Meiosis & Gametogenesis (Current Topics in Developmental Biology)

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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Copyright 0 1998 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1998 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0070-2 153/98 $25.00

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525 B Street, Suite 1900, San Diego, California 92101-4495. USA http://www.apnet.com Academic Press Limited 24-28 Oval Road, London NW 1 7DX, U K http://www.hbuk.co.uk/ap/ International Standard Book Number: 0-12-153 137-6 PRINTED IN THE UNITED STATES OF AMERICA 97 98 9 9 0 0 01 0 2 B B 9 8 7 6

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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

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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

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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

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111. Chromosome Pairing and Spermiogenesis

IV. Summary and Implications 11 1 References

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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

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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

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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.

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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

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Contents

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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

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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

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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

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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

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Contributors

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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

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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-

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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

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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

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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,

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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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1. Recombination in the Mammalian Germ Line

27

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Renwick. J . H., and Schulze. J . ( I 0 6 5 1 hlnle and lemale recomhination fraction\ for the nail-patella: ABO linkngc i n man. Ariri H U I J IG'cwc,/. . 28, 379-392. Reynter\. E.. Van Thienen. M.. Meire. F . De Boulle. K., Devries, K., Kcstelijn, P.. and Willcms, P. ( 1995). Gene conversion betuccii red a n d defective green opsin gene i n blue cone iiionochromacy G ~ J I o ~ 29, ~ I ~323-3%. c\ Rheauine. E.. Sanchcr. R., Simard. J.. C'lianp. Y.. Wang, J . . Pang. S., and 1-abi-ic, F. (1904). Molecular basis of congenital adrrnal hypei-pla\ia in two \iblings with cla\sical non\olt-losing 3P-hydrouysteroid dehydrogenasc deliciency. J . Cliri. Grc/ocrir7. Merch. 79, 1012- I01X. Robinson, W. P., and Lalande. M. ( 1995). Sex-\pecific meiotic recombination i n the PmderWilli/Angelnian syndrome imprinted region. HitrJi. Mol. Grrtet. 4, 801 -806. Rockmill. B., and Rocdcr, G . S. ( 190 I ). A meio\is-specilic protein kina5e homolog required for chromo\ome \ynap\is and recoiiihitiiitioti. Gcrrc\ Dais. 5, 2392-2104. Roderick. T. H., Hillyard, A . L., M a l t a i \ . L. J . . and Blake. C S. ( 1996).Recombinational percentages and chromosomal asaigninciits. Irr "Genetic Variants and Strains of the Laboratory Moure" ( M . F Lyon. S. Rastan. ;ind S D. M. Brown. Eds.). Oxford University Presr. Oxford. Roeder. G . S. (1995). Sex and the \iiiplc cell: Meiosis in yeast. Proc. N u / / . A u i t l . Sci. L;SA 92, 10450- 10456. Rouet. P.. Smith. F.. and Jasin. M. ( lW4a). Exprcs\ion of a \itc-specilic endonucleaw stimulate\ homologous recombination in iiiiiiiiiiialian cell\. Proc. Nhrl. Accid. . k i . USA 91, 6064-6068. Rouet, P.. Smith. F., and Jasin, M. (I904h). Introduction of double-strand breaks into the genome of iiioiise cells by expression 01 ii r;ire-cutiing cndonuclea\e. M d . Cell. Biol. 14, 8096-X106. Roue. 1.. B.. Nadeau, J. H., Turner. R., Franhcl. W. N., Letts. V. A,, Eppig. J. T., KO,hl. S., Thurston. S. J., and Birkenmeicr, E H . (1994). Maps froni two interspecific backcro\u DNA panels a \ ailahle as a community gcnctic mapping resource. Mmrrri. Grrtonir 5 , 253-274. Rubnitz. J., and Subraniani, S . ( 1984). The i i i i n i m u m amotint of homology required for homolo goii\ recombination i n maiiimiilixi ccII\. Mol. Cell. B w l . 4, 2253-2258. Rubnit/, J., and Subrainani. S. ( 19x6). Eutrachroiitosomal and chromosomal gene convenion in mamninlian cell\. M d . Call. 8iol. 6. I6OX-I 614. Rus\ell. L.. Banham. J . , Stelmer, K.. .ind Hun\icker. P. (198X). High frequency of mosaic mutants produced by N-ctIiyI-N-nitrosoiire~iexposure o f mouse [ygotes. Pwc. N d . . A c d . S ~ . I USA . 85, 9 167-9 170. Sant'Angelo. D. B., Lafuse. W. P.. ;itid Pas\morc. H. C. (1992). Evidence that nucleotide \eqiience identity is a requirement for meiotic ci-ossing over within the mouse E b recombinational hotspot. G P ~ ~ O M 13, I ~1334C S 1330. Sato. S.,Hotta, Y., and Tahata. S. ( I W i ) . Structural analysis of a recA-like gene in the gcnome of A ~ l h i d ~ / JI /~I ~i I. l~f ~ ~ DNA t l ~ i . Rr.\ 2, Xtl-93. Sato. S.. Kohayashi, T., Hotta, Y., and Tabnta. S. ( 1995). Characterization of a mouse recA-like gene specifically expressed i n te\ti\. DNA K c i . 2, 147-150. Schirncnti, J.. and Duncan, C. (19x4). Ruininant globin gene structures suggest an evolutionary rolc for Nu-type repeats. Nucl~ic.,.\sop/iilrrrnekirro~~n.\ter. IV. Conjunctive mechanism of the XY bivalent. ('hn~rnosorrrrr 86, 309-3 17. Ault, J . G., and Lin, H:P. P. ( 1984). Bivalent behavior i n Dro.sophi/cr mrlonoficrsrer male5 containing the Iri(/).sc4'.wS~ X chromosome. Chr.otrio,sorrui 90, 222-228. lti I. The question of sepal-ate Ault, J . G., and Rieder, C. L. (1994). Meio\i\ i n / ) r ~ . t ~ p h i males. conjunctive mechanisms for the X Y m d atitosoinal bivalents. Chrornosorncr 103, 352-356. Baker, B. S., and Carpenter, A . T. C. ( 1972). Genetic analysis of sex chromosome meiotic nititants in Droo.sophi/umrlatio~osrrr.C;rnetrcs 71, 155-286. Balakireva. M. D., Shevelyov, Y.Y., Nurminsky, I).I., Livak, K . J . , and Gvoidcv. V. A. (1997) Structural organization and diversitication ol Y-linked sequences comprising .Srr(Sre) gene\ i n Dro.sophi/anwlrrnofitr.\ter. Nuclei(, Ac.id.\ Re.\. 14, 373 1-3736. Besmertnaia, S. L. ( 1934). Abstract\ ot cutmiit work of the Institute Narkonizgrav. Riol. Ztrr.. 3, 221 Cenci, G.. Bonaccorsi, S., Pisano, C., Vei-mi. I;, and Gatti, M. ( 1994). Chromatin and microtubule organization during prerneiotic. meiotic and eai-ly pojtmeiotic stages of Drosophiltr r n r / c r n o ~ ~ ~ ~ ~ fer spermatogenesis. J . Cell Sci. 107, 352 1-3534 Church, K., and Lin, H.-P. P. ( 1985). Kinetochorc microtubules and chromo\ome\ movement d t w ing proinetaphase in Drosophilci r ~ i [ , / ~ i ~ i ~ ~ f i [ ispcrinatocytes \if,r\tudied in life and with the clectron microscope. Chrotnosortici 92, 273-787. Church, K., and Lin, H.-P. P. (1988). Drosophila. A model for the study of aneuploidy. / r r "Aneuploidy. Part B: Induction and Test Sy\teins" ( B . K. Vig and A. A . Sandberg, Eds.), pp. 227255. Liss. New York. Coen. E. S.. and Dover, G. A. (1982). Multiple poll initiation scqtiences in rDNA spacer\ of Drosophila nielanogaster. Nrrcleic Acic/.\ Kc,.\ 10, 701 7-7026. Cooper. K. W. ( 1950). Normal sperniatogenesis in Drosophila. I n "Biology of Drovophiltr" ( M Demerec, Ed.), pp. 1-61. Wiley. Ncw Y o r l . Cooper, K. W. ( 1964). Meiotic conjunctive eleiiiciit~not involving chiasmata. Pro(,. Nail. Ac.crt/. S c i . USA 52, 1248-1255. Craymer. L. ( 198 I ). Techniques for inanipulating chi-oiiiosonie rearrangeinents and their application to Drosophiltr rJielrrnognsrrr. I . Pericentric inversions. G e n ~ i i c99, ~ 75-97. Dcrnburg, A. F.. Daily. D.R., Yook, K. J.. Corbin. J. A,. Sedat, J . W., and Sullivan. W. ( 1006). Selective loss of sperm bearing a compound c h r o m o w n e in the Drosophila leinale. Garrc>/it\ 143, 1629-1642.

