-dense aggregates seen at association sites most likely reflects variation i n the amount of nucleoprotein at these sites and suggests that RNs are not uniform, and certainly are not “bundles” of proteins that arrive on site prepackaged. When SC formation is complete, RADSI foci and RPA foci do co-localize briefly in cytological preparations of late zygotene and early pachytene nuclei (Plug rt al., submitted). However, the antibody studies have provided unexpected insights into the likely activities of these two proteins. Although the postulated
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role 01‘ RADS I is to facilitate recoinbination via participation in heteroduplex formation. RADS 1 foci disappear from the fully formed SCs early in pachynema before MLH 1 is detected and reciprocal recombination is thought to occur. However, RPA remains on the SCs and M L H l foci co-localize with a subset of RPA sites, suggesting that RPA may participate in meiotic reciprocal recombination (Plug et d., submitted).
3. Mismatch Repair Proteins Products of mismatch repair genes are yet another example of an entire class of repair proteins for which other lines of evidence suggest “suspicious activities” and possible involvement in meiosis. As was the case with RecA and its eukaryotic homologs, mismatch repair genes were first described in bacteria, and the first three were designated MiriL, MirtS, and MutH (Modrich, 1991 ). Eukaryotic homologs were soon discovered i n both yeast and mammals and were found to play similar but not identical roles ;IS those reported for bacteria (Modrich. 1991; Modrich and Lahue. 1996). Cells with mutations in two S. c~~reiisirre homologs of MUTS, M S H 4 , and MSH.5 were loiind to be defective in tneiotic recombination but, interestingly, m t in mitotic mismatch repair (Ross-Macdonald and Roeder, 1994; Hollingsworth et N / . , 1995. respectively). Antibody localization studies place M S H 4 in yeast prophase nuclei (Ross-Macdonald and Roeder, 1994). Subsequently, antibody localization o i MLH 1. ;I mammalian mismatch repair protein, to mouse oocytes at diploncma showed that the protein localizes to chiasmata. the cytological sites 01‘ reciprocal recombination (Baker et “/.. 1996). These foci were also seen in mouse spermatocytes at pachynema along the SC. Insofar as recombination is thought to occur within late RNs. the conelation between MLH 1 localization and recombination sites makes MLH 1 a likely component of late RNs. The MLH 1 antibody therefore provides a means of cytological identification of putative sites of recombination i n meiotic prophase. If these sites indeed mark sites of reciprocal exchange, this antibody can provide a means for a direct cytological comparison of recombination in males versus females. Both the number and the location of recombination events differ between the sexes in mammals. In eutherian (placental) mammals, the recombination rate is higher in females, and most of these recombination events occur interstitially (dispersed along the entire lengths of the bivnlents). Not only are there fewer crossovers in males. they are also less randomly distributed, with an elevated number of recombination events near the ends of chromosomes (reviewed in Ashley, 1994). The number of M L H l foci along the SCs in oocytes and spermatocytes reflects these documented differences in recombination rates between males and females (Baker rt d . , 1996) and clearly shows the differences in distribution of these events (compare the number of near-terminal M L H l signals in Fig. 6A. a spermatocyte nucleus, with those i n Fig. hB, an oocyte nucleus). In addition to the generally higher rate of recombination near chromosome ends, males have a
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unique recombination site at the base of the pairing region of the X and Y chromosomes. The recombination frequency within this “pseudo-autosomal region” is 50%, although the region consists of only approximately 2.6 Mb of DNA in humans (Petit et [ I / . , 1988; Rappold and Lehrach, 1988). An MLHl signal is always seen in this region in mouse early pachytene spermatocyte nuclei, although this signal routinely disappears sooner than the MLH l autosomal signals (Fig. 6A, 1 1 o’clock). The timing of appearance and disappearance of MLH 1 in spermatocytes versus oocytes provides a clue to one small cytogenetic mystery. In most organisms there is a close correlation between the number of late RNs observed throughout pachynema and the number of reported recombination events per nucleus (see Carpenter, 1984; Roeder, 1990. for review). However. this is not the case in eutherian male mammals, in which the number of recombination events far exceeds the number of late RNs visualized as barlike structures in late pachytene nuclei (see Ashley, 1994, for review). The number of MLHl loci on SCs in late pachytene mouse oocytes (Baker rt u/., 1995) is similar to the number of reported chiasmata (Polani and Jagiello, 1976). However, in spermatocytes, a closer correspondence between the number of MLHl signals and number of chiasmata is noted during midpachynema. After this substage, the number of signals decreases rapidly until none are left by late pachynema (Baker et ( I / . , 1996). This drop in number of MLH 1 signals parallels the drop in number of bars or late RNs noted in mouse spermatocytes during the same substage (Glamann, 1986). This sexspecific difference in the timing of disappearance of both MLHl and late RNs probably reflects the difference in duration of pachynema (3 days versus 6 days) in oocytes (Speed, 1982) versus spermatocytes (Goetr r t al., 1984). Hence the “deficit” of latc RNs in late pachytene spermatocytes is most likely due to the completion o f recombination-related activities by midpachynema in male eutherian mammals. Further insights into the essential nature of the role of MLHl in reciprocal recornbination will be discussed in Section VI, part D, in which we consider targeted disruption of gene functions. 4. Polymerases
DNA synthesis has been demonstrated to occur during zygonema in plants (Hotta al., 1966; Hotta and Stern, 1971; Hotta et LII., 1984; Stern and Hotta, 1985) and mice (Plug ot c i / . , 1996) and during pachynema in D r ~ m ~ p / z i(Carpenter, /u 198 1) and mice (Moses and Poorman, 1984). Synthesis during zygonema might either be semiconservative replication associated with zygDNA synthesis (discussed above) or repair synthesis associated with a gene conversion event (Carpenter, 1984, 1987). Because recombination occurs during pachynema, DNA synthesis at this time can be predicted to be of the repair type. Whichever type, DNA synthesis requires a large number o f proteins: DNA polymerases and ligases as well as cofactors such as RPA and helicases, to name but a few. Some of these are et
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certain to be involved in meiosis, s o it can be predicted that meiotic antibody localization studies involving some of these proteins will provide interesting new information. However, as discussed earlier in Section VI, part A, in reference to pulling random cDNA sequences from a testis library, a throw-it-on-and-scc-whathappens approach is unlikely to prove the best strategy. Even casual examination of the literature suggests that some of these proteins are hotter prospects for meiotic involvement than others. An interesting example of this Nero Wolfestyle armchair approach to deducing meiotic function leads to polymerase p as a candidate for a role in meiosis. A perusal of the literature shows that, coinpared with other polymerases, the function of POLP is ill-defined. Nonetheles4, it ha5 been found to be necessary for base excision repair (Clairmont and Sweasy, 1996; Sobol et u/., 1996), in the conversion of ssDNA to dsDNA in Xetwpus oocyte extracts (Jenkins e t a / . , 1992; Sweasy and Loeb, 1992), and in gap filling (Sobol et ul., 1996)-alI activities suggesting it might be important in meiotic recombination. Moreover, its highest level of expression is in mammalian testis (Novak et a/., 1990; Alcivar Pt ci/., 1992). Thus, POL@ becomes an intriguing suspect and worthy of further investigation. Antibody localization studies have confirmed the presence of POLP at sites along both asynapsed and newly synapsed axes, a localization that suggests involvement in synaptic events (Plug et a/., 1997).
5. Cell Cycle Progression and Checkpoint Proteins Another gang of characters with suspicious professional activities that is rapidly emerging as important in the meiotic plot is a group of cell cycle proteins. Although they are discussed i n more detail elsewhere in this volume (Chap. 10, by M. A. Handel and J. J. Eppig), they will be considered briefly here. Progression of mammalian somatic cells through the cell cycle is controlled by a series of checkpoint proteins that ensure the successful completion of one stage before allowing the next to commence. Mitotic checkpoint genes (and proteins) fall into two general categories: those that inonitor or participate in the normal progression through the cell cycle, and those that detect damage (usually inflicted) to the genome and halt progression of the cell cycle until repair can be accomplished. Only after repair of the perceived damage is completed do these proteins allow progression to the next step. Although similar controls can be expected to operate in meiosis, the distinction between these two classes of proteins may be less clear-cut than in the mitotic cycle. In mitosis, repair and recombination in most cells occur only in response to unscheduled damage to the genome. However, recombination is an essential part o f the normal progression of meiotic prophase. Therefore, both types of cell cycle proteins are candidates for meiotic involvement and are discussed next.
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C. Family Connections Although homologies to prokaryotic or unicellular eukaryotic genes with known suspicious professional activities have already been mentioned, this discussion will elaborate on the use of these connections as a strategy for meiotic studies and point out the intricacies of family connections within the meiotic process itself. The case in point starts with it human gene with a disease phenotype as the prime suspect. The huinan disease is ataxia-telangiectusia (AT), an aLitosoina1 recessive disorder. Patients with AT have progressive cerebellar degeneration, immune deficiencies. hypersensitivity to ionizing radiation, an increa4cd incidence o f cancer characterized by genomic instability. and gonadal abnormalities, including sterility (see Mcyn, 1995; Shiloh, 1995. for review). Several o f these defects suggest either a direct or indirect role for the gene for AT in mitotic repair and recombination. and the sterility hints at a role in meiosis. When the gene for AT (called ATM, for AT mutated) was isolated and sequenced, i t was found to have homologies to two prcviously described yeast genes: TELI and MEC'l, both members of a family of pliospliatidylinositol 3-kinase (PIK)-like kinases (Savitsky c2t L I I . , 1996). TELl is iiiore closely related to ATM i n primary structure, with 56% identity through the kinase and C-terminal domains, and is required for telomere maintenance (Greenwcll et ( I / . , 1995).I t plays a partially redundant role with M E C l in conferring resistance to DNA damage in S. c e r e \ i ~ i t r e(Greenwell r t d . ,1995). M E C / is structurally and functionally similar to rtrd-3 from S. porribe and r7iei41 from Dr-osop/ii/ci.These three gene products all function as mitotic and meiotic cell cycle checkpoints and in the DNA repair pathway (Greenwell rt d . , 1995; Hari et d., 1995). However. no human homolog had been identified. Therefore, Bentley et ul. (1996) used the sequence of rtrd3 from S. porrihr to fish for the previously unidentified human gene and recovered a gene sequence they call ATR (for AT and m r 1 3 related). Cimprich rt a / . (1996) likewise isolated this sequence and noted its similarity to S. por7ibe r - d 3 . The trail of the isolation of this gene therefore traces from the human ATM gene to MEC//rcic/3 and back to the huinan ATR.Although it has been mapped to human chromosome 3q22-3q23 (Bentley et ( I / . , 19%). no huinan phenotype has yet been ascribed to mutation of
ATR. Several antibodies were made to the ATR gene product and used on a Western blot to show a high level of expression of this protein in testis (Keegan et ( I / . , 1996). Both the known roles of the cross-species homologs and the deinonstrated activity in testis made ATR a prime suspect for meiotic activity. This proved to he the case when antibody studies showed that ATR localized to sites along unsynapsed but not synapsed axes. These ATR foci were particularly intenw when the axes belonged to chromosomes that had experienced synaptic delay (Keegan e t a / . , 1996). However, this is truly a story o f family connections: antibodies to ATM, the original character in this drama and a member of the same family of PIkinases, also localizes on axes of meiotic chroinosoines during zygonema (Keeg-
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an et id., 1996). But like good Malia meinbers, ATR and ATM havc found a way to spread the duties while keeping things in the family. ATM IocaliLation proved to be complementary to ATR, with ATM localization restricted to sites along synapsed but not asynapsed axes (Kccgan r t d., 1996). Although direct proof is lacking, ATR and ATM localimtion suggcsts that both are components of early RNs. The reciprocal localization hints at a rate of turnover of proteins in these structures that was previously unsuspected. Because a turnover of checkpoint proteins would appear unneccssat-y to maintain the status quo, it also implies a change in the state of the chromatin being monitored within early R N s on asynapsed versus synapsed axes.
D. The Body in the Parlor
As mentioned in Section I, mammalian meiotic studies have suffered from a lack of meiotic mutants. Two things have changed this situation: the human genome initiative and knockout mice. Knockouts are induced null mutations that silence expression of the targeted gene. The human genome initiative has led i n only a few years to the idcntification 01' many human disease genes. As we have just seen in the ATRIATM story, corresponding initiatives in other organisms. including S. cewt>isicie,S. poriihe, C. r/c,ptiri.s, D~-osopliii~i, and the mouse, to name but a few, have led to a similar explosion in identification of genes and sequences. Data bases have made it easy to check for homology between species. Prior to the human genome initiative, the actuul gene had not becn identified for most human diseases, such as AT. Again using AT a s an example, the meiotic phenotype of sterility was almost ignored, because an early death from ataxia or cancer made sterility a trivial component o f the phenotype. However, once sequence data became available, the construction o f knockout mice and examination of the full range of the phenotype, including meiosis, in these mice became feasible. When the Atriz gene was disrupted, the knockout mice, as might be expected, were found to have many of the smie dcfects as human patients with AT: ataxia, immune defects, genomic instability. ;in early death from cancer, and sterility (Barlow eta/., 1996; Elson et u/., 1996; Xu et NI., 1996). Examination of surfacespread spermatocytes from Atrti - / - iiiice stained with the COR 1 antibody to visualize the axial/lateral elements by fluorescence microscopy suggested that synapsis during early zygonema is relatively normal, although there appeared to be more interstitial synapsis t h a n us~ialand some fragmentation of axes (Xu et id., 1996). However, by the time spcrmatocytes reached pachynema, things had gone seriously awry. Although homologous chromosomes had synapsed, the axes of the SCs were progressively fragmenting and the cells were degenerating (Xu et ul., 1996).Even by early pachynema, the SCs of each bivalent were already i n multiple pieces. Here it is instructive to compare the knockout meiotic phenotype with that
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from the antibody localization studies of Keegan et (11. (1996). As mentioned above, the ATM antibody localizes to foci along the lengths of synapsed (but not asynapsed) axes in zygonema and early pachynema. The size of the fragments in the Atni-I- mice is similar to the interfocal distances. I t seems likely that fragmentation is occurring at sites of ATM localization. Although it has been suggested that ATM may be functioning as a damage checkpoint (Meyn. 1995), these findings blur the lines between the role of a monitor and a participant. These two studies by Xu et ul. (1996) and Keegan et a / . (1996) illustrate how examination of meiosis in knockout mice and antibody localization studies in meiotic nuclei can complement one another and provide supporting evidence to help define the meiotic role of a protein. Studies on the mismatch repair gene M l h l , mentioned earlier, provide another example of the dual knockout/antibody localization approach. As mentioned above, MLHl was localized to sites of reciprocal recombination (Baker et ul., 1996). In the same study Baker rt (11. showed that synapsis in M l h l - I - mice proceeded perfectly normally (Fig. 7A). The first indication of trouble in these mutants became apparent in midpachynema, when the X and Y began their (usual) precocious desynapsis. In the absence of a functional MLH 1 protein the XY bivalent continued to desynapse until it fell apart into two univalents: the X and Y (Fig. 7B, at 7 o’clock). Similarly, as autosomal desynapsis began, i t became obvious that chiasmata formation had failed and that homologs were also unconnected (Fig. 7C). Unlike A f n - / - mice, most of these desynapsed axes remained intact, but without chiasmata the homologs moved to the metaphase I plate as univalents and the spermatocytes arrested at this point, presumably due to a metaphase 1 checkpoint. The power of this dual approach is evident when this study is compared to a similar study that examined only the knockout phenotype (Edelmann et (11.. 1996). This study, in which the sterility phenotype was studied by EM examination of M I h - I - spermatocytes, concluded only that there was a defect in pachynema that might involve recombination. Knockouts may silence gene expression, but when they are coupled with antibody localization studies, they shout information about who is doing what to whom. In this case, the antibody localization places MLHl at chiasma sites while the knockouts provide evidence that the MLHl protein is required for chiasma formation and the maintenance of bivalents. However, as informative as these studies are, further biochemical analysis is essential to characterize the specific molecular activity of the protein. In closing this section, it seems appropriate to remind the reader of several potential flaws in a knockout study without accompanying antibody localization studies. First, a knockout will arrest development of the mouse at the first point at which the disrupted gene is essential. Rud5I is a striking example. Although yeast RAD51 mutants are viable and encounter difficulties only when the genome has been damaged by radiation or a chemical mutagen, disruption of the mammalian RudSI function by “knocking out” the gene results in early embryonic
Fig. 7 Progression o l meiosi\ in MlhI knockout iiiicc. ( A ) Midpnchynenia. The X Y hivalcnt (lower right) has begun t o desynape, but the di\tal end ( 1 1 the SC is still present and holding the two axes together. ( B ) A slightly later substage of lxic'hgnema i n which the X and Y (lower l e f i ) are no longer attached. ( C j Diplonema. The autosonic\ h;iw de\yiiap\ed and without chiasmata have "fnllen apart" into 40 univalents. The axe\ itre imrnuiio\r;uiied with CORI (white).
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death-generally by the four- to eight-cell stage (Tsuzuki et d., 1996). Here the crime takes place backstage long before the curtain rises on the meiotic act! However, antibody localization in meiotic nuclei can still provide information about the meiotic activity of the protein.
VII. Setting Up a Sting Operation One of the most powerful means of linking an antibody to a specific meiotic activity is the longtime favorite of cytogeneticists: chromosome aberrations that alter the normal progression of events and thereby provide a higher definition of the multistep processes of meiotic prophase. This “tweaking” of the system is possible because chromosome rearrangements such as inversions, translocations, and deficiencies reposition sequences within and between chromosomes. Consequently, in nuclei heterozygous for such rearrangements, homologous synapsis is no longer a simple process of “zipping up” a pair of chromosomes from one end to another. Instead, some of the homologous sequences now reside at different sites on the same chromosome (inversions and duplications), on a different chromosome (translocations), or may no longer even exist within the genome (deficiencies). Such situations can provide different kinds of synaptic outcomes: ( 1 ) asynapsis (failure to pair), which is often linked to sterility, especially in males, and (2) at least two types of nonhomologous synapsis. Studying circumstances surrounding asynapsis can help us separate conditions necessary for preineiotic alignment from those necessary for more precise DNA-DNA homologous recognition and actual formation of the SC, while studying circumstances surrounding nonhomologous synapsis can help us define under what conditions the homology search and synaptic processes can be short-circuited or separated. Both asynapsis and nonhomologous synapsis can also be expected to highlight features of proteins involved in meiosis that might otherwise go undetected. Although chromosome aberrations are also discussed elsewhere in this volume (Chap. 7, by Peter B. Moens and colleagues), we will briefly discuss one type here as we focus on their value in a “sting operation” in conjunction with antibodies. This undercover team is great at catching proteins in the act and at helping us decipher their meiotic functions. The case in point is the mouse T70H/TIWa translocation double heterozygotes. T70H and TI Wa involve the same two chromosomes ( I and 13). They have slightly different breakpoints on Chromosome 1 , but the breaks on 13 are cytologically identical. Double heterozygotes have no normal Chroinosoine 1 or 13 but are heterozygous for the translocation chromosomes: the T70H 1 13 and 13I and the TlWa 1 1 3 and 131. The two 1 1 3 chromosomes form a small heteromorphic bivalent with an SC that loops out in the middle, and the two 131 chromosomes form a large hekroniorphic bivalent, also with an SC that loops out. The DNA associated with these SC loops in the two bivalents is homologous. In early pachytene nuclei, the two heteromorphic bivalents often lie togeth-
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er with the loops facing one another. Their proximity and orientation suggest that some type of presynaptic alignment has occurred. At this point let’s pause a moment to play armchair detective and make some predictions, while keeping in mind the fact that chromatin is organized into loops in which only the chromatin at the base of the loops (not to be confused with the axial loops of the translocation double heterozygotes) is attached to the SC axis. If RADS 1 -coated nucleoprotein is actively engaged in a molecular check for homology during zygonema, there should be a strong RADSI Yignal spanning the chromatin in the region between the homologous axial loops. This is not what is seen. Instead, as is the case in normal spermatocytes, RADS 1 is confined to foci on the axial elements of the loops, suggesting that RADS 1 is relatively “search inactive.” Surprisingly it is the RPA signal, which normally is first evident as foci on fully synapsed axes, that produces a prominent barlike structure in the region between the SC loops. RPA has been implicated in DNA replication, repair, and recombination (Wobbe et al., 1987; Fairman and Stillman, 1988; Wold and Kelly, 1988; Heyer et id., 1990; Moore et id., 199 1; Coverley cJt ul., 1991, 1992) but has no known motifs that would suggest it is capable of facilitating a homology search. However. there is evidence suggesting that i t may be able to do so in conjunction with a second protein, such as homologous pairing protein. or HPP (Moore rt ol., 1991). Despite the fact that RPA’s actual partner i n crime remains unidentified, the differences i n localization patterns ot‘ RADS 1 and RPA suggest that the partner is not RADS I . Although the amount of RPA signal suggests that more ssDNA substrate than normal has been unwound and made available for binding and participating in an extended homology search, an SC is never formed between the two (homologous) loops. It appears that some condition necessary for synapsis is never met. “The case of the loops that refuse to synapse” remains open. However, there are additional lessons to be learned from these translocation double heterozygotes. In normal spermatocytes it is obvious that the number of RADS I foci drops rapidly in early pachynema; however, the dynamics of turnover are more evident on the heteromorphic bivalents. RAD.51 foci disappear from the homologously synapsed axes within a time frame similar to that of normal autosomal bivalents, but remain on the unsynapsed axes of the loops. Nonetheless, the loops can undergo synaptic adjustment, a type of nonhoniologous synapsis that occurs in some heteroLygous chromosome aberrations later in pachynema (Moses, 1980; Moses and Poorman, 1984). When the loops nonhomologously synapse, the RAD.51 foci disappear from the axes (Plug et id., submitted). This observation and the observation that RADS I also disappears from the axes of the asynapsed X chromosome that has no homolog in spermatocytes suggest that the disappearance of RADS 1 is not dependent on successful completion of a hoinology search. As mentioned earlier, asynapsis associated with chromosome aberrations is frequently associated with sterility, especially in males. Over the years, several hypotheses have been advanced to account for this connection. One of the most interesting of these theories, proposed by Miklos (1979), postulates the existence
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of pairing sites that must be saturated by undergoing synapsis; remaining unsaturated leads to death of the spermatocyte involved and, if enough spermatocytes are involved, to sterility. Translated into slightly more sophisticated terms, the Miklos hypothesis postulates a cell cycle checkpoint linking completion of synapsis with meiotic progression. Conceptually, it is not hard to visualize a pairing site as an early RN where homology is checked before synapsis proceeds. Antibody localization studies are now allowing us to identify the protein components of these early RNs and, as discussed earlier, already we are finding different cell cycle proteins associated with asynapsed versus synapsed early RNs. As mentioned earlier in Section VI, part C, ATR. a protein with a postulated role in cell cycle progression, is found on asynapsed axes, whereas ATM, a related PIK, is found on synapsed axes (Keegan et ul., 1996). Interestingly, ATR is first noted as foci on unsynapsed axes, but as synapsis is delayed it accumulates and appears to coat the axis (Plug et d., submitted). Is this an SOS that increases in volume if it goes unanswered (unsynapsed)‘! Regardless of the answer to this specific question, it appears the tools are at hand to begin to solve “the case of death by asynapsis.” See also Chapter 3, by Bruce D. McKee (this volume), for additional hypotheses, and stay tuned for further developments.
VIII. Preliminary Conclusions The obvious goal of meiotic analysis is to explain what is happening at the molecular level. However, as any armchair detective knows, it is exceedingly dangerous to jump to conclusions. Despite Miss Marple’s fictional success at identifying the killer on the basis of characterological similarity to someone in her village, there are obvious perils to such extrapolation in science. Although i t would be intellectually convenient if all organisms negotiated meiosis the same way and all protein homologs exhibited a uniform behavior across broad taxonomic lines, this is already proving not the case. Evolution has provided myriad variations on the basic meiotic theme. These differences can continue to provide important insights into the overall process. For example, although they arc both called yeast, the divergence between Sricx~horornyescerrvisiae (budding yeasl) and Schi,7o.sat.c.haro/n~ce,sponihc (fission yeast) is estimated to be as long as between S. cerrvisirre and mammals. This extensive divergence is reflected in dramatic differences in meiosis in the two species. S. pmnhe does not form an SC but undergoes recombination without any crossover interference. The parallel absence of these two events in S. po/dir supports the suggestion that one function of the SC is to regulate number and sites of crossovers by transmitting an inhibitory signal along its length from one (completed) crossover site to nearby potential sites (Egel, 1978; Maguire, 1988; King and Mortimer, 1990). Zip1 is an SC component of the central region of S. cereiisiae meiotic bivalents (Sym e t a / . , 1993; Sym and Roeder, 1995). Disruption of synapsis in the Zip1 mutant abolishes crossover interference (Sym and Roeder, 1994). I n light of these sim-
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23 1
ilarities, it seems appropriate to summarize some of the apparent differences and similarities between S. crreiisiue and mammalian meiosis already emerging from antibody localization studies.
A. Parallels between Cases (Yeast and Mammals)
Sites o f recoinbination in yeast are dependent on some as yet unidentified property of chromatin structure, not o n the sequences themselves, and moving a sequence containing a recombination hotspot for double-strand break formation changes both the frequency and position of the double-strand breaks (Ohta el d., 1994; Wu and Lichten, 1994, 1995). In mammals, both synapsis and recombination occur preferentially in R-band chromatin (Chandley, 1986; Ashley, 1988), which has a more “open” chromatin contiguration than G- or C-band chromatin (Holm and Rasniussen, 1983). As in yeast, chromosomal rearrangements can alter the recombinational properties of chromosome regions (Ashley, 1988). A stable recombination intermediate is not detected in S. cerevisiae until near the end of pachynema (Davidow and Byers, 1984), although heteroduplex DNA can be detected earlier (Schwacha and Kleckner, 1994, 199.5). One of the mismatch repair proteins, MshZ, binds to such heteroduplex DNA (Prolla er u/., 1994). and both Msh4 (Ross-Macdonald and Roeder, 1994) and MshS (Hollingsworth et d., 199.5) 1ocalic.e as foci on synapsed yeast chromosomes at this time. They have been postulated to be components of late RNs and to be involved in reciprocal recombination leading to chiasmata formation. As discussed above, the mammalian mismatch repair protein MLH 1, which has been convincingly linked to recombination in both mouse spermatocytes and oocytes, does not appear until pachynema. These correlations suggest that even if the mechanisms of recognition of homologous sequences and synaptic initiation differ between S. cereilisiae and mammals, they may share a common pathway.
B. Differences between Cases In S. cerevisiue two copies 01‘ ;I gene inserted into sites on nonhomologous chromosomes recombine almost as frequently as allelic sites on homologs (Jinks-Robertson and Petes, 1985; Lichten et al., 1987). These results attest to the success of a genome-wide homology search in yeast meiotic prophase-a success apparently undiminished by completion of SC formation between homologs. In contrast, rearranged interstitial homologous regions within the mouse genome cannot even synapse (see Section VII). In S. cerevisiue, the rud5OS mutation prevents processing of double-strand breaks to expose single-strand tails (Alani er al., 1990) and abolishes (Alani et ul., 1990) or severely restricts (1,oidl P t a/., 1994) SC formation. Additional studies have shown that the R w A homologs Rad.51 and Dmcl appear (Bishop,
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1994; Rockmill c't a / . , 1995) at about the same time in meiotic prophase as double-strand breaks (Sun et d . ,19x9; Padinore et u / . , 199 I ; Sun, 199 1 ) and SC precursors, while stable recornbination intermediates appear shortly thereafter (Schwacha and Kleckner, 1994, 1995).In mice the time of appearance of doublestrand breaks is not yet known, but RADS 1 is present long before SC precursors or the initiation of chromosomal synapsis. Unless the estimate of relative timing of events is severely off in yeast, RADS 1 may well be playing a different role in mammalian meiosis from the one that it is playing in yeast meiosis. Considering the different mitotic effect of targeted disruption of RADS I in yeast and mice, a different role appears more likely.
IX. Unsolved Cases In the great tradition of mystery stories. it would be ideal if we could conclude by rounding up the entire list of suspects, bring them in, confront them (preferably by clever entrapment), and wring a confession from then). However, in the even greater tradition of science, that would be a tragedy. for one of the greatest biological p u z ~ l e swould be solved and we would all have to retire to read the conclusions rather than continue to seek the solutions. Fortunately, it looks like the fun has just begun and there are plenty of mysteries to keep us all busy for a long time to come!
Acknowledgments We would like to thank the following Ibr providing antihodie\ to the proteins used in the work from our laboratory that i \ hoth preccnted and \ummarirrd here: Chri\la Heyting for SCP3: Peter Moens and Barbara Spyropoulos for COR I ; Charle\ Radding, Etini Goluh. and Gurucharan Reddy for RADSI; Craig Monell (PharMingen) for MLHI: and Zhen-Qiang Pan lor the RPA 70B monoclonal antibody. We aI\o thank Sean Baker and Michael Liskay lor the MlhI knockout inice and Yang X u and David Baltimore for the A / J Hknockout mice. We especially thank Kimberly Colem;in-Hcaley and Joann Swea\y for their comment\ and suggestions o n the manuscript. This work w a s supported by National In\titute\ o f Health grant GM-49779 t o T. A .
References Alani, E., Padmore, R., and Kleckner, N. (1990). Analy\i\ of wild-type and nul50 mutants of yeast suggests an intimate relationship hetwccn meiotic chromosome hynap\is and recombination. Cell 61, 419-436. Alani, E., Thresher. R.. Griffith, J . D., and Kolodner. R. D. (1992). Characterization of DNAbinding and strand-exchange stimulation properties of Y-RPA. a yeast single-strand-DNAbinding protein. J . Mol. B i d . 227, 54-7 I . Albini, S. M., and Jones. G. H. (1987). Synaptoneinal complex spreading in A/;WI ('f'/xr m d A fictrt/o,surn. 1. The initiation and sequence of pairing. Chrorriosorrirr 95, 321-338.
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Rocdcr. G. S. ( 1995). Sex and the single cell: Meiosis in ycnst. P r w . N u / / . Aurtl. % I . USA 92, 10450- 10456. Ross-Macdonaltl, P.. and Rocder. G. G. ( 1994). Mutation of meiosis-\pecilic MutS homolog decrease\ crossing over but not mismatch correction. Cc~ll79, 1069- 1080. Roth. T. F., and 110. M. ( 1967). DNA-rlependent formation of the syn:iptoneinal coi~iplexat meiotic prophaw. J. ('ell B i d . 35, 717-255. Savitsky, K.. Bar-Shit-a, A,, Gilad, S.. Rotinan. G.. Ziv, Y..Vanagnitc, I<., Tagle, D. A,, Smith, S., Uziel. T., S l e i , S.. Ashkenali. M., Pecher. I..Frydinan, M.. Harnik. R., Pataiijali. S. I<., Siinnions. A,, Clines. G. A,, Sarttel. A,. Gatti, R. A,, Chessa, L., Sanal, O., Lavin. M. F.. Jaspers, N. G. J.. Taylor, A. M . R.. Arlett. C. F.. Miki. T., Weissinan, S . M , Lovett, M., Collin\, F. S., and Shiloh, Y. ( 1996). A \ingle ataxia telangtectasia gene with ii pi-oduct similar t o PI-3 ktnasc. S~~if,rIc~O 268, 1749- 1753. Scherthan. H., Weich, S., Schweger, H., Heyting, C.. Hark. M., and Cremer, T. (1996). ('entromere and teloinere tnovetncnts during early meiotic prophase oi mouse and man are associated with the onset of chromosome pairing. J. Cell Riol. 134, I 109- I 125. Schwacha, A,, and Klcckner. N. ( 1991). Identification of joint molecules that form frequently between hoinologs but rarely between sister chromatids during ycast meiosis. Crll 76, 5 1-63, Schwacha, A,. and Kleckner, N. ( 1995). Identification of double Holliday junctions a s inlcrtnediales in meiotic recombination. Cell 83, 783-791. Sherman, J . D., Hcrickhoff, L. A,, and Stack. S. M. (1992).Silver staining two type\ o f meiotic nodules. Gerrorrrr 35, 907-9 15. Shiloh, Y. ( 1095). Ataxia-telaiigicctasia: Closer to unraveling the my\tery. Eur. J. Hiorr. GWI.3, 116-138. Smithies, 0.. and Powers, P. A. (1986). Gene conversion\ and their relation to homologous chromosntne pairing. Philo.r. Dtirr.!.R. Sot.. Lorrtl. IRiol. I 312, 291 -302. Sobol, R. W., Horton, J . K., Kuhn, R., Gu, H., Singhal, R. K., Pra\ad. R., Rajewsky, K.. and Wilsnn. S. H. (1996). Requirement of niaminalian DNA polymerase p in base excision repair. N(Imr-e 379, 1x3- 186. Solari, A. J. (1974). The behavior of the X Y pair in mammals. / t i / . Kc,,.. C?.tol. 38, 273-117. Solari, A. J. ( 19x0). Synaptonernal complcxes and associated structures in microspread human spermatocyte\. Chrornosorrro 81, 3 15-337. Solari, A . J.. and Dresser. M. E. (1995). High-resolution cytological li)cali7ation of the Xho and EcoRl repeat sequences in the pachytene ZW bivalent of the chichen. Chromosorw Rrs. 3, 87-93.
Speed, R. M. (19x2). Meiosis in the lnetel ovary. 1. An analysis at the light microcopy level using hurlace spreading. Chmriio.rorritr 85, 427-437. Spyropoulos. B.. and Moens. P. B. ( 1994). Irr .\r/u hybridization o f meiotic prophase chromo\omes. Iri "Methods in molecular biology" ( K . H. A. Choo, Ed.), pp. I3 1 - 139. Hurnana, Totowa, NJ. Stern, H., and Hotta, Y. (1985). Molecular hiology of nieinsis: Synapsi ociated phenomena. / r i "Aneuploidy: Etiology and inechanisins" (V. Dellarco. P. E. Vnyteh, and A. Hollaender, Eds.). pp. 305-316. Plenum, New York. Struhl, K. (1993). The new yeast genetics. N u i m 305, 391-397. Sun, H. ( 199 I ). Extensive .?'-nver-hanging, single-stranded DNA associated with the meioticspecific douhle-strand hreaks :it the ARC4 recombination initiation site. Cell 64, I I S6- 1161.
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Sun, H. D., Treco, D., Scliultcs, N. P., iiiicl S m \ t a h , J . W. (1989). Double-strand break\ ;it :in i n tiatiori \itc f o ~melotlc gene coiiver\ion. M m w 338, 87-90. Sung, P. ( 1994).Cataly\is of ATP-depeiideiit honiologous DNA pail-ing and \trand exchaiife by yeast RADS 1 protein. Sciericr 265, 1241 1243. Sung, P.. and Robherson, D. L. (1995). D N A \timiid exchange mediated by RadSI-ssDNA n i l cleoprotcin tilairienr with polarity opposite to that o f RecA. C ' c d l l 82, 453-461. Sweaay, J. B., and Loch. L. (1992). Mnmni:ili;iii D N A polymerase p can substitute lor DNII polyniernse I during DNA replication i n /'cc./ic,richici c . o / ~ .J . H it~l.C'/ieui. 267, 1407- I4 10. Syrn, M.. Engebrecht. J.. and Roeder. G . S. (1993). ZIP1 is a syiiaptoneinal complex protein rcquirecl lor meiotic chromosome s y i i q x i \ . ( ' d l 72, 365-378. Sym. M.. and Roeder, G. S. ( 1994). Cro\\oicr interl'erence is abolished i n the ahscnce ot ii \ynaptoiictiiiil complex protein. Cr~ll79, 1 x 3 - 702. Syin, M.. and Roeder. G. S. ( 1995). Zip/-iiiducecl changes in synaptonemal complex \trticttire and polycoinplex assembly. J . Cell H i t ~ l .12X. 455-366. wa. M.. Shinohara, A.. Hottn. Y.. Ogaw,ii. H.. anel Ogawa, T. (1995). Localiiation o f R w A like recombination proteins on chroiiiosonie\ ot lily at various meiotic atage\. Ch7c3 L)c.i.. 9, 925-034. T\uzuki. T.. Fujii, Y., Sakumi. K.. Tomin:ig,i. Y., N,ihao. K., Sekiguehi, M., Matwxhiro. A,. Yoshimura, Y.. and Morita, T. ( 1096). T q e t c d di\rtiption of the RtrtlSI gene leads to Iclhality in embryonic inice. Proc. Nor/. Aced . k i . U S A Y3, 0236-6740. \'on Wett\tein. D.. Rasmu\\en, S. W.. ancl Ilolm, I) H . ( 1984). Thc synaptonemal complex in senetic segregation. Auriir. Rei,. G'cwer. 18, 33 1-4 13. Weith. A.. and Traut, W. ( 1980). Synaptoiieinal complexes uith a\\ociated chromatin in a moth. f$l<,\rff/ k f f 6 ' / i t f ; < ' / / f lz. C ' / i U J f f i O . S f l f f f ( / 78. 275-2'1 I Wobbe, (.. R. I... Wei\\bach. L., Boi-o\\iec. J. A,. Dean. E B.. Murakami, Y.. Bullock, P., nnd Hurwich. J . ( 1087). Replication o f SV40 origin containing DNA in vitro with puritied pi-otcins. Pro(,. Ntrtl. Actrd. .Pi. U S A 84, 1834- I 8 3 8 . Wold, M S., and Kelly, T. ( 1988). I't~rification and charncterimtion of replication protein A. a cellular protein i-equircd 101- i f r virro replication 01 \iiniiin viru\ 40 DNA. Proc. Ntrrl. ( r e / . .S(,i. USA 85, 3 2 3 - 2 5 7 7 , Wu, T.-C'., and Lichteri, M. ( 1994). M c i o s i \ - ~ i r d u c c ddouble-stra~idbreak sites determined by yeast chroinatin structure. SC.rcw(.r 263, 5 15-5 I 8. Wu. T.-C.. and Lichten. M . ( 1995). Factor\ t l i i i ~affect the location and frequency o f incio\i\induced double-strand break\ in Stic c ~ l r t r , . r w x c ww r . c v i s i c r e . Grrieric.\ 140, 55-66, Xu, Y.. A\hley, T., Hraincrd, E. E., Broiiaori. R T.. Mcyn, M. S., and Baltimore, D. (1996).Tai-gcted dt\ruption of ATM leads to growth Ier;irdation. chromosomal fragmcntatioii during iiieiwi\, iiiimune defect\. and thymic I~niplioiiin.G'c,rws Dei.. 10, 24 I 1-2322. ~
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7 Chromosome Cores and Chromatin at Meiotic Prophase Peter B. Moens, Ronald E. Pe,?rlmnn, ,ind Henry H. Q. Heng Department of Biology York University, North York Ontario. Canada M3J I P3
WaIther h u t Institut f u r Biologie Medizinische Universitlit
ZLI
Liibcch
D-23538 Lubeck, Gerniany
1. 11. 111. Iv.
V. VI. VII. VIII.
IX.
1. Introduction Under the assumption that chromatin organization plays a role in the synaptic and recombinant functions of homologous chromosomes ai meiosis, we rcvicw thc
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development of the synaptoneinal complex ( S C ) in the meiotic nucleus. its arrangement in the nucleus. and the association of specific DNA sequences with the cores/SCs. We present evidence for the discriminate utilization of DNA sequences in the ordering and attachment of chromatin loops along the length of meiotic prophase chromosome cores. General evidence I'or a regulatory mechanism conies from the observation that the average size of the loops and the packing density are s p e c k s p e c i f i c . For example, yeast (S. cPrri~i.sinc~) has short, sparsely packed loops, whereas the grasshopper (Chloea1ti.s coii.sp.per-sci)has closely packed long loops (Moens and Pearlman, 1988; Marec, 19%). I t now appears that within a given species, not only are there measurable differences in loop sires (Hen& et ol., 1996), but also the amount of DNA can differ vastly between similarly sized loops. The inechanisin that regulates loop size, therefore. not only measures the amount of DNA from one attachment site to the next, it also appears to take DNA content into account. Additional constraints on loop organization are implied by the location of RADSI, a protein implicated in early recombination. at the base of the loops (Shinohara rt u[., 1992; Ashley et u/., 1995; Haaf rt ~ 1 . . 1995; Terasawa r / d..1995; Ikeya c't (11.. 1996, Moens, in press) and by the recombination-correlated late nodules that are located at the interface of the paired chromosome cores, the S C (Anderson and Stack, 1988: Stack e l 01.. 1989: L. K . Anderson, personal communication). The chromatin organization discussed here is different from the chromatin loop organization observed i n lampbrush chromosomes o f nieiotic prophase in growing amphibian oocytes (Callan, 1986). In the latter, inore than 90% of the DNA lies in the chromosomal axis and the lai-gc loops are transcriptionally active elements. The large Y-chromosome loops i n dipteran spermatocytes are also transcriptionally active elements. Comparisons with mitotic chromatin organization are restricted by the limited characteriation of scaffolds, matrices, the scaffold attachment regions of the DNA (SCARS),and the nuclear matrix equivalent (MARS).
II. SC Structure from Electron Microscopy Synaptonemal complexes arc generally formed by the parallel alignment or synapsis of the axial cores of homologous chromosomes. The many variations-for example, S C components that can form SCs in the absence of chromosonies, and synapsis of axial cores that takes place between nonhomologs-have been reviewed elsewhere (Moens, 1994). This discussion focuses on SC formation between homologs rather than o n any of the exceptions. What brings the pairing chromosomes into some form of proximity is not considered here (see ('hap. 3, this volume, by B. D. McKce). The most detailed inforination on synapsis at the chromosomal level still
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coines from the three-dimensional rcconstruction of electron microscop! ( E M ) on serially sectioned meiotic prophase nuclei. Many such reconstruction4 were published in the 1970s and 1980s. and tlicy have provided the basic information about SC formation in normal and aberrant cases (Rasmussen arid Holm. 1980; Hobolth, 198 I : von Wettstein c’r c i / . , 19x3). I n principle, a given core is recorded in successive sections (Figs. I &I)) and the fragment of‘ each section is nuriibered accordingly. This inforinatioii is superimposed in a summary diagraiii (Fig. 1A). The example of ii locust spcrmatocytc in Fig. 1 shows the coininon feature that thc ends of the cores are attached to the nuclear envelope. In inany organisms, all ends congregate at a specific site on the nuclear envelope (Figs. 7 . 3B). The first visible manifestation 01. synapsis is the presence of transverse Lilaiiicnts between homologous axial corcs, which is established when the cores are still about 300 nm apart, three times the tinal distance. This happens at one o r more places along [he cores at some distance from the nuclear envelope, about 2 p i in this example (Moens, 1969). A composite o f several cores in the process of establishing first contacts (Fig. 2) shows that the initiation of synapsis docs not generally take place at the ends 01‘ the chromosomes. I n plants. the interstitial initiation of \ynapsis is also uell docuiiientcd with EM observation\ (Gillies3 1975; Jenkins. 1983).From dif‘fci-ent pctspcctives. telomcrcs rather than intcrstitiol sites havc been implicated in the inili;ition ol‘ synapsis on theoretical groiiiids (Sen and Gilbert, 19X8), from gcnclic c\$iclcncc (Wicky and Rose. 1996). anti from light microscopic findings ( Ilawc cf ( I / . . 1994). O n balance. interstitial initiation from dircct E M o b s e n ations appears more con\ iiicing. When synapsis is complete. thc niiclcus contains the nionoploid number of SCs. Their three-dimensional cli\trihution in the nucleus can also be btcreoscopically visualized from EM of serial scctions (Fig. 3). This tcchnique permits the testing o f hypotheses regarding thc di\lribution of chromosomes in the meiotic prophase nucleus. For exaiiiplc. it liiis been argued from observations on meiocytes. which have relatively short chroinosonies, that the homologous chroinosome pairs occupy exclusivc domailis within the nucleus. Three-dimerisioii~~l stereoscopy shows that the shorter SCs 01‘ the male rat may be sequestercci (SCs I9 and 20 in Fig. 3A). However, the longer chromosomes can be seen to wind their way though the “domains” of other chroniosmnes (SCs 1. 3, and 5 in Fig. 3 A ) . This observation seems to suggest that perceived “domains“ are a fortuitous consequence of the limited space requii-ed by short chromosoiiies. The iiiiich longer SCs of the gr hopper (’hloctiltis c m s p e m i also fail to display any indication of “domains” (Fig. 3B). Similarly. EM tracking in sections or spreads of long chromosome corex in the process of synapsis does not reveal prcaligninciit other than that expected ticar the site of synapsis. Reconstructions of allohexaploid wheat meiocytes show a consistently high frequency of synapsis between homoeologs, thereby excluding prcalignment as the primary cause for regular bivalent formation between homologs in wheat (Hobolth, 198 I ). Confocal three-dimensional analysis of hetcrochromatic knobs of the long mai7c chro-
Fig. 1 Initiation of chromosome synapsis determined by reconstruction of serial electron micrographs: c: centromere; nm: nuclear envelope; t f transverse filaments; ac: axial core. ( A ) Summary of observations on the cores of a Locusto migruforiu zygotene nucleus. sections 2- 10. (B-D) Electron micrographs of sections 4, 5, and 6. Initiation occurred at a distance of about 2 p n from the nuclear envelope and is characterized by the formation of transverse tilamenis that extend about 300 nm, three times the eventual distance between the lateral elements of the SC. (Reproduced with permission from Moens. 1969).
7. Meiotic chromosomes
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Fig. 2 Syiinptoneiiial complex forniaiion i i i gencral i \ initiated at w m e distance lroin the nuclear envelope. Thc drawing I S a serial recoii\irtictioii, ;I\ iii Fif. I , of sections 1-40 01' the region where iiiost ot' the core attachment sites arc located. Hoinologou\ core\ R, hace thrce synaptic initiation regions. Hoinologous core\ A, C. and I ) each h;ive one intentitial pairing titc
inosonie also failed to give evidence of prealignment (Dawe rt o/., 1994). It can be theorized that in organisms with short chromosomes, a single initiation site effectively coaligns the entire chroinosome pair, giving it the appearance of prealignment. Our observations on fluorescent in situ hybridization (FISH)painted sequences suggest that at early prophase stages, speci tic chromatin segments range widely through the nuclear volume (Section V ) , probably permitting homology searches without a requirement lor chromosomal prealignment.
111. SC Structure from lmmunocytology The development of SCs can also be monitord with silver staining or fluorescent immunocytology of intact o r surfucc-spread meiotic nuclei and by EM o f iiiimitnogold-treated spreads (Counce and Meyer, 1973; Dresser and Moses. 1979; Dobson et u / . , 1994).The antibodica to the synaptic protein SYNl identify the regions where homologous cores are synapsed. In Fig. 4. all 19 pairs of autosomcs are fully paired and are recogniied by the anti-SYN I antibody. I t is evident that i t is the synapsed regions that are detected by the antibody from the X-Y pair. where only the synapsed portion is labeled (arrow in Fig. 4). while the unpaii-cd portions are invisible. Howevt.I. their unpaired ends can be detected by
Fig. 3 Three-dimensional SC distribution in complete pachytene nuclei reconstructed from serial electron micrographs provides evidence that pairs of homologs are not necessarily sequestered in distinct domains. (A) rat; (B) grasshopper, Chlueultis conspersci.
their telomeres, t, and t,, which are visualized by FISH using a telomere probe. The same technique of combined immunofluorescence and FISH techniques was used for the simutaneous visualization of cores, SCs, and specific chromatin segments, as discussed in the following paragraphs. To identify the chromosome cores, we use antibody to the 30-kDa CORl protein of the chromosome core (Dobson et al., 1994). The antibody to CORl recognizes cores before they synapse, the parallel-aligned cores/lateral elements of the SC, and cores when they separate at later prophase stages. Epifluorescent
Figure 4 The antibody to the synaptic protein SYNl recognizes synapsed portions of the SCs and the pseudo-autosoma1 region of the X-Y pair (green fluorescence. arrow), but not the unpaired regions of the Y and X chromosomes. The telomeres are visualized by in siric hybridization with a telomere probe and red rhodamine fluorescence. The combination of immunofluorescenceand FISH was also used for Figs. 13, 14, and 15. 1: telomeres of the paired X-Y chromosomes: I and tx: Y proximal telomeres of the Y and X chromosomes, respectively. Figure 5 The antibody to the chromosome core protein CORl recognizes the parallel-aligned coresflateral elements of the SC. green fluorescent in this confocal microscope image. It also reacts with single cores before they pair (Figs. 6. 16) and when they separate (Fig. ISC).
Figure 6 Synaptic initiation and SC formation as visualized with immunofluorescence. Rabbit antiSC is green fluorescent and mouse anti-SYN1 is red fluorescent, yellow here in combination with green. The many free ends indicate that synapsis is not necessarily initiated at the ends of the chromosomes. Figure 7 Evidence that synapsis is not initiated at the centromeric ends of the chromosomes. Centromeres are red and the synapsed regions are green fluorescent. SC formation is complete in the pachytene nucleus at the upper left. The other nucleus, which is in zygotene, has about 25 synaptic initiation sites. none of which are at the centromeric ends.
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and confocal microscopy of immiitiotluorescen~FITC-labeled cores reveal the parallel-aligned cores as lateral elenients of the SC, with a space of about 0.05 kin between the lateral elements (Fig. 5 ; compare with Fig. 8). The SYNl protein extends from the lateral element t o the central element, where the amino terminal may connect to the SYNl protein amino terminal from the opposing hoinolog (Dobson e/ al., 1994; C. Heyting, personal communication). With these two antibodies, the process of chromosome synapsis in a mouse zygotene nucleus can be monitored immunocytologically (Fig. 6). The unpaired cores and the SCs are green fluorescent with rabbit anti-SC antibody, and the synapsed regions are red fluorescent with mouse polyclonal antibody to the SYNl protein (the combination of green and red produces the yellow color). Although synapsis is extensive, many of the chromosome ends are still frce. As asserted in Section 11, synapsis did not initiate at the ends of the chromosomes. The lack of lelomeric initiation is further supported with the simultaneous visualiution of initiation sites and chromosonie ends. With a human anticentromere antibody, one end of each telocentric mouse chromosome is red fluorescent in Fig. 7. The synapsed regions are green fluorescent. At pachytene. the SCs of the fully synapsed chromosomes are green with a red telomeric centromere. In the zygotene nucleus, the green pairing imitation sites are not at or near the telotneric ends. Evidently synapsis is not initiated at the centrotneric ends. I n s i / u hybridization with a telomere probe also demonstrates that the SYNl protein is not associated with the telomeres at the onset of synapsis in mouse spermatocytes (P. B. Moens, H. H. Q. Heng, personal observations). A sensitive measurement of SC dimensions can be obtained by EM of shadowcast, surface-spread sperinatocytes (Fig. 8 ). For mouse and rat, the chromosome axes, lateral elements, are on average I 1 0 nm wide (range, 80-170 nm, depending on stage and preparation) and thc space between the lateral elements is on the order of SO nm (Dobson ef d., 1994) but wider at late prophase stages. There is a central element between the lateral clements, and late nodules lie either between or on top of the lateral elements (Fig. 8 ) (Moens et LII.. 1987). At early prophase stages, the chromosome axes arc Icss solid and can be associated with what resembles the early nodules seen in plant\ (Fig. 8). Inimunogold labeling shows the prcsencc of COR I protein in the cores, and the presumptive early nodules can be inimunogold labeled with anti-RADS I antibody (Figs. 9, 10). Apparently, they are the equivalent to the IIADS 1 foci of immunofluorescent preparations (see Fig. 16).
IV. Chromatin Loop Attachments to the Meiotic Chromosome Cores The biochemical nature o f DNA in(eracttot1 with the core protein\ has not been establt4hed other than that the rat SC protein\ SCPl (125 kD, SYNI). SCP? ( 190
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Peter B. M o m s et trl.