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Dernburg, A . E, Sedat, J. W.. and Hawley, R. S. (1996). Direct evidence of a role for heterochromatin in meiotic chromosome segregation. Ce// 86, 135- 146. Falk. R. ( 1983). The effect of an unusual chromosome architecture on disjunction and nondisjunction in Drosophila. Getlet. Reh. Curlib. 41, 17-28. Fuller, M. T. ( 1993). Spermatogenesis In "The Development of Drosophila tnelanogosrer" (M. Bate and A. Martiner-Arias. Eds.), Vol. I, pp. 7 I - 147. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Gatti, M., and Pimpinelli, S. (1983). Cytological and genetic analysis of the Y chromosome of Drosoyihilo trir/otio~o.ster:Chrotriosorncr 88, 349-373. Gershenson, S. (1933). Studiea on the genetically inert region of the X chromosome of Drosophila. I. Behavior of an X chromosome deficient for a part of the inert region. J . Genrr. 28, 297-312. Gethmann, R. C. ( 1976). Meiosis in male Dmsophrltr r,ic,/cinoRr/.vrrr.11. Nonrandom segrtgation of compound second chromosomes. Gerieric,s 83, 743-75 I . Grell, R. F. (1976). Distributive pairing fn "Genetics and Biology of Dro.sop/ii/a" (E. Novitski and M. Ashburner, Eds.). Pt. la, pp. 335-486. Academic Press, New York. Grimaldi. G., and Di Nocera, P. P. ( 1988). Multiple repeated units iii Drosophila melanogaster ribosomal DNA spacer stimulate rRNA precursor transcription. Pro(,. Nor/. A u l d . Sci. USA 85,5502-5506. Grimaldi, G., Fiorento. P.. and Di Nocera, P. P. (1990). Spacer promoters are orientationdependent activators of pre-rRNA transcription in Drosophila melanogaster, Mol. Crll. B i d . 10,4667-4677. Handel, M. A. (1987). Genetic control of \permatogenesis i n mice. frr "Results and Prohlems in Cell Differentiation. 1 5. Spermatogenesis. Genetic Aspect\" (W. Hennig, Ed.), pp. 1-62, Springer-Verlag. Berlin. Hardy, R . W., Lindslcy, D. L.. Livak. K . J., Leuis. B., Silversten. A. L., Joslyn. G . L., Edwards. J., and Bonaccorsi, S. ( 1983). Cytogenetic analysis of a segment of the Y chroinosonie of Drorophila me/~iriog~is~er. Gewrirs 107, 59 1-610. Hardy. R . W., Tokuyasu. K. T., and Lind\ley, D. L. (1981). Analysis of spermatogenesis in Dro.tophi/o nir/(rriogcr.\ferbearing deletion5 for Y-chromo\onic fcrtility genes. C/iroi?io.rovicr83, 593-617. Hawley, R. S. ( 1988). Exchange and chromosomal segregation in eucaryotes. Iri "Genetic Reconibination" (R. Kucherlapati and G. R . Smith, Edr.), pp. 497-527. Ainerican Society i o r Mici-obiology. Washington, D.C. Hawley, R. S., lrick, H., Zitron, A . E.. Haddox, D. A,, Lohe. A,, New, C., Whitley. M. D.. Arbel. T., Jang, J.. McKini, K.. and Childs. G. ( 1993). There are two mechanisms of achiamate scgregation in Dro.sophi/n females. oiic of which requires heterochromatic homology. /h*, Gcrirr. 13,440-467. Heyting, C . (1996). Synaptoneinal complex: Structure and function. ( ' I w . Opiri. C(,// Era/. 8, 389-396. Hilliker, A. J.. and Appel5, R. (1982). Pleiotropic effects associated with the deletion o f heterochromatin surrounding rDNA on the X chromosome of Dro.sophi/d. Chromo.sorrirr 86, 469-490. Hilliker, A. J., Holm, D. G., and Appela, R. ( 1982). The relationship between heterochrtrinatic homology and meiotic segregation of compound second autosonies during \permatogencsis i n L)ro.sophi/cr rrie/ririoga.sr~~.r.. Getzer. Res. C m i / i . 39, I 57- 168. Holm, D. G., and Chovnick, A. ( 1975). Compound autosomes i n Dro~ophi/tr~ r J e / c ~ ~ i ~ ~The R[~,~r~,~: meiotic behavior of compound thirds. Grric,ticc 81, 293-31 1 , Jones. G. H. (1987). Chiasniata I n "Genetic Recombination" (R. Kucherlapati and G. R. Smith, Eds.). pp. 2 13-238. American Society lor Microbiology. WashingLon, D.C. Karpen, G. H.. Le, M-H., and Lc, H. (1996). Centric heterochromatiii and the efficiency of achiasniate disjunction i n Drorophiltr female niciosis. S[,irric.r 273. I 18- 122.

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Karpen, G. H.. Schaeffer, J. E., and Laird. C . D. ( 1988). A Drosophila rRNA gene located in euchromatin is active in transcription and nucleolus formation. Genes Dei,. 2, 1745- 1763. nie/ufzogu,sKennison. J . A. ( 1983). Analysis of Y-liiihcd tiiutatioiib to male sterility in Dro.so~phil~r trr. Gmetic.c 103, 2 19-234. Kohorn. B. D.. and Rae, P. M. M. ( 1 9 8 2 ~ 1 )Accurate transcription of truncated ribosomal DNA templates in a Drosophila cell-free \yslc.in. Proc.. Ntrrl. A u l d . Sci. USA 79, 1501-1505. Kohorn, B. D.. and Rae, P. M. M. (1982h). Non-transcribed spacer sequences promote in vitro tran\criptioii of Drosophila ribosomal KNA. Nircleic Acids Res. 10, 6879-6886. Kotani, H., and Kmiec. E. B. (1994). A rolc for RNA synthesis in homologous pairing ewnts. M o l . Ce//.B i d . 14, 6007-6 106. Kremer. H., Hennig, W., and Kijkhof, R . ( 1986). Chromatin organization in the male germ line of Drosophiln hydei. Chrornosomcr 94, 147- I6 I , Le. M.-H.. Duricka. D., and Karpen, G. H. ( 1995). Islands of complex DNA are widespread in Drosophila centric hetcrochrornatin. Grweticx 141, 283-303. Li, X.. and Nicklas, R. B. (1995).Mitotic lorces control a cell-cycle checkpoint. Nuture 373, 630-637. Lichten. M.. and Goldinan. A. S . H. II Y J 51. Meiotic recombination hotspots. Annu. Rei,. G o i e r . 29,423-443. Lifschytz. E., and Lindsley. D. L. (1972). ‘The role of X-chromosome inactivation during \permatogenesi\. Proc,. N d . Accitl. Sci U.Y.4 69, 182- 186. Lifton, R. P.. Goldberg. M. L., Karp. R . W.. and Hogness, D. S. (1977). The organization of the histone genes in Dro.soplii/o nie/moxo\tc,r: Functional and evolutionary implications. Cold Spriiig Horlmr Synzp. Qii(iiit. B i d 42, 1047- I 0 5 I Lin, H.-P. P.. Ault, J . G., Kimhle, M.. a i d Chui-ch. K. (1984). Meio\i\ in Drosophilu ineltiiio,qci.~rer. V. Univalent behavior in /ni/)cc.’i \P~*/R\Y male\. Cun. J . Genet. Cyfol. 26, 445-458. Lindsley, D. L., and Grell. E. H. (I969 ), Spcriniogene\i\ without chromosomes in Drorophilo i } i r / ~ i ~ i [ ~ , qGmctics ~ ~ , s / [ ~ rSuppl. . 61, 60-77 Lindsley, D. L., Pearson, C. A., Rohop. S A,, Jane\. M., and Stem, D. (1979). Genotypic fcacure\ causing sterility of males carrq iiiy B hohbed-deficient X chromosome: Translocations i i i volvirig chromosome 2 or 3. Gerrrri(.\ 91, S69-S70. Lindsley, D. L., and Sandler. L. (1958). ‘The meiotic behavior 01’ grossly deleted X chromosomes in Dro.sophiltr rne/miogtr.s/er.Geneti(.\ 43, 547-563. L.iiid\lcy, D. I.., and Tokuyacu, K. T. ( I O X O ) . Spci-inatogenesis. In “The Genetics and Biology of Urmophilri” ( M . Ashburner and T. K. t’ Wright. Eds.), pt. 2d. pp, 225-294. Academic Pres\. London. Lindslcy, D. L.. and Zimm. G. 11992). “Thc Genoine of Drotolihilo iiir/trnog~i.\trr.”Academic Press, New York. Livak. K. J. (1984). Organization and triappping 0 1 a sequence o n the Drosophiltr iiie/mio,qc/\rer X and Y chromosomes that is transct-ibed during \periiiatogene\is. Gerirtics 107, 61 1-634. Livak, K. J. ( 1990). Detailed structure on the Dro\ophi/o nrchiogtrsrer Stellarc, genes and their transcript\. Genetics 124, 303-3 16. Lohe. A . R., Hilliker. A. J . , and Rohcrts, C A . (1993).Mapping simple repeated DNA sequences in heterochroniatin of Drosophila n i c l t i r i q c i . Gericticc. 134, I 149- I 174. Lohe, A. R., and Roberts. P. A. (1990). Ail utiusual Y chromosome of Uro.soplri/o . s i n i i r / ~ i i icarry~ ing amplified rDNA spacer without rRNA gene\. Griietics 125, 399-406. Lnidl, J . ( 1990). The initiatioii of meiotic chi-omosome pairing: The cytological view. Grriorne 33, 759-778. Lyttle. T. W. ( 1993). Cheaters sometime\ pi-o\per: Di\tortion o f inendelian segregation hy iiieiotic drive. Trrrids Genet. 9, 205-2 10. Mathuo. Y., and Y a n i a A i , T. (1989). tRNA derived insertion element i n histone gene repeating Nirc I t w Acid\ Re,. 17, 225-238. unit of Drorophiln ific~/nrio,~trster.