Fig. 8
Relief image 01' ii rat SC and Iatc nodule ( L N ) lroni a directional shado&-ca\t EM prcparntion. ( A ) A late pachytcne SC with typically enlarged ends and w i t h an L N acro\s tlic SC. t R ) A later \[age than A, with lateral element\ 111 the process o f separation and an LN between the lateral cleiiieiits. ( C ) H i g h magniticatioii ot A . Sculc hnr = 100 nni. The laterd eleimxils arc about 100 iiin wide. A n t i - C O R I iinmunogold lahels the lateral clements (\inall an-owj, but anli-RADS I does n o t lahel the Inte nodule\. ( D ) High magnilicn~ionof H.
kD), and SCP3 (30-33 kD, COR 1 ) have nonspecific DNA-binding properties (Heyting et (11.. 1988; Dobson et ol., 1994; C. Heyting, personal communication). However, several lines of indirect evidence suggest that the bases of the loops are intimately associated with the SC proteins. When isolated meiotic prophase chromosomes are treated with DNase and the residual SCs, stripped of their chromatin, arc collected, they are observed to contain DAPl -positive DNA that evidently was protected from the nuclease digestion. This DNA can be recovered, cloned. and analyzed, as discussed later (Pearlman and Moens. 1992). Additionally, cross-linking experiments with SCP3 protein indicate that there is
7. Meiotic Chrotnosomes
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Fig. 9 Early nodules'? ( A . B ) Previously tinidentilied appendages of the chromosome cores and SC\ at the tiriic of \ynap\i\ are very likely enrly nodule\. which are common in plant SCs but not normolly trccogniLcd in m;immaliaii SC\. The idcntitication i \ hescd on the pre\ence 01' RADS I antigen i n the Lippendazes (Fig. 10). Scale bar = 200 ntii (Reproduced with pertnis\ion from M o m \ ct ( I / . . 1987 )
sequence preference in DNA binding to the protein (C. Heyting, personal c o n niunication). The differential properties of loop verws core chromatin can also be demonstrated with a modified primed ill .siru (PRINS) hybridization technique (Koch et al., 1989). In this technique, the DNA is denatured and the chromosomes are incubated with biotinylated dNTP\, a primer, and DNA polymerase. In addition to the intentionally primed sequences, minor satellite in this case, any singlestrand break or gap can fortuitously serve to initiate polymerization, even in the absence of a primer (Figs. 1 1. 12B). Incorporated biotin is detected with FITCconjugated avidin. Whereas DNA in the loops responds to the PRINS procedure, the DNA of the cores does not serve as a priming template (Fig. 12B, arrows). Because DAPI staining of the same chromosomes demonstrates the presence of DNA in the core (Fig. 12A, arrows). it appears that the DNA i n the cores/SC is protected from the PRINS reaction. Results of both the DNase experiment and the PRINS procedure suggest that the SC-associated DNA is in a special environment that is different from the chromatin organization in general. These experiments demonstrate a distinct SC-DNA status. An interesting aspect of Figs. 1 1 and 12 is that loop-size regulation appears to be chromosome autonomous rather than centrally regulated. Figure 12 shows Miis .spretu.s bivalents with minor satellite chromatin loops at each centromeric region fully labeled with a 13-bp minor satellite specific primer for the PRINS procedure. Mus domesticus, on the other hand, has a lesser amount of minor
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Peter B. Moeiis
ti/.
satellite that is arranged in short. compact loops. I n the hybrid M . dome.\ticmlM. ,spwru.s, each of the chroinosonies o f thc bivalent has kept is parental phenotype, the M . . s p r e f ~partner ~ ~ has a pronounced minor satellite component (Fig. 1 1 , in), and the M . r ~ c r . s c ~ d ud. vm v . ~ f i c uminor . ~ satellite is poorly defined. Apparently, the mechanism that regulates short loops for the minor satellite of M . domesticus chromosomes does not induce short loops of the minor satellite D N A of M . .sprotu.s chrornosomes. The revcrse is probably true too, but is difficult to assess in these preparations. A furthcr example of the regulatory autonomy of the chromosome is given in the section on the alignment of chromosome loops (Section VIII).
V. Sequences Associated with the Core It is a testable hypothesis that specific sequences are involved in the attachment of loops to the core. Karpova et trl. (1989) and we (Pearlman and Moens, 1992)
7. Meiotic Chromosoine~
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Fig. 11 Detnonstriitinn of the radial chi-oinatin loop orgaiiiration in a rneiolic prophnse hivalciit from a Mil\ i ~ i i ~ . \ c i ~ / it ~/ o. sw \ t r c x \ lenialc X M . .\pwfi~\inale F , male. hi .sirit hybridization with ii inninor sntcllitc primer by the PRINS procedure. Den;itui-ed chromosoine\ arc incuhated with a minor \atullite primer, DNA polymerare. and hiotinylated dntp\. FlTC coii.jugatcd to avidin then labels the iniinor satellite ol the M. .spre/i~.\tionnol~~ptic ( i n ) and can fortuitou\ly produce a hackground \taining ( i t the chroinatin loops i n general. Scale ha1 = 5 kin.
isolated pachytene chromosomes that contain SCs. The DNA was digested with DNase I and the “stripped” SCs were found to contain DAPI-positive material, presumably DNA contained within the SC, where it is protected from DNase digestion. The DNA was released from the cores by protease treatment and phenol extraction and the liberated DNA was cloned. No specific core-associated DNA sequences could be identified. Instead. it was found that there was an overrepresentation of certain short repeats when compared to cloned random DNA fragments. PRINS hybridization with these short repeats as primers produces a general labelling of the chromatin but no specific recognition of SCassociated DNA. Apparently, the sequences have a preference rather than specificity for cores or SCs. More indirectly, it is possible to test whether there are sequences that fail to attach to the cores by examining the attachment of different transgenic inserts to the cores. From the examples available so far, it appears that totally foreign sequences, such as a phage A insert, fail to attach to the core of a mouse chromosome but are held in place by the flanking native sequences (Fig. 13) (Heng r t id., 1994). An insert consisting of repeated E. coli pBR plasmids that have a mouse P-globin gene is capable of attaching to the core, presumably by the native sequences, but the loops are longer than average, perhaps because of the presence of pBR DNA (Heng et d., 1994). Native sequences in the megabase range, such as can be detected with a YAC probe (Fig. 14), or long repeated
Fig. 12 Evidence for the differential organization of DNA in the chromatin loops \'crsus the chromosome core. ( A ) DAPl \tailling of Miis .sprmt.\ bivalenls produceh bright fluore~cenceof thc chromosome core and the heterochrornatin (minor hatellite). while the chroinatin loops arc leas bright. ( B ) The PRINS procedure with a minor satellite probe also produces fluorescence of the satcllitc and the chromatin loops but fails to react with the chromosome core (arrows).
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Fig. 13 Phage A repeat\ ( 1-2 M h ) inserted 11) on llic \ynaptonciiial coniplexe\ (SC) ot chromoWIIICS 3 iiiid 3 i n ;I iiiouse spermatocyrc. I t c~ppeaI\ rhai the A sequences are n o t conipetciit t o attiicli to the SC. In\tead. the A loops are lieltl i n place by Ilanking iiiotiw sequences (iirrows). The SCs are Iluore\ceiitly Inbeled M i t h mti-SC ;intihotly and Lhc A phage insert\ w'ith ;I hiotiiiylated A probe. The blach-and-white print is lroni a color slide aiid therefore i i i reve~-\ecoiiIiu\t. Scale bar = 10 urn.
sequences (Fig. 15) have distinct attachments to the SC, as have inserts of native sequences. These obscrvations suggest that there is a discriminate utilization of DNA sequences in the ordering ol' chromatin at meiotic prophase.
VI. DNA Content of the Chromatin Loops There are striking differences in tlic atiiount of DNA that can be present in one or a few chromatin loops of meiotic ptophase chromosomes. In surface spreads, 40kb cosmid probes produce from one to several small fluorescent spots along a single decondensed chromatin loop. Evidently the 40-kb sequences are only a small part of an entire loop. FISH painting with a 640-kb YAC probe illuminates
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Fig. 14 Detection ot chromatin structtire H ith FISH u\ins a 630-kh YAC as a probe ( a r r o w ) . The sinall arr;iy o i fluorewent loops uigge\ts that thc YAC probe probably paints only one loop. and that the four loops ;ire clo\ely ;i\sociated with each other.
a chromatin segment that probably represents not much more than a single loop for each of the four chromatids (Fig. 14, y). Similarly, the modest amount of DNA in SO copies of 7- to 9-kb X-chromosome repeats occupies a region of one or two loops (Moens and Pearlman, 1990). At the other extreme are sequences that contain 50 copies of 100-kb long range repeats (LRR) in the CS7BL laboratory mouse (Winking et d., 1993). Surprisingly, these large, 5000-kb arrays seem to occupy very few loops. I n Fig. IS, there are altogether 20 Mb of LRR at the site in the middle of mouse bivalent 1 . To accommodate this, 5 Mb (one loop) or 2.5 Mb (two loops) of DNA in a loop with a IS-Fm circumference, 120X or 60X condensation is required. Seven times nucleosonie cornpaction combined with 20X or IOX higher order condensation could accomplish the appropriate compaction. In the case of the YAC and the X-chromosome repeats. only a I S X compaction would be sufficient to lit the segment into a single loop. These estimates suggest the possibility that similarly sized meiotic chromatin loops may contain widely different DNA amounts per loop. Mitotic chromosome sizes are noticeably affected by the LRRs, which, in feral mouse populations, can be as high as 1800 copies (Kunze c v d., 1996). Remarkably, at the lcvel of the SC, no synaptic abnormalities were observed in such mice
Fig. 15 Structure o f chromatin Icwps containing SO X Iol)-kh long range repcats (I.RK). ( A ) At cirly pachytenc. the four copies 0 1 the 5-Mb loops iue lairly dispersed atid they scciii to itisen on a single xtiiall rcgioti 0 1 the SC of chromosonic 1 (arrow). (B)Later in pachytcnc stagc. where the X and Y chromosomes are mostly unpaired. the loops are shorter. ( C ) Surprisingly. there is no further condensation as the nucleus progresses 10 the diplotcne stage. (D)At metaphase 1. presumably the most condensed stage o f the chromosome. the LRRs and other sequences are decondcnscd. C: pairs of ccntromen's of hivalcnt I .
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Cr
01.
or in laboratory-bred hybrids with heterozygous LRR sizes (Winking er ul., 1993). A possible reason for thc lack o f synaptic abnormalities may lie in the fact that the vast bulk of LRR DNA is not within the SC but in the loops. An additional regulatory element appears to exist in the geography of the chromosome itself. Telomere sequences that range from 30 to 80 kb in hamster, rat. and tnouse exist in very short loops at the ends of the chromosomes (Fig. 4). However. a telomeric sequence transposed to a medial position of the chroniosome gives a FISH signal close to the size of average interstitial chromatin loops of hamster pachytene chromosomes. Similarly, a survey of sequence-related inserts. varying in length from 30 to 100 kb, introduced randomly into mouse chromosomes suggests a gradient of loop length from short in distal insertions to long in more internal insertions (Heng et al., 1996).
VII. Time Course of Chromatin Loop Development at Meiosis At the time of mouse chromosome core formation and chromosome synapsis, the FISH signals of identifiable sequences are widely dispersed in the nuclear volume and no specific domains are obvious. This is different from what is seen in painted chromosomes or chromosome segments in yeast, which, by virtue of their short loops and short chromosomes, tend to have more localized signals at the same stage (Scherthan er NI., 1992, reviewed by Klein, 1994). The normal time course of chromatin loop development through meiotic prophase is illustrated in Fig. 15 for the LRR, which were described in the previous section. Figure 15A shows an early chromosome synaptic stage, where the LRR in the middle of chromosome 1 is still quite dispersed. Later, at midpachytene. when the XY chromosomes are mostly desynapsed, the autosomal loops are more compact (Fig. 15B). The loops do not become shorter after this point in meiotic prophase. The severe chromosome condensation frequently seen in illustrations of later stages, diakinesis and metaphse I , is probably induced to some extent by commonly used alcohol- and acetic acid-containing fixtures or by high concentrations of CaI+ or MgZ+ used for nuclear isolation. Figure 15C reveals that the LRR is no more condensed at diplotene than it is at pachytene, and Fig. 15D shows that at metaphase I, the LRR is definitely decondensed. The same progression has been observed for a native satellite DNA, YAC probe-visualized native sequences, and various transgenes (Heng et ul., 1994; P. B. Moens and H. H. Q. Heng, personal observations). An exception to this developmental time course was found in the I - to 2-Mb A inserts into chromosomes 3 and 4 (Fig. 13). Unlike in native sequences, the X FISH signal becomes highly concentrated at the diplotene stage of meiosis and regains its dispersed state at metaphase I (Heng et NI., 1994; Moens et ul., 1997).
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VIII. Alignment of Chromatin Loops A simple assumption about the nature of the individual chromatin loops is that they are restrained by the attachment to the chromosome core and extended from there by between-loop osmotic pressure i n the manner of a brush polymer ( J . F. Marko and E. D. Siggia, unpuhlished observations). Because there are four copies of any given sequence i n a prophase bivalent, it is expected that FISH of a cosmid probe should produce four signals. Such regularity, however, is rare. Frequently it appears that two. three, or all four signals have fused. This appearance is not due to inefficient hybridization, because the probe is fairly large, 40 kb, and all nuclei have signals. The observation suggests the possibility that at meiotic prophase, identical sequences or loops are in some manner associated with each other. With probes that paint larger chromosome segments, such as major satellite heterochromatin, LRRs, YAC-related sequences. or phage X inserts, it is equally ineffectual to define four separate sectors. At best, two bundles can be distinguished (Fig. 15B). An ociation of FISH signals has also been reported for spread yeast nuclei in meiosis, and Kleckner in her review of meiosis ( 1996) attributes these interactions to “direct DNA-DNA contacts between intact duplexes with homology searching facilitated by appropriate proteins. No known RecA homologues are involved. however.“ Surprisingly, this phenomenon of cohesion between homologous sequences appears to be chromosome autonomous. Where identical sequences have been incorporated i n different chromosomes. at pachytene there is no evidence for ectopic associations between the identical sequences on the different chromosomes (Fig. 13) (Moens e f al., 1097). Thcrefore, it appears that the association of sequences is regulated within ;I chromosomal context and must depend on factors in addition to simple contacts between identical DNA sequences. In the case of conventional chromosome rearrangements such as translocations, rearranged chromosomal portions appear large enough to permit exchanges of pairing partners at the translocation point. The assessment of chromosomal context. therefore, does not necessarily involve the entire chromosome but does require a substantial portion of a chromosome.
IX. Recombination at the SC In plants and animals, the SC-associated “late nodules,” LNs (Fig. 8). have a convincing correlation with reciprocal recombination events. In the grasshopper, Cliloerrltis consjwrsa, the three large metacentric chromosomes have terminal chiasmata as well as terminally localized LNs, while in Locusfa migratoricl, neither chiasmata nor LNs are localized (Bernelot-Moens and Moens, 1986). Particularly graphic is the presence of an LN near the centroineres of Alliunz ,fistulosum SCs. which correlates precisely with the proximal chiasmata in this
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plant, whereas A / / i w n c e p i lacks localization of chiasmata and LNs (Albini and Jones, 1988). The association of the LN with the SC suggests that the maturation of a recombinant event into a reciprocal exchange occurs at the SC and may involve sequences associated with the SC. The correlation also holds lor some fungi (Zickler, 1977), but in others neither SCs nor LNs appear to be necessary concomitants of reciprocal exchange events. Evidence from Schi:o.sac~hciroriiym poinhe and Asper
7. Meiotic Chromosomes
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Fig. 16 The RADS I protein. a RecA-like protein ilia1 is associated with early recombinant events, double-strand hreah\, hornology search, aiid hirand cxchangc, is located at the chro~iio\oinecore\ ( c ) bcl.orc the chromosoine\ pair. The loci ( I ) \tari to I-educeiii number\ during .;ynnpsis of honiologous chromoroine pairs. The implication I \ thd the I)NA at the base\ or the loops. rather than thc ctironi;itin loop\, is involved in the initiati(rn ol recombination.
At early chromosome pairing stages, thc DNA-binding enzyme, topoisomerase 11, is present throughout the chromatin of the bivalent in accordance with its
normal function in the deconcatenation of sister chromatids. At late stages of prophase, the enzyme is concentrated at the SCs (Moens and Earnshaw, 1989). Its function may be in the resolution o f chromosome interlocks (Rasmussen, 1977, 1986) or topo I1 may function in the resolution of double Holliday junctions following a double-strand break, strand exchange, and the formation of joint molecules (Collins and Newton, 1994; Schwacha and Kleckner, 1994; Stahl, 1996). Gradual separation o f the lateral elements at late prophase due to “chromatin pressure” of the overlapping chromatin brushes ( J . F. Marko and E. D. Siggia, unpublished obscrvations) gives directionality to the topo I1 resolution in favour or unlinking nonsister chromatics. The topo 11 resolution, where it is in addition to the Ruv-mediated resolution of Holliday junctions, would yield an excess of‘ nonrecombinant flanking markers. Such an excess has been reported for the N I H locus in Neirrosportr if closcly linked flanking molecular markers are used (Bowring and Catcheside, 1996).
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Acknowledgments The several projects reported here were completed with the as\i\tance of Barbara Spyropciulos and Mary Lou Ashton at York University and Xiao-Mei Shi of the Department of Genetics. Ho\pital lor Sick Children. Toronto. Ontario. Financial assihtaircc was provided by a grant to PBM and REP from the Natural Sciences and Engineering Research Council of Canada. The ATM nu11 mice were pa17of i~ collaborative project with T. Wynshaw-Boris. C. Barlow. and M. Liyariage at NHGRI. Baltiniore. Thc RadSI analysis wa\ done i n collaboration with D. J . Chen and Z. Shen at Los Alainos National Laboratory.
References Albini, S. M., and Jones. G. H. (19x8). Synaptoncmal complex spreading i n Alliurfr c.cprr and A , f i t r u / o . t u t u . 11. Pachytene observations. The SC liaryotype and correspondence of late rt:combination nodulcs and chiasma. G w w w 30, 399-410. Anderson, L. K., and Stack, S . M. (1988). Nodules associated with axial cores and synaplonemal complexes during zygotene in P \i/o{iuu uudutu. C'lirormtonici 97, 96- 100. Ashley, T.. Plug. A. W.. Xu, J . , Solari. A. J . , Reddy, G.. Golub. E. I..and Ward, D. C. (1995). Dynamic changes i n RadS I distribution on chromatin during meiosis in inale and fcrnale vcrtchratcr. Chroffrotofrrci 104, 19-28. Bahler, J., Wyler, T. G., I.oidl, J. and Kohli. J . (1993) Unu\ual nuclear \tructure i n iiiciotic prophase of fission yeast: A cytological approach. J . C d l Aiol. 121, 241 -256. Bcrnelot-Morn\, C.. and Mocns, P. B. ( 1986). Recombination nodule\ and chiasma l ~ ~ a l i ~ a tini o n two Onhoptera. ('hr-ofno\onitr 93, 220-226. Bishop, I). K. (1994). RecA h o m o l o p Dmcl and RadS 1 interact t o form multiple iiucIe;ir c o n plexes prior to chroiiiosornc \yiiap\i\. Cell 79, I O X I - 1097. Bowing, F. J.. and Catchc\idc, D. E. ( l906). Gene conversion alone accounts lor nrorc than 90% 01 recombination event\ at the m i locus 01. Nrtol~\portr c r u t t / , Getwric,s 143, 129- 136. Callan, H. G. ( 1986). "Larnpbrti\h Chromosome\." Springer, Berlin. Collins. I., and Nculon C. S. ( 1994). Meiosis-spccitic formlation 0 1 joint DNA ~nolcculescontaining wquence\ from hoiiiologou~chromosonics. Cell 76, 65-75. Councc, S . J . , and Mcyer, G. F. ( 1973). Differentiation of the synaptoneinal complex in Locir.\rci spermatocytes studied by whole inount electron microscopy. ~ l ~ f - f ~ f f i f ~ . s [44, ~ f ? i23 ~ r I-2S.i. Dawe, R. K., Sedat, J . W., Agard, D. A,, and Cande, W. Z. (1994). Meiotic chroiiio\orne pairing in maize is associated with a novel chromatin organization. C'rll 76, 901-9 12. Dobson, M. J.. Pearlmnn. R. E.. Karaiskakis. A , . Spyropoulo\. B., and Moens. P. B. (1994).Synaptoneinal complex proteins: Occui-rence. cpitope mapping and chromosome disjunction. J . Cell Sci. 107, 2749-2760. Drmscr. M. E., and Moses. M. J . ( 1979). Silver-staining of synaptoneinal complcxc\ in stnrface spreads for light and electron microscopy. E.tp. Cell Ra.s. 121, 416-419. Egcl-Mitani, M . , Olson, L. W.. and Egel, R. ( 10x2). Meiosis in A.s/wr~illi.\tridukm: Another example for lacking \ynaptonemal coinplexes i n the absence 01' crossover interference. Hercditcit 97, 179- Ix7. Gillies, C. B. (1975). An ultrastructural analysis of chi-ornosoinel pairing in iiiai~e.C. R. .Frcw. Ltrh. Corl.sber.g 40, 135- 16 I . Haaf, T., Golub, E. I., Reddy, G . , Radding, G. M.. Ward. D. C. (199s). Nuclear foci of maniinalian Rad5 I recombination pi-otein i n \omatic cells and its localiration in \ynaptonenial cornplexes. Proc. N a t l . Accid. Sci. USA 92, 2798-2302.
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Heng, H. H. Q., Chamherlain, J. W.. Shi. X-M Spyi-opoulos, B., Tsui, L-C., and Moens, P. B. ( 1996). Regulation of meiotic chrom;itiii loop si/e by chroniosornal position. Prrw. Nrirl. Acrid. Sci. USA 93, 2795-2800. Heng. H H. Q..Tsui, L-C., and M o m \ , P. B. ( l9Y41. Organization of heterologous DNA inserts on the niouse meiotic chromosome core. C ~ / i r f ) f i i ~ ~ . \ f103, ) f i t ~ 40 r 1-407. Heyting. C., Dietrich, A. J. J., Moens, I' B., I>etttner\. R. J., Offenberg, H. H., Redeker, E. J. W., and Vink. A. C. G . (1988). Synapioneiiiel complex proteins. Grrtoriie 31, 81-87, Hobolth. P. ( I98 I ). Chromosome pairing i n allohrxaploid wheat var. Chinese Spring: Trandormation of niultivalents into bivalents. a incchani\m l o r exclusive bivalent formation. Ctrrl.sherg Rrs. C . o i i i i i r i u i . 46, 129- 173. Ikeya, T.. Shinohara, A,, Sato, S., Tabaki. S.. and Ogawa, T. ( 1996). Localization of mouse RadSI and Liin1.5 proteins on meiotic chi-o~iio\onie~ ill late stages of prophase I . Grwr,.\ Cell.\ 1, 379389. Jenkins. G. ( 1983). Chrorno\ome pairing i n Trrriuuri trcctiiwii cv. Chinese spring. C. K . Trrit,,Ltih. Crii-/.dwrq48, 255-283. Karpova. 0. I.. Safronov, V. V., Zaitseva, S. P.. and Bogdanov, Y. E (1989). Some properties 01' DNA isolated from i i i o u s ~synaptoneinal coniplex traction. Mid. H i o l . 23, 57 1-579. Kleckner. N. ( 1996). Meiosis: How could 11 work'! Pinc.. Ntirl. A a i d . Sci. USA. 93, 8 167-8 174. Klcin, S . ( 1991). Choosing your partner: Chrcinioroiiie pairing in yeast iiieio\i\. RioE.s.sct~s16, 869-87 I , Koch, J. E., Kolvraa, S., Petersen, K. B.. Gregerwi. N , and Bolund, L. (1989). Oligonucleotidepriming methods for the chrorno\omc-s~,ccitic1;ihclling of alpha satellite DNA i r i titu. Chrom o s o r t i r i 98, 259-265. Kovalenho. 0. V., Plug, A. W., Haaf. I.. Gonda, I). K., Ashley, T., Ward, D. C.. Radding, C. M., and Golub, E. I. (1996). Mammalian uhi[luitin-conjtigating enzyme Ubc9 inter recombination protein and localize\ i n \yiiaptoneinal complexes. Proc,. Nrrtl. Ar.ot/. St.r . USA 93, 2958-2963. K u n x , B., Wcichcnhan, D., Virks, P.. Tratil.. and Winking, H. (1996). Copy nunihers of a clustered long-range repeat determine C-h;ind \tnining. CJroRener. Cell G'erwr. 73, 86-91 . Marec. I; ( 1995). Chromatin organization and the length or \ynaptonemal complex complement\ in relntion to genome s i x . Chrot~io.toutc~ Re,,\. 3(Suppl. 1 ), 1 1 7. Moens. P. B. ( 1969). The fine mucture o l nieiolic chi-ornosonie polarization and pairing in Locii.\rri riii,yrtr/orro \pernatocytc\. Clirorizo\imrti 28, I -25. Moens, l? €3. ( 1994). Molecular perspectives of cliroinosonie pairing at meiosis. BioEs.scry.s 16, 1 0 I - I 06. Moens. I? B., Chen, D. J.. Shen. Z., KoIa\. N.. Heng. H. H. Q.,and Spyropoulos, B. Rad5I i n munocytology in rilt and mouse spermatocytes and oocytes. Chrotno.so,iitr. i n press. Moens, P. B., and Earn\haw, W. C. ( IWO). Aiiti-topoi\oiiierase I1 recognizes meiotic chroinosome cores. C/tro,rio.somi 98, 3 17-322. Moens. P. B.. Heddle. J . A . M., Spyropoulo\, B., arid Heng, H. H. Q.( 1997). Identical transgene\ on two non-homologous chronio\onies do 1101 promote ectopic synapsis at miohis. Grr t o t i ~ c in , prcas. Moen\, P. B., Heyting, C., Dietrich, A. J . J.. Raain\donk,W. van, and Chen, Q. (1987). Synnptoneinill complex antigen location and conwvation. J . Cell Biol. 105, 93- I ( Moens, P B., and Pearlinan, R. E. (1988). Chromatin cii-ganiiration at meiosis. Rio Moens. P. B.. and Pearlman, R. E. I n .xiti( DNA sequence mapping with surface-spi-ead niouse pachytene chroniosonies. C'\.roCqrrwt. C P / /G w t . 53, 2 1 9-220. M u n i . P. ( 1994). An analysis of interlercnce in llic fi\sion yeast S c ~ i i ~ o , \ ~ c c k r r ~ o ipciirrhe. i i ~ c ~ , ~Gcf I e t i c y 137, 701 -707. Pearlman, R. E.. and Moens, P. B. ( 1992). Synaptonemal complexe\ from DNa\e-treated rat pach) tcne chromosomes contain (GT),, and LINUSINE sequences. Genetic..\ 130, 865-872.
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Raamussen, S. W. ( 1977). Meiosis in Houih\.r m r i lemalcs. Philos. Trtiric. R. Soc Lord. IHiol.1 277, IXS- IXY. Rasmuswn, S. W. ( 1986). Inilialion of synapsis atid interlocking o l ehroinosoines during zygotene in Bonilqi. sperniatocytes. C'ci~l.\hc,r~K ~ T .Cowriwi. 51, 401 -432. Rasinuswn, S. W.. and Holin, P. B. (1980). Mechanic\ 01 ineiosi\. Hereditti.\ 93, 1x772 15. Schei-tIi;iii, H., Loidl. J.. Schuster, T., and Schwei/er. D. ( 1992). Meiotic chroinosonic condensation and pairing in Strc.[.lrci,r,rriv[.(,\ r.er-c,ii\itrc~ \tiitlied by chromosoinc painting. ~ ' / i r - ~ J r ~ , ~ J \ f J i i i ~ i 101, 500-50s. Schwaclia, A . and Kleckncr. N. ( 1994). Identilication o l joint molcculcs that form frequently he~ w e e i ihoinologs but rarely hctwccii aister chromatids during yeast meiosis. Cell 76, 5 1-63, Sen. D.. and Gilbert, W ( 1988). t;oi-mation of pnrallcl lour-\tranded complexe\ by guaniiie-rich motit\ i n DNA and i t s iinp1ic:itions for iiicio\i\. Ntrritrc. i l m ~ l o n 334, ) 365-366. Sliiiiohara, S.. Ogawa, H., and Ogawa. T. ( I W 2 ) . RadS I protein ~ n v o l v c din repair and rccoiiihination iii S. c,e,rcvisitrc, i s :I KecA-like pi-citein. ('rll 69, 457-470. Slack, S. M., Anderwn. I>. K., and Sherman. J . D. ( 1089). C'hiasmata and recornhin;ition nodules in [ 2 f / f f f ! 1 1 / f J J i , y ; / / ~ J t ' i t ! l iGc.Ilor11r . 32, 486-198. Stahl. F. ( 1096). Meiotic reconihination i n yeast: Coronation of the douhle-\trand-bre~i~ icpair model. Cell 87, 96S-96X. Sym. M., and Koeder. C. S. ( 1994). Crossover interferencc I \ aboli\hed in the ab\ence 01' ii synap toncrnal complex protein. C'rll 79, 283-292. T m o u n a s , M . , Pearlman, R. E.. Gasw. P. J . , Pal-k, M. S., and Moen\. P. B. (1997). Proklnprotein inleractions iii the synapti)tienial complex. Mol. H i o l . C'cjll., iii press. Terasawa, M., Shinohara, A,. Hotla, Y., Ogawa. t l . , nnd Ogawa, T. ( IWS). Localization of RccAlike recombination proteins on chroino\oiiies ot the lily at various meiotic stage\. Gr 9,915-914. von Wett\tein, 0.. Rarinu\sen. S. W.. and Holm. P. H . (19x4). The synaplonemal complex in genetic wgrepation. Ariiiit. R n , . C;cvic,f. 18, 33 1-43 I. Wicky, C.. and Rose, A. M . (1996).The role 0 1 chi-omosorne ends during meiosis in C'crc~rior/icihditis rlegcrr1.s. RroGso?J 18, 447-451. Winking, El., Reuter, C., and Traut, W. ( I 1903).Meiotic \ynapsi\ of homogeneously staining regions (HSRs) in chroniosonie I of Mirs rrru.sc.tt/it.r. ClirrJniosorw Rc,.\. I, 37-48. Zickler, D. ( 1977). Development of the ,ynoptonenial ci)mplex and the "recombination n.,dules" during niciotic prophase i n {he \even hivaletits of the fungus Sorrlciriti mrrcrocporo Auer\w. ~'/ir[)iiic).\oiiici61, 289-3 16.
8 Chromosome Segregation during Meiosis: Building an Unambivalent Bivalent Daniel I? Moore and Terry L . O r r - Weaver W h i t e h e a d Institute a n d Department Massachusetts Institute
of
of Biology
Technology
Cambridge. Massachusetts 01,141 1. Inti-oduction II. hlechaii i\ti1 of Chi-oiiiusoiiie 0t-ietit:it ion A . B i \ alent and Dyad Sti-ticturc I\ ('I iticiil t r i Orientation H Rcoi-icntaiion and Recognition (11 I3ipolar Orientation Ill. C'hia\mata A . Chinmiata Deline Point\ ol Atiachiiicni hct\vcen Homolog\ Are Cot-related M i i h C1ii~i~ni;it;iand Di\.junctioii C, l'o\itioti of Crtrssovei- Can He ('rltical t u hiistire Disjunction D. Why Di\t;il Cro\\o\er\ Miflit Fail to l . i i \ i i t e Di\junctioii 1:. Propo\cd Mean\ o f Biiidiiig Chict\iii;il;t F. Pos\ible Meclianisiw of Si\ter (~1iroiii;itid ('ohesioii duriiig Metaphaw 1 Iv. Horiiolo# Attachiiicnt and Segreg;i~i~iiiw i t l i o i i t Chinsniat;i . A Completely Achiasniaic Mctottc 1)i\ i s i o n \ B Notiexchange ('hromo\oiiic.\ 111 Mei(r\i\ \\ t t h Exchange
V. Si\tcr Kinetochore Function A Si\tcr Kinettrchores M~istKcorgani/c hctueeii Meiotic Divi\ion\ B ('ytologicnl Oh\ervation\ 01 S i \ t c i Kinctochorc Duplic;itioli C. I;unctional IXlierentiotion (11 Si\ter Kinetochore\ D. Early Functional Differentiatioil May He Chroinosonie Dependent VI MLloiiitaining Attachment betwccii S i \ l c i Chi oiiiattd\ for M c i o \ i \ II A . Cytology Sliov.\ That Attachiiiclit l'er\t\t\ 111 Proximal Region\ B. Equational Nondi\.junction Kewltiiig ft-otii l'ioximal Exchange C. Po\\ible Mcchnni\ms 01' Coht,\ioii in i h c Cciitromeric Kegions 11. Mutation\ That Dimtpt Cohe\ioii l o r Rlcio\i\ II
V I I S iiinmary References
Faithlul chromosoinc segregaiion duriiig an;ipha\e 1-eqiiires that stable microtubule coti~icciiiid both \pindle poles by inetaphase. Bipolar orientation f o l l o w s ;in active period (11 triiit\itxit coniicctions hetween the hinctochorc\ and pole\. and tension incdiated rhroiigh iitiilchiiiciii\ heiwccn the chromosornc\ stabilizes 11ioe hivaleiits that have c ~ n i i e c t i i ~ ito i s oppo\ite poles This review locuses on h o u the chroniatids arc tied together iii the bi\alcnt to enwre proper wgregation i n the two meiotic divi\ionc. Hoinolog\ are partitioned in nicio\is I . mtl reciprocal ci-ossovcrs, cytologically defined a\ chiasmata, usually hold the hoinolop logeilicr lor this division. The crossovers themselves m i \ t be prevented froin migrating o f t the chroinatitl arms. Binding substance\ localized to the crossover and ci\ter-chrornatid cohc\ioii d i 4 tcr the crossover havc been proposed to pr tion\ arc e\tabli\hctl between chroinowiiie\
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loss of chiasrnata. Spontaneous riondi~junctionevents and niiitation\ that di\rupt the iiiaintcnance 01' chiaaniata are analy/ed in the context of thc\e inodels. Homologs that segrcpate in meiosis I without chiawiata irre brietly discusseil. The hi\,alent niti\t a l w hc con\tructcd that four chrornatids present only t w o fiinctional hinetochores prior to anaphaw I . Cqtology and genetic dnta suggest that the si\ter I\inetochores are duplicated but con\traincd to act a\ a single kinetochorc. Additionally. ceiitroiiiei.ic region\ of bi\ter chromalid\ pre\er\ i' their cohebion until nnaphase I I . even iis coheyion on the Tistcr-chromatid iiriiis I \ lost at anaptiase 1. Mutations that jpecilically disrupt chi\ p r o w \ \ are prewntcd. Copyright C 199X by Academic Prras.
1. Introduction Appropriate partitioning of chromosomes during cell division depends on the arrangement of the chromosomes on the metaphase spindle. Proper segregation of chromosomes is ensured by stable microtubule connections between the chromosomes and opposite poles of the spindle, also called bipolar orientation. The attachments between the chromosomes allow them to resist poleward forces, balancing the connections to opposite poles. Consequently. these paired chromosomes settle at the metaphase plate after a comparativcly unstable and actibe period in prometaphase. The kinetochores of chromosomes and the attachments between chromosomes are vital to achieving bipolar orientation in both mitosis and meiosis. Chromosomes are segregated differently in meiosis than in mitosis and thus must be attached in different ways. In mitosis, recently replicated chromosomes remain bound together along their lengths until bipolar orientation is achieved at metaphase (Fig. IA), and all the cohesion is eliminated between the sister chromatids at anaphase (Fig. 1B). Since homologous chromosomes do not segregate from one another in mitosis, they have no need to be attached. Meiosis presents unique requirements for attachment between chromosomes. Following replication, the cell divides twice, reducing the diploid chromosome content to haploid content. The first meiotic division, the reductional division, segregates homologous chromosomes from one another (Figs. IC,D). In meiosis I, the homologs must be attached to achieve bipolar orientation and segregate reductionally. The second meiotic division, the equational division. segregates sister chromatids (Fig. 1 E). Thus, sister chromatids must remain attached in some manner through all of meiosis I, so that they may be oriented and properly partitioned in meiosis 11. The attached meiotic homologs are called bivalents for historical reasons, although four chromatids are in the structure. A pair of sister chromatids in the bivalent is a half-bivalent. If a bivalent dissociates before anaphase I or if there is no homolog, the pair of sister chromatids is called a univalent. When the pair of sister chromatids has segregated appropriately from a bivalent at anaphase I, it is referred to as a dyad. The kinetochores on the sister chromatids in a univalent, dyad, or half-bivalent are called sister kinetochores. This chapter reviews what is currently known about the ties between chromosomes during meiosis. Bivalent structure requires that homologs be attached, so
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Mitosis Metaphase
\I
'\
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Meiosis Metaphase I
Anaphase I
Metaphase I1
Fig. 1 Chromosome\ during select plia\c\ (it mitotic and nieiotic cell divi\ioiis. Homologous chromomrnes are in different shade\ of griiy. hinetocliore\ in hl inicrotubulc fibers connecting kinetochore\ and \piidle poles. ( A ) During mitotic inetaphase. chroiiio\onw\ align with their kinetochores ;it 1111: inetaph;i\e plate. Sistei-chromatid cohesion extends the length of the chromosomes. ( B ) When iiiitotic ;inapha\e begins. sister chr-oniatid cohesion i s rclea\ed dong the length of the chrorno\omc. ( C )During mct;ipha\e I of mciosia. only a portion of the ar-ins of tlic bivalent i \ aligned on the metaphasc plate. suggc\tiiig that chiasmata act a\ attachment\ hetween hotnolog\. Sister chromatid cohesion e'ttend\ the length of the chromosotnes and may \ e n e to hold the recomhinant chromosomes togethei-.The kinetochore\ of si\ter chromatids are con\traincd to face the same dircction. ( D ) During anapha\c I ( i t tiieio\i\. coticsion between si\ter chromatid\ i\ released along the iiriiis, but ninintained near tlic ceiitroiiicre\. ( E ) During nietaphaae 11. the chroiiiosonies align v, itli their- kinetochore regions on thc tnetaph,i\e plate. Si\ter kinetochore5 are now on opposite \idc\ of the chromatid. (Adapted froni l.u)kk. 1970,)
the role of reciprocal crossovers between homologs, typically the basis of this attachment, will be explored. Reciprocal crossovers by themselves cannot hold homologs together when spindle forces are pulling the chromosomes apart unless a mechanism exists to keep the crossover from sliding off the ends of' the chromosomes. Proposed mechaniwis for maintenance of crossovers as ties between the hoinologs will be examined. Not all chromosomes that faithfully segregate in meiosis I use crossovcrx as attachments between homologs, and we briefly survey alternative methods of holding the chromosomes on the spindle during the reductional division. Attachment between sister chromatids must be preserved through the first meiotic division for proper segregation in the second meiotic division, and we review recently identified proteins responsible for sister chromatid cohesion during meiosis. Finally. we briefly discuss how kinetochore shape aflects the attainment of bipolar orientation, particularly the problem of how sister kinetochores function as a single kinetochore before anaphase I.
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II. Mechanism of Chromosome Orientation A. Bivalent and Dyad Structure Is Critical to Orientation
Structure within the bivalent and dyad. not factors inherent to the spindlc, determine whether chromosomes segregate reductionally or equationally. This was demonstrated by micromanipulation experiments using grasshopper spermatocyte cells fused so that they contained both a meiosis I and meiosis 11 spindle (Nicklas, 1977). Transfer to another spindle did not alter the behavior of chromosomes with regard to bipolar orientation and segregation. Bivalents transferred from a metaphase I spindle to a metaphase I1 spindle oriented and segregated as they would in a reductional division. Dyads from prometaphase I1 spindles were able to orient on prometaphase I spindles, and the sister chromatids segregated from one another as they would in an equational division. Because transfer to a spindle carrying out an entirely different sort of meiotic division did not alter the manner in which the chromosomes segregated, differences in the organization o f a bivalent and of a dyad must determine how they segregate. Bivalents are inherently constructed to facilitate connection to opposite poles. Correct bipolar orientation is generally achieved very quickly. This has been observed, for example, for bivalents during meiosis 1 i n living spermatocytes of the grasshopper species, Me/uiiop/u.s differeriririlis (Nicklas, 1967). Ostergren ( I 95 1) first suggested that initial proper orientation is likely if kinetochores are arranged so that they face opposite directions. Spindle libers from a pole connect most readily with a kinetochore facing that pole, so connection to opposite poles is readily accomplished if two kinetochores are constrained to face opposite directions (Nicklas, 1977). In contrast to the general observation that correct orientation is quickly achieved, long flexible bivalents were found to be maloriented more often than smaller bivalents during prometaphase (White, 196 l ; Nicklas, 197 I ) , presumably because they were less capable of constraining the kinetochores of the bivalent to face opposite poles. The flexibility of these bivalents is thought to be a result of greater distance between the kinetochore and the sites where the homologs are attached, suggesting that the site of attachment is important for the efficiency with which bipolar orientation is achievcd (Nicklas, 197 I ). The shape of the kinetochore is likely to be another element of bivalent structure important for efficiently establishing connections to opposite poles. Kinetochores are typically cupped by chromatin, which may act to hinder access of spindle fibers to the kinetochore itself. Nicklas and Ward (1994) suggested that the cupped shape plays a critical role for the kinetochore that faces neither pole, because the shallower angle of approach of spindle fibers from the more distant pole could favor their attachment over the attachment of fibers from thc nearer pole, even though the density of fibers from the nearer pole is greater. The kinetochores of bivalents in Dro.soplii/ii inrlanogaster spermatocytes arc unusu-
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ally large and protrude such that they are inore exposed to spindle fibers from both poles. In studies of living D u ~ . s o p / ~ ispermatocytes, /~i more than half the bivalents were regularly malorientcd and required unusually long times to achieve bipolar orientation, approximately half the period between initial movement at prometaphase and the beginning 01' anaphase. The unusual shape of the kinetochores has been suggested as an explanation for the lengthy period of reorientation (Church and Lin, 1985). The unusual bivalents that do not quickly achieve bipolar orientation suggest which elements of the bivalent structure are most important for efficiently establishing connections to opposite poles. The sites of attachment and the shape o f kinetochores appear to be critical for the cfliciency with which bipolar connections are made. Understanding how orientation is achieved yields further proof that thcse elements of bivalent and clyacl structure are critical for appropriate segregation.
B. Reorientation and Recognition of Bipolar Orientation Improperly oriented bivalents clo 1-corient m c l do achieve bipolar orientation. At the beginning of proinetaphase I. when interaction with the spindle has just begun, the initial connections between kinetochores and poles are apparently random. All manner of inappropriate microtubule arrangements was observed in electron microscope studies 01' organism\ as divcrse as marine worins, insects, and plantx (Luykx. l96Sa: Church and Lin. 1982, 1985; Jenscn, 1982). Rcorientation i \ a li\ely and active proces\. arid recognition of bipolar orientation is key to attaining the stability ultini:itcly seen at inetaphase. The process of' reorientatioii ha\ been observed in studies of rnaloriented bivalent\ artilicially produced by their removal from the meiotic spindles of grasshopper spermatocytes. Whcn the hivalent is returned to the spindle, typically ii single kinetochore first connectecl to ;I pole, and there was movement of the bivalent toward that pole. Subsequently the other kinetochore connected with the opposite pole, and the bivalenl moved to the inetaphase plate. Connections to the same pole by kinetochores of hoth hoinologs occurred, but these connections were unstable and were quicI\Iy lo\(. The kinetochores ni''ice t new conneclions, until eventually a bipolar ari-angcnicnt w 1967; Nicklas, 1967). Similar initial connection to one pole has been characterized for mitosis as well as meiosis b y observation o f both fixed and living cells of several species (reviewed by Ricder, 1982). Bivalents are relatively stable at the inetaphase plate. Bipolar orientation is recogniLed in xome manner, so that connections between kinetochore and pole d o not continue to be lost. Mechanical tension stabilizes the spindle fiber connection. Ordinarily, bipolar orientation provides this tension, because poleward forces pull the kinetochores in opposite directions and are counteracted by the
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Fig. 2 Tension stabilites spindle fiber coiiiiections between kintochore and pole. ( A ) In the typical bivalent during metaphase 1, 5pindle fiber connections to the pole\ prcwide poleward force (black arrows) and are stabilized by the tension acting through attachments between homolog\. ( B ) A single bivalent during nietaphaae 1 has spindle tiber coiinecticm bctwcen both kinetochores and onc pole stabilired by artificial lorce (white a n o w ) toward the opposite pole. Artificial forcc IS provided by micromanipulation with a needle. (C) Two bivalcnts with connections tn opposite poles are entangled, mimicking attachments between the homolog\, so that tension stahilire\ thew connections. ( D ) Artificial force (white arrow) applicd perpendicular to (he spindle axis stabiliLes the connection between the pole and the kinetochore iiridcr ten\ion. The kinetochore tliiit is not undet- t e n w n iisually reorient\.
bonds that hold the bivalent together (Fig. 2A). The role of tension in creating stable connections was demonstrated experimentally by providing artilicial tension. In one experiment, bivalents with both kinetochores connected to the same pole were stabilized by using micromanipulation to provide an opposing force (Fig. 2B; Nicklas and Koch. 1969). In another type of' experiment, micromanipulation or heat shock during prophase I produced bivalents that were tangled or linked with one another, mimicking attachments between chromosomes. These bivalents also achieved a stable position on the metaphase plate (Fig. 2C; Henderson and Koch, 1970: Buss and Henderson, 1971). Tension on kinetochores apparently stabilized connections made to spindle poles. The kinetochores in the experiments just described directly faced a single pole. If exposure of a kinetochore to spindle fibers from a pole is critical to making a connection with that pole, it could be argued that tension might not have stabilized the connection. Instead, spindle tiber connections with the other pole may have been hindered by the bulk of the chromosomes, reorientation might have
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been inhibited, and the kinetochores might only seemed to have a stable connection. In recent experiments by Nicklas and Ward (1994), micromanipulation was used to apply a force perpendicular to the spindle axis, toward the cytoplasm rather than toward either pole. In this way. kinetochores that did not directly face a single pole could be studied. Kinetochores under tension maintained a connection to a pole. whereas kinetochores without tension frequently reoriented (Fig. 2D). Mechanical tension, rather than exclusion of spindle fibers from the opposite pole, proved to be the stabilking factor. In summary, proper segregation of chromosomes depends on appropriate connections to opposite poles at inetaphase. In prometaphase I, the sites o f attachments between hotnologs and the shape of the kinetochores were shown to be important for attaining bipolar orientation efficiently. Bipolar orientation at metaphase is stable, because spindle fiber connections to the poles are stabilized by mechanical tension. Ties between chroinosomes within the bivalent are essential for tension.
111. Chiasmata A. Chiasmata Define Points of Attachment between Homologs Homologs are attached before anaphasc I, usually through chiasmata. ( Exceptions are addressed later in this chapter.) Chiasmata are observed on the arms of chromosomes in the bivalent in late prophase 1. In early prophase I , during pachytene. the homologs have been paired and, in most species, a structure called the synaptonemal complex (SC) is built between them along their length. The SC consists of latcral elements located berween the sister chromatids and a central element connecting these lateral element\. Before the central region is i n place, the lateral elements are referred to as axial elements. Later in prophase I, at stages termed diplotene and diakinesis, the SC' dissolves. and the homologs repulse one another except at localized points of artachnient located on the arm\ of the chromosomes. The points of attachment are the chiasmata. The cytology of meiotic cells suggests that the role of chiasniata is to hold homologous chromosomes together to provide the tension needed for proper orientation. During metaphase I. [he a m \ of the chromosome rather than kinetochores are aligned on the inetaphase plate (Fig. IC), unlike nietaphasc of m tosis o r meiosis 11, where kinetochores arc aligned on the plate (Figs. I A.E). For bivalents, then. the ties between the chromosomes are not at the kinetochorc but on the arms of the homologs. 6. Crossovers Are Correlated with Chiasmata and Disjunction
It is generally accepted that chiasmata are associated with reciprocal crossovers between the homologous chromatids. txperiments by Tease and Joncs ( 1978)
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Daniel P. Moore and Temy L. Orr-Weaber
A
B
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X
JL
Fig. 3 Chia\inata and c r o s w v r r s are ;it the \ame Iociitioii. Hoinolops are drpictrd 21s white and hlack line\. and open circlcb represent ccntrotiirrcs. ( A . B ) A single cIos\over h r t u e e n "uhlte" and "hlack" chromatids in ;I d i l l e r c ~ i t i i i l l yInbclctl bl\ alent 1 ields il \ islhlc cro\\over point i n the Inetaphaw bivalent Note that ;I hinglc cro\so\cr between s i i i i ~ l :lahelcd ~ chrotiiatid\ w ~ o u l dnot result i n it nietapha\e bivalent u i t h ii vi\ible cIos\ovcr point. CC) If the cros\ovcr niigrntes touai-d the c i i d s o t t h c chromatid\. regions ol similai-ly inheled chromatid uould he \ern hctuccn t h e kinetochorc\ and the chia\ma. Tei-minal iiio\ riiient wii\ not oh\cr\cd.
using spermatocytcs of a locust. 120c.ir.stci mi,qrutoriu, showed that crossover exchange points within the bivalents, when cytologically detected. were located at the same place as the chiasmata. Exchange events were detected by tliffercntially labeling the sister chromatids with 5-brotnodeoxyiiridine incorporated duting replication, so that a crossover between dissimilarly labeled chromatids in the bivalent gave a visible exchange point (Fig. 3A.B ). Similar experiments in other species gave like results, suggesting that the accordance of crossovers with chiasmata is a general phenomenon (Jones, 1987), although the absolute correspondence of crossovers and chiasmata continues to be questioned (see rcview of data from plants by Nilsson c t u/., 1993). Crossovers are generally necessary for proper segregation of homologs. A plethora of mutations that reduce or eliminate exchange result i n high frequencies of missegregation during the reductional division (Jones, 1987; Hawley, 1988; John, 1990). The role of crossovers in ensuring segregation has been examined i n organisms without mutations that reduce the overall level of exchange. Missegregation events occur at low frequencies during meiosis in organisms that are otherwise wild type. The origin of spontaneous missegregation events during meiosis I was assessed by reconstructing the recombinational history of chromosomes I'ound in aneuploid progeny of humans and Dro.sophil~i.Because the disomic gatnete that gave rise to aneuploid progeny could be the result of inissegregation in either of the two meiotic divisions. it was critical that errors in diqjunction during meiosis I be differentiated from errors in meiosis 11 by using centroniere-linked markers (Figs. 4A-C). Chromosomes derived from missegregation during meiosis I had recombinational histories quite different from the histories of chromosomes from meiosis 11 missegregation events.
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Daniel P. Moore and Terry L. Orr-Weaver
The overall frequency of crossovers was reduced in bivalents that underwent spontaneous meiosis I missegregation relative to bivalents carrying out successful meiosis I disjunction. When the recombinational history of chromosomes in human trisomy resulting from meiosis I errors was coinpared with that of chromosomes from successful meiotic segregation, there w n increased frequency of nonexchange and single-exchange events. This was observed for chromosome 16 (Hassold c't [ I / . , 1995). lor chromosome 18 (Fisher et ( I / . , 1995), and for chromosome 21 (Sherman et a/., 1994), using DNA markers to analyze the parental origin of the chrotnosomes and the exchange events that occurred during the meiosis that gave rise to them. The X chromosomes from X X Y atid XXX individuals similarly experienced reduced amounts of exchange. Notably, most meiosis I nondisjunction occurred in nonexchange bivalents (MacDonald et ul., 1994). Spontaneous meiosis I nondisjunction in D. ww/nrzoRtr.rtrr females was surveyed by examining visible markers in progeny conceived from ova disoniic for the X chromosome. The majority of chromosomes were derived from bivalents that had no exchangc event. The rcniainder were derived from bivalents with a single-exchange event (Koehler, Boulton, ot o/., 1996). (The singleexchange bivalents that proved inadequate are discussed further in the next section.) Thus, an appropriate frequency of exchange has been shoun to be important for proper segregation of individual bivalents in organisms from normal populations of humans and Drosophiln, not just i n populations with reduced meiotic exchange due to a mutation. Crossovers are usually sufficient to segregate chromosomes properly in meiosis I. Indeed, a single crossover in a bivalent has been shown to be sufficient to produce disjunction of chromosomes that are only partly homologous. A small pseudo-autosomal region near the teloniere of the X and Y chrotnosomes i n humans and mouse has a genetic length consistent with a single crossover, and this appears to ensure disjunction of thcse mostly nonhomologous chrotnosonies (H. J . Cooke et [ I / . , 1985: Page et ( I / . , 1987). Similarly, rearranged chrotnosomes in 1). nielanog~i.vrerthat carried homologous regions resulting from translocation were shown to ensure disjunction of nonhomologous centromeres (Hawley, 1988). To briefly recapitulate, chiastnata are the cytologically apparent sites of attachment between homologs. Reciprocal crossovers correspond well with chiasmata, and crossovers are usually necessary and sufficient to ensure proper segregation. The exceptional crossovers that arc not sufficient to ensure segregation provide clues to how a bivalent is built.