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McKec, B. ( 1987). X-4 translocations and meiotic drivc in Drosophilri rnrltrrwycrster males: Role of sex chromosome pairing. G'erretic..~116, 409-4 13. McKee, B., and Lindsley, D. L. ( 1987). Inseparability of X-heterochromatic functions responsible for X:Y pairing, meiotic drive. and inale fertility in Dm.sophila rrirlnriogtrster males. G'enrric~.\ 116, 399-407. McKee. B. D. ( 1984). Sex chroniosome nieiotic drive in Drosophila melanogaster males. Gei~rtic~.s 106, 403-422. McKee, B. D. ( 199 I ) . X-Y pairing, meiotic drive, and ribosomal DNA in Drosophila melanogaster males. An?. Not. 137, 332-339. McKce, B. D. (1996). The license to pair: Identitication of meiotic pairing sites in Drosophiln. Chror,lo.conztf105, 135-141. McKee. B. D.. Habera. L., and Vrana, J. A. ( 1992). Evidence that intergenic spacer repeats of Drosophilci melanoKcisfer rRNA genes function as X-Y pairing site\ in male meiosis. and a general model of achiasinatic pairing. G'cvretic.s 132, 529-544. McKee, B. D., and Handel, M. A. (1993). Sex chromosomes, recombination and chromatin conformation. Chron~o.somrr102, 7 1-80, McKee, B. D.. and Karpen, G. H. ( 1990). Drowphila ribosomal RNA genes function as an X-Y meiotic pairing site during male iiieiohis. Cell 61, 61 -72. McKee, B. D., Lum\den, S. E.. and Das, S. ( 1993). The dihtribution of inale meiotic pairing sites on chromosome 2 of Dro.sophiltr rnelmog(ister: Meiotic pairing and segregation of 2-Y transposition\. Chrorriosorrw 102, 180- 194. McKee, B. D., Wilhelm, K., Mcrrill, C., and Rcn. X.-J. ( 1997). Male \terility and meiotic drive aswciated with \ex chromosome rcarrangernents in Drosophila: Role of X-Y pairing. (;enetics, in pres\. Merrill, C. J.. Chakravarti. D., Habera, L., Das, S., Elsenhour, L., and McKee, B. D. ( 1992). Promoter-containing ribosomal DNA fragments function as X-Y meiotic pairing sites i n I). riic4tiriogcirrrr males. /ki>.G'cvwt. 13, 46X-484. Mikloa, G. L. G. (1974). Sex chromosome pairing and male fertility. C ' \ R J ~ W < V . Crll Gerirr. 13, SSX-577. Miller, J. R., Hayward, D. C.. and Glover. D. M. (1983). Transcription of the "non-tramcribcd spacer" of Drosophilo ritelrrriogci\tet- rDNA. Nirr.lric. Acids Kes. 11, I I 19. Murtii, V. L., and Rae, P. M. M. ( 1985). In v i v o transcription of rDNA spacers in Drosophila. Nid 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 . 29, 172- 179. Andersen, B., Pearse, R. V. I., Schlegel. P. N.. Chicon, Z., Shonemann, M . D., Bardin. C. W., and Rosenfeld. M. G. (1993). Sperm 1 : A POU-domain gcnc transiently expressed immediately hefore meiosis I in the male germ cell. Proc. Not/. Acrid. Sc.i. USA 90, 11084-1 1088. Ashley, T., Plug, A. W., Xu, J., Solari, A. J.. Kcddy. G., Goluh, E. I., and Ward, D. C. ( 1995). Dynamic changes in Rad5I distribution o n chromatin during mciosis in male and female vcrtebrates. Chrommmci 104, 19-28. Avallet, O., Vigier, M., Leduque, P., Dubois, P. M.. and Saez, J. ( 1994). Expression and rcgulation of transforming growth factor-p 1 mcshenger ribonuclei id and protein in cultured porcine Leydig and Sertoli cells. Endocririology 134, 2079-2087. Ayer-LeLievre, C., Olson, L., Ebendal, T., Halbook, F.. and P e r s o n , H. (1988). Nerve growth factor mRNA and protein in the testis and cpididymis of mouse and rat. Proc. Natl. Accid. %i. USA 85,2628-2632. Baba, T., Azuma, S., Kashiwabara, S.-I., and Toyoda, Y. (1994). Sperm from mice carrying a targeted mutation of the acrosin gene can penetrate the oocyte zona pellucida and effect fertili/ation. J . B i d . Chem. 50, 3 1845-3 1849. Baba, T., Hoff, H. B., 111, Nemoto, H., Lee. H.. Orth, J., Arai, Y., and Gerton, G. L. (1993). Acro-