C. Position of Crossover Can Be Critical to Ensure Disjunction
Exchange is constrained i n niost organisms such that it is not randomly distributed within the length of the chromosome or randomly distributed among the chromosomes. Crossovers arc usually in the euchroniatin and most corninonly
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occur in the medial portion of the chromosome. The terms proximal, medial, and distal refer to locations relative to the centrotnere of the chromosome. Usually there is at least one exchange event per chromosome, even on chromosomes that are relatively small (Jones, 1987; Hawley, 1988; John, 1990). In the studies of spontaneous meiosis I errors described earlier, the location of an exchange event was vital to its success at ensuring disjunction. Proximal and medial exchanges were underrepresented among bivalents that led to nondisjunction, and single exchanges in a distal location occurred more frequently in bivalents that experienced meiosis I errors than they did in bivalents that disjoined properly. The genetic map lengths of the most distal region of chromosomes from successful segregation events were often smaller relative to that of chromosomes from missegregation. In human ova, this was observed for the short arm of the X chromosome (MacDonald et d.,1994), for chromosome 16 (Hassold ef cil., 1995), and for chromosome 21 (Sherman r t (/I., 1994). Distal exchanges on the X chromosome in Drosophilu ova similarly had less ability to ensure disjunction (Koehler, Boulton, et NI., 1996). The tendency of nonexchange and dihtal exchange bivalents to be highly represented in spontaneous inissegregation has also been observed for Ilro.vophila chromosome 2 (Carpenter, 1973; Gethmann, 1984). Moreover, in the presence of either of two dominant mutations in Drosophila that primarily disrupt scgregation of nonexchange chromosomes in ova. the exchange bivalents that did nondisjoin most frequently had distal exchange events. This has been shown for the X chromosome in Dith females (Moore rt NI.. 1994) and for both the X chroniosome and chromosome 2 in i ~ o t l ~ )females '\~ (Rasooly et al., 1991). In Strcchurornyces cer-e~isici~, distal exchange events were also observed to be less effective for the segregation of engineered chromosomes not required for viability. Yeast model chromosomes carry elements known to be essential for normal replication and segregation, namely, centromere sequence, telomeres, and an origin for replication. Misscgregation of test bivalents can be examined without apprehension about progeny inviable clue to aneuploidy. Exchange events on the artificial chromosomes usually ensured reductional segregation, but distal exchange events were less effective at ensuring disjunction than were proximal and medial events (Ross el ol., 1992). Evidence from species as diverse as humans, Drosophila, and budding yeast demonstrates that distal exchange provides less secure ties between homologs than does medial or proximal exchange.
D. Why Distal Crossovers Might Fail to Ensure Disjunction
Although an exchange is usually sufficient to ensure disjunction. the location of the crossover is also quite important. One possible explanation is that distal events do not provide as stiff a linkage between the kinetochores of the homologs as do medial exchange events, and that this results in nondisjunction (Rasooly et al., 1991 ). At least two arguments may he raised against this hypothesis: ( 1 ) The
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centric heterochromatic regions of homologs have been shown to be paired throughout prophase I in Drosophila oocytes and have been suggested to orient even nonexchange homologs during prometaphase I (Dernburg et al., 1996). so that the location of crossovers on homologs might be expected to have little effect on bivalent organization in the centromeric region of this species. (2) The distal exchanges on the yeast model chromosomes are closer to their centromeres than many exchanges on the natural yeast chromosomes, yet the natural chromosomes segregate appropriately (Ross, Maxfield, et al., 1996). Thus, it is proximity of the exchange event to the end of the chromosome, rather than distance from the centromere, that results in occasional missegregation. Perhaps distal crossovers are inclined to be lost as attachments, because a crossover serves as a connection between the centromeres of the homologous chromosomes only so long as the crossover does not migrate off the ends of the chromosomes. During orientation in prometaphase I, the centromeres of the homologs are pulled in opposing directions, so there must be a mechanism to prevent crossovers from being lost as attachments. Maguire (1974) called this requirement for a contrivance to maintain crossovers "the need for a chiasma binder."
E. Proposed Means of Binding Chiasmata
There are three general proposals for how chiasmata are prevented from migrating off the ends of the chromosomes: ( 1 ) migrating chiasmata are stopped by structures at the terminal ends of the bivalent; (2) binding substances at the site of exchanges hold the chiasmata in place; and ( 3 ) cohesion along the length of the sister chromatids is not released, so migration is not possible. The last two mechanisms are not exclusive of one another and could potentially act redundantly. 1. Terminal Binding
In the first model, if the terminal ends of the sister chromatids cannot be separated, exchange crossovers that occur in medial portions of the chromatid may migrate nearly to the chromosome ends and still serve as a bond between the homologs. One specific proposal suggested that the telomeres of the sister chromatids are not duplicated until the metaphase Uanaphase I transition, so that crossovers might move to the ends of the bivalent chromatids and be caught there to act as an attachment between the homologs (Egel, 1979). Chiasmata terminalization was suggested by early cytological studies of fixed meiotic cells, but i t is no longer generally accepted [see appropriate sections in reviews by either Carpenter (1988) or by Jones (1987)], although it has been argued that the degree of terminalization may be species-specific (von Wettstein et al., 1984). In species
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where chiasmata were directly examined for terminalization in meiosis, it was apparent that the chiasmata were not migrating terminally. Metaphase bivalents with differentially labeled sister chromatids would be expected to display regions of equivalently labeled chromatin if terniinalization brought homologous regions together (Fig. 3C). but this was not observed. Within the extent of resolution in each animal system, no chiasmata movement occurred. Terminalized chiasmata usually correlated with a terminal exchange point in locust spermatocytes (Tease and Jones, 1978), in hamster spermatocytes (Allen, 1979), and in mouse spermatocytes (Kanda and Kato, 1980) and ova (Polani et a/., 1981).
2. Binding Substance Near the Crossover In the second model for a chiasma binder, a binder substance is proposed to hold the exchange event at or near the original site of the exchange. A mutation that disrupts chiasma binding should result in nondisjunction of exchange bivalents, so such mutations will be reviewed here for evidence to support this model. Analysis of the mutations should be approached with the following general considerations. Exchange should occur at normal levels if the mutated gene product is required only as a binding substance to maintain a chiasma. If a mutant exhibits reduced levels of exchange, i t suggests the gene is required for establishment as well as maintenance of functional chiasmata. Cohesion in the proximal region of the half-bivalents would be undisturbed in a mutation that is specific for binding chiasmata; thus cytology should reveal unusual numbers of univalents but should not reveal separated sister chromatids during metaphase I. Separation of sister chromatids before anaphase 1 suggests a more general loss of sister chromatid cohesion. In addition to cytology, genetic assays of nondisjunction events can suggest whether complete loss of cohesion between sister chromatids is occurring before metaphase 1 (Fig. 4D). The "desynaptic" mutations of Zea m r y s best meet the above criteria for a mutation in chiasma binding. Liyl and dsyl are two desynaptic mutations that have been well characterized, although others have been reported (Golubovskaya, 1989). These mutations have not been tested for complementation. Consistent with the expectations of a mutation in binding substance function, both univalents and bivalents with one open arm were observed in metaphase I inicrosporocytes homozygous for the desynaptic mutation, dy1. Crossovers occurred at wild-type levels. Heterochroniatic knobs that are cytologically visible on the arms of maize chromosomes allowed exchange events to be directly assessed in strains heterozygous for the knob. Strikingly, exchange events had often occurred on the arms of univalents and on the open arms of bivalents, providing graphic evidence of exchange events that were not maintained as chiasmata. Cohesion at the univalent kinetochore was maintained until anaphase I, although some equational segregation was observed during this division. However, more monads (single chromosomes resulting from early separation of a
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dyad) were seen during prophasc I1 than could be accounted for by these equational segregation events (Maguire, 197th). Thus, contrary to the ideal expectations of a mutation in chiasma binding substance, d y l seems to disrupt sister chromatid cohesion at some time after the beginning of anaphase I. In addition, the central element of the SC was unusually wide in d y l mutants (Maguire et a/., 1991). Exchange events that failed to bind bivalent arms together were observed cytologically as well, and cohesion at the univalent kinctochore was reported to be maintained until anaphase I1 (Golubovskaya, 1989; Maguire rt a/., 1993). These observations are consistent with our criteria. However, the frequency of exchange was not reported for d s y l homozygotes, and it may be quite reduced. Synapsis was not always complete, and these mutant microsporocytes also had a wide central region in their SC (Maguire et d., 1993). The maize desynaptic mutations remain the best candidates for mutations in chiasma binder, although both d y l and cfsyl mutants also have phenotypes that overlap with more general meiotic functions. dsyl mutants do not achieve full synapsis and have not been shown to have exchange at normal levels, so this gene may be essential for establishment of chiasmata. The failure of d y l mutants to retain cohesion between sister chromatids until metaphase 11 suggests that the gene may be needed for additional functions in sister chromatid cohesion. It may be difficult to identify a mutation that specifically disrupts chiasma binding using such ideal expectations. Separation of maintenance of chiasmata from establishment of chiasmata may require an unusual allele of a gene required for more than one function. Moreover, binding at the site of a crossover might be redundant with sister chromatid cohesion i n the maintenance of crossovers, so that both mechanisms must be disrupted to result in frequent chiasmata failure. The wide central element of the SC reported for both of the maize desynaptic mutations suggest that mature SCs might play a role in establishing or maintaining crossovers that can serve to attach the homologs. Normally, most of the SC dissociates following pachytene. However, remnants of SCs were observed to be associated with chiasniata as late as diplotene in diverse organisms (Jones, 1987), including maize (Maguire, 197%). locust and grasshopper (Moens and Church, 1979), and mouse (Solari 1970). Disassembly of SCs may simply be hindered near sites of attachment, but the possibility exists that SC remnants act to bind chiasmata in place. Several observations argue against a general requirement for the SC in chiasma maintenance. The Zip1 protein of S. cer-rvisiae was localized to the central region of mature SCs and is likely to be a component of the central region. Strains harboring zip1 mutations have a defect in synapsis; full-length axial elements and paired homologs varied in proximity to each other along their lengths. However, crossovers occurred at approximately wild-type levels, and exchange still ensured disjunction. Sister chromatid cohesion does not appear to be defective (Sym and Roeder, 1994). Two organisms. S~.hiio.sr~c.c.hiirr,Invc.rs
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potnhe and Aspergillus nidulans, are unusual in having no SC, although they have structures that look like axial elements. Both species have crossovers in meiosis that ensure disjunction (Olson et ul., 1978; Egel-Mitani et al., 1982). Thus, the central region components of the SC are not essential for crossovers that function as attachments between homologs. Axial SC components, however, could play an important role in establishing and maintaining chiasmata. Exchange bivalents missegregated in S. cerevisiue meiotic cells homozygous for retll, and these mutants failed to make axial elements (Rockmill and Roeder, 1990). Redl protein has been suggested to act in initiating the axial element (Roeder, 1995). Recombination was reduced in red1 meiotic yeast, although the extent to which it was reduced varied widely from region to region. Precocious separation of sister chromatids was seen only at a low level, suggesting that cohesion between the sister chromatids was usually retained until the second meiotic division (Rockmill and Roeder, 1990). The redl phenotype suggests that components of the SC might play a part in producing crossovers that can function as attachments between homologs at metaphase I. Although cytological studies have placed SC remnants near chiasmata late in prophase I, there is no functional evidence that SC remnants play a role in maintenance of chiasmata. N o mutations yet exist that specifically meet the ultimate expectations of a chiasma binder (see also review by Carpenter, 1994), although the maize desynaptic mutations meet many of the criteria. It is not yet clear i f binding substance exists at the site of crossovers.
3. Sister Chromatid Cohesion Cohesion between the arms of sister chromatids has been proposed as a mechanism to maintain chiasmata. A crossover between two homologous chromosomes cannot migrate to the end of the chromosomes if the distal portion of the recombined chromosome is tightly bound to its sister (Fig. 1C). As a corollary, cohesion along the arms of sister chromatids must necessarily be released during anaphase I beyond the most proximal chiasma, so that the recombined homologs are able to segregate from one another (Fig. ID). Sister chromatid cohesion provides the simplest explanation of why distal chiasmata might be less successful than more proximal chiasmata in ensuring disjunction. The more distal the location of the chiasma, the shorter the length of sister cohesion that would be able to maintain it. If binding substance alone holds chiasmata at sites of exchange, then the length of chromosome distal to the crossover should be irrelevant. A slow degradation of sister chromatid cohesion best explains the increase in meiosis I nondisjunction frequency observed as oocytes age in human females. The chromosomes from human trisomy 2 1 progeny exhibited decreased amounts of recombination and often had single exchange events in the distal regions (Sherman et ul., 1994). Recombination occurs prenatally in human fcmalcs.
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followed by a long period of arrest in prophase I until ovulation, a period that can be as long as SO years. Because oocytes are held in prophase 1, well before the meiosis I spindle is built and the bivalents orient, the increase in nondisjunction seems most likely to be due to slow failure of sister chromatid cohesion over time. The more proximal the crossover, the greater is the length of sister chromatid distal to the chiasma, increasing the likelihood that some sister chromatid cohesion remains to prevent loss of the crossover as an attachment between homologs when the forces of the meiotic spindle begin acting on the bivalent. In maize microsporocytes, chiasma-like associations often persist after anaphase I for acentric fragments resulting from exchange events on chromosomes heterozygous for paracentric inversions. These have been used by Maguire (1993) to test the three models of mechanisms that maintain chiasmata. In the most easily interpretable case, an acentric fragment and cytologically distinct homologs result from two exchange events involving three strands, one crossover proximal to the paracentric inversion and one within the inversion (Figs. SA,B). One homolog is a loop dyad and the other homolog is normal. Binding substance localized to the chiasma would give the acentric fragment a tug toward the same pole as the loop dyad. Sister chromatid cohesion distal to the chiasma would cause the acentric fragment to travel with the normal homolog (Figs. SC,D). The latter event happened frequently and was interpreted as demonstrating that cohesion between sister chromatids is most likely to function as a binder. However, this experiment relies on associations that exist after metaphase I. This persistent association does not exclude the existence of a binder substance at the chiasmata that is weaker than sister chromatid cohesion or is simply released earlier than sister chromatid cohesion. Another interpretation of this experiment is discussed in the next section.
F. Possible Mechanisms of Sister Chromatid Cohesion during Metaphase I
Sister chromatid cohesion during meiosis is likely to be even more functionally complex and intricate than during mitosis. Not only must the sister chromatids be held together, but they must be inhibited from interactions that take place in mitotic divisions, such as positioning of sister kinetochores to face opposing poles. In meiosis, crossovers between homologs are preferentially formed relative to crossovers between sisters. Recently, recombination intermediates, identified as double Holliday junctions, at a meiotic “hotspot” of recombination in yeast were shown to favor homolog interaction over sister chromatid interactions (Schwacha and Kleckner, 1994, 1995). Some structural aspect of sister chromatid cohesion in meiosis may serve to direct interactions away from sister chromatids, favoring homologs, either by making one of the sister chromatids inert or by physically restraining the sister chromatids from interacting.
2 79
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45Meiosis I
Fig. 5 Segregation of a particular three-\trand double crossover on
a hcterorygous paracentric inversion. Sinall open circles represent ceiitroiiiere\. ( A ) The aligned hoinologs are depicted in gray and black. One crossover is proximal to the pxiccntric inversion and the other is within the in\ersion. ( B ) Alter cxchange. one homolog is a loop dyad (gray d i d line) while the other homolog is normal (black solid line). An acentric fragment (gmy dotled line) also results from exchange. Reductional segrcgation partitions the loop dyad (C) fwni thc noriiial dyad (D). The accntric fragment frequently m o w s with the nornial dyad to one pole. Regions of hihtci- chromatid c o h e h n that d o n o t experience the spindle forces acting on the dyads iirr \h:idetl lightly. (Adapted from Maguirc. 1993.)
Proposed mechanisms for cohesion between the sister chromatid arms include incompletely replicated chromosomes, unresolved intertwinings between chromosomes after replication, and protein structures that act as a glue between the sister chromatids. These niechanisms are not exclusive of one another, and what binds the sister chromatids along their arms may differ from what binds them near the centromeres.
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Incomplete replication of short DNA stretches is unlikely to be a mechanism for sister chromatid cohesion. Although heterochromatin of some eukaryotic chromosomes is late-replicating (Lima-de-Faria and Jaworska, 1968), S. cerevisiae chromosomes have been shown to replicate completely during S-phase in both mitosis and meiosis (McCarroll and Fangman, 1988; Collins and Newlon, 1994). In addition, pulse labeling in human fibroblasts revealed no replication of DNA in metaphase or early anaphase (Comings, 1966).
1. Catenation as a Cohesive Factor Unresolved intertwinings of sister chromatid strands, also termed catenation, might act as a linkage until anaphase. Control of either access or activity of topoisomerase I1 to the catenated regions would provide linkage between the sister chromatids until anaphase. Topoisomerase I1 is required for the metaphase/anaphase transition in mitosis (reviewed by Holm, 1994). The enzyme is found on pachytene chromosomes in thc axial cores of yeast and chicken (Moens and Eamshaw. 1989; Klein era/., 1992) and has been proposed to be required for formation of the SC, resolving entanglements that arise during this time (von Wettstein er d., 1984). The necessity of topoisomerase I1 in yeast meiosis has been directly investigated using a cold-sensitive mutation, rop2''' (Rose and Holm, 1993). Premeiotic replication, chromosome condensation, and SCs appeared to be unaffected despite the lack of functional topoisomerase 11, but meiosis was blocked and cclls arrested prior to anaphase I with a single nucleus. Meiotic recombination can be eliminated with a rud50 mutation. rcrd.50 r0p2~\ double mutants at restrictive temperature were able to pass through anaphase I and produced binucleate cells. Eventually they went on to produce multinucleate cells. Thus, topoisomerase I1 is required for transition to anaphase I when the honiologs have recombined, presumably because entanglements distal to exchange crossovers must be resolved for the recombined chromosomcs to segregate (Rose and Holm, 1993). To resolve catenated molecules rather than generate additional interlocks, topoisomerase I1 requires directionality provided by other forces. Condensation of sister chromatids provides some directionality to the double-strand passings, and the forces generated by segregation on the spindle at anaphase provide further directionality (reviewed by Holm, 1994). Regions proximal to all the crossovers on a bivalent arm will not experience this force during anaphase I, because the proximal portions of sister chromatids are being pulled in the same direction. Catenations in these regions may not be resolved in anaphase I. Such sister chromatid cohesion on the arms after anaphase I has been dubbed "adventitious" (Kleckner, 1996). The persistent association between sister chromatids observed i n Maguire's classic experiments using maize paracentric inversions (Maguire, 1993) may be
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catenation that is not resolved, and thus an example of adventitious cohesion (Kleckner, 1996). Three-stranded double crossovers in this heterozygous paracentric inversion result in a dyad capablc of segregation in anaphase I without separation of sister chromatids distal to the two crossovers (Fig. 5 ) . The loop dyad has no distal region, and the acentric fragment does not experience spindle forces in a direction opposing the normal dyad. Because catenation may not be resolved without forces to provide directionality, the acentric fragment would bc expected to remain associated with the normal dyad. This adventitious cohesion suggests that catenation in the distal regions of sister chromatids exists. and it does not preclude the possibility that catenation could hold chiasmata at the sites of crossing over, with the provision that topoisomerase I1 activity is inhibited until the metaphase Uanaphase I transition. In mitosis, however, mechanisms other than entanglement must also hold sister chromatids together. Circular minichromosomes were observed to be in close proximity during mitosis (Guacci et al., 1994), although they were not topologically interlocked during metaphase. Despite their lack of catenation, these circular minichromosomes segregated with fidelity (Koshland and Hartwell, 1987). In mitosis, at least, topoisorncrase I1 may only play a role in disentangling the chromosomes and is unlikely to be the sole mechanism holding sister chromatids together.
2. Proteins Potentially Serving to Glue Sister Chromatids Together
a. Mutations Disrupting Sister Chromatid Cohesion Early in Meiosis. In contrast to the phenotypes of the maize desynaptic mutations and of red1 in budding yeast, mutations in three genes from diverse organisms have phenotypes suggesting complete loss of cohesion between sister chromatids well before metaphase I. These are ord, r e d , and .spo76. Cytology provides the best evidence of early sister chromatid separation. Sister chromatid cohesion is required for segregation, so precocious separation of sister chromatids can also be ascertained by genetic criteria (Fig. 4D). In mutants in which exchange occurs, it is informative to know whether crossovers are able to ensure disjunction of homologs. In D. melnnogaster, mutations in the gene ord result in precocious separation of sister chromatids in both sexes. In mutant spermatocytes, separation of sisters was visible during prometaphase 1 (Mason, 1976; Miyazaki and Orr-Weaver, 1992; Bickel et ul., 1997), and sister kinetochores were observed to be separated early in prometaphase I (Lin and Church, 1982). Genetic exchange was reduced, and the remaining exchange events did not ensure disjunction of the bivalent in meiosis I (Mason, 1976). ord' oocytes had reduced exchange along most of the X chromosome, although the reduction was less extreme near the centromere. The few very proximal exchange events that occurred slightly increased the probability of successful reductional disjunction (Mason, 1976). This suggests that
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ord’ does not completely obliterate the ability of an exchange to bind homologs.
Recently the gene has been cloned, and it is predicted to encode a novel protein (Bickel ef d.. 1996). In S. pombe strains that are homozygous for the re(*8-110mutation, precocious separation of sister chromatids has been detected by both genetic assays and fluorescent in siru hybridization (FISH) (Molnar et d . , 1995). Separation of sister chromatids occurred early in prophase 1 in 70% of nuclei: more than 20% showed very wide separation. The chromosome ends had the least separation of sister chromatids (Molnar et d . ,1995). Although S. pornbe lacks SC, there are “linear elements” that are thought to be equivalent to axial elements. Sporadic misalignment in the linear elements was observed in r e d - I 1 0 yeast (Bahler er ul., 1993). Exchange was reduced in a region-specific manner, with as little as a 10-fold reduction at the ends of chromosome I11 and with greater reduction at sites examined on the other two chromosomes (DeVeaux and Smith, 1994). The predicted sequence of rec8 product showed no homology to known proteins (Lin et ul., 1992). In .spo76 homozygotes of the fungus Sordciritr tmcrosporu, precocious separation of sister chromatids was observed cytologically during prophase I, but this cannot be observed genetically, because the meiotic cells arrested and rarely resulted in viable gametes. Regions of the lateral elements appeared split. It is likely, although not yet demonstrated, that recombination is reduced in the homozygous mutant, as it has been shown that recombination was reduced in the heterozygote (Moreau et ul., 1985). In both rec8 and ord homozygotes. exchange between homologs was inadequate for proper disjunction. This is consistent with the hypothesis that sister chromatid cohesion is necessary for crossovers to act as an attachment between homologs. However, these mutations may be required for an early function, such as sister chromatid cohesion immediately after replication, that is a precondition for establishing mature chiasmata without necessarily being required to maintain chiasmata. Moreover, ord is required for cohesion of sister chromatids in male meiosis, a meiotic division that has neither exchange nor SCs, so it is required to ensure reductional disjunction where chiasmata definitely need not be maintained. Early splitting of sister kinetochores also complicates the conclusion that sister chromatid cohesion is required to maintain chiasmata, because inability of the bivalent to ensure proper orientation could be a consequence of sister kinetochores orienting independently.
b. Proteins Identified by Immunocytology as Candidates. Immunocytology has been used to identify candidate sister-cohesion proteins during meiosis. CORl protein is localized to the sister chromatid core during meiosis in hamster, but it is not seen in somatic cells. This antigen is lost along the arms of the meiotic chromosomes at the metaphase I to anaphase I transition, although i t
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continues to be located near the ccntromeres, where sister cohesion is retained until anaphase TI (Dobson er ul., 1994; Moens and Spyropoulos, 1995). However, the protein has not yet been demonstrated to be essential for cohesion. In immunocytological studies of mitotic cells, antigens have been found that are specifically localized to the region along the inner surface of the kinetochore, termed the pairing domain (Rattncr, 1991), and to the inner surfice of the sister chromatid arms. This is likely lo be the site of interaction between sister chromatids at metaphase. Chromatid-linking proteins (CLiPs) are located between the sister chromatids along their length and between the kinetochores during metaphase. Consistent with a role i n sister chromatid cohesion, these proteins are no longer detectable by anaphase. CLiPs were identified by cross-reaction with human autoimmune sera (Rattner et NI., 1988). Inner centromere proteins, INCENPs, also have a localization pattern consistent with cohesion between the sister chromatids. Antibodies to the INCENPs were generated to mitotic chromosome scaffold fractions. The INCENPs are located between sister chromatids during metaphase, remain associated with metaphase plate during anaphase, and are focused in the midbody during telophase (C. Cooke er al., 1987; MacKay er 01.. 1993; Mackay and Earnshaw, 1993; Earnshaw and Mackay, 1994). Recently, an antigen that forms a ringlike structure at the centromere in human and Chinese hamster cells has been proposed to provide sister chromatid cohesion (Holland et al., 1995). Although all of these proteins exhibit localization patterns consistent with sister chromatid cohesion. none of these antigens have been demonstrated to be present on the chromosomes of meiotic cells, nor have they been shown to be required for sister chromatid cohesion in mitosis.
IV. Homolog Attachment and Segregation without Chiasmata Although the usual method of holding homologs together for disjunction in meiosis I involves chiasmata, diverse mechanisms have evolved to allow appropriate partitioning of chromosomes. Wolf ( 1994) recently reviewed a broad range of achiasmate segregational mechanisms. This discussion categorizes a few examples for context before focusing on segregation in the best characterized example of nonexchange segregation, the distributive system of D. riirlarzogaster.
A. Completely Achiasmate Meiotic Divisions
Homolog segregation in meiotic cells can be carried out without any chiasmata at all. Achiasmate meiosis using cytologically observed SCs occurs in oocytes of
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the silkworm, Bornbyx mori. Attachment between homologs is achieved by an adaptation of the SC. Late i n prophase 1, the SC is seen to be augmented rather than dissolved. This “elimination chromatin,” a sort of pseudochiasma, is left behind on the metaphase plate during anaphase I, consistent with a role holding homologs together for reductional division (Rasmussen, 1977). A well-characterized example of achiasmate meiosis without SC occurs in D. rnelanogastrr spermatocytes. The X and Y chromosomes are connected by a threadlike material at metaphase, and molecular analysis has delineated this pairing site to a specific sequence in the rDNA locus. Autosomal pairing sites have also been identified. All of these pairing sites carry out attachment between homologs without any exchange. This work has recently been reviewed by McKee (1996) (see also Chap. 3, this volume).
B. Nonexchange Chromosomes in Meiosis with Exchange
In cells that carry o u t chiasmate meiosis, particular chromosomes may be attached by other means. In particular, heterogametic meiosis involves sex chromosomes that are often largely nonhomologous and do not have exchanges, yet these chromosomes segregate faithfully (John, 1990). A variety of mechanisms have been cytologically characterized, ranging froin cohesive material that is not SC to chromosomes with microtubules attaching them (Wolf, 1994). 1. The Drosophilu Distributive System In D. tnelunogaster oocytes, nonexchange chromosomes have no cytologically obvious physical linkage during metaphase I (Therkauf and Hawley, 1992). Although most of the bivalents are bound by crossovers in meiosis I, the fourth chromosome is much smaller and does not undergo exchange, yet it is disjoined faithfully. The fourth chromosomes are often observed to be off the metaphase plate and located on the meiotic spindle midway between the plate and the poles. Moreover, the X chromosome is nonexchange 10% of the time, and any of the chromosomes are nonexchange when heterozygous for a homolog carrying multiple inversions; yet the oocyte is able to efficiently segregate any of these nonexchange chromosomes. Provocatively, mutations in a single gene, nod, allow nonexchange chromosomes to be lost from the spindle. The nod gene has been cloned and the N-terminal domain of the predicted protein was shown to share homology with kinesin, a microtubule motor (Zhang et ul., 1990). NOD protein has been localized along the chromosome arms and shown to associate with chromatin and microtubules (Afshar, Barton, et al., 1995; Afshar, Scholey, e t a / . , 1995). ‘Thus, a microtobule motor is required to act as an “attachment” between chromosomes
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during metaphase I by maintaining the half-bivalents in position on the metaphase spindle. Interchromosonial microtubules have also been suggested to play a role in linking the chromosomes (Carpenter, 1991). The requirement for NOD protein to stabilize chromosomes on the spindle still leaves open the question of how orientation of nonexchange chromosomes is achieved in prometaphase I . Bipolar orientation might first be achieved with a transient physical association, or orientation might occur without any physical linkage between the chromosomes. Evidence reviewed in the next section suggests that transient linkages may help to orient some, but not all, chromosomes in Drosqihila oocytes.
a. Nonexchange Homologs Associate in Prophase I. Recent results suggest that centric heterochromatin plays a part in orienting homologous nonexchange chromosomes during prometaphase I. In genetic experiments, centric heterochromatin was shown to be critical for segregation: rearranged chromosomes segregated from nonexchange partners that shared homology in centric regions (Hawley, hick, et a/., 1993); and minichromosomes derived from the X chromosome had decreasing ability to segregate from one another as the amount of overlapping centric heterochromatin was decreased (Karpen et al., 1996). FISH for centric heterochromatin of achiasmate homologs was carried out for the obligatory nonexchange fourth chromosome and, additionally, for X chromosomes heterozygous for multiple inversions so that exchange was suppressed. In both cases, the heterochromatic regions of these homologs were tightly associated. I n contrast, FISH for regions on the arms of chromosomes showed random distances, suggesting that diplotene is modified in D. melanogaster oocytes such that the bivalent is connected primarily at crossovers and at centric heterochromatin (Dernburg et a/., 1996). If this association near the centromeres continues into prometaphase I, it could provide an attachment that facilitates bipolar orientation. b. Heterologs Do Not Associate in Prophase I. Heterologous nonexchange chromatids also segregate from one another, but apparently without the benefit of actual pairing. In flat preparations of Drostiphilci oocytes, all of the heterochromatic regions were shown to be associated in a “chromocenter” during diplotene (Nokkala and Puro, 1976). However, FISH for centric heterochromatin of two heterolog~s,a compound second and compound third chromosome, demonstrated that they do not pair (Dernburg et al., 1996). Yet these heterologs segregate from each other seemingly without any physical linkage. A difference in the mechanism by which homologs and heterologs orient in Drosophila oocytes is consistent with genetic dissection of the distributive system. Two classes of mutations predominantly result in nondisjunction of nonexchange chromosomes. Axs, m e i - S S I , and ald disrupted segregation of nonex-
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change homologs only, whereas nod and Dub caused inissegregation of both heterologs and homologs (Hawley and Theurkauf, 1993; Moore et al., 1994). Genetics and cytology suggest that transient physical linkage helps to orient homologs. Heterologs do not have this transient attachment, and both mechanisms require functions by NOD protein to segregate appropriately.
2. Disjunction without Physical Attachment: The “Crowded Spindle” Model The “crowded spindle” model has been proposed to explain how chromosomes might be selected to segregate from one another without physical linkage (Hawley, McKini, e t a / . , 1993). This model was derived from observations on heterologous distributive segregation in Dro.sophi/a: ( 1 ) Disjunction of heterologs occurs in a competitive and preferential manner, so that introduction of a third heterolog disrupts segregation of two heterologs; (2) chromatids with similar sizes and shapes tend to disjoin (Grell, 1976); and (3) the distributive system has a limited ability to sort out chromosomes. It breaks down when more than four unpaired chromosomes are involved. As an example, mutations that reduce exchange result in nondisjunction of the fourth chromosome (Baker and Hall, 1976). In the crowded spindle model, a given nonexchange univalent is more likely to connect to whichever pole is not occupied by another univalent. Smaller chromatids have been observed to move poleward more quickly than larger chromosomes, and movement to a pole is impeded by the presence of other chromosomes on the half spindle (Theurkauf and Hawley, 1992). Chromosomes of similar size and shape with connections to the same pole will be in the most direct competition, so these will tend to reorient to balance the crowding at the poles. As the poles become more crowded with nonexchange chromosomes, this system would have less ability to influence other univalents, consistent with the limited ability of the distributive system. The spindle in Drosophila oocytes is unusually narrow and is organized by the chromatin (Theurkauf and Hawley, 1992), and this may account for some of the efficiency of the distributive system. Other species have been noted to have meiotic spindles that are organized by chromatin (Vernos and Karsenti, 1995). It will be interesting to discover if thesc species also have efficient systems of distributive segregation. The meiotic spindle is organized by spindle pole bodies in S. cerevisiae. In this species, the distributive system is less efficient and has been shown not to follow size and shape rules (Ross, Rankin, et a/., 1996). 117 s i f i i hybridization studies suggest that pairing occurs between nonexchange chromosomes that lack homologs (Loidl et cd., 1994). Perhaps a difference in the structure of the spindle accounts for the differences between the Dm.sopki/a distributive system and that found in budding yeast.
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V. Sister Kinetochore Function A. Sister Kinetochores Must Reorganize between Meiotic Divisions
The kinetochores of sister chromatids do not usually connect to opposite poles in the first meiotic division, yet they clo in meiosis 11, which suggests that there must also be a change in their structure between metaphase I and prometaphase I1 (Fig. 1). Darlington (1932) proposed that sister chromatids share a single kinetochore during meiosis I and thus must move to the same pole during anaphase I. The other possibility, the existence of differentiated sister kinetochores before prometaphase I, has also been proposed. In this model, the sister kinetochores must somehow be constrained in their behavior to make connections with the same pole (Nicklas, 1977). Because replication of DNA is completed well before metaphase I (Collins and Newlon, 1994), unreplicated centromeres cannot account for reductional segregation. If both the underlying DNA and the protein structures that make up kinetochores are duplicated by metaphase I, reductional segregation requires that the duplicated sister kinetochorcs be arranged to act as one functional microtubule attachment site for each half-bivalent. Cytological and functional evidence for sister kinetochore duplication prior to metaphase I are reviewed herc.
B. Cytological Observations of Sister Kinetochore Duplication
Kinetochores are cytologically defined structures. By this criterion, duplication of sister kinetochores has been described as occurring before metaphase I for many species. A progressive differentiation of the sister kinetochores of D. nielanogaster spermatocytes was described during prometaphase I: before microtubule connections are made, there is one structure, and as microtubules attach, there is an amorphous stage and eventually a double disc structure (Goldstein, 1981). A single kinetochore is shared by sister chromatids in early prometaphase I and duplicated by metaphase I in the crane fly, Pules ferrugineu (Muller. 1972), and in the marine worm, Urechis cuupo (Luykx, 1965b). Many species have been noted to have observably duplicated sister kinetochores by metaphase I. Limade-Faria (1956, 1958) observed distinct sister kinetochores in several plant and insect species during metaphase I. In the mouse, paired sister kinetochores were visible in colcemid-arrested metaphase I spermatocytes (Brinkley et ul., 1986). Thus, sister kinetochores appear to duplicate some time before anaphase I. In a few species, kinetochores have a duplicated appearance as early as prophase I and change to a singular appearance during prometaphase I. Two “spindle spherules” were visible in late diakinesis in cells from the salamander, Amphiumu triducr?./um (Schrader, 1936, 1939). Silver-stained chromosomes of several grasshopper species, Chorthippiis jucundus, E. ploruns, and Arcypteru fuscu, had
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clearly duplicated sister kinetochores in prophase I. They appear as two rounded structures, much like the kinetochores in the relatively relaxed period of anaphase and in contrast to the single conical structure shared by the sister chromatids during early metaphase I (Rufas r t a/., 1983, 1989: Suja er a/., 1991, 1992). These studies suggest there may be duplication of sister kinetochores as early as prophase I, although they appear as a single structure during prometaphase 1. a time when they need to act as a single unit.
C. Functional Differentiation of Sister Kinetochores
Does independent function of the sister kinetochores correspond with cytologically observable differentiation? There are instances in which sister kinetochores do function independently in meiosis I. When univalents are present or when mutations disrupt sister chromatid cohesion, the sister kinetochores are seen to be duplicated and capable of making spindle fiber connections with opposite poles. Univalents dividing equationally during meiosis I are frequently reported for a broad spectrum of species from plants to humans (Angel1 ct a/., 1994). In a wheat hybrid with an unpaired chromosome, during prometaphase I, sister kinetochores faced the same spindle pole, but late in nietaphase I, sister kinetochores developed connections to opposite spindle poles that allowed congrcssion to the metaphase plate with an equational bipolar orientation (Wagcnaar and Bray, 1973). In mouse females carrying a single X chromosome that was followed by FISH, the univalent divided equationally in meiosis I about one third of the time (Hunt rt a/., 1995). The behavior of univalents suggests that sister kinetochores can sometimes act as independent units and undergo equational division at anaphase I. Separated sister kinetochores are seen early in prometaphase 1 in ord homozygous Drosophi/u oocytes (Lin and Church, 1982). Genetic assays suggest that the sister chromatids segregate randomly during this division (Mason, 1976: Miyazaki and Orr-Weaver, 1992: Bickel ef d., 1997). Precocious separation of sister chromatids was observed in S. ponzhe that were homozygous for rc.c8-101, and the centromeric regions of chromosomes appeared to be unusually far apart during prometaphase I (Molnar et cil., 1995). These genes are believed to be required for sister chromatid cohesion, and both have functional sister kinetochores before metaphase 1. The simplest explanation is that sister kinetochores are functionally double before prometaphase I and that sister chromatid cohesion is required to constrain their shape into a functionally single kinetochore during prometaphase I. Alternatively, the ord and rec8 gene products may be required for at least two functions: providing cohesion of sister chromatids and preventing early functional differentiation of kinetochores. There is a caveat to these observations that duplicated kinetochores are capable of independent function: in all the examples just cited, anaphase I may be
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delayed an unusually long time, long enough that the sister kinetochores gain independent function. Univalents are known to delay anaphase 1. thereby lengthening metaphase I. Mutations in budding yeast that show low levels of precocious separation of sister chromatids also have been suggested to delay anaphase I past the transition to functionally differentiated sister kinetochores (Carpenter, 1994). For example, inedl, now known to be an allele of dnzcl and renamed d m l - l , causes a reduction in recombination, presumably resulting in univalents that might delay anaphase onset, and this results in low levels of tetrads resulting from precocious separation of sister chromatids (Rockmill and Roeder, 1994). Heterozygosity for a ring chromosome I11 in budding yeast was shown to result in precocious separation of sister chromatids for a normal chromosome VII, perhaps because mechanical probleins i n orienting the heterozygous ring chromosome delay anaphase I (Flatters et u/., 1995). It is possible that mutations disrupting sister cohesion similarly delay anaphase I. Nevertheless, functional duplication certainly can occur before anaphase I begins, and cytological observations of sister kinetochore duplication are seen in metaphase I unperturbed by the presence of univalents or mutations.
D. Early Functional Differentiation May Be Chromosome Dependent
Univalents in the same species show different abilities to orient and segregate equationally. In living spermatocytes of the grasshopper, Eyprepocnemis ploruns, chromosomes that usually exist as univalents, the X and B chromosomes, oriented with different dynamics and segregational results than did autosomal univalents that were induced by heat shock (Rebollo and Arana, 1995). In S. cerevisiae, chromosomal-dependent segregation behavior has been localized to sequences less than 1.6 kb in length that include the centromere (reviewed by Simchen and Hugerat, 1993). In certain mutant yeast strains, the majority of meiotic cells yield two-spored asci rather than four-spored asci, and these spores are diploid. This “single-division meiosis” has been characterized for four mutations, two that are meiosis-specific (spol2 and .spo13) and two that affect the mitotic cell cycle by arresting late in nuclear division (cdc.5 and cdcl4). Regardless of the mutation, mixed segregation of chromosomes occurs during the single-division meiosis, and the chromosomes have inherent tendencies toward equational or reductional segregation without regard for the absence or presence of exchange events on the chromosome. The tendency does not correlate with chromosome size. Replacement of the centromere region changed the chromosome’s inherent tendency, such that the engineered chromosome segregated with the tendency of the replacement centromere region. Heterocentromeric bivalents often yielded trisomic spores, suggesting mixed segregation even within a single bivalent (Simchen and Hugerat, 1993). Future studies of these
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centromere sequences may reveal what makes sister centromeres more or less functionally autonomous during the first meiotic division.
VI. Maintaining Attachment between Sister Chromatids for Meiosis I I A. Cytology Shows That Attachment Persists in Proximal Regions
Kinetochores of sister chromatids are aligned on the metaphase plate in meiosis
IT, suggesting that the attachment between sister chromatids during metaphase I1 is at or near the kinetochore (Fig. 1E). During anaphase I, the portions of sister chromatid arms that are distal to a reciprocal crossover segregate away from each other and must lose cohesion distal to the exchange. Lima-de-Faria (1956) observed for a variety of species that “the structures depicted at anaphase I show the kinetochore divided at this stage, the most proximal regions of the arms being also responsible for holding together the sister chromatids.”
B. Equational Nondisjunction Resulting from Proximal Exchange
Because exchange occurs during meiosis I, it is not obvious that alteration of recombination might result in errors in meiosis 11. However, recent studies in humans and Drosophilu suggest that exchange occurring in the proximal region, or perhaps an increase in number of exchange events, increases the likelihood of equational nondisjunction. Human trisomy for chromosome 2 1 resulting from maternal meiosis I1 errors showed an increase in the overall amount of exchange, and the chromosomes commonly had undergone a proximal exchange (Lamb et al., 1996). Proximal exchanges were even more common in human X-chromosome nondisjunction events, although overall exchange was slightly reduced (MacDonald er al., 1994). Chromosome 18 derived from human trisomy showed an increase in map length, but the locations of the exchanges were not reported (Fisher et al., 1995).In Drosophilu females, the X chromosome had a remarkably similar pattern. Meiosis I1 errors were often correlated with multiple exchanges and exchange in the proximal region (Koehler, Boulton, et al., 1996). Two explanations have been put forward. In the first model (Fig. 6A), the resolution of proximal chiasmata in meiosis I would involve a loss of proximal cohesion, and this might increase the likelihood that sister chromatid cohesion is lost completely, resulting in nondisjunction during meiosis 11. In the second proposal (Fig. 6B), crossovers in the proximal region may result in continued attachment between homologs if sister chromatid cohesion in the region is not released. The bivalent would be unable to separate and thus would segregate in its entirety to one pole. The intact bivalent might segregate reductionally in the second division and yield gametes disomic for sister chromatids. Thus, an appar-
29 1
8. Meiotic Chromosome Segregation
nn
'1
MI
MI1
-+-
Fig. 6 Proposed mechanisms by which proximal exchange might yield gamete\ diaomic for sister chromatids. ( A ) Sister chromatid cohc\ioii i n proximal regions may be lost when proximal crossovers are resolved during anaphase 1. Somc sistci- chromatids may then lack cohesion necessary to segregate pi-operly in meiosis 11. ( B ) S i w r chromatid cohesion in proximal regions may persict so that proximal croswvers cannot be easily resolved during anaphase I . The intact bivalent segregates to one polc. and reductional segregation i n n y occur during meiosis 11.
ent meiosis I1 nondisjunction would actually result from meiosis I nondisjunction (Koehler, Hawley, et al., 1996).
C. Possible Mechanisms of Cohesion in the Centromeric Regions
Attachment between the sister chromatids is maintained through the first meiotic division, although sister chromatids lose cohesion along their arms. Either a mechanism of sister chromatid cohesion is unique to the centromeric region of half-bivalents or sister chromatid cohesion is specifically protected at the centromeric region until anaphase 11. Possible mechanisms of cohesion again include catenation of the DNA or structural proteins. There is not any evidence that catenation binds sister chromatids during metaphase 11. 3. cereiiyiae cells that have undergone meiosis I without exchange and without functional topoisomerase 11 first become cells with two nuclei and then
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eventually become cells with more than four nuclei (Rose and Holm, 1993). These multinucleate cells suggest that topoisomerase 11 is important for the second meiotic division. However, topoisomerase I1 was shown to be required for successful segregation during anaphase I in meiosis with exchange, so it is likely that resolution of catenation on sister chromatid arms is simply delayed until the second division. There is no evidence that catenation in the centromeric regions provides sister chromatid cohesion until anaphase 11. D. Mutations That Disrupt Cohesion for Meiosis II
The Drosophil~iMEI-S332 protein is necessary to maintain the bond between sister chromatids after metaphase I. In mei-S332 mutants, genetic assays of segregation in both males and females revealed low levels of meiosis I nondisjunction and high levels of meiosis 11 nondisjunction. In mutant spermatocytes, meiosis appears cytologically normal until the sister chromatids separate prematurely during anaphase I. Segregation in anaphase I1 is random, the result of the inability to orient in metaphase I1 (Davis 1971; Kerrebrock et al., 1992). The MEIS332 protein localizes to the chromosomes in a manner consistent with a role in maintaining cohesion after the metaphase Vanaphase I transition. As the chromosomes condense and begin prometaphase I, MEI-S332 localizes at discrete loci on the chromosomes. During anaphase I, the protein is clearly located on centromeric regions of segregating chromosomes. MEI-S332 remains on the chromosomes until metaphase 11, but is dispersed or destroyed at the beginning of anaphase 11, when sister chromatid cohesion is released (Kerrebrock et al., 1995). The sister chromatids are presumably attached at their kinetochores before MEI-S332 localizes to the chromosomes, so the protein either augments cohesive structures already present in the proximal regions or acts to protect the cohesive structures until anaphase 11. ME13332 differentiates the regions near the centromeres from the rest of the chromosome arms. It could supplement, replace, or preserve cohesive proteins that extend the length of the sister chromatids, or it could prevent resolution of DNA catenation in the centromeric regions. A phenotype similar to that of mei-S3.?2 was observed in the tomato, Lycopersicon esculenturn. Plants homozygous for the p c mutation are infertile but have no cytologically observable effect on chromosome pairing or chiasmata formation. However, separation of the sister chromatids is visible as early as anaphase I (Clayberg, 1959), pc and mei-S332 are the best candidates for genes encoding cohesive proteins acting at the centromeric regions of dyads.
VII. Summary The structure of the bivalent is critical for successful segregation of chromosomes in meiosis. In particular, attachments between chromosomes and the ar-
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rangement of kinetochores are vital for achieving bipolar orientation on the spindle. Chiasmata usually serve as the attachment between homologs for the first meiotic division. These crossovers between homologs are likely held in place by sister chromatid cohesion along the arms. Binding substances localized to the crossovers may play a role in chiasma maintenance. Current cytology and genetics does not eliminate either of these models, but failure in maintaining sister chromatid cohesion best explains why missegregation most often results from distal crossovers. A variety of mechanisms have evolved for the reductional division that do not require exchange between homologs, and in Drosophila females, some partitioning of chromosomes is carried out without physical attachments. After the first meiotic division, sister kinetochores must reorganize, and attachments between homologs must be relinquished while attachments between sisters are maintained. Sister chromatid cohesion in the centromeric region is preserved for the second division.
Acknowledgments The authors thank Anthony Schwacha, Todd Milnc, Heidi LeBlanc, Andrea Page, and Wes Miyazaki for helpful discussion of the manuscript. This work was supported by grants from the National Science Foundation, the March of Dimes Birth Defects Foundation, and the Council for Tobacco Research. Inc.
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Rasmussen, S. W. (1977). The transformation of the synaptonemal complex into the “elimination chromatin” in Bornhw rnorr oocytes. Chmiirosorncr 60, 205-22 I . Rasooly, R. S., New, C. M., Zhang, P., Hawley, R. S., and Baker, B. S. (1991). The lefhal(/)TW-&’mutation of Drosrphilcr r~relnnogcr.\ter is a dominant antimorphic allele of nod and is associated with a single base change in the putative ATP-binding domain. G ~ J I P I129, ~CS 409-422. Rattner, J. B. (199 I ). The structurc of the mammalian centromere. Rioc~ssnys13, 5 1-56. Rattner, J. B., Kingwell, B. G., and Fritzler, M. J. (1988). Detection of distinct structural domains within thc primary constriction using autoantibodics. Chrornosoriia 96, 360-367. Rebollo, E., and Arana, P. (1995). A coinparative study of orientation at behavior of univalent in living grasshopper spermatocyte\. Chroriiosorrirr 104, 56-67. Rieder, C. L. ( 1982). The formation, structure, and compo4tion of the mammalian kinetochore and kinetochore tiber. In!. R m Cyrol. 79, 1-58. Rockmill, B., and Roeder, G. S. (1990). Meiosis i n asynaptic yeast. Gerierics 126, 563-574. Rockmill, B., and Roeder, G. S. ( 1994). The yeast rrredl mutant undergoes both meiotic homolog nondisjunction and precocious separation of sister chromatids. Generics 136, 65-74. Roeder, G. S. (1995). Sex and the single cell: Meiosis in yeast. /‘roc. Ncrtl. Accrd. Sci. USA 92, 10450- 10456. Rose, D., and Holm, C. (1993). Meiosis-specific arrest revealed in DNA topoisoinerase II mutants. MO/.cell. Bi<J/.13, 3445-3455. Ross, L. O., Maxlield, R., and Dawwn, D. (1996). Exchanges are not equally able to enhance meiotic chromosome segregation in yeast. Proc.. Nor/. Acud. Sci. USA 93, 4979-4983. Ross, L. O., Rankin. S., Shuster. M . F., and Dawson, D. S. (1996). Effects of homology, \ire and exchange on the meiotic segregation of model chrommomes in Srrcc hororri,vce.\ cert~i,i.sioe.Gcnetics 142, 79-89. Ross, L. O., Treco, D., Nicolas, A,, Szostak, J. W., and Dawson, D. (1992). Meiotic recornbination on artificial chrornoaomej in yea\t. generic..^ 131, 541-550. Rufas, J. S., Gosalver. J., Gimenez-Martin, G.. and Esponda, I? ( 1983). Localiration and devclopment of kinetochores and a chromatid core during meiosis in grasshoppers. G~rirtic.cr61, 233-238 Rufas, J. S., Mazrella, C., Sujas, J . A,, and Garcia de la Vega, C. (1989). Kinetochore structures are duplicated prior to the tirst meiotic metaphase: A model of meiotic behavior of kinctochores in grasshoppers. Eur. J . Cell H i o l . 48, 220-226. Schrader, F. ( 1936). The kinetochore or spindle fiber locus in A ~ t i p h i r ~ i /rr&rc/drm. ii Rrol. Bull. 70,484-498. Schrader, F. ( 1939). The structure o f the kinetochore at meiosis. Chrorno.soiii~1, 230-237. Schwacha, A,, and Kleckner, N. (1994). Identification of joint molecule\ that form frequently between homologs but rarely between sister chromatids during yeast meiosis. Cell 76, S I --63. Schwacha, A,, and Klcckner. N. ( 1995). Identification of double Holliday junctions as intermediates in meiotic recombination. Cell 83, 783-79 I . Sherman, S. L., Pctersen, M. B., Freeman, S. B., Hersey, J., Pettay, D., Taft, L., Frantzen, M., Mikkelscn, M., and Hassold, T. J . ( 1994). Non-disjunction of chromosome 21 in iniiternal meiosis I: Evidence for a maternal age-dependent mechanism involving reduced recomhination. Hum. M o l . Gene/. 3, 1529-1535. Simchen, G.. and Hugerat, Y. ( 1993). What determines whether chromosomes segregate reductionally or equationally in meiosis’? Bioc~.\.ccr,v.s1.5, 1-8. Solari, A. J. (1970). The behaviour of chromosomal axes during diplotene in mouse spermatocytes. Chrorrrosorritr 31, 2 17-230. Suia, J. A., de la Torre, J., Gimencr-Ahian, J . F., Garcia de la Vega, C.. and Rufas. J. S. (1991). Meiotic chromosome structure: Kinetochores and chromatid cores in standard and B chromosomes of A r q J t r r r I ,fii.scrr (Orthoptera) revealed by silver staining. Gcworrie 34, 19-27
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Sti,jn. J . A,, Antonio, C., and Rulas, I . S (109’2) Involvement of chromatid cohesivene\\ ;it the centromere and chromosome ai-m\ i n iiieiotic chroinosorne segregation: A ctyological LIPprozich. C/iromo.somf 101, 493-50 I Syin, M.. and Roetler. G. S. (1994). <‘i-o\\ovcr interlcrence is abolished in the absence of a synaptonetnal complcx protein. Cell 79. 2x3-292. Tease. C., and Jones, G. ( 1978). Analysi\ of exch;inge\ in differentially stained meiotic chromosomes of Loc,rt.s~tr rui,qrurorici after 13rdU-\ub\tittition and FPG staining. I. Crossover exchanges in monochiasmate bivalents. Cliroirio.soui(f 69, 163- 178. Thcurhauf, W. li., and Hawley, R. S ( l90’2). Meiotic \pindle assembly in Drosophila females: Beharior of nonexchange chronio\oiiic\ and Ihc eflects of mutations in the iiod kinesin-like protein. J . Cell Riol. 116, I 167- 1 IXO. Vcrno\, I.. and Karseiiti. E. ( 1995). Chroinosome\ take the lead i n spindle assembly. Trerith ( F I I . B i d . 5, 297-301. \ o n Weltstein, D.. Rasrnusaen, S. W.. iind Holm, P. H . ( 1984). The \ynaptonemal complex i n genetic segregation. A m u . Kns. Geucr. 18, 33 1-4 13. Wagenaar, E. B., and Bray, D. F. ( 1973). ‘The ultr;i\tructure of kinetochores of unpaired chromosome\ in a wheat hybrid. Ctrii. J . Gwr. C).ro/ 15, 801-806. White. M. J. D. ( 1961). Cytogenetic\ o f tlie gra\hliopper Morcihu .\curru. VI. A spontaneous pericentric in\-ersion. Alc.rr. J . Zoo/. 9, 7x4-790. Wolf, K. W. ( 1994). How meiotic ccII~rtical with nowexchange chromosomes. Rioessuy.\ 16, 107113. Zhang, P.. Knowlca, B. A,. Goldstein, 1.. S. B.. and Hawley. R . S. (1990).A hinesn-like protein required for di\tributive chroinosoinc wgrcgatioii in Drosophila. Cell 62, 1053- 1062.
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9 Regulation and Execution of Meiosis in Drosophh Males lean Maines and Steven Wasserm'in Department of Molecular Biology and Oncology University of Texas Southwestern Medical Center Dallas. Texas 75235-9148
I . Introduction A . Overview of Sperrnatogene\is €3. Morphological Description o f Meio\i\ C. Identification of Male Meiotic Mutant\ IT. Regulation of the Meiotic Cell Divisions A. Entry into Meiosis B . Regulation of the Cell Cycle Machinery C. Coordination of Meio\i\ with Diflerentiation D. Regulation of the Second Meiotic DiLision Ill. Spindle Formation and Function i n the Meiotic Cell Divisions A . Meiosis-Specific Spindle Ai-chitectui-e €3. Regulation of Spindle Forination IV. Cytokinesis A. Contractile Ring Assembl) and Function B . Cytokinesis in the Drotophilo Germ Liiir and i n Budding Yeast V. Conclusions and Perspective\ References
In this chapter we rcview the regulation and execution of the meiotic cell divisions in the cuntext ol the developmental pi-ogrm that comprise\ Dro.sophi/tr spermatogenesis. Male germ line cells undergoing meinsi\ are readilq identifiable and are of a size and abundance that make\ this system well suited lor rnnrphological characteriLation5 of cell d i v i h n Furthermore. a wide range of molecular genetic techniques are available, facilitating rnechaniqtic investigations. We present an overview of key \rages in spermatogenesis and, in particular, meiosis. We consider the pathways controlling entry into the meiotic divisions in the context of established cell cycle regulators as w e l l as newly identified loci required for meiotic entry. We then review the assembly and function ot both the meiotic spindle and the contractile ring. We conclude with a con5ideration o l que\tioii\ and problems that await further investigation. Copyright 0 199X by Academic Pt-c\s.
1. Introduction In this chapter we review the regulation and execution of the meiotic cell divisions in Drosophila males. In the past 5 yrs a number of new meiotic mutants 301
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have been identified and the gene products of several loci required for male meiosis have been identified. These genetic and molecular analyses have revealed substantial similarities between meiotic regulation in Drosophila and vertebrates and between cytokinesis in Drosophilu and the budding yeast. As a model system for the study of meiosis, Drosophila spermatogenesis is particularly valuable. The isolation and identification of mutations, ordering of gene activities, and molecular characterization of loci of interest are all readily accomplished. Drosophilu meiosis occurs in the context of a complex developmental program and is therefore likely to provide insights into the regulation of meiosis in higher eukaryotes, including vertebrates, distinct from those gathered by studying the corresponding process in yeast. Furthermore, Drosnphila germ line cells undergoing meiosis can be identified easily and are of a size and abundance that makes them well suited to the investigation of mechanisms for the segregation of chromosomes and the partitioning of cytoplasmic contents during cell division. Together, the availability of a range of molecular genetic techniques, the developmental setting in which meiosis occurs, and the accessibility and dimensions of dividing cells make this system a unique and valuable subject of study for probing the regulation and execution of meiosis. We begin our review by presenting the key morphological characteristics of the stages of spermatogenesis and, in particular, of the meiotic divisions. We turn next to the regulation of entry into the meiotic divisions, then to the assembly and function of the meiotic spindle and t o the execution of cytokinesis. We conclude with a consideration of questions and problems that await further investigation.
A. Overview of Spermatogenesis
In presenting our overview of spermatogenesis, we draw heavily on several excellent reviews available i n the published literature (Cooper, 1965; Lindsley and Tokuyasu, 1980; Fuller, 1993), as well as on the elegant characterization of the spermatogenic stem cell populations by Gonczy and DiNardo (1996). In adults, spermatogenesis takes place within each of two coiled testis tubules. The coordinate activities of two types of stem cells, anchored at the apical tip of each tubule, initiate spermatogenesis (Fig. 1). A germ line stem cell divides asymmetrically, generating a blast cell, the spermatogonium, and the regenerated stem cell. Similarly, the asymmetrical division of two somatic cyst progenitor cells produces a pair of cyst cells and a pair of regenerated stem cells. The cyst cells, which do not divide further, encyst the spermatogonium and its progeny until the completion of sperm differentiation. Four mitotic divisions of the spermatogonium result in a cyst of 16 primary spermatocytes. These divisions occur with incomplete cytokinesis, such that all 16 spermatocytes are interconnected by cytoplasmic bridges or ring canals. Following the fourth gonial division, spermatocytes carry out premeiotic DNA synthesis and enter an extended G, or growth phase.
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Fig. 1 Schematic representation of landiiiai-k \t;igc\ of spermatogenesis in I l t n \ f ~ p / f i /!nf l e / n r f f J ~ t r c . rer. Lettered drawings are intended t o convey the appearance of the germ line cells as they appear in phase-contrast microscopy of unfixed testis contents. The twu somatic cyst cells that surround each group of germ cells are also Yhown but arc n o t readily v i s u a l i d by phase contrast. Open circles rcpre5ent nuclei, Germ cell cytoplasm is shown in gray: somatic cells are white. Nucleoli arc shown a\ black spots in (a-d); the black \pots i n niiclei i n i c ) represent protein bodies. The accompanying arrows and line drawing of a testis tubule indicate the approximate location within the testis where each stage begins The apical tip is to the left; the basal end, which in L,iw connects to the seminal vesicle, is shown at the center of the coil. Fully elongated spermatids have their nuclci at the basal end; their tails sti-etch nearly to the apical tip, a distance of about 2 mni. ( a ) Gel-ni line s t e m cells and somatic cysl pi-ogenitor cells are attached to a specialixd structure of wtiiatic origin. the hub (Hardy er u / . , 1979). The gcrm line stem cell is closely associated with two cyst progenitor cells. These stem cells divide ;isymiiictrically. (b) The single rpermatogonium and the two cyst cells arc released, while the parental stem cells (one germ line, two somatic) remain attached to the huh. ( c ) Four rounds of mitotic division with incomplete cytokinesis result in a cyst of 16 early primary qxrmatocytes. These spermatocytes enter i t growth phase, during which time they increase in volume 25-fold. (d) The late primary sperinatocqtes undergo two meiotic divlslons to yield a cyst of 64 haploid spermatids ( c ) The nuclei reform (open circles) and the mitochondria fuse to form the nebenkern (solid circles). At the onion stagc. s o called because of the multilamellate appearance of thc nebenkern in electron microgniphs. hoth of thcsc structures arc closely associated and highly uniform i n size and shape. ( f ) Each spermatid is tr;in\lormed into a mature sperm through a complex proccrs o f cytodifferentiation (Tokuyasu t'r ( I / . . I 972a.h). This process involves nuclear condensation a well as the dramatic elongation of the axoneme and the mitochondria1 derivative t o form the sperm tail. The linal steps of spermatogenesis arc individuali~;ltionand coiling. (Adapted with permission from Ca\trillon f'( d . . 1993.)
During the growth phase, transcription occurs at a high level as spermatocyte volume increases 25-fold. A wide variety of genes active in many aspects of spermatogenesis are transcribed during this time (Olivieri and Olivieri, 1965; Hoyle and Raff, 1990; Schafer et ( I / . , 1990: Eberhart and Wasscrman, 1995). In
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contrast, transcription is nearly undetectable during meiosis and poslmeiotic differentiation (Brink, 1968). Furthermore, analysis of 70 male-sterile and 4200 fertile enhancer trap insertions revealed no examples in which the onset of reporter gene expression occurred after the growth phase (Gonczy e t a / . , 1992; P. Gonczy, personal communication). It is therefore believed that both meiosis and postmeiotic differentiation rely on the RNAs transcribed during this spermatocyte growth phase. The first and second meiotic divisions (MI and MII) occur in rapid succession upon completion of the growth phase. The MI division is reductional, segregating the XY pair as well as the three autosonial pairs. The MI1 division is equational and in general closely resembles a mitotic division. No meiotic recombination occurs in Dr-mophila males. Classic meiotic stages such as leptotene, zygotene, and pachytene are therefore absent. As in the spermatogonial mitotic divisions, cytokinesis in both meiotic divisions is incomplete. The product of meiosis is thus a cyst of 64 interconnected haploid spermatids encysted by two somatic cells. Following completion of meiosis, the haploid germ cells carry out an elaborate program of differentiation. The nucleus in each spermatid reforms and the mitochondria fuse to form the mitochondria1 derivative, or nebenkern. As each spermatid bundle migrates toward the base of the testis, the nuclei are reduced in volume and transformed in shape from spheres to long, thin cylinders. At the same time, the developing axoneine provides the foundation for generation of the sperm tail and is flanked by the elongating nebenkern. Sperm individualization and coiling ensue, followed by the mature spermatozoa exiting the basal end of the testis and entering the seminal vesicle for storage. Morphologically, the overall process of spermatogenesis has been highly conserved during evolution (Tokuyasu et ul., 1972a.b). Moreover, there are many similarities in the regulation and execution of male germ line development among a wide range of species. Common features of spermatogenesis in flies and mammals include, but are not limited to, the maintenance of a germ line stem cell population, a proliferative spermatogonial stage, a functional interaction between germ line and somatic cells in the testis, the packaging of spermatocyte KNA for later translation, the ordering of the reductional and equational meiotic divisions, and the presence of cytoplasmic bridges among clonally related groups of spermatogonia, spermatocytes, and spermatids. Furthermore, evidence has begun to accumulate that the underlying molecular mechanisms are also conserved.
B. Morphological Description of Meiosis The analysis of meiosis in Drosophiln males is facilitated by a regular progression in time and space of the stages of gametogenesis. The number of stem cells, the frequency of stem cell division, and the length of each stage are such that all
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stages of spermatogenesis are often present in a single testis. Furthermore, the stages of spermatogenesis are arrayed in a tixed progression along the length of the testis. Within each cyst, developmental events including meiosis are nearly, but not totally, synchronous, occurring as a wave sweeping across the cyst (Lifschytz and Hareven, 1977; see Fig. 2). The study of meiosis in Dro.sophi/ii inales depends largely on the stereotypical morphology of germ cells before, during, and just after the two meiotic divisions (Tates, 1971; reviewed by Fuller, 1993). No systems are currently available for analyzing meiosis in isolated g e m cells or in testis culture. However, both the course and outcome of the meiotic divisions can be readily observed in live samples prepared from dissected testes (Kemphues et a/., 1980: M. Fuller, personal communication). The meiotic divisions occur along the inside of the first coil of the testis, about one third of the longth from the apical end (see Fig. 1 ). By making an incision near this point in the testis and allowing the contents to spill out, one can release cysts in meiosis, as well as those in slightly earlier or later stages. The preparation is then gently squashed under the weight of a coverslip as liquid is blotted from the edges of the glass. Much of spermatogenesis can be characterized directly in unfixed and unstained cysts under phase-contrast optics, although detailed analysis of chromosome behavior requires staining with aceto-orcein or Hoechst dye (reviewed by Gatti and Goldberg, 1991). The chronology of spermatogenesis presented in the subsequent discussion is based on the riiorphological characteristics of unfixed germ cells, as well as on the results of D N A staining and of immunohistochetnical labeling of tubulin, lamin, and cyclin A (Cenci rt d., 1994; Gonczy ef a/., 1094; Lin et al., 1996). Representative phase-contrast micrographs are shown in Fig. 3; micrographs of imniunostaincd samples are presented in Fig. 3. In the testis, late primary sperniatocytcs at the end of the growth phase have a large nucleus with a prominetic nucleolus (Fig. 2b). DNA staining at this stage reveals the XY pair and the paired bivalent second and third chromosomes as distinct structures at the nuclear periphery (Fig. 3a); the bivalent fourth chromosome is largely heterochromatic and is too small to be readily observed. In early proinetaphase, the nucleus rounds up, the nucleolus becomes smaller and paler, and a cytoplasmic aster-like array appears at one side of the nucleus (Fig. 2c, arrows). The chromosomes begin to condense, frequently at different rates, resulting in one round and one oblong pair of autosomes. Later in proinetaphase, the nucleolus breaks down as the nucleus develops an irregular shape. The centrosome divides in two and moves along the outer nuclear envelope to form the two poles of the meiotic spindle (Fig. 3b). The mitochondria are arrayed along the long axis of the nasccnt meiotic spindle (Fig. 2d). Chromosomes continue to condense and begin to t i i o \ ~from the nuclear periphery toward the inetaphase plate (Fig. 3c). Cyclin A, previously exclusively cytoplasmic, becomes nuclear. At inetaphase, the distinction between the nucleus and the cytoplasm blurs as
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the nuclear lamins disperse (White-Cooper P I ul., 1993; Eberhart and Wasserman, 1995). The bivalent chromosomes condense completely and become attached to spindle fibers emanating from asters located at opposite sides of the nucleus (Fig. 3d, upper cell). Cyclin A degrades (Giinczy et d., 1994) and homologous chromosomes segregate. Chromosomes move rapidly toward the poles i n anaphase of MI (Fig. 9 e ; Fig. 3d, lower cell). The central spindle, composed of interdigitated microtubules, becomes prominent late in anaphase (Fig. 3e). At telophase, the spindle is squeezed into an hourglass shape. Daughter nuclei are again distinct from the cytoplasm, and mitochondria aligned along the spindle are evenly divided into the daughter cells during cytokinesis. The very brief interphase between MI and MI1 is visualized by the decondensation o f the chromosomes and the enlargement of the nuclei (Fig. 2f, arrowhead). Beginning with recondensation of the chromosomes. MI1 proceeds largely as MI (Fig. 2g), with the significant difference that sister chromatids, rather than homologs, segregate. Each of the two meiotic divisions lasts about 1 hrs. counting from early prometaphase to the end of telophase (Cenci et al., 1994). Following MII, chromosomes decondense and spermiogenesis, a process encompassing the postmeiotic stages of spermatogenesis, begins (reviewed by Tates, I97 1 ; Lindsley and Tokuyasu. 1980; Fuller, 1993). The individual mitochondria in each spermatid condense into a dark, spherical nebenkern (Figs. 2h,i). Next to each nebenkern, and roughly equal in size and shape, lies a pale nucleus. Because nuclear diameter in early spermatids correlates with chromosome content (Gonzalez et a / . , 1989), variable nuclear size is indicative of a defect in chromosome segregation. Similarly, since inhibition of cytokinesis
Fig. 2 Meiosi\ in the male germ linc. pharc-conti-a\t light micrographs. The contents o f dissected testes were gently squashed under a coverslip and viewed by phase-contrast microscopy. ( a ) Young spermatocytes (small arrow) and polar priiiiary sperniatocytes (large arrow) froin the apical tip of the adult te\tes. (b) Mature primary spermatocytcs. ( c ) Primary spermatocytej entering the first meiotic division (MI). (d) Cyst of 16 spermatocytcs i n promctnphase (P) to nietaphase (M) ofM1. The spindle and chromosomes are in the clear region in the center of each cell and are outlined by phase-dense layers of membrane and mitochondria (ni) aligncd along the periphery of the spindle. (e ) Cyst of 16 cells i n metaphase ( M ) . anaphase ( A ) . and telopliaw (T) of MI. Note the characteristic gradient of meiotic stages within a cyst. ( f ) Cyst of secondary spermatocytes i n interphase between MI and MII. The nuclei (arrowhead) have reformed, and cytoplasmic components (large arrow) are clustered around them. Some cells have begun tlic \econtl niciotic division (small arrows) (g) Cyst of 32 cells i n MII. As in MI. membranes and mitochondria \urround the clear region that contains the spindle. The two meiotic divisions are distinguishable froin one another by the size and number of cells i n a cyst. ( h ) Cyst of early spermatids at the co;~Ie\ccnc~'or agglomeration stage inimediately after the completion of MIL Mitochondria (ari-ow) have hegun to aggregate to one side of each nucleus. ( I ) Cyst of onion-stage early spermatids. Thc initochondria in each cell ha iggregated to form the mitochondria1 derivative or nebenkern, ii \trtictui-c con\isting of wrapped layer\ of membrane. The nebenkerne (arrow) appear as dark circles iidjaccnt to the white nuclei (arrowhead). Scale hars = 10 p m . (Reproduced with permission froni I~iillci-.1993.)
Phase-Contrast
Tubulin
Hoechst
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Late Meiotic Prophase
Prometaphase
Metaphase
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Fig. 3 Meio\is in the male germ line: Indirect iininunotluore\ccnce. immunocheinical laheling. and phase-contrast micrographs. Testes were disseclcd and hxcd. then stained with antitubulin antibodie\ and the DNA-binding dye Hoechst 332.58 prior to exaniinatioii. ( a ) A niaturc pi-iniary spei-matocyte; the nucleolus (arrow) is \urrwnded by the sex chromosvine chrornatin. ( h ) Cell 111 Iatc n i e i ~ t ~ c prophase of MI. ( c ) Cell i n prometaphase of MI. The nucle;~r~cytoplnsiiiic deniarcatioii is n o longer visible. ( d ) Cells in inetaphase (top) and early niiapha\c (bottom) o f MI. ( e )Cells in late anaphase of MI. In the tubulin panel. note the proininent centiiil spindle: 111 the pha\c-contrast and Hoech\r panels note the characteristic arrangeinent 01' mitochondria along the central \pindle. Scale bar = 10 k m . (Adapted with permission 01' the Coinpany 01 Hiologis~sLld. from Cenci er ( I / . , l W 4 )
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leads to the aggregation of the four nebenkerne into a single mass (Liebrich, 1982). a cytokinesis defect results in early spermatid cells with two, or frequently four, nuclei associated with a single nebenkern. Thus the composition of early spermatid cysts serves as an excellent indicator of any lack of fidelity in the meiotic divisions. C. Identification of Male Meiotic Mutants
Male meiotic mutants can be identified in several ways. Mutations that affect the execution of meiosis are identified by using genetic markers to monitor defects in chromosome disjunction (reviewed by Hawley, 1993). Although such mutants are very useful for identifying genes involved in karyokinesis (see Chap. 8, by D. P. Moore and T. L. Om-Weaver, this volume), this approach only identifies viable and fertile mutants. In another approach, a primary screen identifies male sterile mutants. Mutations that afiect the regulation of meiosis are then selected from the male-sterile set by dissecting testes from each mutant line and examining the germ line cells under the light microscope (e.g., Castrillon et a/., 1993). Traditionally, mutations have been generated with ethyl methane sulfate (EMS), an alkylating agent that generates point mutations. Efficient and random mutagens such as EMS provide the ability to conduct screens to saturation and to create alleles of differing strengths, including null mutations. More recently, insertional tnutagenesis with the P transposable element has been used to generate physically tagged, genetically marked mutations that are easily mapped and analyxd molecularly (Cooley c'r ( I / . . 1988). Although this mutagen shows site selectivity, it allows for straightforward cloning and the ability to induce null mutations by imprecise excision.
II. Regulation of the Meiotic Cell Divisions A. Entry into Meiosis
Genetic evidence has demonstrated that meiotic cell division in Drosophila males is governed by the well-established regulators of the mitotic cell cycle identified in yeast, frogs, and other species (reviewed by Nurse, 1990). Studies by Nurse, Hartwell, and others identified members of the p34/cdc2 kinase family and the cdc25 phosphatase family as being essential in yeast for initiation of mitosis (Hereford and Hartwell, 1974; Nurse and Thuriaux, 1980; Nurse and Bissett, 1981). Similarly, function of Lhictlc2 and the cdc25 homolog tn,irie is essential in Drosophiln males for the onset of the meiotic cell divisions (Jimenez et al., 1990; Lehner and O'Farrell. 1990; Alphey et d., 1992; Courtot et al., 1992: Stern et a/., 1993). The role of the conserved cell cycle oscillator in regulating the spermatogenic meiotic divisions is most readily evident for the tbt.t'inr locus; because this locus is
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not required for mitosis, mutations in tMine do not affect the somatic development or viability of the fly (Alphey et id., 1992; Courtot et al., 1992; WhiteCooper et d . , 1993). Flies mutant for mine are sterile; no meiotic figures are observed in twine males, and no products of meiosis are detected. Premeiotic stages of spermatogenesis, including the mitotic divisions. appear phenotypically normal in tctG/ie mutants. Chromosome condensation begins, centrosomes duplicate, cyclin A moves into the nucleus, and the nucleolus breaks down. However, chromosome condensation is incomplete, cyclin A fails to degrade, and chromosomes remain at the nuclear periphery. In addition, centrosomes do not separate and the spindle does not form. Dmcdc2 is required for mitosis; mutations result in developmental defects and larval lethality (Stern et d.. 1993). Lehner and colleagues therefore generated a conditional Dmcdc2 allele to investigate its role in meiosis (Sigrist et a/., 1995). Using information from yeast studies, they engineered a temperature-sensitive Dincdc2 allele and introduced this transgene construct into a genetic background mutant for Dmcdc2. The meiotic defects in spermatocytes expressing D1ncdc2~$ at the nonpermissive temperature closely resembled those in the tM'ine mutant. Chromosomes partially condensed, but did not congress at the metaphase plate or attach to asters. Cyclin A persisted until well after the onset of spermatid differentiation, indicating an arrest prior to the activation of the cyclin A degradation pathway. Collectively and individually, the twine and Dmcdc2 phenotypes indicate a block in the execution of metaphase of the first meiotic division. The biochemical function of both Dmcdc2 and twine in the onset of the meiotic cell divisions has been inferred from studies of mitotic homologs in other systems. The activity of cdc2 kinases is controlled by their association with a positive regulatory subunit, cyclin, and by a specific pattern of phosphorylation (reviewed by Nurse, 1990: Solomon. 1993). Phosphorylation of threonine 167 (Schi~o.srrcclzai-oin?..c.e.s pornhe numbering) is essential for kinase activity (Could et d . , 1990; Solomon et d., 1990; Krek and Nigg, 1991a,b). Furthermore, cdc2 kinase activity is inhibited by phosphorylation of a residue that lies in the ATPbinding site; tyrosine 15 in S . pornhe (Could et a/., 1990), and threonine 14 in higher eukaryotes (Krek and Nigg, 1991a,b; Norbury et ul., 1991). Removal of these inhibitory phosphates is required for activation of cdc2 kinase and execution of M-phase (Dunphy and Newport, 1989; Gautier et a/., 1989; Labbe et a/., 1989; Morla et al., 1989; Solomon et a/., 1990). Exit from M-phase requires inactivation of the cyclin/cdc2 complex, as catalyzed by the ubiquitin-mediated targeting of cyclin for degradation (Glotzer et a/., 1991). Phosphatases belonging to the cdc25 family remove the inhibitory phosphates on tyrosine 15 and threonine 14 of cdc2 (Dunphy and Kumagai, 199 1 ; Gautier et al., 1991; Strausfeld et a/., 1991). In S. pornhe there is a single phosphatase of this class, cdc25; cdc25 mutants arrest in G2 with no active cdc2 kinase (Russell and Nurse, 1986; Moreno et a / . , 1990). In higher organisms there are two or more cdc25 loci, with a particular isoform often dedicated to the germ line (Sadhu et
9. Regulation and Execution o f hleio\is
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([/.. 1990: Alphey et al., 1903: Wu a i d Wolgenmuth. 199s). In Ilr.o.sophi/ti two
cdc2.5 pliosphatases have been identitied. The String cdc2S is active primarily in mitosis. whcrcas Twine is active primarily in meiosis (Edgar and O’FarreII. 1989, IiWO: Jimcne/. ( I / . , 1990; Alphcy c/ o/.. 1992; Courtot c’t d . , 1997). Based on the structural and functional conservation in cdc35 and cdc3 genes, we and others refer to the point at which /\ciiw rind l l r i ~ ( d c 2are required in spermatogenesis a s a G,/M transition ( White-Cooper c/ u / . , 1993; Eberhart and Wasserman. 1995). We note, however. that although cdc3S and cdc2 activity is thought to be necessary for chroniosoiiie condensation at the G2/M transition in ~ i meiosis. yeast and frogs. this does not appear to hc the case in l l r o , s o p / ~ i /male because chromosomes partially condense in / ~ , i r i rand Diiic~t/c?\ mutants. Ctlc2/cdc2S-indcpcndeiit repul;itiori o f thc condensation state of chroniosonies has also been observed in temiilc meiosis ( l o r discussion. see Murray and Hunt, I 003 ). As ;I result of their failure to initiate the meiotic cell divisions. / i i i / w and / ) / 1 r c d ~ 2 ”mutant testes acciiiiiuI;ite cysts containing 16 tctraploid cells. Howwer. m a n y aspects of‘ spermiogenesis. pirrticularly the programs for sperm head and tail shaping. still occur (White-Cooper u / . . 1993).Consequently. tetraploid spcrniatids. but no motile spctiii. arc pIoduced. Insofx ;is abrogation o f the meiotic divisions does not pre\ cnt the wilil-type postmeiotic program o f diIYerentiation. meiosis aiid spermiogcncsis must he under separate regulation. (I/
B.
Regulation of the Cell Cycle Machinery
1. Control of twine and I~tncdc2Activity
The phenotypic similarities betu,ccn /)r,rcd,2f\and / \ c , i i i c ) mutants suggest that Twine is the phosphatase reyx)iisible I‘or the dephosphorylation and activ;ition of cdc3 at meiotic entry. H o u e \ er, the iiiectiaiiisiii by which the meiotic GJM transition is triggered is unclear. The / \ c . i / i c , gene is transcribed relativelh early during the long G , phase. w e l l bel‘ore tlic onset o f meiosis (Alphey P / ( I / . . 1992: Courtot (’/ u / . , I903). Thus. /\c’irrc, R N A xxxmirlation is not sufficient t o promote entry into the meiotic divisions. ‘l’his situalion differs from that observed i n e x l y embryos. where the transcription 01‘ the .\/r.iri,y cdc3.5 phosphatlrse gene appears to be both necessary and sullicient to drive the GJM transition in some embryonic cell cycles (Edgar nnd O’Farrell. I O X O . IO90).I t is possible that Twine activity is sufficient to drive the GJM trxisition in mciosis. but that this activity is regulated ;it the level of‘ protein accumLilatiori. Alternatively. other I‘actors. such ;is . lin accuiiiuI;itioii or the acti\,ity o f ;I cdc3-activating kinase. may trigger meiotic entry. Upstream tnctors t l i a t rcLgulate the trigger are not know 11. but ciirididatcs ha\,e been identified arid x c described in the next section.
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2. Candidate Regulators of the Cell Cycle Machinery Two genes, pelota and boiile, share the loss-of-function phenotype observed with tbvirw and DmcdcZr5 (Eberhart and Wassernian, 199.5; Eberhart et al.. 1996). Spermatocytes in hniilr and pelotcr mutants carry out chromosome condensation and centrosome duplication but not spindle formation, nuclear lamina breakdown, or chromosome congression at the metaphase plate. Despite their failure to execute meiosis, mutants in either locus still exhibit many aspects of postmeiotic differentiation (Fig. 4B). The phenotypes shared by pelota, hoiile, hvirir and Dmcdc2" define a class of genes required for entry into the meiotic cell division. We term this class the Twine class and believe that these four genes act coordinately to regulate cell cycle progression during male meiosis. Based on the known biochemical functions of proteins in the cdc25 and cdc2 families, we speculate that t+\Yrie and Dmcdc.2 act together to trigger entry into the meiotic cell divisions and that pelota and boule are part of a pathway controlling the expression or activity of tht.r'irie and Dmcdc2. Sequence motifs in Boule and Pelota indicate that both proteins may associate with RNA molecules. The Bode protein contains a conserved RNA-binding domain, the ribonucleoprotein (RNP)-type RNA-binding domain. A 52-amino acid domain in Pelota has substantial sequence similarity to a domain common to a family of eukaryotic polypeptide chain-release factors (Frolova rf a/.. 1994; Koonin Pt (11.. 1994). These release factors are thought to interact directly with RNA, perhaps through this conserved domain. Although b o d e and lidota have a common meiotic phenotypc and may have related biochemical activities, the two loci differ markedly in their patterns of expression and function. The lieloirr gcne is broadly expressed and appears to act in mitosis as well as meiosis. Strong lielotti alleles have a rough eye phenotype, indicative of a disruption in the prccise pattern of cell divisions in the developing eye disc (Eberhart and Wassernian, 199.5). In addition, prlotu mutations reduce fecundity in females and dissection of ovaries reveals defects prior to the meiotic divisions. In contrast, / m i l e is expresscd only in the testis, and hoiile mutations apparently affect only meiosis (Eberhart et a/., 1996). Homologs for both pelota and houlr have been identified and have, i n some cases, been subjected to genetic analysis. Counterparts of pelota have been identilied in humans, plants, worms, yeast, and archaeons (Lalo et a/., 1994;
> Fig. 4 Phenotypes of Twine and Spermatocyte A r m 1 cia\\ nititants. Photographs are ot tinlixed testis contents viewed by phase-contra\t m i c r o s c q y . ( A ) Wild-type testes contents. Spermatocytes, cell5 in meiosi\ I. and onion-stage spermatid5 with dark nebcnkcrne and pale nuclei can be reen. ( B ) pr/ol hoinoiygote 16-cell cysts. The tetraploid \permatid\ each contain a nucleus and nebenkern. Because oi'their 4N chroiiiosoiiial content, the \permatid nuclei are abnormally large. (C) Tr\tes f r o m
wl/Df(sii) I/)fl.?L)Pc-q>/I males coiitaiii plentiful mature pi-imary spermatocytcs hut lach postmt'icitic stapes. Photograph\ are appi-oxirnately the \mie scale. (Adapted with perrni\\ion o f t h c ('ompmiy o f Biologists Ltd. from Eberhart and Wa\serninii, 1995. and Lin ( I / . , l9Y6.) i j t
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Verhasselt et a/., 1994; Wilson et a/., 1994; Eberhart and Wasserman, 1995; Bult et ul., 1996; Ragan et a/., 1996). Mutations inactivating the S. cerevisiae pelota homolog, dotn34, result in defects in both mitosis and meiosis. Mutants i n dom34 execute mitosis slowly, while the meiotic divisions are too rapid, producing fewer spores than wild type. These growth defects can be rescued by expression of pelofa in a doorn3.l mutant yeast (Eberhart and Wasserman, 1995).Together, the molecular and genetic analyses suggest that pelotu represents an evolutionarily conserved function broadly required for cell division. A human locus has been identified that has similarities in sequence, expression pattern, and male sterile loss-of-function phenotype to boule. The Y-linked Deleted in A,-oospermia (DAZ) locus, a candidate gene for the A,-.oosperniiuFactor (AZF), is deleted in one of eight men producing little or no spenn (Reijo et ul., 1995). B o d e and DAZ share similarity with one another and with autosomal vertebrate DAZ homologs in both the RNA-binding domain and a second domain termed the DAZ repeat. The positions of the RNP domain and the first DAZ repeat are conserved in all DAZ family members, including Boule. All of the DAZ-related loci are expressed predominantly or exclusively in the testis. These results indicate that Boule is likely to be a functional homolog of members of the DAZ family and suggest a conservation in the regulation of male germ line development from flies to humans.
C. Coordination of Meiosis with Differentiation
A set of loci distinct from the Twine class has been found to be required for the GJM transition in Drosophila males. Mutations in this second group, the Spermatocyte Arrest class, block both the meiotic cell division and postmeiotic spermatid diferentiation, resulting in the accumulation of cysts of 16 spematocytes. Genes in this class appear to act upstream of the Twine class in the G2/M transition and may play a role in the coordination of cell division with postmeiotic differentiation. Members of the Spermatocyte Arrest class of loci include tneiosis I arrest (mia),cunnonball (can),spermutocyte arrest (su), and alrz/ays early ( a / ? )(Lin et al., 1996). For each gene, except mia, multiple alleles have been isolated, all of which seem to affect male fertility specifically. No meiotic figures or products are observed in the mutants, and there is no evidence of spermatid formation. The cells that accumulate are thus spermatocytes arrested at the end of the growth phase (Fig. 4C). The meiotic entry arrest phenotypes of rnia, can, su, and d y are quite similar to one another and suggest in each case an arrest late in meiotic prophase. Chromosomes begin to condense but remain at the nuclear periphery, and no spindles are formed. The arrest phenotype of u / y mutants is somewhat different from that of
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the other mutants of the Sperniatocyte At-rest class in that the partially condensed chromosomes are less well defined. Two lines of evidence suggest that Sperniatocyte Arrest mutants are blocked at an earlier point in the cell cycle than those in the Twine class. First, distinct dark nucleoli persist in mia, con, .m, and lily spermatocytes, whereas the nucleoli break down in twine, pelotu, boulc., and (,(k2r’mutants (Sigrist et d . ,1995; Lin et id., 1996). Second, whereas cyclin A enters nuclei in mutants of the Twine class, it remains predominantly cytoplasmic in mutants of the Spermatocyte Arrest class (Lin et nl., 1996; Gonczyer u/., 1994: Sigrist et ( I / . , 1995; H. White-Cooper and M. Fuller, personal communication). Although mia, can, sa and a l y have not been characterized at the molecular level, recent data suggest a basis lor the Sperniatocyte Arrest phenotype. WhiteCooper and Fuller have found that RNA transcripts for several genes active in postmeiotic differentiation, including j i c z y onions and janB (Yanicostas et a/., 1989; Fuller, 1993), are absent, or greatly reduced in abundance, in mia, can, sa, and aly, but not twine, mutants ( H . White-Cooper and M. Fuller, personal communication). The Spermatocyte Arrest loci also appear to be required for expression of meiotic regulators, although in this case the mode of action is different for o/y than for can, sa, or m i c r . In o/y mutants the RNAs for cyc/iri B. twine, and b o d e fail to accumulate. In the other Spcrmatocyte Arrest mutants, these RNAs are present, but there are defects at the level of protein expression for Twine and Boule. Specifically, White-Cooper and Fuller find that expression of a chimeric trr*ine-lacZreporter transgene, as monitored by f3-galactosidase activity, is abolished i n mici, can, and .ra mutants. In addition, we have found that at least mia and sa are required for B o d e protein accutnulation ( J . Maines, M. Cheng, and S. Wasserman, unpublished data). The expression data described i n the preceding paragraphs indicate that the Spermatocyte Arrest loci are needed during the spermatocyte growth phase for the expression of genes required for either meiosis or differentiation. The aly gene is likely to act at the level of transcription or message stability. Mutations in miu, crrn, and sa alter protein levels l o r some genes (meiotic regulators) and RNA levels for others (spermatid differentiation factors). These Spermatocyte Arrest loci may therefore be required for transcription, and thereby regulate accumulation of translational factors, or for translation, and thereby control levels of transcriptional factors.
D. Regulation of the Second Meiotic Division
Although MI requires a special mechanism for reductional division, the means for equatorial division must still be available for MII. Furthermore, although MI1 resembles a mitotic division, the checkpoint mechanism that ensures that DNA replication has occurred prior to nuclear division in mitosis must be absent or
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inactivated to allow MIl. The basis for these differences and, more generally, the regulation of MI1 is not well understood. No genes have been identified for which loss-of-function mutations specifically block MU. There is evidence, however, that three loci-Drncdc2, twine, and roughex-play a critical role in control of MIL Experiments with the previously described DmcdcZr' transgene indicate that Dmcdc2 is required for both MI and MI1 in males (Sigrist et al., 1995). D~ncdcZ mutant males that carry two copies of the Dtncdc2r.\ transgene were sterile at all temperatures, with defects in meiosis. A t 18°C they executed MI but failed in MII, producing 32-cell spermatid cysts. When shifted as adults to 27"C, they failed to execute either meiotic division. Two additional copies of the DmcdcZt' transgene restored fertility at 18°C but did not alter the phenotype at the nonpermissive temperature. Two lines of evidence suggest that tccine is also important in the regulation of MIL First, in the wild-type, late primary spermatocytes express h t n e , but no detectable levels of string. In a [wine mutant, background expression of string from a heat-shock promoter is sufficient to rescue entry into MI, but not MI1 (Sigrist e t a / . , 1995). Second, in females, where both string and twine are present at meiosis, mine mutants execute MI but not MII, further suggesting that twine plays a unique role in MI1 (Courtot et d,1992; White-Cooper et al., 1993). Since flies with two copies of the Dt71cdc2~~~ transgene execute MI but fail in MIl, the level of kinase activity may be particularly important for entry into MII. The Twine phosphatase may be important for maintaining this level of cdc2 kinase activity. Alternatively, it is possible that the second meiotic division depends on a product of cdc2 kinase activity formed during the first meiotic division. Support for this idea comes from an analysis of the gene roughex ( r u x ) . Experimental evidence suggests that roiighex acts as a negative regulator of MI1 (Gonczy et a/., 1994). An increase in roughex gene dosage blocks MI1 while allowing normal execution of MI, resulting in 32-cell cysts of postmeiotic germ cells. The X and Y chromosomes disjoin in these cells, indicating that MI occurs while MI1 is blocked. Reciprocally, germ cells from rux mutant flies attempt to execute an extra MII-like division. After normal MI and MI1 divisions, the haploid nuclei of TUX mutant germ cells become less discrete, chromosomes recondense, a monopolar meiotic spindle assembles, and chromosomes randomly distribute into two daughter nuclei. This extra division resembles MI1 in that there is no preceding DNA synthesis or apparent centrosomal duplication. Together, these data strongly suggest that rux negatively regulates MII, with excess rux preventing MI1 and insufficient rux permitting an additional MIl. Although the rux mutant phenotype is observed after a normal MII, there are increases in cyclin A levels prior to MI. Furthermore, lowering the dose of either twine or cyclin A suppresses the extra division, consistent with the hypothesis that excess cdc2 activity is responsible for the extra meiotic division in the rux mutant.
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Because cyclin A is synthesized prior to MI, degraded at metaphase of MI, and not resynthesized for either MI1 or the aberrant M11-like division, rux must be acting at or before the first meiotic division to influence the second division, presumably by limiting the amount of cdc2 kinase activity available for MII.
111. Spindle Formation and Function in the Meiotic Cell Divisions As discussed in the introduction to this chapter, meiosis in Drosophilri males offers the opportunity for a detailed analysis of cell division through the combined use of genetics and cell biology. Among genes required for wild-type chromosome pairing and segregation. a number appear to act specifically in males. For example, the X-linked qiiutioiid producer ( e q ) mutation, discovered by Schultz in 1934, causes rare equational nondisjunction ofthe X chromosome, resulting in sperm carrying either two X chromosomes or no sex chromosome (Morgan er al., 1934). The fact that the eq mutation has little or no effect on disjunction in females (Valentin. 1984) suggests that the eq locus may provide a starting point for dissecting the differences in the regulation of MI1 in males and females. Another X-linked mutation, Rcuwery Disrupfer (RD), results in a 50% reduction in the number of male progeny through an unprecedented mechanism: fragmentation of the Y chromosome during meiosis (Novitski and Hanks, 1961; Erickson, 1965). However, the sub-jects of chromosome pairing and segregation are reviewed elsewhere in this volume (see Chap. 3, by B. D. McKee, and Chap. 8, by D. P. Moore and T. L. On-Weaver), and we will therefore restrict our attention in this section to spindle structure and function. The alignment of the phase-dense mitochondria along the length of the meiotic spindle renders the spindle readily visible by phase-contrast examination during MI and MIL For both meiotic divisions the spindle resembles that of a mitotically dividing cell but is much larger than that in a somatic nucleus. The meiotic spindles are thus especially well suited for immunofluorescence studies (Gatti and Goldberg, 1991). For many loci required for cell division, hypomorphic mutations are sterile and have readily apparent defects in germ line mitoses and meioses. Furthermore, the phenotypic effects of null mutations in essential cell division functions can often be examined in the male germ line. Although mitotic proliferation is necessary for embryogenesis, the maternal contribution of most genes required for cell division is sufficient for this stage of development (Gatti and Baker, 1989). As a result, the larva is typically the first developmental stage in which cell division defects arise, with death at pupation resulting from failure in imaginal disc division. The consequences of a loss-of-function mutation in a cell division gene
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can therefore frequently be characterized by examining germ line divisions in the larval testis.
A. Meiosis-Specific Spindle Architecture
The composition of the spindles in male meioses makes them particularly amenable to genetic analysis. Although comparable in large part to mitotic spindles, male meiotic spindles differ from their counterparts in both mitoses and female meioses in at least one major component. Among the four-P-tubulin isoforms in Drosopida, one, the P,-tubulin, is present only in postmilotic spermatocytes and spermatids and is the predominant P-tubulin in the meiotic spindle. Because the four P-tubulins are encoded by distinct loci, it is therefore possible to specifically disrupt the meiotic spindle by mutation. The testis-specific tubulin subunit, P,-tubulin, is synthesized just prior to meiosis (Kemphues et ul., 1982). Levels of the p,-tubulin present during the germ line mitoses decrease at the same time, such that during meiosis, levels of P2-tubulin protein are an order of magnitude higher than those of p,-tubulin (Kemphues et a/., 1980). A number of mutations in the P,-tubulin gene have been identified that disrupt both meiosis and spermatid differentiation (reviewed by Raff and Fuller. 1984). Recessive alleles that produce an unstable P2-protein (class I alleles) fail to accumulate any tubulin, since the (Y subunit is destabilized in the absence of a p subunit with which to dimerize (Kemphues et a/., 1982). Chromosomes condense normally in such mutants but do not align at metaphase and fail to migrate to opposite poles. The nuclear envelope thickens and the nucleolus breaks down, but spindles are abscnt and no cytokinesis occurs. These defects can be phenocopied by administration of colchicine (Kemphues et a/., 1982), consistent with an absolute requirement for microtubules in spindle formation, chromosome segregation, and cytokinesis. Chromosomes in class I P,-tubulin mutants, while not undergoing segregation, nonetheless decondense and then recondense as if a normal interphase had occurred. Metaphase of the equational MI1 division thus lacks the checkpoint mechanism apparent in metaphase of the equational mitotic divisions (reviewed by Murray, 1992; Sluder and Rieder, 1993). This difference may reflect the fact that MII, unlike a mitotic division, is not directly preceded by DNA synthesis. Although the P-tubulins are highly conserved in sequence, an “isoform swap” experiment suggests that the biochemical properties of the @,-tubdin are significantly different from those of the other isoforms. Taking advantage of‘ the thorough characterization of the P, tubulin promoter in the Raff and Renkawitz-Pohl laboratories (Hoyle and Raff, 1990; Kemphuses el a/., 1982; Michieks et a/., 1993), Hoyle and Raff demonstrated that expression of the P,-tubulin under
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control of the P,-tubulin regulatory elements fails to complement the meiotic defect in a P,-tubulin (Hoyle and Raft', 1990).
B. Regulation of Spindle Formation
The merry-go-round (tngr),polo, and ahtiormal spindle (asp)genes play a role in the function and regulation of the meiotic spindle. All are essential genes, functioning in mitosis as well as meiosis, and mutations in each result in distinct spindle defects. In rngr males, no nuclear or cytoplasmic divisions take place. Instead, the meiotic chromosomes form a circle around the center of the cell and appear to be associated with a monopolar spindle (Gonzalez et ul., 1988). Mutations in polo result in a high frequency of tetrapolar and multipolar spindles in meiosis (Fig. 5) and of monopolar spindles in mitosis (Sunkel and Glover, 1988). In asp mutants the meiotic spindles are irregularly shaped and are associated with an extensive cytoplasmic microtubule network that is absent in wild-type cells. Defects in either polo or L I . S ~result i n an asymmetric distribution of chromosomes and mitochondria among spermatids. Mutations in these two loci show a strong genetic interaction, suggesting that polo and asp act in a common pathway governing spindle formation (Llamazares et a/., 1991). Of the three genes just described, only polo has been cloned (Sunkel and Glover, 1988; Llamazares et ul., 1991 1. The encoded protein, a serine/threonine kinase, has homologs in mammals as well as both fission and budding yeast (Clay et al., 1993; Golsteyn ot cil.. 1994; Holtrich et a/., 1994; Hamanaka et nl., 1995; Ohkura et id., 1995). Mutations i n the yeast homologs have mitotic and, in the case of the budding yeast homolog c.dc.5, meiotic defects (Schild and Byers, 1980). For the fission yeast homolog, p l o l , mutations result in defects in spindle pole body duplication and/or separation (Ohkura et nl., 1995), similar to the aberrations seen in mitotic polo cells. For mitosis, at least one additional protein kinase, the product of the uuroru locus, is required for wild-type spindle lormation. Mutations in uuroro result in spindle defects due to a failure in centrosome separation (Glover et a/., 1995). For meiosis, it is possible that an uurorcr-like protein kinase activity functions in coordination with polo in regulating the centrosome.
IV. Cytokinesis As in other animal cells, cytokinesis in Drosophila spermatocytes begins at anaphase and is first visible as a furrowing of the plasma membrane in a plane orthogonal to the long axis of the spindle (reviewed in Satterwhite and Pollard, 1992; Fishkind and Wang, 1995; Miller and Kiehart, 1995). Cleavage is initiated
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Fig. 5 Meiotic spindles in live cells. ( A , B ) A cyst of wild-type cells undergoing the second meiotic division. (C, D) A tetrapolar spindle in a hoino7ygous polo' cell undergoing meiosis. (E. F) A multipolar meiotic spindle in a hoino/ygous polo' cell. Scale bars =- 10 p,m. (Reproduced with permission of the Company of Biologists Ltd. from Sunkel and Glover, 1988.)
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by a contractile ring, a transient cytoskeletal structure containing a network of antiparallel actin filaments. Cross-linked bipolar myosin I1 filaments are believed to generate the force for contraction of the ring, constricting the cleavage furrow approximately 10-fold. The g ~ i pthat remains is tilled in during complete cytokinesis in the soma but persists during incomplete cytokinesis in the germ line and is transformed into an interccllular bridge termed a ring canal. Little is known about how conlractile rings are assembled, how contraction is regulated, or how the molecular pathways for complete and incomplete cytokinesis differ. Recent experiments in the Dro.rophila germ line have started to provide answers to these questions. Genetic studies have identified a number of loci encoding components of contractile rings or ring canals (Neufeld and Rubin, 1994; Robinson et ul., 1994; Hime e/ c l / . , 1996). Sequencing of cDNAs from these loci has revealed surprising molecular parallels between incomplete cytokinesis in Drosophila gametogenesis and the variant form of cytokinesis observed in the division of the budding yeast (Castrillon and Wasserman, 1994; Sanders and Field, 1995; Longtine r / ul., 1996). Finally, antibodies raised against the products of the Dro.sophilu genes have enabled cell biologists to begin to dissect the organization of these cytoskeletal structures.
A. Contractile Ring Assembly and Function
Two critical issues with regard to assembly of the contractile ring are how the position of the cleavage furrow is determined and how components of the contractile ring are recruited into a functional apparatus. There is substantial evidence that positioning of the cleavage furrow is mediated by the spindle asters (Rappaport, 1986). Characterization of germ line cell divisions in wild-type and mutant males indicates that the spindle may also play a role in ring assembly. During wild-type meioses and mitoses the protein product of the KLP3A (Kine.,in-Likr-Prorein-ut-SA)gene is highly concentrated in the midbody, a structure surrounding the interdigitated microtubules that constitute the central spindle (see Fig. 3e). Dissection of males carrying a null mutation in KLP3A mutant males revealed defects late in anaphase of meiosis I: the central spindle and midbody were absent (Williams P / ul., 1995). Cytokinesis was also affected. KLPSA spermatids frequently contained an abnormally large nebenkern associated with two equally sized nuclei; rarer spermatids had a single nebenkern and four nuclei. Since in both cases the nuclei were always of equal size, there appeared to be a defect in cytokinesis but not in chromosome segregation. Togelher, the molecular and phenotypic characterizations of KLP3A indicate that this gene plays a key role in assembly of the midbody and central spindle, and that these structures are in turn critical for proper assembly or function of the contractile ring.
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B. Cytokinesis in the Drosophila Germ Line and in Budding Yeast
Whereas cytokinesis in Dmsophila male meiosis involves a contractile stage followed by stabilization of an intercellular gap, cytokinesis in S. cerevisiue has no contractile stage, but rather comprises a membrane fusion event and septum formation at the neck between mother cell and bud (Sanders and Field, 1995). It was therefore quite unexpected that two Drosophiki loci active in meiotic and mitotic cytokinesis would have homologs in the budding yeast. The diuphanous (dia)and peuiiut ( p u t ) genes are apparently required only for cytokinesis (Castrillon and Wasserman, 1994; Neufeld and Rubin, 1994) Null alleles give rise to polyploidy in hoinozygous larvae; imaginal discs are severely reduced, with many multinucleate cells. Cleavage furrows are absent. Despite the polyploidy resulting from the cytokinesis defect, chromosome segregation appears to be relatively normal. In many hyperploid diu cells, anaphases involve bipolar spindles and chromosomes are segregated equally, with no lagging chromosomes or other abnormalities. In other, more extremely hyperploid cells, spindles are multipolar, as has been observed in other mutants that produce hyperploid cells (Gatti and Baker, 1989: Karess et ol., 1991). Defects in cytokinesis during meiosis are readily apparent in males homozygous for a hypomorphic din mutation (Fig. 6). Most tlia' spermatids contain two or four nuclei of normal size associated with a single large nebenkern, the size of which correlates with the number of nuclei. The presence of rare spermatids containing eight nuclei (Fig. 6, inset) indicates that cytokinesis in din' inales can also fail in the mitotic divisions preceding meiosis. Diaphanous is a one of a group of proteins termed the formin homology (FH) family, which includes the S. ceret~isiueprotein BNII (Bud Neck Involved 1 ) (Castrillon and Wasserman, 1994). Peanut belongs to the highly conserved septin protein family, which has four members in the budding yeast and at least three in Drosuphilu. Although pnut is the only Drosophiln septin gene for which mutations have been identified, all four S. crrrvisiae septims-CDC3, CDCIO, CDCI 1, and CDCI2-have been shown to be necessary for cytokinesis (Hartwell, 1971; Byers and Goetsch, 1976; Longtine et al., 1996). Intriguingly, B N l l was first identified on the basis of a synthetic lethal interaction with a mutation in the CDC12 septin (Longtine et ul., 1996), which suggests that the two genes act in the same pathway and perhaps interact.
> Fig. 6 Cytokinesis defect in dirr' testis. Photographs are of untixed testis contents visualized by phasecontrast microscopy. ( A ) Part o f a 64-cell cyst of wild-type (permatids Each spermatid contains ii \ingle pale nucleus (arrowhead) and a single dark nebenkern (arrow). Although this cyst is intact, spermatid cysts typically rupture into smaller groups of cells owing to the absence o f a tixation step. ( B ) Group of six din' spermatids, each containing four nuclei (arrowheads) associated with a single lai-ge nebenkern (arrow). Inset: Single din' spermatid containing eight nuclei. Scale basis = 10 Km. (Reproduced with permission of the Company of Biologists Ltd. from Castrillon and Wasserman, 1994.)