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granin, an acrosomal cysteine-rich glycoprotein. is the precursor of the growth-modulating peptides, granulins, and epithelins, and is cxpr d in somatic as well as male germ cells. Mol. Reprod. L)ei,. 34, 233-243. Baker. J., Hardy, M . P., Zhou. J., Boiidy. C., Lupa, F., BellvC, A. R., and Efstratiadis, A. (1996). Effects of the l g f l gene null inutatioii on mouse reproduction. Mol. Endocritiol. 10, 903-9 18. Baker. S. M.. Bronner. C. R., Zhang. L., Plug. A. W., Robatzek, M., Warren, G., Elliott, E. A,, Yu, J., Ashley, T., Arnheim, N., Flavell. R. A,, and Liskay, R. M. (1995). Male mice defective in the DNA mismatch repair gene PMSZ exhihit abnormal chromosome synapsis in meiosis. Cell 82, 309-319. Baker, S. M., Plug, A. W., Prolla, T. A,, Bi-oiiiicr. C. R., Harris, A. C., Yao, X.. Christie, D.-M., Monell, C.. Arnheim, N., Bradley, A,. Ashley, T , and Liskay, R. M. (1966). Involvement of mouse M l h l in DNA mismatch repair and meiotic crossing over. Nature Gerier. 14, 3 3 - 3 4 ? , Barlow, C., Hirotsune, S., Paylor. R., 1,iyaiiagc. M., Eckhaus, M.. Collins, F.. Shiloh, Y., Crawley, J. N.. Ried, T., Tagle, D., and Wynshaw-Boris, A. ( 1996). Arm-deficient mice: A paradigm of ataxia telangiectasia. Cell 85, 159- 17 I Becker, W., Kentrup. H., Heukelbach, J . , and Joost, H . 4 . (1996). cDNA cloning and characterization of rat Clk3, a LAMMER kinase predominantly expressed in testis. Biochim. Biop/i~.\. Ac.t e ~ ~ l o p i i i e n tand a l cell type specificity of LINE-I expression in inouse testis: Implications lor tranhpo5ition. Mol. Crll. Biol. 14, 2.584-2592. Brocefio. C., Ruiz, P., Vilaro, S.. and Pons. G. ( 1995). The muscle-specific phosphoglyceratc inutase gene is specifically expressed i n testis during spermatogenesis. Eur. J . Biodiem. 227, 629635. Brock, W A,, Trostle, P. K., and Meistrich. M. L. (1980). Meiotic synthesis of testis hirtones in the rat. Pmc. Ntrtl. Actrd. Sci. USA 77, 37 1-375. Burns, N.. Grimwade, B.. MacDonald. P. H., Choi. E. Y., Finberg, K., Roeder, G. S., and Snyder, M. ( 1 994). Large-scale analysis of genc expression, localimtion, and gene disruption in Sric c h o r m i w r s cerei>isicie.Gerir.v Dev. 8, 10x7- I 1 0 5 . Caldecott, K. W., McKeown. C. K., Tucker, J . D., Ljungquest, S.. and Thompson, I-. H. (1994). An interaction between the inaminiiliiui D N A repair protein XRCCI and DNA ligase 111. M ol. Cell. B i d . 14. 68-76.

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Nishiyama, S., Masamune, Y., and Nakanishi, Y. (1994). The silencer of mouse fgk-2 consists of two discrete DNA elements that individually have no effect. Gene 141, 261-266. Nowak, R., Siedlecki. J . A,, Kaclmarek, L., Zmudzka, B. Z., and Wilson, S. H. (1989). Ixvels and size complexity of DNA polymerase p mRNA i n regenerating liver and other orgms. Biochirn. Biophjs. Actcr 1008, 203-207. Nowak, R., Woszczynski, M., and Siedlecki. J. A. (1990). Changes in the DNA p~lyiiiera~e p gene expression during development of lung. brain, and testis suggest an involvernent of the enzyme in DNA recombination. Exp. Cell Res. 191, 5 1-56, O’Brien, D. A,, Gabel, C. A,, and Eddy, E. M. (1993). Mouse Scrtoli cells secrete mannose 6-phosphate containing glycoproteins that are endocytosed by sperinatogenic cells. Biol. Reprod. 49, 1055- 1065. O’Brien, D. A,, Gabel, C. A,, Rockctt, D. I.., and Eddy, E. M. (1989). Receptor-mediated endocytosis and differential synthesis of mannose 6-phosphate receptors in isolated spermatogenic and Sertoli cells. Eridocrinology 125, 2973-2984. 0’Brien, D. A,, Magyar, P. L., Hanes. R. N., and Tsuruta, J. K. (1993). Expression of the IGFII/cation-independent mannose 6-phosphate receptor i n the mouse seminiferous epithelium (abstract). Mol. Biol. Cell. 4, 137a. O’Brien. D. A,, Welch, J. E.. Fulcher. K. D., and Eddy. E. M. (1994). Expression of inaiinose 6-phosphate receptor messenger ribonuclcic acids in mouse spermatogenic and Sertoli cell\. B i d . Reprocl. 50, 429-435. Ogura, T., Takenouchi, N., Yamaguchi, M., Matsukage, A,, Sugimura, T., and Esumi. H. ( 1990). Striking similarity of the dirtribution patterns of the poly(ADP-ribose) polymerase and DNA polymerase beta among various niouse organs. Biocherrt. Biop/?~.s.H r s . Comituri. 172, 377384. Ohsako, S., Bunick, D., Hess, R. A., Nishida. T., Kurohniaru, M., and Hayashi, Y. (19941.Characterization of a testis specific protein localized to endoplasmic reticulum of spermatogenic cells. Anal. Rec. 238, 335-348. Ohsako, S., Hayashi, Y., and Bunick, D. (1994). Molecular cloning and sequencing of calnexin-t: An abundant inale germ cell-specilic calcium-binding protein of the endoplasmic reticulum. J . Biol. Chem. 269, 14140-14148. @yen, O., F r ~ y s a ,A., Sandberg, M.. Eskild. W., Joseph, D., Hansson, V., and Jahnsen, T. (1987). Cellular localization and age-dependent changes in mRNA for cyclic adenosine 3’,5‘-inonophosphate-dependent protein kinases in rat testis. Biol. Reprod. 37, 947-956. @yen. O., Myklebust, F., Scott, J . D., Cadd, G. G.. McKnight. G. S., Hansson, V., and Jahnsen, T. (1990). Subunits of cyclic adenosinc 3’.5’-monophosphate-dependentprotein kinase show differential and distinct expression patterns during germ cell differentiation: Alternative polyadenylation in germ cells gives rise to unique smaller-sized inRNA species. Biol. Reprod. 43, 46-54. Parmentier, M . , Libert, F., Schurmans, S., Schiffniann. S., Lefort. A,. Eggerickx, D., Ledent, C.. Mollereau, C., Gerard, C., Perret, J., Grootegoed, A,. and Vassart, G. ( I 992). Expression of members of the putative olfactory receptor gene family in inainnialian germ cells. Ntrrrtre 355, 453-455. Parvinen, M., Pelto-Huikko, M., Soder, O., Schultz, R., Kaipia, A,, Mali. P.. Toppari, J., Hakovirla, H., Lonnerberg, P., Ritzin, E. M., Ebendal, T., Olson, L., Hokfelt. T.. and Person. H. (1992). Expression of p-nerve growth factor and its receptor in rat seminiferous epithelium: Specific function at the onset of meiosis. ./, Cell Biol. 117, 629-641. Paulssen, R. H., Paulssen, E. J . , Gordeladze, J. O., Hansson, V., and Haugen, T. B. (1991), Cellspecific expression of guanine nuckotide-binding proteins in rat testiculnr cells. B i d . Reprod. 45, 566-57 I. Pawlak, A., Toussaint, C., Levy, I., Bulk, E, Poyard. M., Brouki, R., and Guellaen, G . (1995). Characterization of a large population of mRNAs from human testis. Ge/ionzics 26, I5 I - 158.