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Diaphanous and Peanut, as well as their yeast counterparts, are found in regions of the cell where cytokinesis occurs. Peanut and Diaphanous localize to the contractile ring and intercellular bridge of dividing cells (Neufeld and Rubin, 1994; B. S. Gish and S. Wasserman, unpublished results), while BNIl (Longtine et a/., 1996) and the yeast septins localize to the region of the mother-bud neck. There the septins appear to be components of the ring of 10-nm filaments (Haarer and Pringle, 1987; Ford and Pringle, 1991; Kim et a/., 1991). It is possible that Diaphanous and Peanut are not required for the contractile phase of cytokinesis but rather are necessary to maintain the cytoskeleton in the contracted state. The existence of S. cerevisiae homologs for these proteins would then be less paradoxical. Two lines of evidence support this hypothesis. First, it has recently been shown that Peanut protein persists at the cleavage furrow after arrest, localizing to the ring canals of spermatocyte and spermatid cysts (Hine et al., 1996). Second, other members of the FH family, to which Diaphanous belongs, are not limited in function to cytokinesis but participate in a variety of cytoskeletal-mediated processes in a wide range of organisms (Nurse et a/., 1976; Emmons et a/., 1995; Petersen et al., 1995; Chang et a/., 1996).
C. Additional Cytokinesis Factors
A number of genes in addition to diuphanous and peunut have been shown to
encode components of the cleavage furrow or contractile ring. For the products of many of these loci, immunolocalization studies have been carried out in wildtype spermatocytes as well as in spermatocytes with specific defects i n cytokinesis. For a subset, mutations have been isolated and analyzed. These studies have provided insights into the mechanisms for assembly and function of the contractile ring and, in addition, have contributed to the increasing number of immunological and genetic reagents available for dissecting these mechanisms. Studies in other species have shown that cofilin, an actin-binding protein, localizes to cleavage furrows (Nagaoka et a/., 1995). A member of the actin depolymerization family, cofilin competes with tropomyosin, myosin, and villin for actin binding in vitro (Nishida et ul., 1984; Nishida, 1985; Pope et al.. 1994). Mutations in a Drosophila cofilin locus, winstar ( t s r ) , were identified among a collection of recessive lethal mutations exhibiting mitotic abnormalities in larval brains (Gunsalus et a/., 1995). Examination of primary spermatocytes in tsr males revealed a failure of cytokinesis, as well as defects in centrosome migration and separation at prometaphase of both MI and MIL During prophase of MI, aggregates of actin were found associated with centrosomes. In addition, during anaphase of both meiotic divisions, misshapen F-actin-containing structures were observed at the normal site of contractile ring formation. Gatti, Goldberg, and colleagues argue that the function of twinstcir is 10 regulate the assembly of actin into cytoskeletal structures (Gunsalus et LJI.. 1995). They speculate that in the absence of tsr activity, there is an uncontrolled accu-
9. Regulation and Execution of Meio
325
mulation of actin at nucleation centers situated near centrosomes. In addition, they suggest that a disorganized recruitment of actin into the contractile ring precludes a wild-type disassembly of the ring. Anillin, a component of both contractile rings and ring canals, was first identified by actin affinity chromatography (Miller et ul., 1989; Field and Alberts, 1995). This Drosophilu protein, which has no known homologs, bundles actin filaments in vitm and is found specifically in dividing cells. Immunofluorescence studies have demonstrated a cell cycle-dependent compartmentalization of anillin (Field and Alberts, 1995). Anillin is nuclear during interphase, whereas during anaphase and telophase it is concentrated in the cleavage furrow. In spermatocytes, anillin localizes to contractile rings and, later, to ring canals, whereas actin is lost following constriction of the contractile ring (Hime et ul., 1996; M. Gatti, personal communication). Together, the pattern of localization of anillin in vivo and the biochemical properties of the protein in virro suggest two possible functions in meiotic cytokinesis (Field and Alberts, 1995). First, anillin may play a role in organizing the contractile apparatus, perhaps serving as part of the link between the plasma membrane and the developing actin-based ring. Second, anillin may help to stabiliye the intercellular ring canals (Hime rt al., 1996).
V. Conclusions and Perspectives Although a number of genes involved in the regulation and execution of meiosis in Drosophilu males have been cloned and characterized, we understand the basic biochemical function for only those few, such as twine and P,-tubulin, for which there has been extensive characterization in other systems. Furthermore, there are many loci for which no molecular studies have been conducted. This set of loci includes the Spermatocyte Arrest class of meiotic entry genes and the cytokinesis loci shunk, fumble, and four w h r c ~ drivc l (Castrillon et a/., 1993; Fuller, 1993). It is clear, therefore, that much remains to be learned about the known meiotic loci. Genetic screens aimed at identifying additional factors required for meiosis hold considerable promise. There have been only a few large-scale screens for male-sterile mutations affecting spermatogenesis or, more specifically, meiosis (Lifschytz and Hareven, 1977; Hackstein. 1991; Castrillon et ul., 1993; C. Wu, M. Fuller, and S. DiNardo, personal communications). Furthermore, none of these screens has achieved saturation, and the number of genes identified to date is likely to be small relative to the total number of relevant loci (for discussion, see Castrillon et ul., 1993). Efforts directed at identifying chromosome-specific disjunctional mutations have also been quite limited (reviewed by Hawley, 1993). Further screens for mutations affecting male fertility or meiotic chromosome disjunction should therefore provide a substantially expanded basis for analyying the molecular mechanisms governing meiosis. Although work on the molecular foundations of Drosophilu male meiosis is
326 Jean Maines and Steven Wasserman still in an early stage, two general themes have become apparent. One is that the meiotic divisions constitute a program that is in large measure independent of the developmental events that either precede or follow MI and MII. For example, the onset of the meiotic cell divisions is apparently indifferent to the faithful execution of the preceding mitotic divisions. Lifschtyz has found that mutations that reduce the number of mitotic gonial divisions do not block meiosis (Lifschytz and Meyer, 1977), and we and others have found that meiosis can occur following a failure in cytokinesis during the gonial mitoses (see, e.g., Fig. 6, inset). Similarly, the initial phase of the program for spermatid differentiation is unaffected by mutations that disrupt chromosome segregation (e.g., class I P,-tubulin alleles) or that block the meiotic cell divisions altogether (e.g., Twine class mutations). Another theme emerging from the studies reviewed in this chapter is that mechanisms governing the regulation and execution of meiosis have been broadly conserved. As discussed earlier, components and regulators of the cell cycle oscillator in Drosophilu male meiosis, such as twine and hnule, have counterparts in the male germ line of vertebrates, (e.g., Cclc25C and DAZ), and components of the contractile apparatus in flies (Diaphanous and Peanut) have homologs in the budding yeast (BNllp and Septins). A number of questions remain unexplored regarding the regulation and execution of the meiotic cell divisions in males: Are thc waves of meiosis within cysts triggered or coordinated by a single cell? If so, is this spermatocyte, like the oocyte, one of the two cells among 16 that are directly interconnected with four other cells? What role, if any, do the somatic cyst cells play in the control of meiosis? Why is the onset of MI regulated by a testis-specific gene when the striking difference between meiosis in males and females occurs at MII? The answers to these and other questions will, we hope, become clear as we learn more about the genes, the gene products, and the pathways governing Dm.wphilu male meiosis.
Acknowledgments We thank Helen White-Cooper, Minx Fuller, Maurilio Gatti, Pierre Ciinciy. and Stephen DiNardo for communication of unpublished results; Pierre Gonczy. Stephen DiNardo, and Dennis McKearin for critical reading of the manuscript; and Gianni Cenci, Minx Fuller, and Alisha Titenor for assistance in preparation of figure\. This work was supported by a grant to S.A.W. from the Excellence in Education Fund.
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o,strc.(~lrciro,ri?.c~rs potfibu: A dcvclopmc~itallyconirolletl function needed for con.jugation. Mol. CC//. H J f ) / . 15, 3697-3707. Pope. B . Way, M.. Matsudaira, P.T.. aiid Weeds. A . (1994. Characterisation of the F-actin binding domains of villin: Classitication 01 I - x t i i i h i d i n g proteins into two group.; accoi-ding to their h i d i n g sites 011 actin. FERS Lcr/ 338, 5X-02. Kaff, E. C., and Fuller, M. T. (1984). (icnetic aiinl)sis of microtuhule function in l ~ r m o / i h i k iI.f f “Molecular Biology of the Cytoskclcton” (G Boi.isys, Ed.), pp. 293-304. Cold Spring Harbor Laboratories, Cold Spring Harbor. K:rgan. M . A,, Logsdon. J. M . , Sensen, C. W., Charlehois, R. L., and Doolittle, W. F. (1996). An archaehacterial homolog o f Pclotn, a meiotic cell division protein in eukaryotes. FEMS Micro. Lrrr. 144, 151-155. Kappaport, R. ( 1986). E\tablishment of thc mechanism of cytokinesis in animal cells. lrit. Rei,. Cyrol. 105, 245-28 I. Keijo. K . . Lee, T. Y., Salo, P.,A ~ ~ i g a ~ ~ pK; i. .nBrc)v,n. . L. G., Rosenberg. M., RoLcn, S., Jalfe, T., Strau\, D., Hovatta. O., r t cr/. (l9OSl. I)iver\e \pei-matogenic defects in humans caused by Y chromo\ome deletions encompassing ;I novel KNA-binding protein gene. Neiture Gcric,r. 10,
3 8 - 393. Robinson, D. N., Cant, K., and Coolcy, I .. ( I0941 Morphogenesis of Dro.\o/ih,ltr ovarian ring canals. I ) e i , e l o p r f i e r i / 120, 201 5-2025. Kus\ell. P., and Nurw, P. (1986). cd[ 3.5 ’ tunctiotis ;I\ an inducer in the mitotic control of fission yea\t. (‘c,/ 45, 145-53. Sadhu. K., Reed, S. 1.. Richnrdwn, H , and Ku\\cll, t’. ( I 990). Human homolog of fission yeast cdc25 mitotic inducer I \ predoniinnntly cxprcs\ed in G2. P r o ( . N u t / , Accrd. Sci. USA 87, S 1395 14.3. Sanders. S. L., and Field, C. M. (1995). (’ell di\ihion Bud-sire selection is o n l y hkin deep. CiJrr. H i o i . 5, 1213-1215. Sattcrwhite. L. L.. and Pollard, T. D i I W 2 ) Cytokinesis. C‘itrr. Opiii. Cell B i d . 4, 43-53. Schild. D.. and Byer\, B. (1980). Dipkiid \pore loiiiiation and other meiotic effects of two celldivision-cycle mutations of S f t c c h f i ~ - ~ , ~ ~c i. c~r cc, ,i ,ci \\i r r e . G ( w ~ / ~ 96, c . s 859-876. Sigri\t. S.. Ried, G.. and Lehncr. C. I . ( lr)O5) Diiicdc2 kinase is required for both meiotic divi \ion\ (luring I~r0.sophiltrspermatoyie\is ;itid is aciivated by the Twindcdc25 phosphata\e. M r c / i . Dn: 53, 247-260. Sludei-, G., and Kieder, C. L. (1993). I‘lie events iind I-egulation of anaphasc onset. IJJ “Chromo\ o m Segregation and Aneuploidy” (13. V. Vig\. Ed.). pp. 21 1-224. Springer, New York. Solomon, M. J. ( 1993). Activation ol tlic vat-ious cyclin/cdc? protein kinases. C~O-K Opiu Cell RifJ/. 5, 180-186. Solomon. M. J.. G l o t m , M . , Lee, T. H., l’liilippc. M., and Kirschner. M. W. (1990). Cyclin activation of p34cdc2. C r / / 63, 1013-1024. Stern, B.. Ried, G., Clegg, N. J . , Grigliatti, ‘I. A . and Lehner, C. F. ( 1993). Genetic annlyhis o f the I ~ J - o . s o ~ cdc2 ~ ~ / o homolog. Dci.e/opwtrr 117, 2 19-232. Strausield, U.. Labbe. J . C., Fesquct. I).. (’;ivadoIc. J. C.. Picard. A,, Sadhu. K.. Russell, I?. and Dorec. M. ( 1991). Dephosphorylatim iind a c t i v i i t i ~ i nof a p34cdc2/cyclin B complex i n vitt-o by human CDC25 protein. N c ~ I J 351, ~ ~ c ,241-145. Sunkel. C. E., and Glover, D. M. (I9XX) l i d o , ‘I mitotic mutant of Dro.wphilo displaying ahnortnal \pindle poles. J . Cell Sci. 89, 25-3X. Tales, A . D.( I97 1 ). Cytodifferentiation chi-ing Spermatogenesis i n Dro.wphi/fr Jifc,/uJf~,gcr.\rc,r: An Electron Microscope Study. Ph.D. thc\t\. Ri;k\iiiiivcrsiteit, Leiden. Tokuyaw. K. T., Peacock. W. J., and H a t t l y , R. W. i 1072a). Dynamics of spermiogenesis in Uro. s o / J ~ J J/ ~J ~I ~ / ~ ~ ~ I O I.~ UIndividualiiniiori . T ~ P ~ . procc\\. Z. Zrl//ir.wh. Mikrmk. A w r . 124, 479-506. Tokuya\u, K.T., Peacock, W. J . , and Hardy, K . W. ( 1972b). Dynamics of spermiogencsis in Dro.~o/i/riluriirkrffo,~arrrr. 11. Coiling process. %. % ~ / / o J - . T ~ I .Mikrosk. Ancrf. 127, 402-525.
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Valentin, J. ( 1984). Genetic control of meiosis: The eq gene in Drowphilcr nirlnriogtrstrr. Hrruclifm 101, 11.5-117. Verhasselt, P., Aert, R.. Voet, M.. and Volckaert, G. (1994). Twelve open reading frame\ revcaled i n the 23.6 kb segment Ranking the centromere on the .Strcchuroni?.c.rs c.rrr\~isirie~ chromosome XIV right arm. f i w s r 10, 1355-1361 White-Cooper, H., Alphey, L., and Glover, D. M. (1993). Thc cdc2.5 homologue rkt'iiw IS required for only some aspects of thc entry into meiosis in flrc~sophilr~. J . ('ell S c . i . 106, 1035-- 1044. Williams, B. C., Riedy, M. F., Williams, E. V.. Gatti, M.. and Goldberg, M. L. (1995). The I l m .rophiltr kinesin-like protein KLP3A is a midhody component required for central spindle assembly and initiation o f cytokincsis. J . Cell N i o l . 129, 709-723. Wilson, R.. Ainscough, R.. Anderson, K., Bayncs, C., Berks, M.. Bonfield, J., Burton, J.. Connell, M . , Copsey, T., Cooper, J., rr ( I / . (1994). 2.2 Mb of contiguous nucleotide sequence from chromosome 111 of C. elegms. Nrrture 368, 32-3X. Wu. S., and Wolgemuth, D. J . (1995). The distincL and developmentally regulated patterns of expression of members of the mouse Cdc25 gene family suggest diflerential functions during gametogenesis. Dei,. Biol. 170, 195-206. Yanicohtas, C., Vincent, A,, and Lepcsant. J . A. ( 19x9). Trnnscriptional and posttransxiptional regulation contributes to the sex-regulated expression of two sequence-related genes at the ,jciriir.\ locus of Dro.so/~hrlarrrrltrriogtrcter. Mol. Cell. Biol. 9, 2526-2535.
10 Sexual Dimorphism in the Regulation of Mammalian Meiosis M a r y Ann Handel Department of Biochemistry. Cellular and Molecular Biology University of Tennessee Knoxville. Tennessee
John J. @pig Jackson Laboratory Bar Harbor. ME
1. Introduction and Overview 11. Regulation of the Onset 01 Meiotic Prophaw 111. Genetic Evcnts of Meiotic Prophaw: A Kt.gulatory Role in Gametogcne\i\" IV. Regulating G,/M Tr;in\ition and Meiotic Di\ i\iom A . The G,/M Transition during Oogcnc\i.; H . The G,/M Tran\ition during Si"rrii"t(~ferie\is
V. Gnmetic Function of Meiotic I'n)phaw VI. Suinrnary and Perspective\
Reference\
1. Introduction and Overview Meiosis is overtly sexually dimorphic i n mammals, with several key features that differ markedly between males and females. In females, meiosis is initiated once, more or less synchronously i n a finite and limited pool of cells, but in males, meiosis is initiated continuously in cells derived from a mitotically proliferating stem cell population. In females, all chromosomes exhibit equivalent transcription and recombination activity cluring meiotic prophase, whereas in males, the sex chromosomes are largely excluded from transcription and recombination during meiotic prophase. In females. the gametogenic differentiation to form fully grown oocytes occurs prior t o the cnd of meiosis, in the diploid phase, but in males, the cytological steps 01' gainetogcnic differentiation occur after the end of meiosis, in the haploid phase. In iemales, the progress of meiosis is arrested at the end o f t h e first meiotic prophase and reinitiated at a later time period when an extremely small number of cells are selected for growth and meiotic maturation, stimulated by hormonal signaling. In contrast, in males, meiosis and gametogenesis proceed continuously without cell cycle arrest or interruption. In females, 333
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there is one gametic product per meiosis; in males, there are four. In mammals, meiosis in females requires more time than in males. In the male, meiosis can be initiated and completed in a matter of days, but in the female it takes much longer. and completion of meiosis can be delayed for years. This chapter focuses on sexual dimorphism in the regulation of the progress and tempo of the cell cycle during meiosis. For convenience, we use the term meiotic cell cycle, although it is not strictly accurate because meiosis, by definition. is not cyclic. We use the term to encompass events from the determination of the onset of meiotic prophase, the rate of progress through meiotic stages (particularly prophase I), and the regulation of the transition from meiotic prophase into the meiotic division phases. In this context, we also consider those events that differentiate meiosis from the mitotic division cycle. Most important of these is the homologous recombination occurring during the first meiotic prophase. Because chiasmata are required for accurate anaphase segregation of homologous chromosomes (Nicklas, 1974), we presume that the ganietocyte somehow monitors the progress of recombination to ensure that metaphase chromosome condensation and anaphase chromosome segregation do not occur before the completion of recombination events. Thus, progress through the meiotic cell cycle is tied not only to accurate replication of DNA (as is the mitotic cycle), but also to accurate recombination. Differences exist in rates and distribution of recombination in males and females (see Chap. I . by D. L. Pittman and J. C. Schimenti, this volume); how important these are to the ultimate success of meiosis and gametogenesis is not clear. In spite of the genetic difference between the meiotic and mitotic cell cycles, evidence to date suggests remarkable similarity between the two in the regulation of the transition from prophase, or the end of the G2 phase of the cell cycle, to metaphase (the G,/M transition). In fact. investigation of the oocyte GJM transition provided much of the foundation for the discovery of universal mechanisms of cell cycle control (Murray and Hunt, 1993). The key molecular complex involved is metaphase-promoting factor, MPF (formerly known as maturationpromoting factor). MPF consists of a 34-kDa protein, homologous in all species to the p34 product of the fission yeast d c 2 + gene, and its partner cyclins. The catalytic component, p34cdc2, is a serine-threonine protein kinase whose activity is controlled by its association with the regulatory component, cyclin B, as well as by its phosphorylation status (Murray and Kirschner, 1989; Lewin, 1990; Nurse, 1990; Murray and Hunt, 1993). Because key residues of the p34ccic2must be phosphorylated and others must be dephosphorylated for activity, both kinases and phosphatases are important for regulation of the cell cycle and the G,/M transition, as are a host of other proteins, some known and some as yet unknown. Progress from metaphase to anaphase and division is regulated by proteolytic destruction of cyclin B, mediated by the anaphase-promoting complex (Murray and Kirschner, 1989; King et ul., 1996). We shall consider the role of these
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proteins in the context of sexual dimorphism in the regulation of the exit from meiotic prophase and progress through the division phases.
II. Regulation of the Onset of Meiotic Prophase The first overt sexual dimorphism of mammalian meiosis is the time of initiation and the selection of the population in which meiosis is initiated. This occurs in females during development of the fetal gonad and encompasses the entire pool of mitotically proliferating oogonia. I n contrast, meiosis in males is initiated in the adult gonad from a select population o f proliferating, yet specifically differentiated, spermatogonia. Meiotic prophase is initiated in oogenic cells in the fetal mouse ovary over a period of several days, beginning at Day 13.5 of gestation. In the fetal testis, the spermatogenic cells cease mitotic proliferation but do not enter meiosis. However, fetal spermatogenic cells are not incapable of entering meiosis. In fact, meiosis can be induced in mouse fetal testicular germ cells when cocultured with fetal ovaries containing germ cells initiating meiosis. This suggested that cells in the fetal ovary produce meiosis-activating substance(s). Similar activity has been reported i n extracts of adult testis (Gondos rt a/., 1996) and in ovarian follicular fluid, and there is evidence that these substances are steroids (Byskov r r ( I / . , 1995). Furthermore, fetal male gonocytes arc capable of initiating meiosis when in ectopic locations, such as the adrenal gland (Upadhyay and Zamboni, 1982; Hogg and McLaren, 1985). The fact that male germ cells do not initiate meiosis when within the testis has given r i s e to the idea that there i s a meiosis-preventing substance in the fetal and neonatal testis. Organ culture with testicular extracts leads some experimental support for this idea (Byskov, 1979; Gondos rr a/., 1996). However, there is no information on the nature of this substance. Some progress is being made toward iniderstanding events leading up to the induction of the meiotic cell cycle during spermatogenesis. This occurs only in type B differentiated spermatogonia. Because a set number of mitotic divisions are involved in differentiation of spcrmatogonia as they become type A, intermediate and type B (Meistrich and van Beek, 1993), it is likely that the male germ cell becomes committed (a1though perhaps not irreversibly) to meiosis as it leaves the stem cell pool and differentiates as a type A spermatogonium. These stages of spermatogenic differentiation are correlated with the expression of the Kit gene, coding for a growth factor receptor, on spermatogonia (Dym, 1994; Dym et al., 1995; Orth er i l l . , 1996). The ligand that binds to KIT. the product of the Mgf’gene, is produced by thc Scrtoli cells (reviewed in Kierszenbaum, 1994). It is not yet known if the KIT ligand (KL) i s essential for differentiation and/or proliferation and/or survival of spermatogonia (since all these functions may be related). and the possibility that KL is involved in directly regulating the onset
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of meiosis has not yet been investigated. Nonetheless, in the normal sequence of events, differentiation of spermatogonia is directly linked, by a set number of mitotic divisions, to onset of meiosis. These data implicate a role for Sertoli cellsynthesized KL in the process. Otherwise, factors initiating commitment to and onset of meiosis in the testis are as elusive as they are in the ovary.
111. Genetic Events of Meiotic Prophase: A Regulatory Role in Cametogenesis? A most dramatic aspect of meiosis is the first prophase. Mammalian meiosis is not sexually dimorphic with respect to the relative length of time devoted to prophase (days) as opposed to the division phases (hours). We can assume from this allocation of time for the phases of meiosis that the events of meiotic prophase during gametogenesis are significant and require considerable time. How much of this time is for functions pertaining more to gametic differentiation than for meiotic events is not known. We consider this issue later in this chapter, and here focus on the genetic events of meiosis. The broad outline of steps unique to meiotic prophase I has been established for many species. After replication of DNA, meiotic prophase begins with condensation of chromosomes during the leptotene and zygotene substages. During zygonema, homologous chromosomes begin to pair, with pairing fully established by the pachytene stage. Pairing can be recognized cytologically in pachytene gametocytes by the presence of the synaptonemal complex (SC), a proteinaceous structure whose function is thought to involve mediation of chromosome pairing and/or recombination (see Chap. 7, by P. B. Moens, R. E. Pearlman, W. Traut, and H. H. G. Heng, this volume). Although standard dogma has been that pairing precedes recoinbination, recent molecular evidence from analysis of synchronized yeast meiotic cells suggests that the earliest molecular events of recombination, double-strand DNA breaks, may precede or occur concurrently with the initiation of homologous pairing during the leptotene or zygotene stages (Padmore ef a/., 1991; Hawley and Arbel, 1993). but it is not known if this is the case i n higher eukaryotes. The remaining events of recombination are thought to occur during pachynema and in the context of the SC, but the exact nature of these events at the molecular level and how much of pachynema they occupy are not known. With respect to higher eukaryotes. the time of completion of recombination is not known, nor is it known how the molecular events of recombination are resolved as cytologically visible chiasmata. nor is it known if the molecular events of recombination are wholly completed before chiasmata are visible. Thus the pachytene stage of mammalian meiotic prophase remains a black box. with the beginning defined by homologous pairing of chromosomes and SCs and the end defined by disassembly of SCs and visualization of the chiasmata holding the
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homologous chromosome pairs, o r bivalents, together at crossover sites. In female mice, the events of the pachytene stage encompass several days toward the end of fetal development and culminate in cell cycle arrest at the end of the first meiotic prophase, in an extended diplotene, or dictyate phase. In the male mouse, these events take about a week, with progress directly to the division phases. Events occurring during the period of chromosome pairing are likely to be essential for gametogenesis because male mice with pairing defects fail to complete meiosis, with consequent infertility. Arrest can occur at almost any point during meiosis I prophase, from the zygotene/pachytene transition to the late prophase transition to metaphase (Handel, 1987; de Boer and de Jong, 1989). This apparent checkpoint is another interesting example of sexual dimorphism in meiosis. Females with the same pairing defects also experience germ-cell loss at pachytene, but, unlike males, enough germ cells complete meiosis to result in a limited period of fertility for thcse mutant females (Burgoyne e t a / . , 1992; Mittwoch and Mahadevaiah, 1992; see also Chap. 11, by P. A. Hunt and R. LeMaireAdkins, this volume, for more extensive discussion). These sex-specific differences may possibly derive from a more stringent prophase checkpoint recognizing meiotic abnormalities in male germ cells, or perhaps there is considerable sexual dimorphism in control by a late prophase or metaphase checkpoint. To what extent the tempo of meiosis is regulated and determined by progress and completion of the genetic events of meiotic prophase is not known, and there are only circumstantial data relevant to this issue. It seems likely that there should be meiotic checkpoint incchanisms that ess the fidelity and completion of recombination processes, but the evidence for either signal or effector elements is limited. In female germ cells, acquisition of competence for the meiotic division phase is not temporally linked to meiotic recombination, since competence arises in arrested dictyate cells long after disassembly of the SC. However, the existence of a possible link between completion of recombination events and cell cycle progress may be more easily investigated in male germ cells, where the two arc temporally more closely related. The requirements for onset of the division phase in cultured mouse spermatocytes were studied by making use of premature induction of the G,/M transition by okadaic acid (OA) (Wiltshire et a /., 1995). In this assay, OA is used to abrogate normal cell cycle controls in order to determine when spermatocytes acquire competence to condense chiasmate bivalents, an indication of' completion (or near completion) of recombination. Early pachytene and midpachytene spermatocytes were capable of responding to OA treatment with induction of a GJM transition, evidenced by both SC disassembly and condensation of chiasniatc bivalents in a metaphase-like configuration (Handel and Wiltshire, 1995; M. A. Handel, unpublished observations). Leptotcne and Lygotene spermatocytes responded to OA treatment with condensation of chromatin, but individualized chromosomes and chiasmata were not visible; these early prophase spermatocytes are therefore not competent to condense chiasmate bivalents. Ability to progress into meiotic metaphase arises in
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the context of tripartite SC and chromosome pairing. Cells positive for the Synl antigen, found only in tripartite synaptonemal complex (see Chap. 7, by P. B. Moens e f a/., this volume) are competent, while cells lacking the antigen, the leptotene and zygotene spermatocytes, are unable to condense metaphase chromosomes in response to OA treatment. It is logical to hypothesize, based on this evidence, that onset of competence to condense metaphase chiasmate bivalent chromosomes is likely to be controlled in part by genetic events of recombination. Further evidence that the progress of the meiotic cell cycle may be dependent on completion of genetic events of meiotic prophase derives from the study of mice genetically null (produced by homologous recombination) at the Atm (mutated in ataxia-telangiectasia) locus (see also Chap. 6, by T. Ashley and A. Plug, this volume). The protein encoded by this gene is a member of a family of phosphatidylinositol-3-kinase-likeproteins implicated in DNA metabolism and cell cycle checkpoint control (Hawley and Friend, 1996). The mouse Armencoded protein localizes to regions of the SC of paired chromosomes but not to the lateral axes of unpaired regions (Keegan e t a / . , 1996). The Atm-null knockout mice are sterile, with arrest of spermatogenesis at the zygotene or pachytene stage; and no primary oocytes or follicles were found in the ovaries of adult females, although the stage of oogenic arrest is not known (Xu et a/., 1996). Chromosomes in spermatocytes of these mice exhibit abnormal synapsis and fragmentation. These data suggest a meiotic role for the Armencoded kinase, although the role may well be a direct enzymatic function in recombination, because the mutation does not abrogate a checkpoint; rather, the phenotype reflects a seemingly intact checkpoint mechanism that halts meiosis in response to apparent DNA damage. Clear sexual dimorphism in events linked to chromosome pairing and recombination is seen in effects of the null knockout mutation for a protein ot the SC, HSP70-2 (see also Chap. 5, by E. M. Eddy and D. A. O'Brien, this volume). The HSP70-2 protein is associated with the SC in pachytene spermatocytes but not in oocytes (Allen et al., 1996); thus the SC is also sexually dimorphic with respect to its composition in mammalian meiocytes. Male, but not female, mice homozygous for the induced null mutation in the Hsy70-2 gene are infertile (Dix et ol., 1996). THe HSP70-2 protein seems to act as a chaperonin, associating with p34cdc2 and potentiating its regulatory interaction with cyclin B (Zhu et al., in press). The SC assembles in germ cells of males homozygous for the null mutation but becomes fragmented by diplotene substage, with arrest of germ-cell development and apoptosis (Dix et a/., 1996). These observations also constitute evidence for the importance in regulation of meiotic progress of proteins intimately linked, structurally and temporally, to chromosome pairing and recombination. However tantalizing, the evidence for signal elements that couple cell cycle progress to the genetic events of meiosis derives from the mutations and condi-
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tions described above and is indirect only. Although there is much evidence for and speculation about the role of meiotic checkpoints, thus far we have limited evidence for mammalian mutations with the classic hallmark of meiotic checkpoint mutations (those that permit, rather than abrogate, cell cycle progress in the presence of DNA damage). Experimental resolution of these issues awaits not only discovery of more mutations but also a deeper understanding of the molecular genetic events of recombination and how the gametocyte might both monitor and signal completion and fidelity of these events in order to exit prophase and enter the meiotic division phase.
IV. Regulating G,/M Transition and Meiotic Divisions Far more is known about the regulation of exit from meiotic prophase to the division phase during oogenesis than is known about the same transition during spermatogenesis. Oocytes arrest at the dictyate stage of late prophase, and the ability to manipulate experimentally that arrest and its release led to insights into cell cycle progress that have had universal implications. Here we first review the meiotic G,/M transition as manifest during oogenesis, with an emphasis on features unique to oogenesis rather than on features common to all cell cycles. Unfortunately, the same extent o f knowledge is lacking with respect to the G,/M transition during spermatogenesis. Although there is clear sexual dimorphism in female meiotic arrest and male meiotic progress through the GJM border, there is insufficient information about male meiotic progress to discern whether or not differences in key enzymes and regulatory molecules exist at this critical transition.
A. The GJM Transition during Oogenesis
Although oocytes in nonatretic follicles are arrested at prophase I until the periovulatory period, the mechanisms that maintain meiotic arrest undergo fundamental changes during follicular development. Oocytes in large antral follicles are usually competent to resume meiosis spontaneously, without gonadotropic stimulation, and do so when isolated from this stage of follicular development and cultured (Pincus and Enzmann, 1935; Edwards, 1965), but oocytes from smaller, preantral follicles are not (Szybek, 1972; Erickson and Sorensen, 1974; Sorensen and Wassarman, 1976; Wickratnasinghe et ul., 1991). Thus, the oocytes from antral follicles are maintained in meiotic arrest by factors arising from the oocytes’ companion somatic cells, the follicle cells, whereas oocytes from preantral follicles are arrested at prophase by autonomous regulatory mechanisms. The oocytes of preantral follicles are thercfore incompetent to resume meiosis. Nevertheless, competence to resume meiosis does not necessarily ensure compe-
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tence to complete the first meiotic division and progress to metaphase 11, where meiosis in oocytes is normally arrested once again. Although oocytes isolated from small antral follicles are often able to resume meiosis in vitro, the progression of meiosis becomes arrested in metaphase I, rather than advancing to metaphase I1 (Sorensen and Wassarman, 1976; Wickramasinghe et a/., 1991; De Smedt et al., 1994). Thus, competence to complete meiotic maturation is acquired in at least two steps; first, oocytes in small antral follicles become competent to reinitiate meiosis, undergo germinal vesicle breakdown (GVB), condense chromosomes, and progress to metaphase I. Further development of oocytes at the GV stage, however, is necessary for oocytes to become competent to advance beyond metaphase I, enter anaphase, and proceed to metaphase 11. These final changes in oocyte meiotic competence usually occur during the later stages of antral follicle development. Oocytes that become arrested in metaphase I are referred to as partially competent to undergo meiotic maturation.
1. Factors Participating in the Acquisition of Competence to Resume Meiosis in Oocytes Oocyte competence to resume meiosis results both from processes controlled by an autonomous cell cycle-regulating program and from signals received from somatic cells. Incompetent mouse oocytes isolated from small antral follicles of mice and cultured without any association with somatic cells do not undergo significant growth and do not acquire competence to resume meiosis. In contrast, when cultured with intact gap junctional associations between the oocytes and their companion granulosa cells, the oocytes grow and become competent to resume meiosis. Importantly, incompetent oocytes denuded of their companion granulosa cells but cocultured with them, or in somatic cell-conditioned medium, become competent to resume meiosis and progress to metaphase I even though they do not undergo significant growth (Eppig, 1977; Canipari et a/.. 1984; Chesnel e f a/., 1994). Oocyte growth is therefore not required for oocytes to acquire competence to resume meiosis, even though these processes are normally correlated. Moreover, this observation suggests that a paracrine factor(s) from somatic cells promotes the acquisition of competence to resume meiosis. Insofar as similarly functioning factor(s) are produced by fibroblasts, and not just by granulosa cells, they may be cell-division regulatory factors common to both meiotic and mitotic pathways. Although oocytes do not become competent to resume meiosis without signals from somatic cells, they autonomously acquire some of the components necessary to drive meiosis. Cyclin B and ~ 3 4 ~ d kinase c2 are components of MPF, a key factor in driving mitotic or meiotic cell division. Activation of MPF occurs before GVB and, in mammalian oocytes, continues to increase until metaphase I (Hashimoto and Kishimoto, 1988; Choi et a[., 1991; Hampl and Eppig, 1994). Production of cyclin B and ~ 3 kinase 4 is not ~ synchronous ~ ~ in growing ~ oocytes.
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The concentration of cyclin B is not the limiting factor in the acquisition of competence to resume meiosis, because it reaches its peak in incompetent oocytes. In contrast, the concentration of p34cdc2 kinase increases during the time when oocytes are acquiring competence to undergo GVB (Chesnel and Eppig, 1995; de Vantery et al., 1996; Mitra and Schultz, 1996). In fact, the concentration of p34c.dc2 kinase increases autonomously in oocytes without stimulation by factors from somatic cells (Chesnel and Eppig, 1995). Even though the concentration of p34cdc2 increases, the oocytes remain incompetent to undergo spontaneous maturation unless they are cocultured with somatic cells (Chesnel et a/., 1994). However, exposure to the phosphatase inhibitor OA, promotes the resumption of meiosis by incompetent oocytes expressing (Chesnel et al., 1994; Chesnel and Eppig, 1995), even if that level is relatively low (de Vantery et a/., 1996). Studies on pig and rat oocytes have also demonstrated that the concentration of ~ 3 4 kinase ~ ~ is~ similar 2 in both GVB-incompetent and GVB-competent oocytes (Christmann et a/., 1994; Goren et a/., 1994). Taken together, these studies indicate that, although the production of cyclin B and ~ 3 kinase 4 is ~ necessary for the acquisition of GVB competence, their production alone is insufficient. The synthesis and activity of upstream regulator of MPF critical for the acquisition of GVB competence is probably induced by paracrine factors from somatic cells. Two well-characterized direct regulators of MPF activity are the cdc25 and weel proteins. cdc25, a protein phosphatase, activates MPF, and weel, a tyrosine protein kinase, inactivates MPF. The concentration of cdc25 phosphatase increases and that of wee 1 kinase decreases as mouse oocytes acquire competence to resume meiosis (Mitra and Schultz, 1996). Moreover, the increasing accumulation in the GV of cdc25 phosphatase, as well as cyclin B and p34cdc2, correlates with acquisition of meiotic competence (Mitra and Schultz, 1996). Thus, the location and organizational assembly of these proteins in the GV may be more critical for meiotic competence than their concentration in the oocyte as a whole. The activity of molecules involved in cell cycle regulation is usually controlled by phosphorylation-dephosphorylation mechanisms. I t has long been established that the arrest of oocytes competent to resume meiosis is sustained by elevated protein kinase A (PKA) activity (see Schultz 1991, for review). It is not clear, however, exactly how or where PKA functions. Can it directly phosphorylate components of MPF, or are its substrates far distal to MPF in the phosphorylation-dephosphorylation cascade'? For example, there is evidence that PKA may negatively regulate MPF by mediating the phosphorylation of p34cdL.2 in Xenopus oocytes, thus antagonizing the effect of cdc25A (Rime et a/., 1994). In contrast, some evidence from rat oocytes suggests that PKA does not directly phosphorylate p34cdc2,but that PKA may function upstream to regulate the activity of a tyrosine protein phosphatase whose substrate is p34cdc2 (Goren and Dekel, 1994). I t is also possible that the substrates of PKA are necessary for the regula-
~
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tion of meiosis, but outside the immediate MPF pathway. Besides functioning to sustain meiotic arrest in competent oocytes, PKA may also function in the acquisition of competence to resume meiosis, because incubation of incompetent oocytes, without somatic cells, in medium containing the membrane-permeable CAMP analog dibutyryl cyclic adenosine monophosphate (dbcAMP), augments the competence-acquiring program (Chesnel et al., 1994). The phosphorylation of centrosomal proteins is correlated with the acquisition of competence to resume meiosis by mouse oocytes (Wickramasinghe et al., 1991). Treatment of the oocytes with an inhibitor of protein synthesis results in centrosomal dephosphorylation and loss of competence to resume meiosis. Promoting PKA activity by treatment with a CAMP phosphodiesterase inhibitor, 3-isobutyl- 1-methylxanthine, rephosphorylates centrosomes and restores competence to resume meiosis (Wickramasinghe and Albertini, 1992). Exactly what role the centrosomes play in the acquisition of competence or activation of MPF remains to be determined.
2. Why Do Some Oocytes Arrest in Metaphase I? Oocytes become competent to undergo meiotic maturation in at least two steps. In the preceding paragraphs we discussed step I , the acquisition of Competence to resume meiosis and progress to metaphase I. In step 2, oocytes acquire competence to complete anaphase I and progress to metaphase 11. In mitotic cell cycles, the onset of anaphase occurs via ubiquitin-mediated proteolysis of cyclin B in conjunction with the anaphase-promoting complex (APC); preventing the destruction of cyclin B with a nondegradable mutant form of cyclin B results in inability to exit metaphase (Murray et al., 1989; Huchon et al., 1993). Oocytes isolated from small antral follicles usually become arrested in metaphase I and cannot activate the mechanisms that drive entry into anaphase I; these oocytes are referred to as partially competent to undergo meiotic maturation. Anaphase I in mammalian oocytes coincides with the degradation of cycle B and a decline in p34cdc2activity (Hashimoto and Kishimoto, 1988; Choi et a/., 1991; Fulka et al., 1992; Hampl and Eppig, 1995). Perhaps it is not surprising, therefore, that metaphase I arrest of partially competent mouse oocytes is correlated with high stability and hyperaccumulation of cyclin B and sustained MPF activity (Hampl and Eppig, 1995). The underlying cause for the failure to degrade cyclin B in this situation, however, is not known. Partially competent oocytes apparently possess the necessary proteolytic components for cyclin B degradation since insemination activates the resumption of meiosis and the production of a polar body (Eppig et ul., 1994). Thus, partially competent oocytes seem to be deficient in a mechanism, specific to meiosis I, that activates the cyclin B degradation pathway, since they can enter anaphase after sperm penetration, which generates a signal that otherwise normally promotes entry into anaphase 11. Perhaps partially competent oocytes have not yet produced a fully functional checkpoint at meta-
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phase 1 that can either assess the establishment of a normal metaphase 1 or signal the activation of the cyclin B degradation pathway. Alternatively, the checkpoint may be fully developed but a meiosis I-specific cyclin B degradation pathway is incomplete. Regardless, it is clear that the system that normally responds to sperm penetration and inactivates MPF develops despite arrest at metaphase 1.
3. Mechanism of Metaphase I1 Arrest Two principal factors are essential for the maintenance of metaphase I1 arrest, MPF and cytostatic factor (CSF). High MPF activity is necessary to maintain metaphase I1 arrest, and the function of CSF is to sustain MPF activity. Like MPF, CSF is not a single molecular entity; rather, CSF activity is the coordinated function of at least two molecules, MOS and MAP-kinase. MOS is the product of the M o s proto-oncogene. MOS mRNA in produced in a translationally inactive form throughout mouse oocyte growth (Goldman et a/., 1987; Mutter and Wolgemuth, 1987; Keshet et ul., 1988). MOS is present, but in barely detectable amounts, in fully grown, GV-stage mouse oocytes, but synthesis and accumulation increase dramatically during oocytc maturation (Paules et id,1989). MOS does not appear necessary for GVB in mouse oocytes as it is in frog oocytes (Sagata et d., 1989; Yew et a/., 1992), since GVB occurs in oocytes of Mos-null mice produced by homologous recombination (Colledge et al., 1994; Hashimoto et ul., 1994; Verlhac et nl., 1996). In contrast, MOS is necessary for metaphase I1 arrest in both amphibian and mammalian oocytes. That MOS is required for metaphase I1 arrest in mouse oocytes is demonstrated most dramatically in oocytes from Mas-null mice, in which there is no arrest at metaphase 11. Rather, most oocytes continue directly into anaphase I1 and the next cell cycle (Colledge e t a / . , 1994; Hashimoto et a/., 1994; Araki p t ul., 1996; Verlhac et ul., 1996). The role of the Mos gene is apparently yet another example of sexual dimorphism in the regulation of meiosis since reproduction appears normal in male Mos-null mice (Colledge et ul., 1994; Hashimoto er a/., 1994); see subsequent discussion. How does MOS function to sustain metaphase I1 arrest'? MOS is necessary for MAP kinase activity, and MAP kinase is an essential component of the metaphase 11-arresting system; little or no MAP kinase activity is detected in oocytes of Mos-null mice (Araki et ul., 1996; Verlhac et a/., 1996). Despite a decrease in MPF activity between meiosis I and 11, chromosomes do not decondense and a network of microtubules typical of interphase never forms (Kubiek et al., 1992). In fact, the characteristics of the microtubular network are more correlated with the pattern of MAP kinase activity than with that of MPF (Verlhac et ul., 1994), and some of the MAP kinase in inetaphase 11 oocytes is associated with the microtubular organizing centcrs (Verlhac et ul., 1993). Likewise, MPF activity in metaphase 11-arrested oocytcs also appears associated with the spindle (Kubiak et ul.. 1993). Metaphase I1 arrest depends on an equilibrium of cyclin B synthesis and
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degradation that sustains a critical level of MPF activity (Kubiak et al., 1993). When protein synthesis is inhibited, there is a release from metaphase I1 arrest by a calcium-independent mechanism (Siracusa et al., 1978; Clarke and Masui, 1983; Bos-Mikich et al., 1995; Moses and Kline, 1995; Moos et al.. 1991). Inhibition of protein synthesis probably tips the equilibrium in favor of cyclin B degradation and consequent loss of MPF activity and release from metaphase 11 arrest. Nevertheless, inhibition of protein synthesis is clearly not the physiological mechanism for resumption of meiosis I1 since the kinetics of this process is markedly retarded in the presence of protein synthesis inhibitors compared to the kinetics of calcium-mediated release from metaphase I1 (Moos rt nl., 1996). Preventing the destruction of cyclin B sustains a level of cyclin B necessary to maintain MPF activity, and it appears that the MOS-MAP kinase cascade functions to inhibit cyclin B degradation. There is no MAP kinase activity in Mosnull mice and no metaphase I1 arrest. Moreover, metaphase I1 arrest is released by addition of a specific MAP kinase-inactivating phosphatase to extracts of Xenopus eggs (Minshull rt al., 1994). Thus, MOS probably functions to maintain metaphase I1 arrest by promoting MAP kinase activity, which in turn either inactivates the cyclin B degradation system or prevents its activity from being increased. In either case, the net result is to tip the cyclin B concentration equilibrium above the critical level necessary to sustain MPF activity.
4. Release from Metaphase I1 Arrest Fertilization is the key event that signals the release from metaphase I1 arrest. The sperm-mediated signal transduction mechanisms that activate the egg are beyond the scope of this review, but the critical event is the release of the egg’s intracellular calcium (see Carroll etal., 1996, for review). Although the activity of CSF is clearly involved in maintaining metaphase I1 arrest, degradation of cyclin B occurs before degradation of MOS and inactivation of MPF (Lorca etul.. 1991; Watanabe et al., 1991; Weber et al., 1991). The decrease in MPF activity also precedes decreased MAP kinase activity (Fen-ell et al., 1991; Lorca et al., 1993; Verlhac er d., 1994; Moos et ul., 1995). The crucial mechanism that initiates cyclin B degradation and consequent MPF inactivation is not well understood, but the activation of type 11 calcium-dependent protein kinase (CaM KII) is probably involved. For example, studies using extracts of Xenopus eggs found that cyclin B degradation and the metaphase to anaphase transition could occur on addition of a constitutively active CaM KII, even in the absence of calcium (Lorca rt al., 1993, 1994; Morin rt al., 1994). Moreover, the activity of CaM KII is elevated after stimulation of parthenogenetic activation of mouse eggs (Winston and Maro, 1995). Thus, the activation of CaM KII is probably a specific target for calcium mobilized at fertilization and for release from metaphase I1 arrest. In the absence of MOS and MAP kinase activity, microtubules are not sustained in the normal metaphase-like configuration and normal spindles are not
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maintained (Araki et al., 1996; Verlhac et (I/., 1996). These observations establish a link between the MOS-MAP kinase cascade and microtubule organization. Proper maintenance of the spindle may play some role in the maintenance of metaphase I1 arrest (Kubiak et ( I / . , 1993), but spindle maintenance is clearly very important for egg activation after sperm-egg interaction. Disruption of the metaphase 11 spindle with microtubule-depolymerizing agents, such as colcetnid or nocodazole, prevents activation by sperm, as indicated by failure of the eggs to decondense chromosomes and enter interphase (G. Schatten et a/., 1985; Maro et a/., 1986; H. Schatten et a/., 1989).Nevertheless, the mobilization of intracellular calcium induced by either fertili~itionor parthenogenetic activation is not prevented by spindle dissociation (Winston P I al., 1995), and neither is the increase in CaM KII (Winston and Maro, 1995). Thus, release from metaphase 11 arrest requires some function of the spindle to utilize the calcium signal to initiate cyclin B degradation and consequent reduction of MPF activity. The reason for this can be only speculated, but several of the molecules essential for either the maintenance of metaphase I1 arrest, such as MPF, MOS, and MAP kinase, or required for calcium-mediated release from arrest, such as CaM KII, are reported to be associated with spindles/microtubules (Ohta el NI., 1990; Zhou et ( I / . , 1991; Kubiak et nl., 1993; Verlhac et ( I / . , 1993). Entry into anaphase I1 requires more than simple reduction of MPF activity and termination of metaphase 11 arrest. Addition of a nondegradable cyclin B to Xenoprs egg lysates prevents inactivation of MPF but does not block sister chromatid separation (Holloway et u / . , 1993). Moreover, inhibition of ubiquitinmediated proteolysis in the lysates delays sister chromatid separation (Holloway ef ul., 1993). Thus, the protein “glue” (Wells, 1996) that holds sister chromatids together may require ubiquitin-mediated proteolysis before entry into anaphase is possible. regardless of MPF Ievcls. This is supported by observations of Drosophilcr mutations yirizyles and rl7rc.e OM'S that produce defects in sister chromatid separation during mitosis without preventing the degradation of cyclins (Stratmann and Lehner, 1996). It is proposed that the Pirn-encoded protein functions at the metaphase/anaphase transition and is involved in the release of sister chromatids (Stratmann and Lehner, 1996). Furthermore, genes have been identified in budding yeast that are important for proteolysis of cyclin B; yeast carrying mutations in these genes fail to undergo the metaphase/anaphase transition (Irniger et d., 1995). It is thought that the products of some of these genes might also be critical for proteolysis of proteins leading to sister chromatid separation. Taken together, studies such as these indicate that successful metaphase II/anaphase I1 transition in oocytes requires proteolysis of both cyclin B and molecules that function in sister chromatid adhesion. Both may be initiated by a checkpoint located at the kinetochore or spindle apparatus. (See also Chap. 8, by D. P. Moore and T. L. Om-Weaver. this volume.) This may explain, in part, why disruption of the spindle by niicrotiibule-depolymerizing agents prevents egg activation.
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B. The CJM Transition during Spermatogenesis The G2/M transition during spermatogenesis is of considerable interest because it is temporally closely linked to events of recombination (see preceding discussion), but it has been difficult to study experimentally. There is no arrest at the end of prophase and consequently no accumulation of cells at the GJM boundary. The progress of meiosis is continuous and rapid from the end of meiotic prophase through the division phases. Although prophase takes somewhat more than a week in mouse spermatocytes, the late prophase (diplotene stage) and meiotic divisions are accomplished in two days or less. Thus, at any given time, the numbers of cells entering meiotic divisions are not high relative to meiotic prophase spermatocytes or postmeiotic round spermatids. Additionally, because these cells do not have unique size characteristics, they cannot be separated by common methods employed for enrichment of the large pachytene spermatocytes or the small round spermatids (BellvC e t a / . , 1977; BellvC, I993), further impeding their accessibility for analysis. Consequently, much of what we know about the G,/M transition during spermatogenesis derives from studies on steady-state levels (blotting methods and in situ hybridization) of transcripts and proteins deemed likely candidates as participants in the cell cycle transition, and from an experimental assay for competence to undergo the GJM transition (Wiltshire et al., 1995).
1. Proteins That Might Be Involved in Spermatogenic G,/M Transition The developmentally regulated pattern of transcription during meiosis of various genes encoding cell cycle-related proteins is reviewed by E. M. Eddy and D. A. O'Brien (see Chap. 5, this volume). We briefly review here those proteins most likely to be candidates for involvement in the G,/M transition by virtue of their presence in the critical cells at the right time, namely, in late pachytene spermatocyte. Simply because it is the most universal, the prime candidate for mediating the onset of spermatogenic meiotic metaphase is the p34cdc2protein kinase that is the catalytic component of MPF. Highest levels of transcript for ~ 3 4 were ~ 'found ~ ~ ~ in pachytene spermatocytes (Rhee and Wolgemuth, 1995),as were high levels of p34~"2 protein and immunoprecipitable H 1 kinase activity (Chapman and Wolgemuth, 1994), when levels in pachytene spermatocytes were compared with those in round spermatids and cytoplasmic fragments and residual bodies. To explore further the accumulation of this protein throughout meiosis I prophase, itnmunoblotting of proteins from highly enriched fractions of leptotene/zygotene and pachytene spermatocytes was used to demonstrate that ~ 3 4 ' ~protein ~' is present in both leptotene/zygotene spermatocytes that are incompetent to condense metaphase chiasmate bivalent chromosomes, and in higher levels in the competent pachytene spermatocytes (J. Cobb and M. A. Handel, unpublished observations). I t has been shown that mouse spermatogenic cells express both cyclin B1 and
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B2 transcripts. The level of c y . H / transcript is higher in postmeiotic cells than in pachytene spermatocytes, while (,!cB2 is most abundantly expressed in pachytene spermatocytes (Chapman and Wolgeinuth, 1992, 1993). In contrast to transcript levels, the highest levels of cyclin B 1 protein were found in pachytene cells (Chapman and Wolgemuth, 1994). Additionally, the mouse cyclin Al gene is highly expressed in late pachytcne cells, suggesting possible involvement i n the GJM transition (Sweeney et ( I / . , 1996). while the mouse cyclin A2 is expressed in a pattern suggesting G, /S or S involvement but not a role in the onset of meiotic metaphase (Ravnik and Wolgemuth, 1996). A novel serine/threonine kinase that has been implicated in G,/M regulation is the NIMA protein, essential for mitosis i n A.~p~r-gillu.s nidulatzs. A mouse homolog is the protein product of the N r k / gene. This gene is highly expressed in pachytene spermatocytes (Letwin c ~ (t I / , , 1992) and there is evidence that it could be a participant in the meiotic G,/M transition of spermatocytes (Rhee and Wolgemuth, 1997). The phosphatase product ot the Ctlc2SC gene is expressed in the testis and is found in pachytene spermatocytes. but in higher levels in round spermatids (Wu and Wolgemuth, 1995). By comparison to its role in other systems, the Cdc2SC phosphatase might be expected to play a key regulatory role in the onset of sperniatogenic meiotic metaphasc. Although there is no direct evidence for its role, it is found in the right place at the right time. A major conundrum involves the role of the MOS protein. As reviewed earlier, this protein is a critical component of CSF and necessary for maintenance of MI1 arrest i n oocytes. Results from two different knockout mutations demonstrate that the Mo.r gene product is essential for female fertility in mice but not for male fertility (Colledge r t a!., 1994; Hashimoto et ul., 1994), a clear sexual dimorphism in the use of this protein, which might be most simply explained by the lack of a metaphase arrest in spermatocytes. There is some inconsistency in the literature with respect to expression of transcript and protein product of the Mo.s gene. Transcripts of the M o s gene are produced during spermatogenesis; some authors tind them restricted to haploid cells (Goldman rt ( I / . , 1987; Sorrentino et al., 1988), whereas others find them in pachytene cells but most abundantly in haploid cells (Mutter and Wolgemuth, 1987). The 43-kDa MOS protein has been detected by Western blotting as expressed in mouse pachytene sperinatocytes (Herzog rt (I/., 1988) and also in the rat, again, only in predivision phase pachytene spermatocytes (van der Hoorn P t ( I / . . 1991). An interesting and largely unexplained observation is that overexpression of a c-mos transgene driven by a pachytene-specitic promoter results i n an apparent increase of mitotic proliferation of spermatogonia (cells not expressing the transgene); this may have resulted from an alteration in germ cell-somatic cell interactions in the testis (Higgy et d., 1995). The function of the MOS protein in male germ cells is unknown, but clearly it is not critical in meiotic regulation or can be compensated by other proteins, since the null mutation does n o t impair male fertility. Taken together, these studics suggest that many universal cell cycle compo-
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nents that function during the oogenic GJM transition are also present in the relevant spermatogenic cells before the GJM transition, although their function has yet to be demonstrated. However, because cells undergoing the GJM transition cannot be purified from mouse testes, it has not been possible to assess the presence or activity of these proteins at the critical time.
2. Spermatogenic G,/M Transition in vitro Although it has not been technically feasible to isolate and study testicular germ cells undergoing the GJM transition, pachytene spermatocytes maintained in short-term cultures experimentally treated with OA undergo a precocious GJM transition involving both disassembly of SCs and condensation of chiasmate bivalent chromosomes (Wiltshire er a/., 1995). This suggests that disassembly of SCs and chromatin condensation are linked and can both be activated by common cell cycle controls, although they may well proceed subsequently by independent pathways. The configuration of metaphase chromosomes after OA treatment is attained through the usual substages of late pachytene, diplotene and diakinesis; the condensed bivalents are morphologically indistinguishable from those of cells undergoing a normal GJM transition; and the bivalents exhibit the normal numbers of chiasmata (Wiltshire et a/., 1995). This experimental paradigm allows assessment of proteins involved in the induced G,/M transition as well as of factors contributing to spermatocyte competence to undergo the transition. Spermatocytes treated with OA consistently undergo activation of histone H 1 kinase concurrently with transition to a meiotic metaphase-like configuration of chromosomes (Wiltshire er a/., 1995). The induced G,/M transition and activation of HI kinase are blocked by staurosporine, a broad-spectrum inhibitor of protein kinases, including MPF (J. Cobb and M. A. Handel, unpublished observations). Although this evidence is strongly suggestive of a role in this cell cycle transition for the universal cell cycle regulator, MPF (most commonly assayed by its histone HI kinase activity), it has not been possible to demonstrate that the G,/M transition induced by OA is actually dependent on the concurrent activation of MPF. Synthesis of ~ 3 is not4 a controlling ~ ~ factor ~ in the ~ spermatocyte’s acquisition of competence to undergo the OA-induced G,/M transition. insofar as leptotene and zygotene cells incompetent to undergo the G,/M transition contain p34cdc2, although in lower concentration than the competent pachytene spermatocytes (J. Cobb and M. A. Handel, unpublished observations). Although accumulation of a critical concentration of p34cCic2may be responsible for the acquisition of competence to undergo the induced transition to metaphase, it cannot be the factor controlling the onset of metaphase in vivo. Pachytene spermatocytes have maximal levels of p34cdc2 and are competent to undergo the induced cell cycle transition, but do not spontaneously enter meiotic metaphase. The critical control over the entry into meiotic metaphase irz vivo may lie not in
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accumulation o r activation of cell cycle regulatory protein but rather in their subcellular localization, which may change during the acquisition of competence for the division phase. The "OA assay" has also permitted assessment of the requirement for topoisomerase I and I I activity i n the induced GJM transition. demonstrating a requirement for the latter. Although the topoisomerase I inhibitor camptothecin does not prevent the induced < ; , / M transition. two diffcrently acting inhibitors o f topoisomerase 11, teniposidc ond IC'KF- 193, completely inhibit the G,/M transition (Cobb et d . , 1997). Interestingly. the activity o f topoisomerase I I is required for some but not all aspects 01' the induced transition to meiotic metaphase. Condensation of chromatin to form individualized bivalent chromosomes requires topoisomerase 11 activity. hut the concurrent disassembly o f the S C occurs even when topoisomerase I I i s inhibited (Cobb et ( I / . . 1997). The requirement for topoisomerase I I activity in anaphase separation of chromatids has previously been demonstrated in ;I number of cell types, and meiotic division arrest induced by inhibition 01' topoisoinerase I I has been observed in cultured X w o p s spermatocytes ( Morsc-<;audio and Risley. 1994). Not surprisingly. this requirement for topoisomerase I I activity is probably not an example of sexual dimorphism, l'or. although a requirement for topoisomerase I1 has not been yet demonstrated lor the oocytc's GJM transition. it i s implicated in the transition from melaphase to anaphase in the separation of honiologous chromosomes (Fulka ('t o/.. 1994). Interesting implications about nieiotic competence can be derived from these analyses of induced metaphase transition hy cultured pachytenc spermatocytes. Both male and female ganietocytes undergo considerable growth a s they progress toward meiotic metaphase. However. ;IS i n the female, growth per se of the spermatocyte i s not required for ;icquisition o f competence t o undergo the induced G,/M transition, because small early pachytene cells are competent (Handel and Wiltshire, 1995; M. A. Handel, unpublished observations). Accumulation of appropriate cell cycle regulators (e.g., MPF, phosphatases. topoisomerases) must occur in the acquisition of competence but may not be the determining regulator. Instead. competence appears to be related most closely to the progress of the genetic events of mciotic prophase. for complete pairing o f chromosomes must be accomplished before acquisition of competence ( Handel and Wiltshire, 1995; M . A. Hanciel. unpublished observations). Even so, competence to undergo the induced G 2 / M transition arises early in the pachytene stage, at a time when events of recombination iire probably still in progress. Either recombination is completed earlier than might be anticipated. or the completion of these events is tied to cell cycle regulatory mechanisms and is brought about hy O A treatment. Thus far, it has been possible to utiliLe experimental induction of the GJM transition to assess some aspects of acquisition of spermatocyte meiotic conipetence. Informative as this experimental analysis has been, it has not yet yielded
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insights into what controls the end of meiotic prophase and the onset of the division phase in viva. That is, cells that have acquired competence to undergo the OA-induced G,/M transition in culture are cells that do not enter meiosis in iivo or, indeed, in culture without OA. Thus, it remains an enigma how the spermatocyte knows that it has reached the end of meiotic prophase and that it is time for the division phase. It is possible, as is the case for oocyles, that both factors intrinsic to the spermatocyte, which may be those assessed in culture, and those emanating from the surrounding Sertoli cells are required.
3. Metaphase-Anaphase Transition during Spermatogenic Meiosis Next to nothing is known about the transition from nietaphase I to anaphase and the factors involved in the spermatocyte’s rapid progress directly through the second meiotic division. The GJM competence assay described earlier does not permit analysis of anaphase, because OA interferes with assembly of the meiotic spindle apparatus. There has been only limited success in analysis of this transition in cultured spermatogenic cells, and no consistent success utilizing nontransformed cells. However, some systems for in vitm analysis of meiotic progression have used transformed cells. In one study (Rassoulzadegan et d., 1993), immortalized Sertoli cells were used as a substrate for culture of a mixed population of germ cells. Analysis by flow cytometry suggested the appearance, in a population of diploid cells, of 4C and then 1C cells, suggesting that meiotic division might be occurring in some of the germ cells. There was, however, no analysis of these cells to determine the presence of SCs (a hallmark of meiosis) or meiotic metaphase chromosomes or segregation of homologs (Rassoulzadegan et a/., 1993). Hofmann et d.(1994) reported meiosis in immortalized germ cells, but the cells capable of meiosis have not been successfully propagated in culture (Wolkowicz et al., 1996).Thus, although these reports provided hints of meiotic divisions in vitro, neither the culture parameters nor the cytogenetic capabilities were amenable to definitive conclusions about meiotic divisions, and we remain without an appropriate experimental system for analysis of this important step of the spermatogenic meiotic division phase.
V. Gametic Function of Meiotic Prophase A nonsexually dimorphic feature of mammalian meiosis is the gametocyte growth that occurs during meiotic prophase. In the mouse, oocyte growth occurs at the end of meiotic prophase, in the extended diplotene, or dictyate, phase, and results in an increase in cell diameter from 20 p m to 80 pm, whereas mouse spermatocyte growth occurs throughout meiosis I prophase, resulting i n an increase in cell diameter from 8 p m to 18 pm. Such growth is a common feature in animal meiosis and apparently serves a gametic function. In the female, the
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growth represents the accumulation of maternal factors (e.g., stored mRNAs, proteins) necessary for early embryonic development, and can be quite dramatic in cases when all or most of embryonic development depends on stored egg nutrients, as in amphibians. The future success of the embryo is ensured further by unequal cytoplasmic divisions in meiosis, discarding three of four meiotic products in favor of one with all the accumulated maternal factors for early embryogenesis. In the male, growth most likely reflects the accumulation of factors required for subsequent haploid spermiogenic differentiation (see Chap. 5 , by E. M. Eddy and D. A. O’Brien, this volume). Even though haploid gene expression of mRNAs for spcrmiogenesis is well documented, many mRNAs used in the haploid phase are transcribed during the diploid phase in the spermatocyte. Dramatic growth during the diploid phase is a strategy that appears to be universally advantageous in gametogenesis and ensures garnetidembryonic benefits of the entire parental genome. Additionally, evolutionary conservation of this feature of meiotic prophasc may also lie in the fact that the open and extended chromatin configuration characteristic of meiotic prophase provides a template not only for recombination but also for abundant transcription over an extended period of time, facilitating the accumulation of parental factors for successful gametogenesis and early embryogenesis. Whether this is related temporally or mechanistically to the establishment of appropriate parental imprint is not yet known, although it is clear that parental imprint is established anew in the germ line.
VI. Summary and Perspectives This review of regulation in the mammalian meiotic cell cycle has emphasized both common and sexually dimorphic features of oogenesis and spermatogenesis. The existence of common features has led to synergism in two areas of endeavor: the study of regulation of oogenesis and the regulation of spermatogenesis. For instance, it is already clear that the identification of evolutionarily conserved molecules regulating oogenic progress into the meiotic division phase will continue to be informative about the mechanism of meiotic progression in spermatogenesis. On the other hand, key features of mammalian meiosis are sexually dimorphic. As we continue to explore these differences experimentally, we should not be surprised to discover that these dimorphisms may account for sex differences in the roles of even those proteins common to both oocytes and spermatocytes; perhaps common regulatory mechanisms are not at work. For example, meiotic arrests are a characteristic of oogenesis and can occur at varied stages of the meiotic cycle, at the GV stage or metaphase I or metaphase 11 (Sagata, 1996). Why such meiotic arrests occur only in the female gamete is purely conjectural at this point: Are they a means of synchronizing the parental genomes? Do they facilitate accumulation of maternal factors? In con-
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trast, meiotic arrests are not a feature of spermatogenesis. This renders experimental dissection of regulation of the meiotic division phase in spermatogenesis more difficult, but greater understanding of this important dimorphism may shed light on the sex-specific gametogenic requirement for the Mos gene product, as well as other key regulatory differences. Another potentially important dimorphic feature of mammalian meiosis lies in the differences in anatomical relationships of somatic cells to germ cells in the ovary and the testis. In the ovary, many follicle cells surround a single germ cell until the meiotic division phase; there is bidirectional communication, and thc coordinated activities of the follicle cells provide some of the signals to the oocyte for meiotic progress. Disruption of the communication between follicle cells and the oocyte can signal the end of meiotic prophase and entry into the division phase. In contrast, a single Sertoli cell is in contact with several generations of germ cells in the testis. Furthermore, disruption of communication between the Sertoli cells and spermatocytes does not appear to signal entry into the meiotic division phase. There is ample evidence for germ cell-Sertoli cell communication, but as yet no identification of signals regulating meiosis. In fact, it will require considerable ingenuity to understand how the Sertoli cell, receiving information from several generations of germ cells, could send meiotic signals recognized by only one of the several kinds of germ cells that it nutures. Although i t is likely that the Sertoli cells provide such signals, they clearly are not sufficient, and germ cell-autonomous signals are also required for spermatogenic meiotic progression. In these respects the spermatogenic cell is perhaps more similar than the oocyte to simpler systems for analysis of meiosis, such as yeast (Giroux rt a/., 1993). Because in higher organisms meiosis is both unique to and a defining event of gametogenesis, understanding the common and sexually dimorphic features of regulation of meiotic progression and division in germ cells will not only enhance our knowledge about meiosis in general but may also lead to practical benefits for control of gametogenesis.
Acknowledgments Work in the authors’ laboratories was supported by NIH grants HD31376 and HD33816 to MAH and CA62392 to JJE. We are grateful to Yugi Hirao, Patricia Hunt. Barhara Knowles. John Schimenti, and Richard Schultz for thoughtful comnients on thc manuscript.
References Allen, J. W., Dix, D. J., Collins, B. W., Merrick. B. A., He, C., Selkirk, J. K., Poorman-Allen, I?, Dresser, M. E., and Eddy, E. M. (1996). HSP70-2 is part of the synaptonemal complex in inouse and hamster spermatocytes. Chromosornc~104, 4 14-42 I . Araki, K., Naito, K., Haraguchi, S., Suzuki. R., Yokoyaina, M., Inoue, M., Aizawa, S.. Toyoda,
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Verlhac. M.-H., Kubiak, J. Z.. Claihe. H. J., and M x o . B. (1994). Microtuhule and chromatin behavior follow MAP kinase activity but i i ~ MPF t activity during meiosih in mouse oocytes. Dul,f4o/""e/lt 120, 1017 - 1025. Verlhac, M.-H., Kubiak, J. Z.. Weber, M., Geraud, G., Colledge, W. H., Evan\, M. J., and Maro, B. (1996). Mos is required for MAP kinase iictivation and is involved i n microtubule organization during meiotic maturation i n the mouse. Dwdoprwti~122, 8 I5-X22. Watanabe. N., Hunt, T., Ikawa. Y., and Sagata, N . (199 I ) . Independent inactivalion of MI'F and cytortatic factor (Mos) upon fertiliiation of X r m p u eggs. N o t w e 352, 247-248. Wcbcr, M., Kubiak, J. Z., Arlinghauh, R. R . , Pines, J., arid Muro, R. (1991). c-mos protooncogene product is partly degraded after release from meiotic arre\t and persists during inter. w . Biol. 148, 393-397. phase in mouse ~ y g o t c \ D Wells, W. A. E. (1996). The spindle-a\sembly checkpoint: Aiming lor a perl'ect mitosis, cvci-y lime. Trerids Cull B i d . 6, 228-134. Wickramasinghc. D., and Albertini, D. F. ( 1992). Centrouomc phoaphorylalion and the dewlopmental expression of meiotic competence i n mouse oocytes. Dm: Bwl. 152, 62-74. Wickramasinghe, D.. Ebert. K. M., and Alhertini, D. E (1991L Meiotic competence acquisition i\ associated with the appearance 01' M-phase chiiriicteristic\ in growing mouse oocyte\. D n : B i d . 143, 162-172. Wiltshire, T., Park. C., Caldwell, K. A.. and Handel. M . A. (1995). Induced premature G Y M transition i n pachytcne spermatocyte\ includes events unique to meiosis. Dui: H i d 169, 557567. Winston, N. J . , and Maro. W. ( 1995). Calmoduliri~dependentprotein kinase I I activated tr;inricntly in ethanol-stimulated mouse oocyte\. Dri: B i d . 170, 350-352. Winston, N . J . , McGuiniiess, 0..John\on, M. H.. and Maro. B. (1995). The exit of niouw oocytes from meiotic M-phase requires an intact spindle during intracellulai- calciunl release. .I Cell Sci. 108, 143-151 Wolkowicr, M . J., Coonrod. S. M., Reddi. P. P.. Millan, J . L.. Hofiiiann. M . C.. and Herr. J. C. ( 1996). Relinement 0 1 the differentiated phenotype of tlie \permatogenic cell lnic GC-lapd(ts). B i d . Rc,prod. 55, 923-932. Wu, S., and Wolgemuth, D. J. ( 1995). The d i d n c t and developmentally regulated pattern, of expression of members of tlie niousc cdc25 gene family \ugge\t differential function\ dui-ing gametogene\iu. Dci: B i d , 170, 195-206. Xu. Y.. A\hley, T., Brainerd, E. E., N r o n m , R. .I., Meyn. M. S . , and Baltimore, D. ( l9Yfi). Targeted disruption of ATM leads t o growth retardation, chromosomal fragmentation during nicioai\, immune defects, and thymic lymphonla. Curie.\ /lei: 10, 241 1-2422. Yew, N., Mellini, M. Id.,and Vande Woude. G. F. ( 1992). Meiotic initiation by the t r i m protein i n Xariopuc.. Nrittrr-c.355, 640-652. Zhou, R. P.. O\harsson, M., Paules, R. S.. Schult/, N., Cleveland. D., a i d Vande Woude. G. E ( 199 I). Ability of the c-rrio.~product to asxx-ialc with arid pho5phorylate tuhulin. Scrrrrc.r 251, 6 1 1-675. Zhu, D.. Dix, D. J.. and Eddy, E. M. (1997). HSP 70-2 i \ required for CDC2 kinase acti\lty in meiosi\ I of mouse spermatocytcs. D u i ~ u / o p r ? ~ (~i n~ rpress) i/
11 Genetic Control of Mammalian Female Meiosis Patricia A. Hunt and Renee LeMaire-Adkins Department of G e n e t i c s and the C e n t e r for Hiinmi Genetics Case Western R e s e r v e University University Hospitals of Cleveland Cleveland, Ohio 44106-4955
I . Inti-oduction 11. The Human Fernale Meiotic Pro 111. Female Meiosis I\ Initiated diiriiis 1,etal De\cltipinent IV. A Quality Control Checkpoint Opci-ate\ at l'acliylene A Loss o f Germ Cell\ ;it the Pacliytciic S t q c I \ ;I Characteri\tic 01 Female Meiosis B. Yeast Mutants Provide Insight into the Nature ot the Pachytcne Checkpoint ;itid the Gcnes Involved C . Studie\ o l Mice with Numcric;il ant1 Structui-al Ahiiormulitie< pi-ovidc Evidcncc That a Pachytene Checkpoint Operates i i i I3otli Mnlc\ atid Feinale\ V. The Ability to Resume Meiosi\ I\ Acquired (1 A . Meiotic Competence I s Acquii-ctl i n S t c p of Oocyte Growth K Meiotic Resumptioil Occurs iii Ke\poii\c t o 1'eriovulatot.y Horrnoiial Stirnuli C. Evidence That thc P r o c e s ~t i t I,'(illiculogciic\is 15 Coiiiproiiiised i n the Reproductively Aged Human Ovary I>New Technique\ for the iir \';[/.(I Gi-o\bth (11 Oocytc\ PI-wide a New Approach to Studying the Factors That Iiilluencc Mciotic Chroinosome Segi-egation VI. Chroiiio\oiiic\ Play ; i n Active Kolc iii the Forinntion o f the Meiotic Spiiidlc VII. The Mctnphase/Anapli~iseTraii\itiriii A . 'The Role of Cliia\iiiata in M c ~ i o ~Clit.oino\oinc ic Segregation H. Evidence for a Spindle A\seiiihl> /Chroiii~i\ome--Medi;itedCheckpoint C . Mammalian Fein;ilc Mciosi\ l.ack\ Chtiiiiio\oine-Mcdiated Checkpoitit Coiitrol VI11. AIrest at Second Meiotic Metaphn\c: Ilo Chroiiiosoinz\ Play a Rolc'l IX. The Future: Mammalian Meiotic Mutant\ Will Pi-wide Important Iiihights into the Conti-ol ot Mainmalisn Feinalc Meiosi\ Kelct.ence\
1. Introduction Mammalian female meiosis has two unicliie features: the division is extremely protracted-in long-lived species, completion of meiosis can take decades-and the tnciotic divisions, especially i n the human, are particularly error prone. The lack of naturally occurring meiotic mutants has severely hampered the study of
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mammalian meiosis. Because the basics-homologous chromosome pairing, recombination, and segregation-are highly conserved among species, i t seems safe to assume that many of the genes that control the meiotic process will also be conserved. Although our current understanding of meiotic cell cycle control in mammals is derived largely from studies in yeast and Drosophila, gene targeting and transgenic technology now make possible direct studies of the role of individual genes in the mammalian meiotic process. As in mitotic cells, the meiotic process is subject to checkpoint control mechanisms that regulate cell cycle progression. However, unlike mitotic or male meiotic cells, the meiotic process in females requires additional cell cycle controls to ensure arrest at two independent points in the cell division because, with few exceptions, mammalian female meiosis is characterized by a protracted arrest at prophase of the first meiotic division (MI) and a subsequent arrest at metaphase of the second meiotic division (MII). This chapter considers the control of chromosome segregation during mammalian female meiosis. In reviewing this process, we place particular emphasis on the complexities of mammalian meiosis and on differences between male and female mammals, for we believe that an understanding of these differences may provide insight into the high error rate in the meiotic process in the human female. Sexually dimorphic aspects of the regulation and tempo of the mammalian meiotic cell cycle are also discussed by Handel and Eppig (Chap. 10, this volume).
II. The Human Female Meiotic Process Is Error Prone The human female appears to be particularly prone to meiotic errors: at least 1520% of all human conceptions are chromosomally abnormal as a result of errors in chromosome segregation during meiosis (Jacobs, 1992). The past decade has brought a significant increase in our understanding of the origin of human aneuploidy and of factors associated with human nondisjunction. For example, we now know that the vast majority of human aneuploidy is maternal in origin, resulting from errors in chromosome segregation during female meiosis (Hassold et a/., 1994); that the incidence of maternal meiotic errors is strongly correlated with maternal age (H old et ul., 1994); and that aberrant genetic recombination is an important component of human female nondisjunction (Hassold et a/., 1995; Lamb et a/., 1996). The correlation between maternal age and the incidence of meiotic errors is especially striking. Current risk figures suggest that, by the fourth decade of life, approximately half of the oocytes a woman ovulates may be chromosomally abnormal (Hassold, 1986). Little is known about the basis of this effect. It is commonly believed to originate in maternal MI because, in the human female, oocytes enter meiosis during the fetal period and remain suspended in prophase of MI at the diplotene (dictyate) stage until ovulation. As a result, completion of
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MI may take 40 years or longer. The duration of the division has led to speculation that the basis of the age erfect on trisomy is due to events occurring either prenatally at the time of entry into MI (Henderson and Edwards, 1968), during the interval between meiotic anesl and rcentry (e.g., Penrose, 1965), or during the periovulatory period at the time of reentry into MI (Crowley et 01.. 1979; Sugawara and Mikamo, 1983; Eichcnlaub-Ritter et a/., 1988; Warburton, 1989); or it may even be due to changes in the uterine environment that influence the survival of the chromosomally abnormal conceptus (Ayme and Lippman-Hand, 1982). Although the weight of evidence from human studies argues against changes in the uterine environment (the so-called "relaxed selection" model) as a causal factor (reviewed in Hassold e t a / . , 1993), the present data do not allow us to distinguish between factors acting prenatally (at the time of meiotic entry) or postnatally (during the prolonged resting phase or at the time of resumption and completion of MI). As discussed i n Sections V and VI1, the most recent evidence suggests that human age-related aneuploidy may reflect a decline in the process of oocyte growth as well as differences in cell cycle control between male and female meiosis.
111. Female Meiosis I s Initiated during Fetal Development In the majority of mammals, mitotic proliferation of oogonia is limited to the prenatal period, and all oogonia enter meiosis before or shortly after birth. The prenatal differentiation of germ cells has been best characterized in the mouse: the germ cell lineage is though1 to be established during early gastrulation. when an estimated 40 or so cells are segregated to the extraembryonic mesoderm (Lawson and Hage, 1994). Germ cells colonize the developing genital ridge at approximately 10.5 days of gestation and continue to proliferate mitotically for the next several days. The first cells enter meiosis at approximately 13.5 days of gestation, and the rest of the population initiates the division over the course of the next several days (McLaren, 1983). The signals that control the earliest events in germ cell differentiation remain poorly understood. Available evidence suggests that X-reactivation occurs in XX germ cells in the developing ovary shortly after their arrival at the genital ridge and several days prior to meiotic entry (Monk and McLaren, 1981; Tam rt ol., 1994). Because XX germ cells thac fail to reach the genital ridge do not appear to undergo X-reactivation, it has been postulated that the signal for reactivation emanates from the developing ovary (Tamrr a/., 1994). However. the subsequent entry into meiosis is apparently independent of external signals, because in both male and female embryos, germ cells that fail to enter the genital ridge and are found in the developing adrenal glands initiate meiosis prenatally on a normal female schedule (Zamboni and Upadhyay. 1983; reviewed in McLaren, 1985). The behavior of these ectopic germ cells suggests that prenatal meiotic initiation
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is not induced by the differentiating ovary but rather is a germ cell-autonomous event that, in the male, is inhibited by signals produced in the developing testis. Recent studies have demonstrated that, under the influence of appropriate growth factors, murine primordial germ cells can be induced to proliferate indetinitely in vitro. The resultant cell lines, named EG cells, behave like pluripotent ES cell lines (Matsui et ul., 1992; Resnick et a/., 1992). Interestingly, attempts to establish EG cell lines from the genital ridges of 12.5-day inale and female embryos demonstrated a sex-specific difference in the in vitro proliferative potential of primordial germ cells: germ cells from normal male fetuses proliferated during the culture period, yielding multiple colonies, whereas those derived from normal females failed to proliferate, yielding few, if any, colonies (Matsui etnl., 1992).The difference in proliferative potential has been suggested to reflect an intrinsic difference in the ability of XX and XY germ cells to respond to multiple growth factors. However, subsequent comparisons of the in vitro proliferation of primordial germ cells of individual XX female, XY male, XO female, and XXY male fetuses also indicate reduced proliferative potential of germ cells isolated from the 12.5-day fetal ovary (Hunt et al., in press; P. A. Hunt, unpublished observations). Hence it seeins more likely that germ cells in the developing ovary undergo a commitment event at least 1 day prior to meiotic entry that renders them incapable of mitotic proliferation in response to growth factors in vitro. Insofar as both XX and XO germ cells demonstrate similar restricted proliferative potential, meiotic commitment apparently is neither a consequence of X-reactivation nor is influenced by the chromosome constitution of the germ cells themselves. In summary, we currently have little understanding of the signals that control early events of female germ cell differentiation, but available evidence suggests that two events occur prior to meiotic entry: primordial germ cells lose their potential for mitotic proliferation and, in response to signals from the differentiating ovary, reactivation of the inactive X chromosome occurs. Although both events appear to require stimulus from the differentiating ovary, the decision to initiate meiosis during the prenatal period is itself apparently independent of external signals and occurs in all germ cells unless they are exposed to an inhibiting signal in the differentiating testis.
IV. A Quality Control Checkpoint Operates at Pachytene A. Loss of Germ Cells at the Pachytene Stage Is a Characteristic of Female Meiosis
In most mammalian species, oocytes have reached the diplotene stage and entered meiotic arrest by the time of birth. However, the population of arrested oocytes in the ovary of the newborn female represents only a subset of the germ cells that initiated meiosis. The loss of oocytes due to atresia during the perinatal period has been recognized as a feature of normal ovarian development for many
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years. In the human female, germ cell attrition at the pachytene stage has been estimated to result in the loss of more than one third of the oocyte population (Speed, 1988). As detailed below, germ ccll loss at the pachytene stage is thought to reflect the action of a checkpoint control mechanism that monitors the process of synapsis and/or recombination.
B. Yeast Mutants Provide Insight into the Nature of the Pachytene Checkpoint and the Genes Involved
Studies of sporulation-defective mutants in the budding yeast, Swchlirornyces cerevisine, suggest that a similar cell cycle checkpoint mechanism functions during meiosis in lower eukaryotes. Four different meiotic mutants arrest at pachytene: z i p l , which encodes a synaptonemal complex protein, and dnzc-1, top2, and sepl, which are involved i n the processing of DNA double-strand breaks. Interestingly, in the presence of il mutation that blocks the initiation of recombination, pachytene arrest is overcome in three of the four mutations (dnzcl. top2, and z i p ] ) ; hence, arrest at pachytene is thought to be triggered by the accumulation of intermediates i n the recombination and/or synapsis pathway (reviewed by Roeder, 1995). In somatic cells, cell cycle arrest i n response to DNA damage appears to be the mitotic equivalent of the pachytene checkpoint control mechanism. However, in mitotic cells, DNA damage can cause cell cycle arrest at three different stages (the G , / S transition, GJM transition. and S-phase) of the cell cycle. The genes that have been identified in DNA damagc cell cycle control in eukaryotes and their proposed mechanism of action havc been extensively reviewed recently (Lydall and Weinert, 1996). The meiotic control mechanism is clearly related to the mitotic DNA damage checkpoint, because several of the yeast mitotic proteins also function in the meiotic checkpoint process (Lydall et d., 1996). Nevertheless, the meiotic checkpoint clearly differs in significant respects, owing to the action of meiosis-specific genes involved in recombination. The study of these genes in lower eukaryotes will be of particular interest in understanding meiotic cell cycle control in mammals.
C. Studies of Mice with Numerical and Structural Abnormalities Provide Evidence That a Pachytene Checkpoint Operates in Both Males and Females
Studies of mice with structural and numerical chromosome abnormalities that impede homologue synapsis provide evidence that the pachytene checkpoint in mammals detects disturbances in the synapsis and/or recombination of even a single pair of homologues. For example, all X/autosonie translocations in the mouse are male sterile and most show clear evidence of spermatogenic inipair-
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ment at or around pachytene. Similarly, a number of autosomal rearrangements, insertions, and aneuploid conditions have been reported t o show male-limited sterility (reviewed in de Boer and de Jong, 1989). In contrast to the situation in males. female carriers of structural abnormalities produce viable gametes, although many are semisterile due to loss of unbalanced progeny. Thus, it may seem that female meiosis lacks similarly stringent checkpoint control. However, detailed studies of female carriers of a reciprocal translocation (Mittwoch et ( I / . , 1981) and an insertion (Mittwoch et N/., 1984) suggest that this is not the case. These studies revealed a marked reduction in ovarian size in female carriers, and subsequent studies demonstrated a marked reduction in oocyte numbers in the translocation carriers (Burgoyne et a/., 1985; Setterfield et d., 1988). Hence it appears that both male and female meiosis are subject to cell cycle arrest at pachytene when the normal process of homologous pairing and/or recombination is disturbed. Further evidence that both male and female meiosis share this checkpoint comes from studies of XO females and XO, Sxr (Sex reversed) male mice, in that substantial germ cell loss at pachytene has been documented for both (reviewed in Burgoyne and Mahadevaiah, 1993). However, it is after this checkpoint that sex-specific differences begin to emerge. That is, in both sexes a significant proportion of cells progress beyond pachytene, apparently escaping the pachytene checkpoint by partial or complete self-synapsis (Speed, 1986) of the univalent X chromosome. Interestingly, an almost total block during meiotic metaphase renders the XO, S.ur male sterile (Sutcliffe et al., 1991). In contrast, the XO female mouse produces viable gametes, although her reproductive life span is significantly shortened, owing to the increased loss of oocytes at pachytene (Burgoyne and Baker, 1985). In summary, the combined data from meiotic studies of mice with numerical and structural chromosome aberrations provide evidence that a pachytene cell cycle control mechanism that monitors homologue synapsis or recombination (or both) functions in both sexes. However, the striking difference in fertility typically observed between male and female carriers has caused many investigators to question why perturbations in meiotic chromosome behavior have a more severe effect on male meiosis than female meiosis. Recent data suggest that the answer lies not in differences in checkpoint control during meiotic prophase but in a subsequent checkpoint that monitors the alignment of chroinocomes at metaphase (see Section VII).
V. The Ability to Resume Meiosis Is Acquired during Follicle Growth When the mammalian oocyte rcaches the diplotene stage it enters a period of meiotic arrest and remains in a quiescent state until it is stimulated to undergo growth and maturation. This type of Cz arrest is a common feature of female meiosis in a broad range of species, although the developmental stage of the
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oocyte at the time of arrest varies among species: in many nonmammalian species the oocyte is fully grown or nearly so at the time of arrest, although some transcription or protein synthesis may be required prior to resumption of meiosis. In mammals, virtually all of oocyte growth occurs after the cell has entered meiotic arrest. Because the ability of’ the mammalian oocyte to resume and complete meiosis is not acquired until relatively late in the growth process, an understanding of meiosis in mammals requires at least rudimentary knowledge of the process of folliculogenesis and oocyte growth.
A. Meiotic Competence Is Acquired in Stepwise Fashion during the Final Stages of Oocyte Growth
Although there is some variation among species, in most niammals follicle formation occurs shortly after birth, when the prophase-arrested oocyte becomes surrounded by a flat layer of nonproliferating granulosa cells. This primordial follicle may remain in a quiescent state for years or may initiate the process of follicle growth almost immediately. Because the resumption of meiosis and the first embryonic mitotic divisions occur before the initiation of zygotic transcription, the machinery and regulatory components necessary for these early events must be stockpiled in the oocyte. Folliculogenesis, therefore, is an intense period of oocyte growth coupled with the growth and differentiation of the follicle. The mature mammalian oocyte is a huge cell (the ovulated murine oocyte is approximately 80 k m in diameter) and, by comparison with somatic cells, is a veritable storehouse; the full-grown murine oocyte has been estimated to contain on the order of 0.5 ng of total R N A and some 30 ng of protein (Shultz. 1986). The growth of the oocyte is coordinated with the growth and differentiation of the somatic cells of the follicle. Gap junctions provide bidirectional communication between the oocyte and its surrounding somatic cells throughout the growth process, supporting the development and function of both the oocyte and the follicle in which it grows. The complex interaction of hormonal influences and cellular signaling pathways, both betwcen the oocyte and somatic cells and between somatic cells, that controls the transformation of the primordial follicle into a preovulatory follicle in mummals is only beginning to be understood (e.g., Hirshfield, 1991; Eppig, 1993; Gougeon, 1996). The basis of follicle recruitmcnt remains a mystery. Furthermore, dissecting the process of oocyte growth in the adult ovary has been complicated by the fact that the growth process is extreinely protracted: it has been estimated that from initiation of growth by a primordial follicle to ovulation of a mature oocyte takes on the order of seven cycles in the human female (Gougeon, 1996) and over 20 days in the mouse (based on studies of oocytes grown in LVtro, see Eppig et ( I / . . 1996, for review). Hence, at any given time, follicles o f a range of maturation stages are present in the adult ovary. However, the first cohort of follicles begins to grow in the sexually immature female. The lack of a mature endocrine envi-
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ronment results in atresia of' the entire group of such follicles before growth is ever completed. Nevertheless, in contrast to follicles in the adult ovary, this cohort provides a nearly synchronous population of growing follicles; thus, studies of this early wave of folliculogenesis have provided valuable insight into the process of oocyte growth. The study of oocytes obtained at successive stages of follicle growth during the first wave of folliculogenesis in the murine ovary has demonstrated that the ability to resume and complete the first meiotic division is acquired by the oocyte in stepwise fashion during the late stages of follicle growth. Thus, oocytes obtained from small antral follicles are meiotically immature and, when released from the follicle, will spontaneously undergo nuclear envelope breakdown and chromosome condensation but are unable to complete MI. With further growth, the oocyte becomes fully meiotically mature and will spontaneously resume and complete MI and arrest at metaphasc of MI1 when isolated from the follicle. Hence, during the growth process the oocyte undergoes a transformation from a small oocyte incapable of meiotic resumption to a large. meiotically competent oocyte that is maintained in meiotic arrest by inhibitory signals generated in the follicle. The ability of the oocyte to undergo fertilization and support embryonic development is similarly acquired during the late stages of oocyte growth. AIthough the programs controlling the nuclear (meiotic) and cytoplasmic (developmental) maturation of the oocyte are clearly linked, experimental evidence from the study of murine oocytes suggests that they are distinct processes (Eppig rt al., 1994; see also Handel and Eppig, Chap. 10, this volume).
B. Meiotic Resumption Occurs in Response to Periovulatory Hormonal Stimuli
In the sexually mature mammalian female, the midcycle gonadotropin surge results in the ovulation of oocytes from any fully mature follicles present in the ovary. The dramatic and complex process of follicle maturation and ovulation has been the subject of extensive reviews (e.g., Espey and Lipner, 1994; Downs, 1995; Ledger and Baird, 1995; Gougeon, 1996). During the ovulatory cycle, follicles that have reached the appropriate developmental stage respond to the elevated follicular phase levels of follicle-stimulating hormone (FSH) and become functionally mature. Functional maturity means, among other things, the acquisition of luteinizing hormone (LH) receptors by somatic cells, so that the subsequent midcycle surge of LH can trigger the cascade of events that results in luteinization of the follicle. From a meiotic standpoint, the most important consequence of the hormonal stimulus and extensive changes in the somatic cells of the periovulatory follicle is that the meiosis-inhibiting influence of the follicle is abolished and the oocyte is stimulated to resume and complete MI and arrest at MI1 metaphase (see Downs, 1995, for a discussion of the mechanism).
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The final stages of oocyte growth are characterized by coordinated modifications in nuclear and cytoskeletal organization in preparation for meiotic resumption (reviewed in Wickramasinghe and Albertini, 1993). Two distinct populations of microtubules are present i n the meiotically mature oocyte: As the oocyte enters the final stages of development, microtubule organizing centers (MTOCs) become associated with the nuclear envelope in a process that is thought to be influenced by the change in follicular steroids. The appearance of these perinuclear MTOCs has been correlated with the acquisition of meiotic competence, and it is this population of MTOCs that acts as the source of microtubules for the formation of the meiotic spindle. A second set of microtubule foci can be observed moving to the periphery of the oocyte during the final stages of oocyte growth. This subset of centrosomes functions as a microtubule reservoir, remaining inactive during the resumption and completion of meiosis but participating in the formation of the mitotic spindle during the early cleavage divisions of the embryo (Albertini, 1992).
C. Evidence That the Process of Folliculogenesis I s Compromised in the Reproductively Aged Human Ovary
Interestingly, studies of oocytes obtained from antral follicles of unstimulated human ovaries in our own laboratory suggest the meiotic competence of the oocytes is influenced by donor age (P. A. Hunt, unpublished observations). Previous studies have suggested that approximately 50% of human oocytes recovered from unselected follicles will spontaneously progress to metaphase I 1 when liberated from the follicle (Jagiello et ( I / . . 1976; Uebele-Kallhardt, 1978; Van Blerkom, 1990; Gras er a/., 1992). Although we observed no significant difference in the rate of meiotic resumption, as indicated by germinal vesicle breakdown, polar body extrusion was delayed i n oocytes obtained from donors over the age of 35 yrs. Furthermore, thc majority of oocytes that extruded a first polar body and arrested at second meiotic inetaphase had aberrations in spindle formation and chromosome alignment, consistent with an age-related reduction in meiotic competence. Differing success rates for in vitvo fertilization pregnancies in women over the age of 40 with self and donor eggs indicate a similar agerelated decline in the developmental competence of the human oocyte (e.g., Navot ct a/., 1991; Wood ct ( I / . , 1992). Taken together, these data suggest that advancing reproductive age aflects both the meiotic and the developmental competence of the human oocyte. We interpret these observations to mean that agerelated changes in the ovarian environment compromise the process of folliculogenesis, resulting in a reduction i n oocyte quality. This suggests that the well-characterized age-related increase i n meiotic nondisjunction in the human female may actually be a symptom of a more global effect of age on the oocyte growth process.
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Studies of the structure of the meiotic spindle in MII-arrested human oocytes from different-aged donors provide support for this hypothesis. Aberrations in the metaphase alignment of the chromosomes were observed in the majority (79%) of MI1 oocytes obtained from older donors (40-45 yrs). I n contrast, similar abnormalities were observed in a minority ( 17%) of oocytes obtained from younger donors (20-25 yrs) (Battaglia et a/., 1996). These observations support the proposal by Hawley that, as human females age, the capacity of the oocyte to form a normal spindle diminishes (Hawley et d., 1994). However, instead of a time-dependent degradation of some essential spindle component (Hawley et al., 1994), we suggest that an age-related decline in the process of oocyte growth results in inadequate stockpiling during development, with resultant defects in both the meiotic and developmental competency of the human oocyte.
D. New Techniques for the in Vitro Growth of Oocytes Provide a New Approach to Studying the Factors That Influence Meiotic Chromosome Segregation
Recent advances have made possible the in vitr-o growth and maturation of murine oocytes derived from immature follicles. Oocytes from oocyte-granulosa complexes isolated from preantral follicles of neonatal mice (Eppig and Schroeder, 1989; Eppig ef a/., 1992), from intact primary follicles (Spears et ( I / . . 1994), or from primordial follicles (Eppig and O’Brien, 1996) can be successfully grown, matured, and fertilized in vitr-oand, on transfer to foster mothers, can give rise to liveborn individuals. However, a crucial and to date largely ignored question is whether in i i w o culture conditions have an effect on the genetic quality of the oocyte. Not only is this of fundamental importance in optimizing existing culture conditions but, given the high meiotic error rate in the human female, analysis of the meiotic process in an iiz \itro system provides a unique opportunity to begin to understand the control of the female meiotic process and the factors that influence it.
VI. Chromosomes Play an Active Role in the Formation
of the Meiotic Spindle The essential feature of the first meiotic division is the segregation of homologous chromosomes, a process that requires the maintenance of associations between homologues and proper attachment to the spindle apparatus. The classic view of spindle assembly holds that specialized cell organelles (centrioles, centrosomes) mediate the process. However, studies suggest that microtubule nucleation and spindle assembly is a cooperative effort on the part of centrosomes,
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chromosomes, and motor proteins. The role of the chromosomes in the organization of the meiotic spindle is visually apparent in the oocyte. For example. in Di-osophilu oocytes, the chromosomes are held in a tight mass called a karyosome following recombination. With the transition to metaphase, microtubules begin to polymerize outward from the karyosome, eventually tapering at the ends to form spindle poles. Similarly, in the murine oocyte, with the breakdown of the nuclear envelope the population of MTOCs formerly associated with the nuclear envelope begins to nucleate inicrotubulcs around the condensing chromosome bivalents. In contrast, the population of MTOCs at the periphery of the oocyte shows no evidence of microtubule nucleation. The spindle-promoting properties of chromosomes were experimentally demonstrated over a decade ago in studies of murine oocytes (Maro et al., 1986; Van Blerkorn and Bell, 1986). Treatment of MII-arrested oocytes with the microtubule-depolymerizing agent nocodamle was found to completely disrupt the inetaphase alignment of chromosomes, yielding clusters of small groups of chromosomes dispersed throughout the oocyte cytoplasm. On removal of the nocodazole, the individual chromosome clusters were found to promote microtubule polymerization and MTOC recruitment, forming multiple MI1 spindles in a single oocyte cytoplasm and undergoing division, as evidenced by the extrusion of multiple polar bodies. The spindle-promoting ability was demonstrated to be a property of the individual chroniosotne clusters and not an artifact of spindledepolymerizing effects of nocodamle, since, on transfer to recipient germinal vesicle stage oocytes, the transplanted chromosome clusters were similarly able to cause the formation of an independent spindle and extrusion of an independent polar body. The recent demonstration that plasmid DNA without centromeric sequences is sufficient to govern spindle acsenibly (Heald et al., 1996) provides new insight into the spindle-promoting properties of chromosomes. By introducing magnetic beads coated with plasmid DNA into X m o p u s egg extracts, Heald et ul. found that chromatin is sufficient to promote microtubule polymerization. Although the presence of intact chromosomes was previously known to favor polymerization by altering the catastrophe frequency of tubulin (reviewed in Hyman and Karsenti, 1996), these experiments demonstrated not only that intact chromosomes and centromeric sequences are unnecessary, but that centrosomes and centrioles are also unnecessary for microtubule nucleation. Although chromosomes clearly play ;in important role in the formation of the spindle, the chromosomes themselves do not organize the bipolar spindle structure. A class of proteins known as microtubule motor proteins function as both the spindle architects and the conductors of chromosome movement during cell division. These proteins, which convert the energy from the hydrolysis of adenosine triphosphate into force and movement, include members of the kinesin superfamily and the dynein family. The role of motor proteins in the separation and maintenance of the spindle poles, the formation and maintenance of the
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bipolar spindle structure, the control of microtubule dynamics, and the movement of the chromosomes during cell division has been extensively detailed in several reviews (e.g., Barton and Goldstein, 1996; Varnos and Karsenti, 1996; Walczak and Mitchison, 1996; Yen and Schaar, 1996). During prophase, motor proteins act to shuffle polymerized microtubules into a bundle with distinct polarity (i.e., plus ends directed toward the metaphase plate and the minus ends facing outward), and to taper the minus ends of the microtubules into spindle poles. The combined action of different motor proteins localized along the length of the microtubules, at the kinetochore (the proteinaceous structure that forms at the centromere of condensed chromosomes undergoing both mitotic and meiotic division and that functions as a mechanical link between the spindle microtubules and the chromosome), and along the chromosome arms results in the congression of the chromosomes to the spindle equator in a characteristic metaphase configuration.
VII. The Metaphase/Anaphase Transition A. The Role of Chiasmata in Meiotic Chromosome Segregation
The classic metaphase alignment of chromosomes at the spindle equator during mitotic cell division is the result of the action of opposing forces: the movement of sister kinetochores toward opposite spindle poles, counterbalanced by the cohesive forces that continue to maintain an association between the chromosome arms. Chromatid cohesion is released at anaphase, allowing sister chromatids to move to opposite spindle poles. In contrast to mitosis, MI results in the segregation of homologous chromosomes rather than sister chromatids. In most organisms, paired homologues, or bivalents, are stabilized at the spindle equator at the first meiotic metaphase by chiasmata, the physical interlocking that results from recombination between homologous chromosomes during meiotic prophase. Chiasmata are thought to function in two ways to ensure the proper segregation of homologues at the first meiotic division: first, by maintaining the homologous chromosomes in a paired orientation that promotes the capture of their kinetochores by opposite spindle poles, and second, by counterbalancing the forces directing homologous centromeres toward opposite spindle poles and thus allowing the congression of the chromosomes to the spindle equator (Nicklas, 1974; reviewed in Hawley, 1988, and Carpenter, 1994). The role of chiasmata in ensuring proper chromosome segregation at MI is best illustrated in recombination-deficient mutations. In yeast and Drosophila a number of different mutations have been described that reduce the level of recombination, and all show a corresponding increase in the incidence of meiotic nondisjunction. Furthermore, in studies of spontaneous nondisjunction in Drosophila and humans, abnormally positioned chiasmata (either too close to the
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centromere or too close to the telomeres) also increase the likelihood of nondisjunction (Hawley, 1988; Hassold c’t a/., 1995; Lamb et al., 1996). The central role of chiasmata in chromosome segregation suggests that the meiotic segregation of unrecombined hoinologues or univalent chromosomes will be impaired, and indeed, the study of univalent chromosomes in a number of species has demonstrated their inherent instability (e.g., Darlington, 1939; Callan and Jacobs, 1957; Maquire, 1987). However, in certain organisnis meiosis is characterized by the complete lack of recombination (e.g., male Drosophila) and in others one or more pair of chromosomes is always achiasmate (e.g., female Drosophila and a few mammalian species in which the sex chromosomes are achiasniatic). Despite the absence of recornbination, proper meiotic chromosome segregation occurs in these organisms, and the study of these meiotic exceptions has led to an increased understanding of the backup mechanisms that exist to ensure proper segregation in the absence of recombination (reviewed in Wolf, 1993). The best characterized example is the achiasmate segregation system in female Drosophila. Although recombination occurs in Drosophila female meiosis, the small fourth pair of chromosonies is ulwuys achiasmate. Segregation of chromosome 4 homologues, and of any other homologous pair that fails to undergo recombination, is achieved through the action of the meiosis-specific microtubule motor protein, NOD. NOD is localized along the length of the chromosome arms at metaphase and acts to push the chromosomes toward the spindle equator, counterbalancing, as do chiasmata, the poleward forces acting at the kinetochore (Hawley, 1988; Theurkauf and Hawley, 1992; Hawley and Theurkauf, 1993; Afshar et al., 1995). Separate mechanisms for the scgregation of nonexchange chromosomes have evolved in a variety of species, including a few mammals (Wolf, 1993). However, despite the fact that a human nod homologue has been cloned (Tokai ct ul., 1996), it remains unclear whether any such backup meiotic segregation mechanisms exist in the two best characterized tnammals, the mouse and human. The answer to this question is of obvious importance in understanding the high incidence of meiotic nondisjunction in our own species.
B. Evidence for a Spindle Assembly/Chromosome-Mediated Checkpoint In addition to influencing spindle assembly, the chromosomes also influence the progression of the cell division. A cell cycle checkpoint control mechanism that regulates anaphase onset has been described in both mitotic and nonmammalian meiotic cells. This metaphase/anaphase transition checkpoint has been described as a spindle assembly or a chromosome-mediated checkpoint, because defects in either spindle formation (reviewed in Wells, 1996) or the alignment of chromosomes at metaphase (e.g., Rieder et d . ,1994, 1995; Li and Nicklas, 1995; Wells and Murray, 1996) can delay the onset of anaphase. By ensuring that all chromo-
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somes are properly attached to spindle microtubules and aligned at the midzone of the bipolar spindle before the cell proceeds into anaphase, this cell cycle control mechanism functions to prevent the mis-segregation of chromosomes to daughter cells. The kinetochore apparently provides the source of the signal that triggers activation of the checkpoint, although the mechanism by which this occurs remains unclear. Studies of chromosome alignment and anaphase onsetpredominantly the elegant studies in grasshopper spermatocytes by Bruce Nicklas (Nicklas, 1967; Nicklas and Kock, 1969; Nicklas and Kubai, 1985)provided the basis for the hypothesis that checkpoint control involves a tensionmediated mechanism based on the attachment of kinetochores to opposite spindle poles (Mclntosh, 1991). Although apparent exceptions to a simple tensionmediated mechanism have been described (e.g., Rieder et al., 1995),the hypothesis is supported by observations in both mitotic and meiotic cells in a number of different species (reviewed in Pluta et ul., 1995). Furthermore, studies of the phosphoepitope-specific monoclonal antibody, 3F3/2, have provided evidence of a biochemical change in the kinetochore with microtuble attachment. The 3F3/2 antibody produces a strong signal at the kinetochores of chromosomes that have not formed a stable bipolar spindle attachment (Gorbsky and Ricketts, 1993). With microtubule attachment, the signal becomes greatly diminished. Recent studies of grasshopper spermatocytes demonstrate that physically detaching one kinetochore of a bivalent from the spindle results in increased signal intensity at the unattached kinetochore. If this unattached kinetochore subsequently forms an attachment to the same spindle pole as the attached kinetochore, creating a situation in which both kinetochores of the bivalent arc attached to microtubules but the centromere is not placed under tension, the staining intensity does not diminish. However, if the unattached kinetochore is placed under artificial tension using a microneedle, a corresponding decrease in 3F3/2 staining intensity is observed (Nicklas et al., 1995). The identification of yeast mutants defective in this type of checkpoint control has allowed several of the components of the feedback signaling mechanism that activates the checkpoint to be determined (Hardwick et al., 1996).One important player is the MpsIp protein kinase. This protein is required for spindle pole duplication during G I , but recent evidence suggests that its overexpression at metaphase results in activation of the spindle assembly checkpoint even in the presence of an intact spindle (Hardwick et al., 1996).