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Persengiev. S. P., Robert, S., and Kilpatrick. D. 1,. ( 1996). Transcription of the TATA binding protein gene is highly up-regulated during spermatogenesis. Mol. Enclocrinol. 10, 742-747. Persson. H., Ayer-LeLievre, C., Sodcr, 0.. Villar, M . J . . Metsis, M., Olson, L., RitzCn, M.. and HBkfelt, T. (1990). Expression of p-nerve growth factor receptor mRNA in Sertoli cells downregulated by testosterone. Science. 247, 704-707. Persson, H., Pelto-Huikko, M., Metsi\, M., Siidei-, O., Brenk, S., Skog, S., Hokfelt, T., and RitzCn, E. M. ( 1990). Expression of the neurolransmiltcr-synthesizing enzyme glutamic acid decarboxylasc in male germ cells. M o l . Cell. B i d . 10, 470 1-47 1 1 , Persson. H., Rehfeld, J . F., Ericsson, A., Schalling, M., Pelto-Huikko, M., and Hokfelt, T. (1989). Transient expression of the cholecystokinin gene in male germ cells and accumulation of the peptide in the acrosomal granule: Possible role of cholecystokinin in fertili7ation. Proc.. Ntrrl. Acnd. Sci. USA 86, 6166-6170. Pines, J . ( 1993). Cyclins and cyclin-dependent kinases: Take your partners. Trr.ridv B i o c h m . Sci. 18, 195-197. Primakoff, P.. Hyatt, H., and Tredick-Kline. J. (19x7). Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion. J . Cell Biol. 104, 141149. Rao, B. J., Brahmachari, S. K . , and Rao, M . R. S. (19x3). Structural organization of the meiotic prophaw chromatin in the rat testis. J . Biol. Clirni. 258, 13478-13485. Rao, B. J., and Rao, M. R. S. (1987). DNase I site mapping and micrococcal nuclease digestion .i d . Cliem. 262, 4472-4476. of p a c h y m e chromatin reveal novel structural teattires. .IB Rao, M. R. S., Rao, B. J . . and Ganguly. J . (19x2). Imalization of testis-variant histones in rat testis chromatin. Biocheni. J . 205. I 5-7 I Reed, L. J . . and Yeaman, S. J. (1987).Pyruvate dchydrogenase. /ti “The Enzymes” (P. D. Boyer. Ed.). volume 18, pp. 77-95. Academic Pi-ess. New York. Reines, D. ( 1992). Elongation factor-dependent transcript shortening by template-engaged RNA polymerase I I . J . Biol. Clwrn. 267, 3795-3800. Reitmair, A. H., Schmits, R., Ewel, A.. Kedstoii. M.. Mitri, A,, Waterhouse, P.. Mittrucker, H.-W., Wakeham, A,. Liu, B., Thomason, A,. Griesscr. H., Gallinger, S.. Balhausen, W. G., Fishel, R., and Mak, T. W. (1995). M S H 2 delicient mice arc viable and susceptible to lymphoid tumors. Nufirre Geiier. 11, 64-70. Richlei-. C., Ast, G., Goitein, R.. Wahrman. J.. Spcrliiig, R.. and Sperling, J . (1994). Splicing c o n ponents arc excluded froin the traii\criptionally inactive XY body in male meiotic nuclei. M o l . Biol. Cell. 5, 1341- 1352. Rivkees. S. A. ( 1994). Localization a n d char rimtion of adenosine receptor expression in rat te,ti\. O i d ~ ~ c r i 135, ~ k ~2307-13 g~ 13. Rogers. M. B., Hosler, B. A,, and Guda\, I..J . ( I W I ) . Specific expression of a retinoic acid-regulated. iinc-tinger gene. Rex- I , i n preiiiiplaiitntion embryos, trophoblasts and sperniatocytes. Dei,rlo~pirri~ 113, 8 15-824. Rosario. M . O., Perkins, S. L., O’Bricn. D. A,, Allen, R. L., and Eddy, E. M. (1992). Identilication of the gene for the developmentally expressed 70kDa heat-shock protein (P70) of inotise sperrnatogenic cells. Dev. Biol. 150, I I I Rotter, V.. Schwartz, D., Almon, E., Goldtinger, N.. Kapon, A,. Meshorcr, A,. Donehower. I.. A,. and Levine, A. J . (1993). Mice with reduced lc\el\ of p53 protein exhibit the testicular giantcell degenerative syndrome. Proc,. Nor/. Actid. Sci. (JSA 90, 9075-9079. Rubin, M. R., Toth, L. E., Patel, M . D., D‘Eu\tachio. l’., and Nguyen-Huu, M. C. (1986). A mouse homeo box gene is expressed i n spermatocytes and embryos. Science 233, 663-667. Ruppert, S . , Cole, T. J . , Boshart, M.. Schiiiid, E., and Schtitz, G. (1992). Multiple mRNA ISOforms of the transcription activator protein CRt:B: Generation by alternative splicing and specific expression in primary spermatocytes. EMNO J . 11, 1503- 15 12. Sancar, A,, and Sancar, G. B. (1988). DNA repair ciiiyiiies. Annu. R n . . Biochem. 57, 29-67, -