C. Mammalian Female Meiosis lacks Chromosome-Mediated Checkpoint Control
Recent studies in our laboratory suggest that this checkpoint is absent or only partially functional in mammalian female meiosis. Studies of the XO female
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mouse have been particularly instructive; analysis of MI in oocytes from XO female mice demonstrated that the presence of an unpaired chromosome has a disruptive effect on meiotic chromosome behavior (Hunt et al., 1995). The X chromosome failed to form a stable bipolar spindle attachment and align at the metaphase equator in approximately 20%' of metaphase cells. Furthermore, meiotic abnormalities in oocytes from XO female mice were not limited to the X chromosome. The presence of a univalent chromosome at MI appeared to affect chromosome alignment of other chroniosomes in the complement in a significant proportion of oocytes at both MI and Mil. In previous cytogenetic studies o f preiinplantation stage embryos from XO females, we had observed an extremely high frequency of hyperploidy (Hunt, 1991). The high frequency of ancuploidy for chromosomes other than the X chromosome suggested that failure of alignmcnt at metaphase was causing segregation errors at anaphase. However, this was contrary to expectation, because failure of one or more chromowrnes to align at the spindle equator should trigger the chromosome-mediated checkpoint, causing a delay in the onset of anaphase until proper alignment is achieved. To determine if, in fact, we could detect cells that were delayed or arrested at metaphase I, we initiated meiotic progression studies of oocytes from XO females and XX sibling controls. Surprisingly, we not only found no evidence for a dclay or arrest of cells at metaphase I in oocytes from XO females. but the onset of anaphase I was significantly uccelercited by comparison with oocytes from X X sibling control females (R. LeMaire-Adkins and P. A. Hunt, unpublished observations). Although the reason for the accelerated cell cycle kinetics remains unclear, the absence of delayed or arrested cells in XOs suggests that female meiosis lacks the normal checkpoint control mechanism that monitors the alignment o i chroiiiosomes at inetaphase. These results have important implications, for thcy suggest a fundamental difference in meiotic cell cycle control between male and female mammals. In retrospect, the fact that female meiosis lacks or has impaired chromosomemediated metaphase/anaphase checkpoint control is not surprising. As discussed in Section IV, a variety of situations in the mouse have been described in which structural or numerical abnormalities result in male-limited sterility. The available data suggest that stringent pachytene checkpoint control operates during both male and female meiosis to cull cells with disturbances in the process of pairing and/or recombination. Thus, the difference in fertility between males and females must reflect differences i n the fate of those cells that escape the pachytene checkpoint. Male and female mice possessing only a single X chromosome provide the best characterized example: the XO female mouse is fertile, whereas the XO, Sxr male is sterile (reviewed in Burgoyne and Mahadevaiah, 1993). Both XO females and XO, Sxr males sustain considerable germ cell loss at pachytene, but in both sexes a significant number of cells survive the pachytene checkpoint by self pairing of the X chromosome (Speed, 1986). Our meiotic studies demonstrate
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that, in the XO female, the meiotic divisions proceed in the face of gross disturbances in chromosome alignment, although the end result is a dramatic increase in aneuploidy. In contrast, in the XO, Sxr male, the vast majority of cells that survive pachytene do not progress beyond metaphase, presumably as a result of the action of the metaphase/anaphase checkpoint. The few sperm that are formed are diploid (Levy and Burgoyne, 1986), suggesting that these cells survive meiosis by omitting one of the meiotic divisions. The difference in the fate of metaphase cells with misaligned chromosomes represents a fundamental difference in meiotic cell cycle control between male and female mammals. Furthermore, the apparent lack of a meiotic chromosome surveillance mechanism during female meiosis may provide an explanation for the astonishingly high frequency of chromosome errors during human female meiosis. If aberrant chromosome behavior at MI is simply better tolerated during mammalian female meiosis, then age-related changes that increase the frequency of univalents or the premature segregation of bivalents during meiosis will be evident only in female gametes; the more sensitive cell cycle checkpoint that monitors chromosome behavior during male meiosis will ensure that such cells do not complete meiosis.
VIII. Arrest at Second Meiotic Metaphase: Do Chromosomes Play a Role? The second meiotic division is characterized by two unique features: a normal interphase stage is lacking, and cell cycle arrest occurs at metaphase. The chromosomes remain condensed following the segregation of homologues at MI, and formation of the second meiotic spindle and alignment of the chromosomes at the spindle equator occur rapidly. The oocyte remains arrested with a fully formed second meiotic spindle until fertilization. Although the nature of the signal that initiates metaphase arrest remains unknown, the way in which cell cycle arrest is maintained is at least partially understood (for a detailed discussion, see Handel and Eppig, Chap. 10, this volume). Whether or not chromosome behavior plays a role in maintaining MI1 arrest remains unclear. In Drosophila, oocytes arrest at metaphase of the first meiotic division, and arrest at this stage has been demonstrated to be mediated by the chromosomes (Jang et a/., 1995). One or more recombinational events between homologous chromosomes are required for arrest. However, it is not genetic exchange per se that mediates arrest, but rather the tension that is created when an exchanged bivalent forms a bipolar attachment to the meiotic spindle. Chiasmata (the cytological manifestation of recombination) act to counterbalance the poleward forces acting on a bivalent. That is, sites of recombination serve as an anchor, maintaining the association between homologous chromosomes and
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allowing the bivalent to align at the spindle equator by counterbalancing the poleward forces at opposing kinctochores of the bivalent. Chiasmata are resolved at anaphase of MI, allowing homologous chromosomes to segregate to opposite poles (see Section VII). At MII, as in mitotic divisions, cohesion between sister chromatids serves to maintain an association and allow alignment at the spindle equator. Whether the tension resulting from the attachment of kinetochores to opposite spindle poles and the maintenance of cohesive forces between sister chromatids is necessary for maintenance of MI1 metaphase arrest remains unknown. However, if sister chromatid separation is inhibited in murine oocytes by exposing MII-arrested oocytes to a topoisomerase 11 inhibitor, neither fertilization nor artificial activation can induce the inactivation of MPF, and the cell remains arrested at MI1 (Fulka et ul., 1994). Similarly, the onset of anaphase in the fertilized oocyte is dependent on the presence of an intact MI1 spindle. If the spindle is disrupted by exposure to microtubuledepolymerizing agents, high levels of active MPF are sustained in the fertilized or artificially activated oocyte, preventing the onset of anaphase (Kubiak el d., 1993; Winston er a/., 1995). Hence a spindle assembly checkpoint appears to function at MI1 to maintain metaphase arrest in the absence of an intact spindle.
IX. The Future: Mammalian Meiotic Mutants Will Provide Important Insight into the Control of Mammalian Female Meiosis In lower eukaryotes, the study of'meiotic mutants has provided tremendous insight into the control of the meiotic process and has allowed the identification of inany of the important meiotic genes. Mammalian homologues for some of these genes have been identified, and gene targeting methodology in the mouse has made possible the production of the first mammalian meiotic mutants. Null mutations have been created in several murine mismatch repair genes (Baker et a/., 1995; de Winder ul., 1995; Reitmair et ul., 1995; Baker et ( I / . , 1996; Edelman eta/., 1996). In addition to its role in somatic tissues, mismatch repair fulfills an important function in meiotic recombination in the recognition and repair of mismatches in heteroduplex DNA. Thus, it might be expected that mismatch repair mutants would have meiotic as well as mitotic abnormalities. Preliminary studies of mouse knockouts suggest that this is indeed the case, although there is considerable variation in the meiotic phenotypes. Although the studies to date have focused on male meiosis, comparative studies of male and female niciosis in these mutants will provide important information about differences in meiotic cell cycle control between oogenesis and spermatogenesis. Recent instructive examples have been provided by the studies of the Prm2 and
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Mlhl knockout mice. Targeted disruption of the Pnzs2 gene results in severe synaptic defects at early prophase in male mice homozygous for the mutation. A corresponding loss of germ cells at pachytene and later stages is evident (Baker et ul., 1995),suggesting that synaptic defects trigger the pachytene checkpoint control mechanism. In contrast, homozygous deficient females are reported to be fertile but have not yet been extensively studied. Studies of female meiosis will determine if the fertility differences reflect differences in the role ofthis gene in male and female meiosis or differences in cell cycle control. Specifically, it will be important to determine if the absence of the PMS2 protein also results in synaptic defects in female meiosis. The lack of such defects would suggest fundamental differences between male and female meiosis in the role of this protein in synapsis and recombination. However, the more likely scenario, that females homozygous for the Pms2 gene disruption show synaptic defects and the loss of a significant number of germ cells at pachytene, as in the male, would provide further evidence for differences in the checkpoint control mechanisms, as discussed earlier. Unlike the situation for the Pins2 null mutant, both male and female homozygous carriers of the M l h l gene disruption are sterile (Baker el d., 1996; Edelman et al., 1996). In males, initial meiotic chromosome pairing appears normal but homologous chromosomes prematurely separate and by metaphase most chromosomes appear as univalents. This suggests that Mlhl promotes normal chiasma formation and/or stabilization; and consistent with this interpretation, Baker et ril. (1996) reported a 10-fold or more reduction in chiasma frequency in MLH 1-deficient spermatocytes. Later stage germ cells are never identified in MLH 1 -deficient males, suggesting arrest at metaphase I. In MLHl-deficient females, corpora lutea are identified, although rarely, and no information on oocytes is available. Preliminary studies of female meiosis suggest that, despite the dramatic decrease in recombination and the resulting premature separation of homologous Chromosomes, female meiosis-unlike male meiosis-proceeds with the first meiotic division (P. A. Hunt, unpublished observations). In summary, animals carrying targeted disruptions of DNA mismatch repair genes as well as of other genes known to play a role in meiosis in lower eukaryotes provide the first of the long awaited mammalian meiotic mutants. Detailed analysis of the meiotic process in male and female mutants will provide valuable insight into the role of these genes in mammalian meiosis and will allow the first systematic approach to understanding the differences in the control of the meiotic process in mammalian males and females.
Acknowledgments P.A.H. is supported by NIH grant HD3 I X66 and March of Dimes grant FY96-062 I . We thank John Eppig, Mary Ann Handel, Terry Hassold, and the niemhers of our laboratory for discussions and helpful coninients on the manuacript.
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Hunt. P. A. (1991). Survival of XO niou\c Ictuscs: Effect of parental origin of the X chroinosome or uterine environment. D e i v l o p w ~ i r111, 1137-1 141. Hunt, P. A,, LeMaire, R., Embury, P., Mrtr/. K., and Sheean, L. (1995). Analy\is of chromosome behavior in intact inaininalian oocytes: Monitoring the segregation of a univalent chroinosome during mammalian female meiosis. Hum. Mol. Gcwet. 4, 2007-2012. Hunt P. A,, Worthman, C., Levinson, H., Slallings. J.. LeMaire, R., Mroz, K., Park, C., Handel, M . A , : Germ cell loss in the XXY niiilc mouse: Altered X-chromosome dosage affects prenatal development. M o l . Reprod. D e v , in pre\s. Hyman, A. A,, and Karsenti, E. (1996). Mot-phopcnetic properties of microtubulcs and mitotic spindle assembly. Cell 84, 401 -410 Jacobs, P. A. ( 1992). The chromosome coinplemenl of human gametes. Oxford Rei,. Reprod. Biol 14, 48-72. Jagiello. G.. Ducayen, M., Fang, J.-S.. iind Gralfeo. J. (1976). Cytogenetic observations in mainmalian oocytes. In "Chromosome\ Today" (P. L. Pearson and K. R. Lewis, Eds.), pp. 43-63. John Wiley, New York. Jang, J. K., Messina, L., Erdnian, M. H., Arhel, T.. and Hawley, R. S. (1995). Induction of metarrest in Drosophila oocytes by chia\ina-based kinetochore tension. Scierice 268, 19 171919.
Kubiah. J. Z., Weber, M., de Pennart. H. Winston, N.. and Maro, B. (1993). The rnetaphase I1 iirrest i n mouse oocytes is controlled through inicrotubule-dependent destruction of cyclin B in the prcsence of CSF. EMRO J . 12, 3773-3778. Lamb, N., Freeman, S. B., Savage-Au\tiii, A,, Pcttny, D., Tart, D., Hcrsey, J., Gu. Y.. Shen, J., Saker, D., May. K. M., Avamopoulos, I).. Petersen. M. B., Hallberg, A,, Mikkelsen. M., Hassold, T., and Sherman, S. L. ( 1996). Non-disjunction of chromosome 21: Evidence for initiation of all maternal errors during ineio\is I. Ntrmw Genet. 14, 400-405. Lawson. K. A,, and Hage, W. J. (1994). Clonal analysis of the origin of primordial germ cells in the niousc. C i h Fortr7d. Syrnp. 182, 68-9 I . Ledger, W. L., and Baird, D. T. (1995). Ovulation 3 Endocrinology of ovulation. /ti Y h i e t e s The Oocyte" ( J . G. Grudiinskaq and I . 12. Yovich, Eds.). pp. 193-209. Cambridge University Press, Cambridge. Levy, E. R.. and Burgoync, P. S. ( 19x0) Diploid \permatids: A manifestation of sperniatogenic impairment in XOSxr and T31H/ t iiiiile mice. C m g e n e t . C d / Grnet. 42, 159-163. Li, X.. and Nicklaa, R. B. (1995). Mitolic force\ control a cell-cycle checkpoint. Nofirre 373, 630-632. Lydall, D., Nikolsky, Y., Bishop, D. K., and Weinert. T. ( 1996). A meiotic recombination checkpoint controlled by mitotic checkpoint penes. Ntrtitre 383, 840-843. Lydall, D., and Weinert, T. (1996). Froin DNA damage to cell cycle arrest and suicide: A budding yeast perspective. Curt: Opin. Gewr. /lei: 6, 4- I 1. Maguirc, M. P. (1987). Meiotic behavior 01 ii tiny fragment chromosome that carries a transposed ccntromcrc. Genoriic 29, 744-746. Maro, B.. Johnson, M. H., Webb, M . , iiiid Flach. G. ( 1986). Mechanism of polar body formation in the mouse oocyte: An interaction hetwccn the chromosomes, the cytoskeleton and the plasma membrane. J . E t h - y l . E.YI,. Mtwp/io/.92, I 1-32, Matsui, Y., Zsebo. K., and Hogan, B. L. ( 1992). Derivation of pluripotential embryonic s t e m cell\ froin inurine primordial germ cell\ in culturc. Cell 70, 841 -847. Mclntmh, J . R. ( I991 ). Structural and mechanical control of mitotic progression. Cold Spring Horlior Symp Qurrrit. B i d . 56, 61 3-6 10. McLaren. A . ( 1983). Primordial germ cells in mice. Bih/iot/wtr A m . 24, 59-66. McLarcn. A. (1985). Relation of germ ccII sex t o gonadal differentiation. /ti "The Origin and Evolution of Sex," pp. 289-300. Li\\. Ncw York.
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Mittwoch, U., Mahadevaiah, S., and Olive. M . B. (1981). Retardation of ovarian growth i n malesterile mice carrying an outoronial translocation. J . M d . G P w . 18, 414-417. Mittwoch, U., Mahadevaiah. S.. and Setterfield, I.. A. ( 1984). Chromosomal anomalies that cause inale sterility in the mouse a l w reduce ovary m e . Grrrer. Re.\. 44, 2 19-224. Monk. M., and McLaren. A. ( 1981 ). X-chromosome activity in foetal semi cells of the mouse. J . Embrwl. Erp. Morphol. 63, 75-84. Navot, D.. Bergh, P. A., Williams, M. A., Garrisi, G. J . , Guznian, I . , Sandlcr. B., and Grunfeld, L. ( 19911. Poor oocytc quality rather than implantation failure as a caure of age-related decline in female ferlility. L t m r t 337, 1375- 1377. Nicklas, R. B. (1967). Chromosome micromanipulation. 11. Induced reorientation and the cxperiniental control of segregation i n meiosis. Climmosomtr 21, 17-50. Nicklas, R. B. ( 1974). Chromosome segregation mechanisms. Gmrrics 78, 205-2 13. Nickla.;, R. B., and Kock, C. A. (1969). Chromosome micrornanipulatioii. 111. Spindle fiber tension and the reorientation of mal-oriented chroinosornes. J. Cell R i d . 43, 40-50. Nicklas, R. B., and Kubai. D. 1‘. ( 1985). Microtubule\, chi-omosome movement, and reorientation after clironiosonies are detached from the spindle by tnicromanipulation. Chmvrocomtr 92, 3 13-324. Nicklas. R. B., Ward, S. C., and Gorhrky, G. J. ( 1995). Kinetochore chemistry IS sensitive to tension and may linh mitotic forces to a cell cycle checkpoint. J . Cell Biol. 130, 929-939. Penrore, L. S. (1965). Mongolism as a problem in human biology. /ti “The Early Conceptus, Normal and Abnormal,” pp. 94-97. University of St. Andews, Dundee. Pluta. A. E, Mackay. A. M.. Ainsztein, A . M.. Goldberg, 1. G., and Earnshaw, W. C. (1995). The centromere: Hub of chromosomal activities. Scirncr 270, 159 I 1594. Reitinair, A. H., Schniits, R., Ewel. A., Bapat, B.. Redston, M., Mitri. A,, Waterhouce, P., Mittrucker, H.-W.. Wakeham, A.. Liu. €3.. Thomason, A,. Griesser, H., Gallinger, S., Ballhauren, W. G., lishel, R.. and Mak, T. W. ( 1995). M S W delicient mice are viable and susceptible to lymphoid tumors. Noritre Grrirr. 1 I , 63-70. Resnick, J. L., Bixler. L. S., Cheng, L. and Donovan. P. J. ( 1992). Long-term proliferation 0 1 mouse primordial germ cells in culture. Ntrritr-c, 359, 550-55 I . Ricder, C. L., Cole. R. W., Khodjakov. A., and Sluder. G. ( 1995). The checkpoint delaying anaphase i n response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. J . Cell Biol. 130, 941 -948. R i d e r , C. L., Schultz, A., Cole, R., and Sluder, G. (1994).Anaphase onset in vertehrate wmatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle. J . Cell Biol. 127, 130 I - 13 10. Roeder. G. S. (1995). Sex and the single cell: Meiosis in yeast. /‘roc.. Ntrrl. A u r d ki. USA 92, 10450- 10456. Schultl. R. M. ( 1986). Moleculai- aspects of inaniinaliiin oocyte growth and maturation. Irr “Experimental Approaches to Mammalian Embryonic Development” ( J . R o s a n t and R. A. Pederren. Eds.). pp. 195-237. Cambridge University Press, New Yorh. Setterfield, L. A,, Mahadevaiah, S., and Mittwoch. U. ( 1988). Chromosome pairing and _perm cell loss i n male and female mice carrying a reciprocal translocation. J . Reprod. Fertil. 82, 369379. Spears, N., Boland, N. I . , Murray, A. A,. and Gosden, R. G. (1994). Mouse oocytcs derived from in vitro grown primary ovarian follicles are fertile. Hum. Reprod. 9, 527-532. Speed, R. M. (1986). Oocyte development i n X O foetuses of man and mouse: The possible role of heterologous X-chromorome pairing in germ cell survival. Chrnrriocor,io 94, I I S - 121. Speed, R. M. (1988). The possible role of meiotic pairing anotnalicr in the atresia of human fetal oocytes. Hum. Genet. 78, 260-266. Sugawara, S., and Mikamo, K . ( 1983). Absence of correlation between univalent fommatioii and meiotic ncmdisjunction in the aged female Chinere hamster. Cuo,qrrier. C’ell Gwer. 35, 34-40. -
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381
Sutcliffe. M. J., Darling. S. M., and Burgoyne. P. S (1991). Spermatogenesis in XY, Sxr’ and XOSxr.’ mice: A quantitative analy\is o l \perni;itogcncsis throughout puberty. Mol. Reprod. Dei: 30, 8 I -x9. ( IW4). X-chromosome activity of the mouse primordial Tam, P. P. L., Zhou, S . X.. and Tan, germ cells revealed by the expression 01 ail X-linked lacZ transgcne. Ilei~eloprrirrit120. 29252932. Thcurkauf. W. E.. and Hawley, K. S. ( 1991). Meiotic \pindlc assembly in Drowphila females: Beha\ ior of noncxchange chroinc)\oiiir\ and the ellects o f mutations in the riod kineaiii-like protein. J. Cell Riol. 116, I 167- I 180. Tokai, N.. Fujimoto-Nishiyama, A., Toyo\hiiii:i, Y.. Yonemura, S., Twkita. S., Inoue. J., and Yaniamoto. T. (1996). Kid. a novel hincsiii-lihe DNA binding protein, is localized to chrornosome\ and the mitotic spindle. E M H O ./. 15, 457-467. Uehele-Kallhardt, B.-M. ( 1978). “Human Oocytc\ and Their Chroniosomea: An Atlas.’’ Springer, Berlin. Van Blerkom, J. ( 1990). Extrinsic and intrinsic inAucncc\ on human oocyte and early embryonic developmental potential. I n “Element\ of Maiiinialian Fcrtiliration” (P. M. Was\erman. Ed.). pp. 8 I - 109. CRC Press, Boca Raton, l:12. Van Blerhom. J.. and Bell, H. (1986). Kcgul;ition 01 development i n the fully grown mouw 00cyte: Chroinosome-mediated temporol atid spatial differentiation of the cytoplasm and pla\ma membrane. J . E i d i n o l . E q i . Morphol. 93, 7 I3 -73X. Varnos, I..and Karsenti. E. (1996).Motoi\ involved in spindle aa\embly and chromosome segregation. Cur.,: Opiri. Cell Biol. 8, 4-c). Walczak, C. E.. and Mitchison, T. J. ( 1996). Kine\in-i-elatcd protein\ at niitotic spindle poles: Function and regulation. Cell 85, 943- 046. Warburton. D. ( 1989). The effect of matcrnal age on the frequency of trisomy: change in meio\is o r in utero selection’? /r? “Molecular ;in11 Cytogcnetic Studie\ oi Non-Disjunction” (T. J. Hassold and C. J . Epstein, Eda.), pp. 165- 1 X I . Alan K. Liss, Ncw York. Wells, W. A. E. ( 1996). The spindlc-;i\wiiihly checkpoint: Aiming for a perfect niito\i\, every time. 7rend.> Cell Bio. 6, 228-234. Well\, W. A. E., and Murray. A . W. ( IWh) Aherrnntly xgregating centromere\ activate the spindle a\semhly checkpoint in budding yca\t. J . c‘cll H i o l . 133, 75-X4. Wickramasinghe. D., and Alhertini, L) k. ( 1993). Cell cycle control during mammalian oogenesis. Curr: fiipics Drir B i d . 28, 125- 153. Winston. N. J., MacGuinness, O., Johnson, M. H.. mid Maro, B. ( 1995). The exit of mouse oocytcs from meiotic M-phase requires ail intact q n d l c during intracellular calcium release. J . Cell Sci. 108, 143-151. Wolf, K. W. (1993). How mciotic cell\ deal with nowexchange chromosomes. BioEsstr~c 16, 107-1 14. Wood, C., Calderon, I., and Cronibie. A . ( IW?).Age and fertility: Resulta of a\\i\ted repi-oductive technology in women over 40 year\. J . A.\.\i.c/ Rt,/imd. G r w t . 9, 482-484. Yen, T. J.. and Schaar, B. T. ( 1996). Kinetochot-e lunction: Molecular motors, awitches and gates. Cur?: o/Jiil.CC// B i d . 8, 38 1-388. Zamboni. L.. and Upadhyay, S. (1983). Gerni cell dilterentiation in mouse adrenal glands. J . Ekp. ZOO/. 228, 173-193.
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12 Nondisjunction in the Human Male Terry J. Hassold Department of Genetics and the Ccnter for Human Genetics Case Western Reserve University and the University Hospitals of Cleveland Cleveland. Ohio 44106
I. Introduction: An Overview of thc Prohleni 11. Approaches to Studying Male Meiotic Nondis.junction: Methodology and Results A. Studies of Trisomic Fetuses and Livehorn\: The Contribution 0 1 Paternal Nondisjunction to Human Aneuploidy B. Studies of Aneuploidy in Male Germ Cell\ 111. The Etiology of Male Nondis~junction A. Age and Nondis,junction i n the Human Male H . Aberrant Genetic Recombination and Malc Nondisjunction C . Aneuploidy and Infertility in 1111' Human Male D. Environmental Component\ ol Male Nondi\junction Iv. Summary and Future Directions
References
1. Introduction: An Overview of the Problem Aneuploidy is the most common class of chromosome abnormality in humans and, according to some, the most important genetic hazard facing man (Bond and Chandley, 1983). No less than 0.3%' of all newborns are trisomic or monosomic (Hassold and Jacobs, 1984), most with significant physical or intellectual abnormalities. Considered as a class, aneuploidy is the most common known cause of mental retardation. Most aneuploidy derives from errors in maternal meiosis, and accordingly, most nondisjunction research is directed at understanding abnormalities in oogenesis. However, paternally derived aneuploidy is also extraordinarily common. Most cases of sex chromosome monosomy (45,X) involve loss of the paternal sex chromosome, and as this condition is associated with 10% of all spontaneous abortions, paternally derived aneuploidy is a leading genetic cause of pregnancy wastage (Hassold et ul., 1092). Further, approximately 1 in 500 liveborn males have either a 47,XXY or a 47,XYY chromosome constitution. The former condition is a common genetic cause of sterility and, in about 50% of cases, involves an additional paternal sex chromosome (Hassold et al., 1991); the latter has been associated with intellectual and/or behavioral abnormalities and is always due to Y-chromosome nondisjunction. Finally, at least 1 in 7000 newborns has an addi383
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Terry J. Hassold
tional paternal chromosome 2 1 , making paternal nondisjunction an important genetic cause of mental retardation (Antonarakis, 1993). In addition to its clinical importance, male meiosis has several features that make studies of male nondisjunction an attractive alternative to studies of female nondisjunction. These include the ability to collect products of meiosis without resorting to invasive procedures, the availability of a virtually limitless supply of gametic products, and the ability to study nondisjunction free from the confounding effects of increasing maternal age. For these reasons, several investigators have suggested that analysis of male nondisjunction may serve as a useful model for several aspects of human nondisjunction, for example, systematic searches for meiotic mutants, the possible effect of environmental exposures on nondisjunction, and the possibility that variation in chromosomal structures predisposes to nondisjunction. This review summarizes the current understanding of human male nondisjunction, first considering studies aimed at determining the incidence and stage of origin of nondisjunction and then describing recent information on etiological factors associated with the male nondisjunctional process.
II. Approaches to Studying Male Meiotic Nondisjunction: Methodology and Results Two general approaches have been used to study nondisjunction in the human male. One involves a retrospective analysis of abnormal meioses, in which DNA polymorphisms are used to study the parental origin of aneuploid feluses or liveborns and, in those cases found to be paternal in origin, to examine the stage of origin of the abnormality. The other involves analyses of the sperm cells themselves, using either a direct cytogenetic approach to study pronuclear chromosomes in interspecific fertilization events, or cytological stains to specific chromosome regions or fluorescence in siru hybridization (FISH) with chromosome-specific probes to analyze intact sperm heads.
A. Studies of Trisomic Fetuses and Liveborns: The Contribution of Paternal Nondisjunction to Human Aneuploidy
Over the past decade, considerable information has accrued on the origin of aneuploidy in humans. By using chromosome-specific DNA polymorphisms to study the inheritance of the additional or missing chromosome in monosomic or trisomic conceptuses, it is a straightforward exercise to determine the parental origin of the abnormality. Furthermore, for trisomic conceptuses, DNA markers located at or near the centromere can be used to determine the meiotic stage of origin of the extra chromosome.
385
12. Nondisjunction in the Human Male
This approach has now been used to study over 1000 monosomic or trisomic conceptuses (Table I). The results indicate that most cases of sex chromosome monosomy-the only monosomic condition identified in appreciable frequency in clinically recognized human pregnancies-involve loss of the paternal sex chromosome. This appears to be the case among liveborn individuals with Turner syndrome, as well as for those instances of 45,X that terminate in spontaneous abortion. In both categories, paternally derived errors account for approximately 70-80% of the cases. Other than this, little is known about the origin of sex chromosome monosomy, for a very simple reason: the “relevant” chromosome is missing and unavailable for further study. Nevertheless, from a consideration of the relative frequencies of sex chromosome monosomy and trisomy, it seems unlikely that the two categories of sex chromosome aneuploidy have a common origin. That is, sex chromosome monosomy is an extremely common abnormality in humans, occurring in an estimated 1-2% o f all clinically recognized pregnancies (Hassold, 1986). If it were attributable to simple meiotic nondisjunction, it would be expected that the reciprocal products of meiotic malsegregation-the 47,XYY, 47,XXY, and 47,XXX condition-would also be common. In fact, cumulatively sex chromosome trisomies occur in fewer than 0.1 % of all clinically recognized prcgnancies. This is unlikely to be due to differential survival of 45,X conceptuses, because available evidence suggests that 45,Xs are at a selective disadvantage by comparison with the sex chromosome trisomies (Hassold, 1986). Thus, it
Tdhk 1 Molecular Smile\ of Parenral Ol-igin o f Autosoma1 and Sex Chroniowme Aneuploidy No. 01 caw\ Sex chi-oniosonie monosomy Trisornl
4 I0 I1
13 13
Maternal
Paternal
(k Paternal
I37
29
IOX
79
16 6 6 5
10 6 6 5 I 22
6 0 0 0 0 3 2 4 0 8 60 2 58 3
38 0 0 0 O 12 17 15 0
I
25 12 27
I0
15 16 IX
I04 105
?I 22
642 20
XXY
133
xxx
23 104 97 5 82 18 7s
47
44
Data from Fi\her cf crl. 1995: Ha\\oltl P / ol. 1902; Kupke and Muller. 1989; Lamb Ya-GansC/ trl. 1993; Zal-agoia r’f crl. IW1, and I-la\5old, unpublished ohsenation\.
X
9 10 43 6 PI
ol. 1996;
386
Terry J. Hassold
seems reasonable to conclude that the mechanisms of loss of a paternal sex chromosome are different from those associated with the generation of an additional X or Y chromosome. In contrast to sex chromosome monosomy, approximately 90-95% of trisomic conceptuses result from maternal nondisjunction. However, as the data in Table I indicate, there is considerable variation among chromosomes in the likelihood of identifying a paternally derived trisomy. For certain conditions, such as trisomy 16, paternal nondisjunction appears to be a minor or nonexistent contributor to trisomy. For most other conditions, such as trisomy 18 or 21, patemally derived cases are identified, but in only 5- 10% of conceptuses. Finally, for a few conditions the level of paternal nondisjunction is much higher: approximately 40-50% of cases of trisomy 2 and the 47,XXY condition are attributable to paternal errors, while all 47,XYYs derive from paternal Y-chromosome nondisjunction. What is the source of this variation in the level of paternally derived trisomies? It is commonly thought to reflect chromosome-specific variation in paternal meiotic nondisjunction frequencies, but there are at least two other plausible explanations. First, it may be that the level of paternal nondisjunction is relatively similar among different chromosomes but that chromosome-specific levels of maternal nondisjunction vary. For example, if maternal meiotic errors are common for chromosome 16 but rare for chromosome 2 , the perceived level of paternal nondisjunction for trisomy 2 will be higher than that involving chromosome 16. Alternatively, the data on trisomic conceptuses involve studies of a population of “survivors”-that is, fetuses that survive long enough to be recognized as clinical pregnancies. If genomic imprinting effects result in the differential likelihood of survival of paternally versus matemally derived trisomies for specific chromosomes, the perceived level of paternal nondisjunction may be quite different from the level at the time of fertilization. Ultimately, the analysis of nondisjunction in sperm, using methodologies such as FISH, should make it possible to distinguish between these different possibilities (see later discussion). In addition to determinations of the parental origin of trisomy, DNA polymorphism analysis has been used to study the stage of origin of nondisjunction. Most maternally derived trisomies result from errors at the first meiotic division (e.g., Abruzzo and Hassold, 1995), but, surprisingly, this docs not appear to be the case for paternally derived trisomies (Table 11). For example, for trisomy 21, the most extensively studied condition, meiosis I1 errors are more common than meiosis I abnormalities, with a small proportion of cases apparently resulting from postfertilization mitotic nondisjunction. For other autosomal trisomies, for which less information is available, meiosis I1 and mitotic errors also appear to be quite common. Thus, the available data suggest that the mechanisms of nondisjunction associated with paternal nondisjunction are different from those responsible for maternally derived trisomies.
387
12. Nondisjunction in the Human Male Table I1 Molecular Studies 01
[lie S t q c of Origin in Paternally Derived
Autosomal Trimiiiites"
Trisoiny
2 13-1s 18
21 22
Meio\i\ I
Meiosis II
3 I 0 I3
0 4 0 21
I
0
Mitotic
"Sex chromosome trisoiiiies have heeii excluded Iroin this table, since all paternally derived 47,XXYa originate ill meiosis I and all 47,XYYs at meiosis 11 or po\t-fertili~a~toii. Data from Abrurm and l-la\\i)ld. iO95: Fisher C I rrl. 1995; Lamb C I ti/. 1996; and Hassold. unpuhli\hed oh\er\arions.
B. Studies of Aneuploidy in Male Germ Cells
Three general approaches have been taken to the study of aneuploidy in male germ cells. The first involves analysis of the number of "spots" in sperm nuclei, using cytological stains specific for chromosomal regions. Initially this technique was employed with a quinacrine staining assay to estimate the incidence of Y-chromosome aneuploidy (Pearson ct d., 1970; Barlow and Vosa, 1970). Subsequently, modified C- or G-banding protocols were used to visualize chromosomes 1 and 9 in interphase nuclei, including sperm (Bobrow et ul.. 1972; Gcraedts and Pearson, 1973). When this approach has been used, estimates of chromosome-specific aneuploidy of I-5% have been reported (e.g., Sumner er a/., 1971). However, these values us~iallyhave been dismissed, for two reasons. First, the staining procedures are not entirely chromosome specific; thus, in some proportion of cells, two or more nonhomologous chromosomes will stain positively and the cell will be scored incorrcctly as aneuploid. Second, the values imply an extraordinary level of nondisjunction in human sperm. That is, if the values are representative of all human chromosomes, a large minority, if not a majority, of human sperm must be aneuploid. This seems highly unlikely, insofar as studies of trisomic conceptuses have shown that most are maternally derived (Table I). Thus, the practical value of cytological staining for sperm aneuploidy screening is unclear, although investigations using this approach are occasionally still reported (e.g., Bibbins et d., 1992). The second general approach involves cross-species fertilization of golden hamster oocytes with human sperm. which allows visualization of the chromosome complements of both species. Originally described by Rudak et ul. (1978), this technique has been extensively used over the past 15 years to characterize both numerical and structural chromosome abnormalities in human sperm. To
388
Terry J. Hassold
date, over 25,000 sperm chromosome complements have been examined (for reviews, see Martin rt d.,1991; Jacobs, 1992), with the overall rate of aneuploidy (calculated as twice the hyperhaploid rate) being nearly 2% (Table 111). Disomy for most chromosomes has been identified, but there is also evidence for significant heterogeneity among chromosomes. That is, chromosomes I , 9, 16, 2 I , and the sex chromosomes are overrepresented and cumulatively account for over 50% of the hyperhaploid sperm that have been identified (Table IV). This suggests the existence of chromosome-specific mechanisms of paternal nondisjunction, perhaps related to chromosome structure (e.g., chromosomes I , 9, and 16 differ from all other autosomes in containing variable blocks of pericentromeric heterochromatin) or possibly to variation in recombination (e.g., the XY bivalent and chromosome 2 I are normally held together by one chiasma in male meiosis, whereas most other chromosomes are joined at two or inore points of exchange [Laurie and Hulten, 19851). However, these suggestions are based on observations from a small number of hyperhaploid sperm and need to be confirmed on a larger sample. This cannot easily be accomplished by additional human-hamster studies, because it has taken several laboratories over 15 years to identify fewer than 250 hyperhaploid cells (Table Ill). Thus, investigations of possible chromosome-specific mechanisms of paternal nondisjunction require an alternative method. Fortunately, the recent introduction of fluorescence it1 situ hybridization (FISH) has provided such an approach (see Fig. 1, for example). Using FISH, an extremely large number of sperm can be scored quickly, and the availability of chromosome-specific probes makes it possible to evaluate aneuploidy levels for all human chromosomes. Furthermore, the use of FISH makes it possible to analyze all sperm in an ejaculate, not just those capable of fertilizing in an itz i,itro situation. Beginning in the early 1990s. several groups initiated FISH studies to evaluate aneuploidy in sperm (e.g., Guttenbach and Schniid, 1990; Cooner et a/.,
Table I11 Summary of Cytogenetic Studies of Aneuploidy in Human Sperm, Assayed Using thc Human-Hamster Fusion A s w y
Study Jacob\ er (I/.( I992 l" Benet et ti/. ( 1992) Ro\enbusch and !jter.uk (1994) Tcmplado ('I ti/. ( 1996) Total
No. of spei-ili analyzccl 20.895 so5 867 3,346 25,713
Hypoploidy
Hypcrploidy
No.
%
No.
%
448 36 12
2.1 9I 1.4
146 10 I0
07 2.0 1.2
"')
57
1'7
,:. .3
3.1
723
0.9
I .7
802
E\tiiiiated % aneuploidy (IxHqprrploidy) I .4 4.i)
:.4
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I ? . Nondkjunction in the H u m a n Male
x
5.11
i
,
4 0 0 I
I I0 -1 3
4 -I -I h
I0
o 0 -I (1
Ih i
1991; Han r t ( I / . , 1992; Holmcs mil Martin. 1993: Martin c't ( I / . , 1993; Rohhins or 1993; Wyrobek et ( I / . . 1990) and reported disomy frequencies ranging from 0. 1 to 2.05t l o r the sex chroniosoiiics and chromosomes 1. 12, 15. 16. and 17. Houc\.cr. virtually a11 of these stuclies rclicd o n one color FISH. that is. analysi\ o f o n e chromosome-specific prohc at ;I tiiiic, This practice mikes it impossible to d i s t i n g i s h between disomic x i i l diploid sperm, and thus the mcaninglulncss of' these early reports is uncertain. iMore recently investigators have begun to LIK t\vo- o r three-color FISH. This appi-oach not only makes i t possible to discriminate disomic from diploid .spcriii. hut ;iIso m i k e s it possible to distingiiihh between sex chromosome disomy I-csirlting from meiosis I (i.e.. X Y disomy) o r meiosis I 1 (i.e.. Y Y o r X X discmi! ) nondis,junction (Williams t't [ I / . . 1003). [I/..
390
Terry J . Hassold
Unfortunately, it is not yet possible to distinguish between meiosis I and meiosis I1 nondisjunction of autosonies, although methods are now being developed for this purpose (O’Keefe et al., 1996). Results from many two- or three-color FISH studies of sperm aneuploidy are now available, and data from several of the larger ones are summarized in Table V, to facilitate comparisons among laboratories, different studies from the same laboratory have been grouped together. Several points are clear from these data. First, not surprisingly, the data indicate that meiotic nondisjunction is substantially lower in the human male than in the female. That is, most studies suggest chromosome-specific levels of disomy of approximately 0.05-0.15%. considerably lower than the estimated value of about 0.3-0.5% for the human female (e.g., Jacobs, 1992). Second, studics in which both autosomal and sex chromosome disomy levels have been estimated indicate a higher level of nondis.junction involving the sex chromosomes. In most studies, the combined incidence of XU, XX, and Y Y disomy is at least two- to threefold that of individual autosomes. Third, there is suggestive evidence for variation among autosonies in the frequency of nondisjunction. Spccifically, in three studies in which the frequency of disoniy 21 has been examined, disomy 21 levels are significantly higher than those involving other autosomes (Griffin et id., 1996; Spriggs et ul., 1996; Blanco et ul., 1996). Thus, the results of these initial FISH studies are consistent with molecular studies of the parental origin of trisomy and with previous human-hamster studies in two important respects: observed levels of paternal nondisjunction are lower than those estimated for maternal nondisjunction, and there appears to be significant among-chromosome variation in the level of nondisjunction. However, there are also several important discrepancies between the FISH studies and the earlier studies, as well as inconsistencies among the different FISH studies. First, most of the FISH studies imply levels of paternal nondisjunction far in excess of those observed in the human-hamster studies. For example, FISH studies conducted in the laboratories of Martin (Martin et al., 1995; Martin and Rademaker, 1995; Spriggs et (11.. 1996) and Hassold (Williams et a/.. 1993; Griffin et al., 1995, 1996) suggest chromosome-specific levels of disomy of 0.1% or more, implying a total frequency of hyperploidy of at least 2.3% (i.e., 0.1 % X 23 chromosomes), over twice the 0.9% value observed in the human-hamster studies (Table 111). The overall levels of disomy implied by the studies of Bischoff et a / . (1994) and Pellestor et a1 (Pellestor, Quenesson, Coignet, Girardet, Andreo, and Charlieu, 1996; Pellestor, Quenesson, Coignet, Girardet, AndrCo, Lefort, et al., 1996) are even further elevated, to approximately 6% and 7577, respectively. Only in the studies of Wyrobek’s laboratory (e.g., Wyrobek et a / . , 1993; Van Hummelen et a/., 1996) are the values obtained by FISH consistent with those obtained from the human-hamster fusion assays. Second, the chromosome-specific levels of disomy observed in FISH studies are difficult to reconcile with available data on the frequency of paternally
X
0
5
r!
5'
-
m
392
Terry J. Hassold
derived trisomies in clinically recognized pregnancies. The sex chromosome trisoinies provide a good example of this point. Table VI summarizes information on the incidence and parental origin of sex chromosome trisoiny in clinically recognized pregnancies and provides an estimate of the amount of paternal nondisjunction needed to account for these cases. These values are then compared with data from three of the larger FISH sperm studies in which values for sex chromosome disomy have been reported. For 47.XXYs, the observed levels of disomy are 1 . 1 to 4.3-fold that of the estimated frequency of paternal nondisjunction, and for 47,XYYs 0.8 to 5.3-fold. Differences of this magnitude are perhaps not surprising, because only a relatively small number of sperm and donors have been studied with FISH and because the data from clinically recognized pregnancies depend, in part, on uncertain assumptions regarding levels of fetal wastage. However, for 47,XXXs all three estimates ofdisomy far exceed the incidence figures from clinically recognized pregnancies. This suggests either that the different studies have systematically misscored normal sperm as XX disomics or that XX sperm are at a selective disadvantage by comparison with normal sperm, or that 47,XXX fetuses (and perhaps paternally derived cases in particular) are much more likely to perish in u t r m than is currently appreciated. Presently there is little basis for discriminating among these possibilities. How ever, the considerable among-study variation in the estimates of all three categoTable VI
Correlation between E\tiiiiated Frequency o f Paternally Derived Sex Chroniomme Trisomieg in Clinically RecogniLed I'i-egnancies and Frequency of Sex Chroinosoine Disomy Observed in FISH Studies Parameter ebaluatrd Estimated frequency ( % ) in c1inic;illy recognized prcgnancics (Haswld ;ind Jacobs. 1984) Estimated proportion (% j of paternally derived case\ (Ahrum) and Hassold. I995 j Frequency ( % j of diwmic \perm needed to account for paternallyderived case\ Frequency (a) disomic sperm identified i n Wyrobeh ('1 ( I / . (1993) Griftin t'i ( I / . ( 1995, 1996) Spriggs CI ( I / . ( 1996) Excess of disornic sperm identitied in: Wyrobek c't ( I / . (1993) Griffin C I ( I / . ( 1995, 1996) Sprigg\ rf a/. ( 1996)
47.XXY
47,xxx
47.XYY
0.08
0.0s
0.04
44
5
100
0.035
0.0025
0 04
0.04 0. I0
0.04
0. I S
0.07
0.09 0.03 0.2 I
1.1
16.0 8.0 28.0
2.3 0.8 5.3
2.0 4.3
0.02
12. Nondisjunction in the Human Male
393
ries of sex chromosome disomy indicate that at least some of discrepancies are artifactual in origin. Finally, there is extraordinary variation among the different FISH studies in the observed levels of disomy. Among those summarized in Table V, the implied overall frequencies of sperm disomy range from about 1% (Wyrobek et al.. 1993; Van Hummelen et al., 1996) to about 7% (Pellestor, Quenesson, Coignet, Girardet, Andreo, and Charlieu, 1996; Pellestor, Quenesson, Coignet, Girardet, Andrko, Lefort, ef al., 1996). It is difficult to imagine any plausible biological explanation for this variation, as it seeins unlikely that different laboratories would, by chance alone, have studied donors with such widely varying frequencies of meiotic nondisjunction. Instead, it seems likely that the scoring criteria differ among the various laboratories and that the variation is artifactual. This is troubling, because it brings into question both the reliability of the FISH procedure and the biological relevance of the results. As suggested previously by other investigators (e.g., Van Hummelen rt al., 1996), it would be useful to develop stringent scoring guidelines that would be shared by all laboratories and to conduct trial experiments in which different laboratories score preparations from the same set of donors. Nevertheless, even if such measures are adopted and the estimates of disomy become more uniform, it should be recognized that the FISH approach will always be more subjective than conventional cytogenetic analysis; that is, identification of two “spots” in a sperm head will never be as satisfying as identification of two chromosomes in a metaphase; nor can the FISH analysis easily be confirmed. Thus, it is unrealistic to think that FISH will ever provide error-free estimates of disomy in sperm. This does not mean that FISH has no place i n studies of male nondisjunction. Rather, it means that it will be more efficient if used to answer questions related to differences i n frequency of aneuploidy (such as differences among chromosomes, among donors. or between exposed and unexposed populations) than questions requiring exact estimates of the frequency of aneuploidy.
111. The Etiology of Male Nondisjunction Despite years of intensive study, we still know little about factors that influence the frequency of trisomy in humans. For example, efforts to identify important environmental components to trisomy or to identify trisomy-prone families have met with little success (Abruzzo and Hassold, 1995). I n part, this may be due to the design of the previous studies, virtually all of which involved analyses of trisomic fetuses or liveboms. In such studies, the ability to identify environmental agents or rare meiotic mutants affecting chromosome disjunction depends in large part on the background level of trisomy in the general population. In humans. at least 4% of all clinically recognized pregnancies are trisomic, and for women above 40 years of age, this value increases to 30-35% (Hassold et a/.,
394
Terry J. Hassold
1996). Thus, traditional attempts to identify environmental or genetic susceptibilities to nondisjunction may be hindered by the “noise” attributable to sporadic, maternally derived cases of trisomy. A potentially useful alternative involves the direct analysis of male gametes, an approach that has recently been used to study several aspects of male nondisjunction. This section presents recent observations in four such areas: aging and male nondisjunction, aberrant genetic recombination and male nondisjunction, infertility and male nondisjunction, and environmental influences on male nondisjunction.
A. Age and Nondisjunction in the Human Male The relationship between advancing maternal age and increasing incidence of trisomy in humans has long been established, but the possibility that there might also be a paternal age effect has been somewhat contentious. Most epidemiological studies of Down syndrome have concluded that there is no such effect (e.g., Penrose and Smith, 1996; Hook et ul., 1990). However, Stene and colleagues (Stene and Stene, 1978; Stene et al., 1981) have consistently reported the existence of a paternal age effect in Down syndrome, and this difference of opinion has led to considerable controversy in the literature (e.g., Carothers, 1988). More recently, the ability to distinguish molecularly between maternally and paternally derived trisomies has made it possible to ask directly whether paternal age is elevated in trisomies of paternal origin. Several groups have reported on this question, with conflicting results. For example, Petersen et al. (1993) recently analyzed the parental ages of paternally derived cases of trisomy 2 1 and noted an increase in paternal age in cases of meiosis 1 origin. Similarly, Schinzel and colleagues (Lorda-Sanchez et ul., 1992) reported significant increases in paternal age in paternally derived 47,XXYs, paternal uniparental disomy 15 (Robinson et a/., 1993), and paternal trisomy 18 (Ya-gang et d., 1993). However, MacDonald et al. (1994) observed no effect of increasing paternal age in their analyses of paternally derived 47,XXYs and 47,XXXs, or did Zaragoza et ul. (1994) in a small series of paternally derived acrocentric trisomies. Thus, neither epidemiological nor molecular studies have resolved the question of whether there is a paternal age effect on male nondisjunction, and it seems unlikely that they will ever do so. That is, epidemiological studies typically include all trisomies, and since at least 80-90% are maternal in origin, such studies have limited power to detect a paternal age effect on the remaining, paternally derived cases. Similarly, molecular studies are limited by the relative rarity of paternally derived cases: despite several years of work by many laboratories, fewer than 200 paternal trisomies have been identified (Table I). An obvious alternative approach involves direct analysis of sperm (Table VII).An initial cytogenetic study of age and paternal nondisjunction in which the human-hamster fusion approach was used found no evidence of an increase in
Table VII
Direct Studies o f Aneuploidy in Human Sperm to Examine the Association of Age and Male Nondisjunction Method of analysis
Study
No. of males
Age range (yrs)
Summary of results Significant decrease in hyperploidy with increasing age, from 3.7% for males 20-24 to 0% for males 40 and older Significant age-related increaw i n disomy 1 and Y Y d i m n j . with about a 2 X difference in frequencies between the youngest and oldest \tudy subject\ Significant effect for XY and XX disomy and borderline significant effect for YY disomy, with each condition being about 2 X as common in men 50 and older as men under 30 Significant increases in XX and YY disomy, with 2-3X increases in each in men in the older age group
a/.(1987)
Standard cytogenetic studies using humanhamster fusion analyses
30
22-55
Martin e f a / . (1995)
FISH sperm studies of chromownes I . 17. and sex chromosomes
10
2 1-52
Griltin
FISH sperm studieb of chromosome 18 and sex chromosomes
21
18-60
FISH sperm studies of chromosome 8 and sex chromosomes
14
22-59
Martin
el
e f (I/. (1995)
Robhins rt a/.( I 995)
396
Terry J. Hassold
hyperploidy with age; indeed, a significant decrease was observed (Martin and Rademaker, 1987). However, only 23 of a total of 1582 sperm was hyperploid, limiting the ability to detect subtle differences and eliminating the possibility of analyzing individual chromosomes. More recently, three groups have initiated large FISH sperm studies of paternal age and aneuploidy (Table VII). In Martin et d . (1995), over 225,000 sperm from 10 donors aged 21-52 years were studied. A significant age-related increase was observed for disomy 1 and YY disomy, although no obvious effect of age was detected for disomy 12 or XY or XX disomy. In Robbins et ( I / . (1995), a study involving 205,000 sperm from 14 men aged 22-59 years, a significant increase in the incidence of both YY and XX disomy was observed, although no effect was observed for the single autosome studied. Griffin et (11. (1995) studied disomy levels of chromosome 18 and the sex chromosomes in approximately 400,000 sperm from 24 males aged 18-60 years; significant age-related increases were identified for XY and XX disomy, with a borderline significant increase in Y Y disomy and a nonsignificant increase for disomy 18. Thus, each of the three FISH sperm studies identified significant increases in one or more types of sex chromosome disomy, and in one of the studies (Martin et ul., 1995) a significant increase in an autosomal disomy was identified. These results provide the first direct evidence that meiotic nondisjunction increases with age of the human male, and. as the results are based on analyses of over 1,000,000 sperm, they are difficult to dispute. However. there are still a number of caveats to this interpretation. First, the results are based on samples from a relatively small number of males (no. of cases = 48), and need to be confirmed in a more extensive series of individuals. Second, relatively few individual chromosomes have yet been examined, so that the possible association of paternal age with aneuploidy for other clinically important chromosomes such as chromosome 21 remains to be determined. Third, the results pertain only to gametes, and any clinical relevance depends on aneuploid sperm being equally likely as euploid sperm to participate in fertilization. Finally, in each of the three studies the effect that was observed as small, typically involving only twofold differences in disomy rates in men in the youngest and oldest age groups. For example, in Griffin et al. ( 1 9 9 3 , the increase in XY disomy was from 0.08%;:in men aged 1829 years to 0.19% in men SO years and older, an age-related increase of only 0.1 I %. Thus, there is little basis for suggesting that older men, like older women, be offered prenatal testing for age-related nondisjunction. Nevertheless, the combined results of these studies suggest that older men, like older women, have an increased likelihood of producing aneuploid offspring.