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Sano. M., Ohshima, A,-S., Kawamura, N., Kitajima, S., and Mizatani, A. (1987). Immunohistochemical study of calinodulin i n developing mouse testis. J . Exp. Zoo/. 241, 5 1-59, Sarge, K. D.. Park-Sarge, 0.-K., Kirby, J. D.. Mayo, K. E., and Moriinoto, R . 1. (1994). Expression of heat shock factor 2 in mouse testis: Potential role as a regulator of heat-shock protein gene expression during spermatogenesis. B i d . Reprod. 50, 1334- 1313. Sarge, K. D., Zimarino, V., Holm, K., Wu, C., and Morimoto. R. 1. ( 1991 1. Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability. Genes Dev. 5, 1902-191 1. Sausaine, C., Gabriel-Robez, 0.. and Rumpler, Y. (1994). Patterns of ribonucleic acid synthesis in human primary spermatocytes. Andrologia 26, 139- I 1 1 . Schmckel, K.. Meuwissen, R. L. J., Dietrich, A. J . J . , Vink, A. C. G., van Mark, J., van Veen, H., and Heyting. C. ( 1996). Organization of SCPl protein molecules within synaptoneinal coniplexes of the rat. E x p . Ce/l Rrs. 226, 20-30. Schmidt, E. E., and Schibler, U. (19%). High accumulation of components of the RNA polymerase 11 transcription machinery in rodent spermatida. Drw/opmen/ 121, 1373-2383. Schmitt, M. C., Jainison, R. S., Orgebin-Crist, M.-C., and Ong, D. E. (1994). A novel, testis-specitic member of the cellular lipophilic transport protein superfamily, deduced from a complimentary deoxyribonucleic acid clone. B i d . Reproti. 51, 239-245. Schumacher, J. M.. Lee, K., Edelhoff, S.. and Braun, R. E. ( 199Sa). Distribution of Tcnr, an RNA-binding protein. in a lattice-like network within the sperrnatid nucleus in the mouse. Biol. Reprod. 52, 1274- 1283. Schumacher. J. M.. Lee, K., Edelhoff. S.. and Braun, R. E. (199Sb). Spnr. a rnurine RNA-binding protein that is localized to cytoplasmic microtuhules. J . Cel/ B i d . 129, 1023- 1032. Schwartz, D., Goldtinger, N., and Rotter, V. ( 1993). Expression of pS3 protein in spermatogenesis is confined to the tetraploid pachytene primary spermatocytes. Oncogene 8, 1487- 1494. Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D.. Feunteun, J., Ashley, T., Livingston. D. M. (1996). Association of BRCAl with RADS I in mitotic and meiotic cells. Cell 88, 265-275. Seidah, N. G., Day, R., Hamelin, J., Gaspar, A,, Collard, M. W., and Chritien, M. (1992). Testicular expression of PC4 in the rat: Molecular diversity a t a novel germ cell-specific Kex2/subtilisin-like proprotein convertase. Mol. Ozdocrinol. 6, 1559- 1570. Shannon, M., and Handel, M. A. ( 1993). Expression of the Hprt gene during spermatogcncsis: Implications for sex-chromo\ome inactivation. Biol. Reprod. 49, 770-778. Shinohara, A,, Ogawa, H., Matwda, Y., Ushio, N., Ikeo, K., and Ogaua, T. (1993). Cloning of human, mouse and tission yeast recombination genes homologous to RADSI and J-KA. N m , m Genet. 4, 239-243. Sirnhulan-Roaenthal, C. M., Rosenthal, D. S., Hilz, H., Hickey, R., Malkas, L., Applegren. N.. Wu, Y., Bers, G.. and Smulwn, M. E. (1996). The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the mulriprotein DNA replication complex. Biochemi.s/ry 35, I 1622- I 1633. Singer-Sam, J., Robinson, M. 0.. BellvC, A. R., Simon, M. 1.. and Riggb, A. D. (1990). Measurement by quantitative PCR of changes in HPRT. PGK-I, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis. Nuclric Acids Rrs. 18, 1255- 1159. Slaughter, G. R., and Means, A. R. (1989). Analysis of expression of multiple genes encoding calmodulin during spermatogenesis. Mol. Entlocrino/. 3, 1569- 1578. Soderstriim, K:O. ( 1976). Characterization of RNA syntheais in mid-pachytene spermatocytes of the rat. Exp. Cell Res. 102, 237-245. Sorrentino, V., McKinnery, M. D., Giorgi, M., Geremia, R., and Fleissnei-. E. (1988). Expression of cellular protooncogenes in the mouse male germ line: A distinctive 2.4-kilobase p i n - f transcript is expressed in haploid postmeiotic cells. Pro(,.Ntitl. Actrd. s(,;. USA 85, 2191-1195. Sosa, A., Altamirano, E., Hernandey. P., and Rosado, A. (1972). Developmental pattern of rat testis hexokinase. Life Sci. 11, 499-510.

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Sperry. A. O., and Zhao, L.-P. ( 1996). Kinesin-related proteins in the mamnialian testes: Ciindidate motors for meio. 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

265

8. Meiotic Chromo$ome Segregation

Mitosis Metaphase

\I

'\

Anaphase

C

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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C

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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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A

B

C

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.

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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

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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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385. Maguire, M. P. (1974). The need for a chiasma binder. J . Theor. Biol. 48, 485-487. Maguire. M. P. (1978a). Evidence for separate genetic control of crossing over and chiasma maintenance in maize. Chrornnsorticr 65, 173- 183. Maguire, M. P. (197%). A possible role for the synaptonemal complex in chiasma maintenance. Exp. Cell Res. 112, 297-308. Maguire, M. P. (1993). Sister chromatid association at meiosis. Muydi(xi 38, 93-106. Maguire, M. P., Paredes, A. M.. and Riess, R. W. (1991). The desynaptic mutant of maize as a combined defect of synaptonernal complex and chiasma maintenance. Geriorne 34, 879-887. Maguire, M. P., Riess, R. W., and Paredes, A. M. (1993). Evidence from a maize desynaptic mutant points to a probable role of synaptonemal complex central region components in provision for subsequent chiasma maintenance. Genome 36, 797-807. Mason, J . M. (1976). Orientation disruptor (ord):A recombination-defective and disjunctiondefective meiotic mutant in Drosophilu mrlcrnognsrer. Genetics 84, 545-572.