B. Aberrant Genetic Recombination and Male Nondisjunction From studies of yeast and Drosophilri. it has long been recognized that mutants that reduce meiotic recombination typically have increased frequencies of non-
12. Nondisjunction in the Human Male
397
disjunction (e.g., Hawley et a/., 1994; Rockmill and Roeder, 1994). Advances in human genome research have finally made it possible to ask whether this is true for human nondisjunction as well. That is, the availability of highly polymorphic DNA markers makes it possible to determine the amount and position of recombination on nondisjoined chromosomes in families with trisomic conceptuses (Chakravarti and Slaugenhaupt, 1987) and to compare these data with comparable information from normal meioses. In an early analysis of trisomy 2 I , Warren et al. ( 1987) provided the first direct evidence that reduced genetic recombination is indeed associated with human trisomy. Subsequently, several investigators extended these observations to other, maternally derived trisomies (for a review, see Hassold et a/., 1996) and demonstrated significant reductions in recombination in maternal sex chromosome trisomy, maternal trisomies 16, 18, and 2 1, and maternal uniparental disomy 15. Thus, aberrant genetic recombination is an important contributor to human female meiotic nondisjunction. What about male meiotic nondisjunction? Presently, too few cases of paternally derived autosomal trisomies have been identified to allow any detailed analysis of recombination and autosomal nondisjunction. However, preliminary studies of paternally derived 47,XXY indicate an effect of aberrant recombination on male sex chromosome nondisjunction. Specifically, in studies of 39 paternally derived 47,XXYs, Hassold et ( I / . (1991) found only 6 that showed evidence of crossing-over in the XY pairing, or pseudoautosomal region; subsequently, Lorda-Sanchez et a / . ( 1992) reported that they were unable to detect any pseudoautosomal exchanges in 10 paternal 47,XXYs. These results are in sharp contrast to normal male meioses, i n which a single exchange ordinarily joins Xp and Yp, and suggests that-at least for the X and Y chromosomesfailure of recombination is an important component of paternal meiotic nondisjunction.
C. Aneuploidy and Infertility in the Human Male It has long been recognized that constitutional chromosome abnormalities predispose to male infertility. For example, individuals with a 47,XXY chromosome constitution (Klinefelter syndrome) are typically azoospermic and are frequently ascertained because of infertility (e.g., Bond and Chandley, 1983). Further, histological studies of testes of males with a 47,XYY chromosome constitution suggest variable levels of spermatogenic impairment, although such individuals are not overrepresented in infertility clinics. The ability to directly analyze the chromosome makeup of human sperm has led investigators to pose a different question regarding male infertility: Are males with idiopathic infertility are at increased risk of producing chromosomally ahnormal gametes? Over the past decade, two general methodologies-cytogenetic
398
Terry J. Hassold
studies of sperm in interspecific fusions and FISH sperm analyses-have been used to address this question. Results from several of the largest such studies are summarized in Table VIII. In one set of interspecitic fertilization studies, the possible association between morphologically abnormal sperm and aneuploidy was examined. In humanhamster fusion assays involving 30 fertile males, Martin and Rademaker (1988) correlated donor-specific rates of aneuploidy with rates of abnormal sperm forms. Subsequently Lee et ul. (1996) microinjected morphologically normal sperm or sperm with abnormally large, small, or unusually shaped heads into mouse oocytes and cytogenetically examined the sperm-injected oocytes. Neither of these two studies identified an association between sperm morphology and the rate of chromosome abnormality, which suggests that the genetic complement of the sperm does not affect cellular morphology. In other human-hamster fertilization studies, Moosani et al. ( 1 995) and Rosenbusch and Sterzik ( 1994) compared the frequency of chromosome abnormalities in fertile and subfertile or infertile males. In studies of five infertile males, Moosani et al. (1994) observed a twofold increase in the frequency of numerical abnormalities by comparison with controls. However, most of the abnormal cells were missing one or more chromosomes-occurrences that can have technical as well as biological causes-and thus the meaningfulness of the increase is uncertain. In Rosenbusch and Sterzik (1994), no obvious difference in chromosome abnormalities was observed between case and control individuals; however, only 146 metaphases were available from the infertile individuals, which limited the ability to detect moderate differences in frequency between the case and control subjects. Thus, the studies of interspecific fertilization have provided little evidence that either sperm morphology or male infertility is associated with large increases in aneuploidy in sperm. However, the correctness of this interpretation has recently taken on new importance, owing to the increasing use of intracytoplasmic sperm injection (ICSI) in human in vitro fertilization (IVF) procedures (e.g., Toumaye e f al., 199.5); indeed, in many IVF centers ICSI is the procedure of choice in male-factor infertility cases. There are at least two concerns regarding the chromosome makeup of these ICSI-initiated pregnancies. First, the results of the human-hamster studies cited in the preceding paragraphs give little re‘‘1 1 assurance that aneuploidy rates are the same in infertile as fertile men; in fact, the results reported by Moosani et al. ( 199.5) suggest an increase among the infertile males. Second, there are preliminary-if controversial-results from studies of ICSI pregnancies that suggest an increase in chromosome abnormalities in the conceptuses. In’t Veld et al. (1995) reported an extraordinary effect, as they identified sex chromosome abnormalities in 5 of 15 fetuses from ICSI pregnancies referred for prenatal diagnosis because of maternal age. Tournaye et a/. (1995) found no evidence for an effect of this magnitude; nevertheless, they identified 5 chromosome abnormalities (including 4 sex chromosome trisomies)
'lable V l l l
Surririiary at Cytngcnetic and I:ISI I S t u J i c s ot Spcrm in Male lnt'titility
Manin and Rademahcr ( 19x7)
Human-hamster
Lec el nl. (1996)
Micruiii,ic.ction 0 1 hrini;in sperm into
Roscnbusch and Stcrzih (1991) Mooaani f'c ( I / . ( 1995)
Human-hamster a\\ay Human-hamster
assay
30. all of proven
Not stated; at Icasl 900
No difference in numerical anomalies between
200
Nri ditlircncc i n niiincricill unorrialics hctwvcii morphologic;illy nornial and
3.:. including S
867
So difterence in numerical anomalir,
whfertile male\ 5 infertile males
518
krIihty
mules with low and high rates o r
mr~rpholopicallyahnoi-nial sperm
abnormal hperm
mouse oocyte.;
Ltlxl?
Miharu ef u/. (1994)
FISH sperm study o f sex chromosomes and I . 16; 17. 18 FISH sptrrri study ol %ex chromosotncs and
1 and 11 HSH sperm study of ses chromosomes and 10 autosomes
9 fertile and 13 rnfcrtilc m a b
450.580
I0 iriicrtilc 1 1 i i l l ~ k , I0 controls ( b of pl'o\.cn tcrkili t y 5 inftnilr malev and 1 fertile males
205,2 I3 frotii test sub ci-t s Not stated
between test and contrtol individuals Significant increase in numerical anomaliei by crmparison with laboratorq. control\ (k..8.9% vs. 4.2%) N o significant difference in rate of dimmy
bctween infertile and fertile males SigiiiliL.ilnt incrcasus i n disorriy I and X Y
disolny Hi$Iy sipnilicant increase i n o ~ r m l lle w l of autosomal disomy, from 2 - 0 5 in controls to 15.5% in infertile individuals
400
Terry J. Hassold
in 371 prenatal diagnoses, a large excess over the expected number of sex chromosome trisomies among livebirths. Thus, it will be important to monitor the chromosomal status of fetuses of ICSI pregnancies, but more extensive analyses of aneuploidy in fertile and infertile males are also needed. Preliminary results obtained using the FISH sperm approach are now available from a few groups, and in two studies (Martin, 1996; Pang et ul., 1995) signifcant increases in disomy have been reported in the infertile individuals (Table VIII). However only a relatively small number of sperm from a limited series of test subjects have been investigated. Additional FISH studies will be important to characterize the increase, if any, in meiotic nondisjunction in these individuals. D. Environmental Components of Male Nondisjunction
There is an extensive literature on aneuploidy and the environment, but, surprisingly, hard data linking environmental factors to human meiotic nondisjunction are lacking. In part, this lack is a result of the difficulty in obtaining or testing the appropriate study material. That is, studies of aneuploid zygotes must be coupled with studies of fetal wastage, insofar as virtually all aneuploid conceptuses are eliminated early in pregnancy; direct studies of female gametes are hampered by the inability to obtain sufficient numbers of oocytes; and, until recently, studies of male gametes were limited by the lack of appropriate biomarkers. However, the availability of FISH spcrm technology now makes it possible to obtain and to screen a large number of sperm from appropriate populations of exposed and nonexposed individuals. Preliminary studies from Wyrobek and his colleagues suggest that this will be a profitable approach. For example, initial studies of cigarette smoking involving studies of over 300,000 sperm in 15 heavy smokers and 15 nonsmokers indicated a significant increase in some (disomy 8 and YY disomy) but not all (XU and XX disomy) types of disomy (Wyrobek et ul., 1995); however, as smoking is confounded with other lifestyle habits such as alcohol and caffeine consumption, it will be important to confirm these initial observations. Other preliminary studies from this same group indicate possible aneuploidy-inducing effects of the sedative Valium (Baumgartner et d.,1996) and suggest that certain anticancer agents may also increase the likelihood of meiotic nondisjunction (Robbins et ul., 1994). These studies are still in their infancy, but more extensive analyses of larger series of individuals may well lead to the first demonstration of environmental consequences on meiotic chromosome segregation.
IV. Summary and Future Directions In the past, most human nondisjunction research focused on the conceptus. By combining epidemiological, cytogenetic, and molecular approaches to analyze
12. Nondkjunction in the Human Male
40 1
aneuploid fetuses and liveborns, we have learned a great deal about the incidence of trisomy and monosomy i n dirferent types of human populations, about the parent and meiotic stage of origin of different aneuploid conditions, and about the relationship between increasing parental age and nondisjunction. These studies in turn have led to the identification of the first “molecular” correlate of human nondisjunction: aberrant genetic recombination. By their very nature, however, studies of aneuploid conceptuses provide only a retrospective window on a long since completed meiotic event. Future analyses of nondisjunction will likely focus on tbc underlying molecular abnormalities and will require more immediate access to the abnormal process, which might be achieved by monitoring the meiotic process in virro or by analyzing mature gametes. The availability of large numbers of male gametes suggests that genetic analyses of sperm can play an important role in these studies, and this is consistent with the preliminary results of the FISH sperm studies. Furthermore, by combining FISH sperm studies with other molecular approaches such as singlesperm PCR (Schmitt et d.,1994), it should be possible to address questions about nondisjunction that are presently intractable. For example, one of the most interesting questions concerning human aneuploidy is whether or not there arc nondisjunction-prone individuals. There have been a number of attempts to identify such individuals-there have been studies of the incidence of trisomy in inbred human populations (Alfi et d..1980), studies of recurrence rates in individuals with trisomic liveborns or fetuses (Warburton et d.,1987), and studies of the parental origin of trisomy in families with multiple trisomic offspring (e.g., Pangalos et al., 1992)-but these studies have provided little evidence for a genetic component to trisomy. Studies of human sperm may provide a valuable alternative in the search for meiotic mutants. For example, one potentially useful approach could involve analysis of males known to have fathered a trisomic offspring. In an initial set of studies, FISH could be used to determine whether sperm disomy rates are elevated in such males, and, if so, whether the frequency of disomy is elevated for all chromosomes, consistent with a meiotic mutation having a “global” effect on chromosome segregation, or elevated for the chromosome present in triplicate in the donor’s trisomic offspring, consistent with a chromosome-specific predisposition to nondisjoin. Subsequent studies would depend on these initial results. If individuals with global increases in disomy were identified, an obvious question would be whether this was attributable to alterations in the recombinational process; this can now be assayed by using single-sperm PCR to assess recombination directly. Alternatively, if individuals with chromosome-specific increases in disomy were identified, subsequent molecular analyses would focus on features of the chromosome itself, such as possible size or sequence variation in the centromere. Similar approaches may be useful in addressing other issues in human nondisjunction research and, eventually, in identifying factors important in the genesis of aneuploidy in our species. However, it is also important to sound a cautionary
402
Terry J. Hassold
note regarding this approach. Molecular cytogenetic studies of sperm aneuploidy are still in their infancy, and whereas their potential is clear, relatively few hard data have yet been obtained. It will be important to monitor the studies over the next few years and to determine whether this approach can live up to its billing in providing a useful model system for studying human nondisjunction.
Acknowledgments Research conducted in the Hassold laboratory and summariied in this publication was supported by NIH grants HD2 134 I and HD32 1 I I .
References Abruzzo, M. and Hassold, T. (19%). The etiology of non-diqjunction in humans. En\'iroii. Molec. Mutcir. 25, (Suppl. 26), 38-47. Alfi, 0..Chang, R., and Azen, S. (1980). Evidence for genetic control of nondisjunction in man. Am. J . Hum. Gener. 32, 477-483. Antonarakis, S. E. (1993). Human chromosome 21: Genome mapping and exploration circa 1993. Trendy Genet. 9, 142- 148. Barlow, P.. and Vosa, C. G. ( 1970). Thc Y chromoaoine in human spermatozoa. Norure 226, 96 I 962. Baumgartner, A,, Czeizel, A. E., Adler, I.-D., Lowe, X., Schniid, T. E., and Wyrohek, A. J. (1996). Chromosomes X, Y, and 21 aneuploidies in sperm of men who ingested ultra-high doses of diuepam. Ani. J. Hum. Geiier. 59, (Suppl.). A I 1 I . Benet, J., Genesca, A,, Navarro, J . , Egozcue, J., and Teinplado, C. (1992). Cytogenetic studies in motile sperm from normal men. Him. Genet. 89, 176-180. Bihbins, P., Ward, J., Jonathan, B., Lipshultz, L., Hokanson, J., and Legator, M. (1992). Incidence of sperm with two fluorescent bodies i n men with impaired fertility. Ferril. Steril. 57, 102407. Bischoff, F. Z., Nguyen, D. D., Burt, K. J., and Shaffer, L. G. (1994). Estimates of aneuploidy using inulticolor fluorescence in situ hybridization on human sperm. Cjtogmer. CelI Genet. 66, 237-243. Blanco, J., Egozcue, J., and Vidal, E (1996). Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization. Hum. Reprod. 11 (4), 722-726. Bobrow, M., Madan, K . , and Person, P. L. ( I 972). Staining of some specific regions of human chromosomes, particularly the secondary constriction o f no. 9. Nature Neti. Biol. 238, 122124. Bond, D., and Chandley, A. (1983). "Aneuploidy." pp. 86-91. Oxford University Press, Oxford. Carothers, A. D. (1988). Controversy concerning paternal age effect in 47, f 2 1 Down's syndrome. Hun,. Genet. 78, 384-385. Chakravarti, A,, and Slaugenhaupt, S. (1987). Methods for studying recombination on chromosomes that undergo nondisjunction. Genoinics I, 35-42. Chevret, E.. Rousseaux. S . , Monteil, M.. Pelletier, R., Cozzi. J., and W e , B. (1995).Meiotic segregation of the X and Y chromosomes and chromosome I analyzed by three-color FISH in human interphase spermatozoa. Cyrogmer. Cell Gener. 71, 126- 130. Coonen, E., Pieters, M., Dumoulin, J., Meyer, H., Evers, J., Ramaekers, F., and Geraedts, J. -
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Williams, B. J., Ballenger, C. A,, Maltcr, H. E., Bishop, E, Tucker, M., Zwingman, T. A,, and Hassold, T. J . ( 1993). Non-disjunction in huinan sperm: Results of fluorescence in .ri/rc hyhridization studies using two and three probcs. Hum. Mol. Genet. 11, 1929-1936. Wyrobek, A., Alhborn, T., Ballhorn, R.. Stanker. L., and Pinkel, D. (1990). Fluorescence in situ hybridimtion to Y chromosomes in decondensed human sperm nuclei. Mol. Reprod D P ~27, : 200-208. Wyrobck, A. J., Rohhins, W. A., Tang, C.. Kohayashi, A,, Baulch, J., Wcier, H.-U., and Pinkel, D. (1993). Hierarchical organi7ation of human sperm chromatin is a critical factor in the detection of chromosomal aneuploidies by fluore\cence in situ hybridization. Am. J . Hun?. Gewr. 53, (Suppl.), A130. Wyrobek, A. J . , Rubes, J., Cassel, M.. Moore, D.. Perreault, S., Slott, V., Evenson, D., Zudova, Z., Borkovcc, I.., Selevan. S., and Lowe. X. (1995). Smokers produce more aneuploid sperm than non-smokers. hi.J. Hum. Gmei. 57 (Suppl.). A131. Ya-gang, X., Robinson. W., Spiegel, R., Binkert, F., Ruefenacht, U., and Schinzel, A . (1993). Parental origin of the supernumerary chromosoiiie in trisomy 18. Clin. Genet. 44, 57-61. Zaragoza, M . V., Jacobs, P. A., James, R. S., Rogan, P., Sherman, S., and Hassold, T. (1994). Non-disjunction of hurnan acrocentric chromosomes: Studies of 432 trisoinic fetuses and liveborns. Hurn. Grner. 94, 41 1-41 7.
Index
A A s folliculogenesis compromise, 367-368 human male nondisjunction relationship, 394-396 Anaphase, metaphase-anaphase transition chiasmata role, 370-371 female meiosis regulation, 370-374 mammalian spermatogenesis G,/M phase transition regulation, 350 Aneuploidy, human male chromosome nondisjunction environmental components, 400 etiology, 383-384 future research directions, 400-402 overview, 383-384 study methods, 384-393 livebirths, 384-386 male germ cells, 387-393 trisotnic fetuses, 384-386 Antibodies, see a/so ImmunoHuorescence microscopy; Immunolocalization mammalian protein meiotic function analysis, 21 1-214 sister-cohesion protein analysis, 282-283 Ataxia-telangiectasia, meiotic protein function analysis, 224-226, 230
C CDC2 protein, meiotic expression, 163-164 Cell cycle Drosophilu male meiosis regulation study, candidate regulators, 31 1-314 mammalian meiotic protein function analysis, 223 regulation, meiotic gene expression, 162- I65 Cellular communication regulators, meiotic expression, 165-168 growth factors, 166-167 neuropeptides, 167 receptors, 168
Centromeres, chromosome segregation role, sister chromatid cohesion mechanisms, 291 -293 Checkpoint proteins mammalian female meiosis quality control human genetics, 372-374 pachytene role, 362-364 germ cell loss, 362-363 mouse mutant studies, 363-364 yeast mutant studies, 363 mammalian protein meiotic function analysis, 223 Chiasmata, chromosome segregation cohesion factor catenation, 280-28 1 crossover correlation, 269-272 crossover failure, 273-274 crossover position, 272-273 disjunction, 269-274 homolog attachment points, 269 metaphase-anaphase transition role, 37037 I sister chromatid cohesion, 277-283, 293 terminal binding, 274-278 Chromatids, chromosome segregation mammalian female meiosis regulation, 368 sister chromatid attachment maintenance centromeric region cohesion mechanisms, 29 1-293 chiasmata role, 277-293 equational nondisjunction, 290-29 1 meiosis I1 cohesion disruption mutations, 292 proximal exchange, 290-291 sister kinetochore function, 287-290 duplication, 287-288 functional differentiation, 288-290 reorganization, 287 Chromatin prophase loop alignment, 257 associated DNA sequences, 250-253 attachments, 247-250 development time course, 256
407
408 Chromatin (corn,) DNA content, 253-256 recombination hotspot identification, DNA accessibility, 5 1-52 Chromosomes, see ulso DNA bivalent segregation achiasmatic division, homolog attachment, 283-286 chiasmata cohesion factor catenation, 280-281 crossover correlation, 269-272 crossover failure, 273-274 crossover position, 272-273 disjunction, 269-274 homolog attachment points, 269 metaphase-anaphase transition role, 370-371 sister chromatid cohesion, 277-283, 293 terminal binding, 274-278 orientation mechanisms, 266-269 bipolar orientation recognition, 267269 bivalent structure, 266-267 dyad structure, 266-267 reorientation, 267-269 overview, 263-265, 292-293 sister chromatid attachment maintenance, 290-293 centromeric region cohesion mechanisms, 29 1-293 equational nondisjunction, 290-291 meiosis I1 cohesion disruption mutations, 292 proximal exchange, 290-29 1 sister kinetochore function, 287-290 duplication, 287-288 functional differentiation, 288-290 reorganization, 287 crossing over chiasmata association, 269-274 mammalian germ line recombination, 8 12 physical versus genetic distances, 10- I I recombination hotspots, 11-12 sex differences, 9- 10 DNA repair, see DNA, double-stranded breaks mammalian protein meiotic function analysis, aberrations, 228-230
Index nondisjunction human males, 383-402 aberrant genetic recombination, 396397 age relationship, 394-396 aneuploidy. 384-393, 397-400 environmental components, 400 etiology, 393-400 future research directions, 400-402 infertility, 397-400 male germ cells, 387-393 overview, 383-384 study methodology, 384-393 trisomic fetuses, 384-386 sister chromatid attachment maintenance, 290-291 pairing male Drosophila pairing sites, 79-96 autosomal pairing sites, 8 1-85 female pairing sites compared, 95-96 function mechanisms, 89-94 implications, 109-1 10 molecular composition, 89-92 sex chromosome pairing sites, 85-89. 94-95 spermatogenesis, 79-8 I transcription relationship, 92-94 overview, 77-79 spermiogenesis, 96- 109 chromosomal sterility, 100- 103 implications, 1 10- 1 1 1 meiotic drive, 96- 100 metaphase mitotic model, 106-109 pairing site saturation, 103- I05 X-inactivation, 105-106 prophase cores chromatin loop alignment, 256-257 associated DNA sequences, 250-253 attachments, 247-250 development time course, 256 DNA content, 253-256 overview, 24 1-242 synaptonemal complex electron microscopic structure analysis, 242-245 immunocytological structure analysis, 245-247 recombination site, 257-259 recombination dynamics, 39-40
Index
409
sterility, 100-109 metaphase mitotic model, 106-109 pairing site saturation. 103- 105 X-autosome translocations, 100- 10 I X-inactivation, 105- 106 Y-autosome translocations, 100- 1 0 1 j'+Y m d + chromosome, 102 Cohesion, chromosome segregation role chiasmata cohesion factor catenation, 280-281 sister chromatid cohesion, 277-283, 293 sister chromatid attachment maintenance centromeric region cohesion mechanisms. 29 1-293 meiosis I1 cohesion disruption mutations, 292 Communication regulators, see Intercellular communication regulators Competence, female meiosis regulation, 340342, 365-366 Contractile ring assembly, Drosophila male meiosis regulation, 321 Coprinm cinereus epistasis group model. double-stranded DNA break repair pathway analysis, 128- 134 Crossing over chiasmata association, 269-274 mammalian germ line recombination, 8 1L
physical versus genetic distances, 10-
It recombination hotspots, 1 1 - 12 sex differences, 9-10 Cyclin, meiotic expression, 163- 164 Cytokinesis, Drosophila male meiosis regulation, 3 19-325 contractile ring assembly, 321 germ line, 322-324 Cytoskeleton, meiotic protein gene expression, 174-175
D Disease, mammalian germ line recombination association, 18-22 Disjunction, see Chromosomes, bivalent segregation Disomy. see Chromosomes, nondisjunction Dmcdc2 gene, Drosophila meiosis regulation, 31 1
DNA, see d s n Chromosomes double-stranded breaks chromatin DNA accessibility, 5 1-52 recombination hotspot activation, 50-5 1 repair gene function Coprinus cit2ereu.s epistasis group model, 128-134 genetics, 123-125 homology-based repair event abundance, 126- 128 overview, 117-119, 132-134 pathways, 119-123 physiology, 1 17- I I9 hypervariable minisatellite DNA Holliday junction site resolution, 6065 recombination hotspot assay, 48-50 prophase chromatin loop association DNA content, 253-256 DNA sequences, 250-253 recombination, see Recombination repair proteins meiotic expression, 152-155 mismatch repair genes, 221 -222 Z-DNA, recombination hotspot assays. 4648 DNA methyltransferase, meiotic expression, I77 DNA-protein binding, recombination hotspot activation, 52-56 Drosophila chromosome pairing studies male pairing sites, 79-96 autosomal pairing sites, 81-85 female pairing sites compared, 95-96 function mechanisms, 89-94 implications, 109-1 10 molecular composition, 89-92 sex chromosome pairing sites, 85-89, 94-95 spermatogenesis, 79-8 1 transcription relationship, 92-94 overview, 77-79 spermiogenesis, 96- 109 chromosomal sterility, 100-103 implications, 1 10- 1 1 1 meiotic drive, 96-100 metaphase mitotic model, 106-109 pairing site saturation, 103- 105 X-inactivation, 105-106
41 0 Drosophilu (cant.) meiosis regulation studies cell cycle machinery regulation, 3 1 1 314 candidate regulators, 312-3 14 DrncdcZ mutant activity control, 31 1 rwinr mutant activity control, 31 I , 325326 cytokinesis, 3 19-325 contractile ring assembly, 321 germ line, 322-324 differentiation coordination, 3 14-3 I5 meiosis entry, 309-31 1 overview, 301-309 male meiotic mutant identification, 309 morphology, 304-309 spermatogenesis, 302-304 second meiotic division regulation, 3 15317 spindle formation, 3 17-3 19 nonexchange chromosome distributive system, 284-286
E Electron microscopy mammalian protein meiotic function analysis, 204, 207-21 I synaptonemal complex structure analysis, 242-245 Evolution, mammalian germ line recombination evidence, 13
F Fertility, human male nondisjunction, 397400 Fetal development, mammalian female meiosis regulation, 361 -362 Fluorescence in siru hybridization DNA chromatin loop content analysis, 253256 human male meiotic nondisjunction analysis. 388-393, 401 Follicles age effects, 367-368 female meiosis regulation, 364-365 Follicle-stimulating hormone, mammalian female meiosis regulation, 366-367
Index G Gametogenesis mammalian germ line recombination study problems, 3-7 experiment size, 6-7 meiotic product recovery, 3-4 meiotic gene expression, 179 prophase role, 336-339, 350-35 I Gz phase mitotic phase transition regulation, 339-350 oogenesis, 339-345 competence initiating factors, 310-342 metaphase I arrest, 342-343, 351 metaphase I1 arrest, 343-345 spermatogenesis, 346-350 metaphase-anaphase transition, 350 regulating proteins, 346-348 in vitro studies, 348-350 Gene conversion, mammalian germ line recombination, 12-18 evolutionary evidence, 13 gene conversion measurement strategies, IS-I8 major histocompatibility complex, 13- 14 Gene expression, mammalian meiosis expressed genes, 148- 178 CDC2 protein, 163- 164 cell cycle regulators, 162-165 cyclin, 163-164 cytoskeletal proteins, 174- I75 DNA repair proteins, 152-155, 271-222 energy metabolism enzymes, 172- 174 growth factors, 166- 167 heat-shock 70-2 protein, 163-164 histones, 149- 15 I intercellular communication regulators,
165-168 kinases, 169- 170 lamins, 149 neuropeptides, 167 nuclear structural proteins, 149- I52 phosphodiesterases, I70 promoter-binding factors, 157- 160 proteases, 175-177 receptor proteins, 168 regulatory proteins, 170-172 RNA processing proteins, 160- I62 signal transduction components, 169- 172 synaptonemal complex components, 15 1 152
41 1
Index transcriptional machinery, I60 transcription factors, 155- 157 tumor-suppressor proteins, 164- I65 overview, 142- 146, 178- 18 1 RNA synthesis, 147-148 Germ cells Drosophilu male meiosis regulation, cytokinesis, 322-324 human male chromosome nondisjunction, 387-393 mammalian female meiosis quality control checkpoint, 362-363 mammalian recombination, 1-26 crossing over, 8- 12 physical ver.sus genetic distance\, 1011
recombination hotspots, 1 1 -12 sex differences, 9-10 disease, 18-22 gametogenesis study problems, 3-7 experiment size, 6-7 meiotic product recovery, 3-4 gene conversion, 12- I8 evolutionary evidence, 13 gene conversion measurement strategies, 15- 18 major histocompatibility complex, I 3 14 genetic control, 22-26 early exchange genes, 22-23 early synapsis genes, 23-24 late exchange genes, 24-26 overview, 2-3 Glutamic acid decarboxylase, meiotic expression, 178 Glutathione S-transferase, meiotic expres\ion, 177 Gonadotropin, mammalian female meiosis regulation, 366-367 Growth factors, meiotic expression, 166- I67
H Heat-shock 70-2 protein, meiotic exprcsslon, 163-164 Histones. meiotic expression, 149-1 5 I Holliday junction hypervariable minisatellite DNA site resolution, 60-65 aynaptonemal complex recombination \ite, 259
Human genetics female meiosis regulation, 359-376 checkpoint control, 372-374 chromosome role, 368-370, 374-375 competence, 365-366 fetal development role, 36 1-362 follicle growth period, 364-368 future research directions, 375-376 initiation, 361 -362 meiosis resumption, 364-367 meiotic errors, 360-361 metaphase-anaphase transition, 370-374 inetaphase I1 arrest, 374-375 oocyte growth period, 365-366, 368 overview, 359-360 pachytene quality control mechanisms, 362-364 periovulatory hormonal stimuli, 366-367 spindle formation, 368-372 male nondisjunction, 383-402 etiology, 393-400 aberrant genetic recombination, 396397 age relationship, 394-396 aneuploidy, 397-400 environmental components, 400 infertility, 397-400 future research directions, 400-402 overview, 383-384 study methodology, 384-393 aneuploidy, 384-393 male germ cells, 387-393 trisomic fetuses. 384-386
I Inmunofluorescence microscopy sister-cohesion protein analysis, 282-283 synaptonemal complex structure analysis, 245-247 Immunolocalization, mammalian meiotic protein function analysis, 201 -232 candidate protein selection, 216-228 biological activity involvement, 2 17-223 cell cycle progression, 223 family connections, 223-225 knockout comparisons, 225-227 meiotic involvement, 216-217 mismatch repair proteins, 221 -222 mitotic proteins, 223 polymerases, 222-223
41 2 Immunolocalization (cont.) Rod51 protein, 217-219 Rpu protein, 219-221 chromosome aberration analysis, 228-230 marker antibodies, 21 1-214 meiotic process, 203-207 observation methods current methods, 208-2 1 I historical perspectives, 207-208 overview, 201-203, 230-232 spatial resolution, 214-216 temporal resolution, 2 14-2 I6 yeast comparison, 23 I Infertility, human male nondisjunction. 397400 Intercellular communication regulators, meiotic expression, 165-168 growth factors, 166- I67 neuropeptides, 167 receptors, 168 K Kinases, meiotic expression, 169- 170 Kinetochore, chromosome segregation role, 287-290 duplication, 287-288 functional differentiation, 288-290 overview, 264-269 reorganization, 287 Knockout genes mammalian female meiosis regulation studies, 375-376 mammalian protein meiotic function analysis, 225-227
I, Lamins, meiotic expression, 149 Light microscopy, mammalian protein meiotic function analysis, 207-208 Long terminal repeat element, recombination hotspot identification, 45, 50 Luteinizing hormone, mammalian female meiosis regulation, 366-367
M Major histocompatibility complex mammalian germ line recombination conversion, 13-14 recombination hotspots, 44-46
Index Mammals germ line recombination, 1-26 crossing over, 8- I2 physical versus genetic distances, 10II recombination hotspots, I 1 - I2 sex differences, 9- 10 disease, 18-22 gametogenesis study problems. 3-7 experiment m e , 6-7 meiotic product recovery, 3-4 gene conversion, 12-18 evolutionary evidence, 13 gene conversion measurement strategies, 15- 18 major histocompatibility complex, 1314 genetic control, 22-26 early exchange genes, 22-23 early synapsis genes, 23-24 late exchange genes, 24-26 overview, 2-3 human male nondisjunction, 383-407etiology, 393-400 aberrant genetic recombination, 396397 age relationship, 394-396 aneuploidy, 397-400 environmental components, 400 infertility, 397-400 future research directions, 400-402 overview, 383-384 study methodology. 384-393 aneuploidy, 384-393 male germ cells, 387-393 trisomic fetuses, 384-386 meiosis regulation female genetic control, 359-376 checkpoint control, 372-374 chromosome role, 368-370, 374-375 competence, 340-342, 365-366 fetal development role, 36 1-36? follicle growth period, 364-368 future research directions, 375-376 initiation, 361-362 meiosis resumption, 364-367 meiotic errors, 360-361 metaphase-anaphase transition, 370374 metaphase I1 arrest, 374-375 oocyte growth period, 365-366, 368
Index overview, 359-360 pachytene quality control mechaniains, 362-364 periovulatory hormonal stimuli, 366367 spindle formation, 368-372 sexual dimorphism, 333-352 gametogenesis role, 336-339 metaphase arrest, 342-345, 35 I oogenic G2/M phase transition, 339345 overview, 333-335 prophase gametic function, 350-35 1 prophase onset regulation, 335-336 spermatogenic gap,/mitotic phase transition, 346-350 meiotic gene expression expressed genes. 148-178 CDC2 protein, 163-164 cell cycle regulators, 162-165 cyclin, 163-164 cytoskeletal proteins, 174- 175 DNA repair proteins, 152- 155 energy metabolism enzymes, 172- 174 growth factors, 166-167 heat-shock 70-2 protein, 163- 164 histones, 149-151 intercellular communication regulators, 165-168 kinases, 169-170 Iarnins, 149 neuropeptides, 167 nuclear structural proteins, 140- I52 phosphodiesterases, 170 promoter-binding factors, 157- 160 proteases, 175- 177 receptor proteins, 168 regulatory proteins, 170- 172 RNA processing proteins, 160- 162 signal transduction components, 169- 172 synaptonemal complex components. 151 - 152 transcriptional machinery, 160 transcription factors, 155- I57 tumor-suppressor proteins, 164- 165 overview, 142-146, 178-181 RNA synthesis, 147-148 meiotic protein function analysis, 201 -232 candidate protein selection, 216-228 biological activity involvement, 21 7223
41 3 cell cycle progression, 223 family connections, 223-225 knockout comparisons, 225-221 meiotic involvement, 216-217 mismatch repair proteins, 22 1-222 mitotic proteins, 223 polymerases, 222-223 RudSl protein, 217-219 Rpa protein, 219-221 chromosome aberration analysis, 228-230 marker antibodies, 21 1-214 meiotic process, 203-207 observation methods current methods, 208-21 1 historical perspectives, 207-208 overview, 201 -203, 230-232 spatial resolution, 214-216 temporal resolution, 214-216 yeast comparison, 231 Markers mammalian germ line phenotype, 7 mammalian protein meiotic function analysis, 21 1-214 recombination polarity effects, 40-44 Meiosis chromosome cores, see Chromosomes, prophase cores chromosome nondisjunctions, see Chromosomes, nondisjunction chromosome pairing, see Chromosomes, pairing chromosome segregation, see Chromosomes, bivalent segregation DNA repair, see DNA, double-stranded breaks function, 3 17-3 I9 mammalian protein immunolocalization function analysis, 201 -232 candidate protein selection, 2 16-228 biological activity involvement, 2 17223 cell cycle progression, 223 family connections, 223-225 knockout comparisons, 225-227 meiotic involvement, 2 16-21 7 mismatch repair proteins, 22 1-222 mitotic proteins, 223 polymerases, 222-223 Rad51 protein, 217-219 Rpa protein, 219-221 chromosome aberration analysis. 228-230
41 4 Meiosis (conr.) marker antibodies, 2 1 1-2 14 meiotic process, 203-207 observation methods current methods, 208-21 I historical perspectives, 207-208 overview, 201 -203, 230-232 spatial resolution, 214-216 temporal resolution, 2 14-2 16 yeast comparison, 23 I meiotic drive, 96- I09 chromosomal sterility, 100-109 metaphase mitotic model, 106-109 pairing site saturation, 103- 105 X-autosome translocations, 100- 10 I X-inactivation, 105- I06 Y-autosome translocations, 100- 101 y+ Yrnul+ chromosome, 102 distorted sperm recovery ratios, 96-98 sex chromosome rearrangements, 96-98 spermatid elimination effects, 98 sperm dysfunction, 98 target specificity, 98-99 recombination, see Recombination regulation cell cycle machinery regulation, 31 1-314 candidate regulators, 3 12-3 14 Dmcdc2 mutant activity control, 3 I I mine mutant activity control, 3 I I , 325326 cytokinesis, 319-325 contractile ring assembly, 321 Drosophila germ line, 322-324 yeast budding, 322-324 differentiation coordination, 3 14-315 mammalian female genetic control, 359376 checkpoint control, 372-374 chromosome role, 368-370, 374-375 competence, 340-342, 365-366 fetal development role, 36 1-362 follicle growth period, 364-368 future research directions, 375-376 initiation, 361-362 meiosis resumption, 364-367 meiotic errors, 360-361 metaphase-anaphase transition, 370-374 metaphase I1 arrest, 374-375 oocyte growth period, 365-366, 368 overview, 359-360 pachytene quality control mechanisms, 362-364
Index periovulatory hormonal stimuli, 366367 spindle formation, 368-372 meiosis entry, 309-3 1 1 overview, 301-309 male meiotic inutant identification, 309 morphology, 304-309 spermatogenesis, 302-304 second meiotic division regulation, 3 15317 sexual dimorphism, 333-352 gametogenesis role, 336-339 metaphase arrest, 342-345, 35 1 oogenic gap,/mitotic phase transition, 339-345 overview, 333-335 prophase gametic function, 350-35 1 prophase onset regulation, 335-336 spermatogenic gap,/mitotic phase transition, 346-350 spindle formation, 317-319, 368-372 Metabolism, energy metabolism enzymes, meiotic expression, 172- I74 Metaphase chromosome segregation achiasmatic division, homolog attachment, 283-286 chiasmatd cohesion factor catenation, 280--281 crossover correlation, 269-272 crossover failure, 273-274 crossover position, 272-273 disjunction, 269-274 homolog attachment points, 269 metaphase-anaphase transition role, 370-37 I sister chromatid cohesion, 277-283 terminal binding, 274-278 orientation mechanisms, 266-269 bipolar orientation recognition, 267269 bivalent structure, 266-267 dyad structure, 266-267 reorientation, 267-269 overview, 263-265, 292-293 sister chromatid attachment maintenance, 290-293 centromeric region cohesion mechanisms, 29 1-293 equational nondisjunction, 290-291 meiosis I1 cohesion disruption mutations, 292
41 5
Index proximal exchange, 290-291 sister kinetochore function, 287-290 duplication, 287-288 functional differentiation, 288-290 reorganization, 287 spindle assembly-chromosome-mediated checkpoint, 371-372 G,/M phase transition regulation oogenesis, 342-345 spermatogenesis, 350 metaphase I1 arrest, 343-345, 374-375 mitotic chromosome sterility model, 106I09 Microscopy, see specific types Minisatellites, hypervariable DNA Holliday junction site resolution, 60-65 recombination hotspot assay, 48-50 Mitosis Dro.sophila chromosome pairing studies metaphase mitotic model, 106- 109 G,/M phase transition regulation, 339-350 oogenesis, 339-345 competence initiating factors, 340342 inetaphase I arrest, 342-343, 35 1 metaphase I1 arrest, 343-345 spermatogenesis, 346-350 inetaphase-anaphase transition, 350 regulating proteins, 346-348 in vitro studies, 348-350 mammalian germ line expansion, 4-5 mammalian protein immunolocaliration function analysis candidate mitotic protein selection, 223 Mouse, female meiosis regulation studies knockout genes, 375-376 pachytene quality control checkpoint, 363364 N Neuropeptides, meiotic expression, 167 Nondisjunction, see also Chromosomes, bivalent segregation human males, 383-402 etiology, 393-400 aberrant genetic recombination, 396397 age relationship, 394-396 aneuploidy, 397-400 environmental components, 400 infertility, 397-400
future research directions, 400-402 overview, 383-384 study methodology, 384-393 aneuploidy, 384-393 male germ cells, 387-393 trisomic fetuses, 384-386 sister chromatid attachment maintenance, 290-29 1 Nuclear proteins, meiotic expression, 149- 152 Nucleolus, chromosome pairing relationship, 94
0 Oogenesis G,/M phase transition regulation, 339-345 competence initiating factors, 340-342 metaphase I arrest, 342-343, 351 metaphase I1 arrest, 343-345 mammalian female meiosis regulation, 365366, 368 mammalian germ line mitotic expansion, 56 Ornithine decarboxylase, meiotic expression, 177
P Pachytene, mammalian female meiosis quality control checkpoint, 362-364 germ cell loss, 362-363 mouse mutant studies, 363-364 yeast mutant studies, 363 Pairing, see Chromosomes, pairing Phosphodiesterases, meiotic expression, 170 Phosphoribosylpyrophosphate synthetase, meiotic expression, 177 Plasmids, recombination hotspot assays, 4650 hypervariable minisatellite DNA, 48-50 retroviral long terminal repeat element, 50 Z-DNA, 46-48 Polymerases, mammalian protein meiotic function analysis, 222-223 Promoter-binding factors, meiotic expression, 157-160 Prophase chromosome cores chromatin loop alignment, 256-257 associated DNA sequences, 250-253 attachments, 247-250
41 6 Prophase (cont.) development time course, 256 DNA content, 253-256 overview, 24 1-242 synaptonemal complex electron microscopic structure analysis, 242-245 immunocytological structure analysis, 245-247 recombination site, 257-259 gametogenesis regulation, 336-339, 350-35 1 meiotic chromosome segregation heterolog association, 285-286 nonexchange chromosome homolog association, 285 onset regulation, 335-336 Proteases, meiotic expression, 175- 177 Protein, see specific types Protein-DNA binding, recombination hotspot activation, 52-56
R RudSl protein mammalian protein meiotic function analysis, 217-219 synaptonemal complex recombination site, 258 Receptor proteins, meiotic expression, 168 Recombination hotspots chromatin DNA accessibility, 5 1-52 chromosome dynamics, 39-40 cis-trans control mechanisms, 56 crossing over, 1 1- 12 double-stranded DNA breaks, 50-5 I genetic identification, 40-50 major histocompatibility complex role, 44-46 marker effects, 40-44 physical versus genetic maps, 40 plasmid recombination assays, 46-50 polarity, 40-44 overview, 38 protein-DNA binding role, 52-56 recombination initiator models, 57-65 biochemical-genetic convergence, 57-60 Holliday junction resolution, 60-65 human male nondisjunction, 396-397 mammalian germ line, 1-26 crossing over, 8- 12 physical versus genetic distances, 10- 1 1
Index recombination hotspots, 1 I - 12 sex differences, 9- 10 disease, 18-22 gametogenesis study problems, 3-7 experiment tire, 6-7 meiotic product recovery, 3-4 gene conversion, 12- 18 evolutionary evidence, 13 gene conversion measurement strategies, 15-18 major histocompatibility complex. 1314 genetic control, 22-26 early exchange genes, 22-23 early synapsis genes, 23-24 late exchange genes. 24-26 overview, 2-3 synaptonemal complex-associated late nodules, 257-259 Regulatory proteins, meiotic expression, 170172 Repair proteins DNA double-stranded break repair gene function, 117-118, 126-128, 132-134 meiotic expression, 152- 155 mismatch repair genes, 22 1-222 Repeat elements, recombination hotspot identification, 45, 50 Resting phase, see G , phase Retroviral long terminal repeat element, recombination hotspot identification, 45, 50 RNA meiotic synthesis, 147-148 processing proteins, meiotic expression, 160- I62 Rpa protein, mamnialian protein meiotic function analysis, 21 9-221
S Succhurornyces cerevisiae cytokinesis, 322-324 DNA double-stranded break repair gene function, 117-118, 126-128, 132-134 female meiosis regulation, pachytene quality control checkpoint, 363 meiotic chromosome segregation, 289-290 meiotic protein function analysis, 21 4-2 16, 230-232 Segregation, see Chromosomes, bivalent segregation
Index Signal transduction, meiotic gene expression, 169-172 kinases, 169- I70 phosphodiesterases, 170 regulatory proteins, 170- 172 Sister chromatids, chromosome segregation attachment maintenance centromeric region cohesion mechanisms, 29 1-293 chiasmata role. 277-293 equational nondisjunction. 290-29 I meiosis 11 cohesion disruption mutation\, 292 proximal exchange, 290-29 1 sister kinetochore function, 287-290 duplication, 287-288 functional differentiation, 288-290 reorganization, 287 Sister-cohesion protein, immunofluorescence microscopy analysis, 282-283 Spermatogenesis Drosophila chromosome pairing studies, 96- I09 chromosomal sterility, 100- 103 implications, 110-1 1 I meiotic drive, 96- I00 metaphase mitotic model, 106- 109 pairing site saturation, 103- 105 X-inactivation, 105- 106 male meiosis regulation study, 302304 human male nondisjunction, 383-402 etiology, 393-400 aberrant genetic recombination, 396 397 age relationship, 394-396 aneuploidy, 397-400 environmental components, 400 infertility, 397-400 future research directions, 400-402 overview, 383-384 study methodology, 384-393 aneuploidy, 384-393 male germ cells, 387-393 trisomic fetuses, 384-386 mammals GJM phase transition regulation, 346350 metaphase-anaphase transition, 350 regulating proteins, 346-348 in v i m studies, 348-350 germ line mitotic expansion, 4-5
41 7 meiotic gene expression CDC2 protein, 163- 164 cell cycle regulators, 162- 165 cyclin, 163-164 cytoskeletal proteins, 174-175 DNA repair proteins, 152-155, 221222 energy metabolism enzymes, 172-174 growth factors, 166- 167 heat-shock 70-2 protein, 163- 164 histones, 149-151 intercellular communication regulators, 165- 168 kinases, 169-170 lamins, 149 neuropeptides, 167 nuclear structural proteins, 149- 152 overview, 142-146, 178-181 phosphodiesterases, I70 promoter-binding factors, 157- I60 proteases, 175- I77 receptor proteins, 168 regulatory proteins, 170- 172 RNA processing proteins, 160- 162 RNA synthesis, 147-148 signal transduction components, 169I72 synaptonemal complex components, 151-152 transcriptional machinery, 160 transcription factors, 155- I57 tumor-suppressor proteins, 164-1 65 Spindle assembly meiotic cell division function, 317-319 meiotic chromosome segregation crowded spindle model, 286 mammalian female meiosis regulation, 368-372 Synaptonemal complex chromosome prophase cores electron microscopic structure analysis, 242-245 immunocytological structure analysis, 245-247 recombination site, 257-259 chromosome segregation achiasmatic meiotic division, 283-284 chiasmatic homolog attachment points, 269 mammalian protein meiotic function analysis, 220-22 I meiotic gene expression, 15 I - I52
41 8 T Transcription chromosome pairing relationship, 92-94 regulation, meiotic gene expression, 155I60 promoter-binding factors, 157- 160 transcriptional machinery components, 160 transcription factors, 155- 157 Transcription factors, meiotic expression, 155157 Transduction, see Signal transduction Translocation, chromosomal sterility role, 100101 Trisomy, see Chromosomes, nondisjunction
Index Tumor-suppressor proteins, meiotic expression, 164- 165 Twine gene, Drosophila meiosis regulation, 31 I . 325-326
W Western blot, meiotic protein function analysis, 216, 224
Y y+ Ymd' chromosome, synthetic chromosomal sterility, 102
Contents of Previous Volumes
1 The Role of SRY in Cellular Events Underlying Mammalian Sex Determination Blanche Capel
2 Molecular Mechanisms of Gamete Recognition i n Sea Urchin Fertilization K a y Ohlendieck nnd W i I l i ~ m / Lennarz
3 Fertilization and Development in Humans Alan Trounson and Ariff Bongso
4 Determination of Xenopus Cell Lineage by Maternal Factors and Cell Interactions
Sally Moody, Daniel V Bailer Alexandra M Hainski, and Sen Huang
5 Mechanisms of Programmed Cell Death in Caenorhabditis elegans and Vertebrates Masayuki Miura and lunyrng
Minri
6 Mechanisms of Wound Healing in the Embryo and Fetus Paul Martin
7 Biphasic Intestinal Development in Amphibians: Embryogenesis and Remodeling during Metamorphosis Yun Bo Shi and Atsuko Ishizuyc+Oka
1 MAP Kinases in Mitogenesis and Development
lames E. Ferrell, Jr. 2 The Role of the Epididymis in the Protection of Spermatozoa Barry T. Hinton, Michael A. Palladino, Daniel Rudolph, Zi lian Lnn, and Jacquelyn C. Labus 41 9
420
Contents of Preview Volumes
3 Sperm Competition: Evolution and Mechanisms T. R. Birkhead
4 The Cellular Basis of Sea Urchin Gastrulation
leff Hardin
5 Embryonic Stem Cells and in Vifro Muscle Development Robert K. Baker and Gary E. Lyons
6 The Neuronal Centrosome as a Generator of Microtubules for the Axon Peter W. Baas
1 SRY and Mammalian Sex Determination Andy Greenfield and Peter Koopman
2 Transforming Sperm Nuclei into Male Pronuclei in Vivo and in Vifro D. Poccia and P Collas
3 Paternal Investment and lntracellular Sperm-Egg Interactions during and Following Fertilization i n Drosophila Timothy L. Karr
4 Ion Channels: Key Elements in Gamete Signaling Albert0 Darszon, Arturo Lievano, and Carmen Beltran
5 Molecular Embryology of Skeletal Myogenesis Judith M . Venuti and Peter Cserjesi
6 Developmental Programs in Bacteria Richard C. Roberts, Christian D. Mohr, and Lucy Shapiro
7 Gametes and Fertilization in Flowering Plants Darlene Southworth
1 l i f e and Death Decisions Influenced by Retinoids Melissa B. Rogers
2 Developmental Modulation of the Nuclear Envelope Jim Liu, lacyueline M . Lopez, and Marinna F. Wolfner
421
Content5 of Previous Volumes
3 The €GFR Gene Family i n Embryonic Cell Activities Eileen D Adarnson and Lynn M Wiley
4 The Development and Evolution of Polyembryonic Insects Michael R Strand and M ~ o r l r ~Crht g
5 p-Catenin I s a Target for Extracellular Signals Controlling Cadherin Function: The Neurocan-GalNAcPTase Connection / x k Lilien, Stanley Hoffnian, Carol Eisenberg, and Janne Balsnmo
6 Neural Induction in Amphibians Horst Grunz
7 Paradigms t o Study Signal Transduction Pathways in Drosophila Lee Engstrom, Elizabeth N o / / ,
Cellular and Molecular Procedures in Developmental Biology Guest edited by Flora de Pablo, Alberto Ferrus, and Claudio D. Stern
1 The Avian Embryo as a Model in Developmental Studies Elisabeth Dupin, C~therineTiller, ,
i d
Nicole M. Le Douarin
2 Inhibition of Gene Expression by Antisense Oligonucleotides in Chick Embryos in Vitro and in Vivo Aixa b! Morales and Flor
3 Lineage Analysis Using Retroviral Vectors Constance L . Cepko, Eliz;lbeth Rydcr, Chistopher Austin, Jeffrey Golden, and %awn Fields-Berry
4 Use of Dominant Negative Constructs t o Modulate Gene Expression Giorgio Lagna and Ali Hernr11,1ti-Briv,ii1lou
5 The Use of Embryonic Stem Cells for the Genetic Manipulation of the Mouse Miguel Torres
6 Organoculture of Otic Vesicle and Ganglion Jiidn J.
Garrido, Thomas .Sdiin~iiidng,loan Represa, and Fermndo Giraldez
422
Contents of Previous Volumes
7 Organoculture of the Chick Embryonic Neuroretina Enrique 1. de la Rosa, Begona Diaz, and Flora de Pablo
8 Embryonic Explant and Slice Preparations for Studies of Cell Migration and Axon Guidance Catherine E. Krull and Paul M . Kules'i
9 Culture of Avian Sympathetic Neurons Alexander v. Holst and Hermann Roher
10 Analysis of Gene Expression in Cultured Primary Neurons Ming-Ji Fann and Paul H. Patterson
1 1 Selective Aggregation Assays for Embryonic Brain Cell and Cell Lines Shinichi Nakagawa, Hiroaki Matsunami, and Masatoshi Takeichi
12 Flow Cytometric Analysis of Whole Organs and Embryos lose Serna, Belen Pimentel, and Enrique J. de la Rosa
13 Detection of Multiple Gene Products Simultaneously by in Situ Hybridization and lmmunohistochemistry in Whole Mounts of Avian Embryos Claudio 0. Stern
14 Differential Cloning from Single Cell cDNA Libraries Catherine Dulac
15 Methods in Drosophila Cell Cycle Biology Fabian Feiguin, Salud Llamazares, and Cayetano Gonznlez
16 Single CNS Neurons in Culture Juan Lerma, Miguel Morales, and M x i n de 10s Angeles Vicente
17 Patch-Clamp Recordings from Drosophila Presynaptic Terminals Manuel Martinez-Padron and Alberto Ferrus