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McCarroll. R. M.. and Fangman, W. L ( I%3X). Time o f replication of yeast cenlromere\ and telomeres. Cell 54, 505-5 13. McKee, B. D. ( 1996). The license to pair: Identilication of meiotic pairing sites in DrO.\Ophi/CI. C/mrriosor?icr 105, 135-141

Miyazaki. W. Y., and Orr-Weaver, T. I_. ( 1902). Sister-chromatid misbehavior in Drosophila ord mutant\. Gewrics 132, 1047- 106 I M t m . I? B., and Church, K. (1979). The di\trihution of synaptonemal complex matenal in melaphase I bivalents of Loc'usftr and Chlocwlri\ (Oitlioptern. Arcididae). Clinmosottia 73, 247-254. Moms, P. B.. and Earnshaw. W. C. ( 19x9). Anti~topoisorneraseII recognixs meiotic chromosome core\. Chmrrrosornrr 98, 3 17-32?, of chiasmata and chromosorn;il di\Moens. P. B., and Spyropoulo\. B. ( 1995) Iniintin~~cytology iunction at mouse meiosis. Chroniotorritr 104, 175- 182. Molnar. M., Bahler, J . . SpicLki, M., and Kohli, J . ( 1995). The r e d gene of S(.lri;oFacchrr~-orn!cr.s powhe IS involved in linear element formation, chromosome pairing and sister-chromatid cohesion during meiosis. Gerre/ic.c 141, 6 1-73, Moore, D. P., Miyazaki, W. Y., Tomkiel, J . t.,and Orr-Weaver, T. L. ( 1994). Oortble o r riofhirig: A Drosophila mutation affecting niciotic chromosoine segregation in both females and males. Gonfvic'.r 136, 9.53-964. Morcau. P. J. E. Zickler, D., and Leblon, C;. ( 19x5). One cla\s of mutants with disturbed centromcrc cleavage and chromosome pairing in .Sord~rr-icrrrinc.ro.\porcc. Mol. Gcw. Gerrcv. 198, 1x9- 197.

Muller, W. ( 1972). Elektronenmikroshopi\clie Untersuchungen zum Formwechsel der Kinetochoren wahrend der Sperinatocytentcil tinge" \ o n Pule.\ ferrufiiriecr (Nerncitoc~rtr).Chrrmoc o r r i n 38, 139- 172. Nickla\. K. B. ( 1967). Chromosome iiiicroin;iniptil~ition.11. Induced reorientation and the cxperimental control of segregation in niciosi\. (Berlin)21, 17-50. Nickla\. R. B. (1971). Mitosi\. Ad\'. Ccll Siol. 2. 225-297. Nicklas. R . B. ( 1977). Chromo\ome d~stribution:Experiments on cell hybrids and iri \Yfro. Philos. Trotr\. R. So 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.)

9. Regulation and Execution of M e h i s in Dro.wp/ii/a Males

323

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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 r ~ . ~ ~ pnic,lono,~ti\rc,,-: single P element rnutagenesi\. Grrirtic.~135, 180-505. C a h l l o n , D. H., and Wasserman, S. A ( 1994). / ) I ~ J / J / I ~ I U O Uis. Srequired for cytokinesis in Dro.sophfl(rand shares domains of siniilai-ity with tlic products of the limb &eforuiit! gene. DevelO/"" ('I If. 120, 3 367 -3377. Cenci, G., Bonaccor\i, S., Pisano, C., Vcrni, F., and Gatti, M. (1994). Chromatin and microtubule organi/ation during premeiotic. meiotic and early postmeiotic stages of Drasophilo ~nrltrriogtr.\f c r spcrmiitogenesis. J . Cell Sci. 107, 352 1-3534. Chanp. F.. Hajihagheri, N., and Nurse. P. (1996). I-i\\ion yeast contractile ring protein cdcl2p: Initiation of ring formation from a single point. ./. Cell Biol., in press. Clay, F. J.. McEwcn, S. J.. Bertoncello, I.. Wilk\. A . F.. and Dunn. A . R . (1993). Identification O of Droand cloning of a protein kinas-encoding mouse gene. Plk. related to the ~ C J ~gene sop/i;/~I. PrOC,. Ntrf/. ActId. .%,i us/\ 90. 4881-4886. Cooky. I-., Kelley, R . , and Spradling, A . ( 19x81.lnrertional mutagenesis of the Drosoph,lti genome with single P element\. Scir,uro.sophiltr. / t i "Biology" (M. Demerecs, Ed.), pp. 1-61. Hafner, New York. Courtot. C., Fankhauwr, C., Sirnanis. V.. and Lehner. C. F. (1992). The Drosoplrilo cdc25 homolog f i i , i u e i \ requircd for ineiosis. / > c i . c . l r p w , i r 116, 405-41 6. Dunphy, W. G . and Kumagai, A ( I09 I ) . The ctlc25 protein contains an intrinsic phosphatase activit). Cell 67, 180- 196. Dunphy, W. G.. and Newport. J. W. ( l 9 8 9 ) , Fi\\ion yeast p13 block\ mitotic activation and tyrosine dephocphorylation of the X w i o p r l , \ cdc7 lprotein kinase. Cell 58, I8 I -10 I . Eherhart. C. G., Maines, J. Z., and Wirssei-man, S. A. (1996). Meiotic cell cycle requirement for a fly hoinologue of human Deleted i n Aioosperniia. Ntrrure 381, 783-785. Eberhart. C. G., and Wasserman, S . A . ( 1095). Thc pcdorti locus cncodea a protein required for meiotic cell divition: An analysis o f G Y M iti-i-c\t in f>ro.sop/irlnspermatogenesis. De1ylopmeuf 121. 3477-3486. Edgar. B. A.. and O'Farrell, P. H. ( 19x9). Genetic control ol cell division patterns in the Drosophilo embryo. Cell 57, 177- 1 X7. Edgar, B. A.. and O'Fnrrell, P. H. ( 1900).The three postblastodcrm cell cycle\ o f Dro.\ophilo e n bryogenesis are regulated in G2 by \triti,q. Crll 62, 469-480. Emmons, S.. Phan, H., Callcy. J., Chen. W.. James, B., and Manseau, L. (1995). C t r p p w i n o , a Drosophiltr maternal effect gene requircd !'or polarity of the egg and embryo, is related to the vertebrate limh tlrfijnniry locus. Gtvu,.\ Dew. 9. 2482-2494. Erickson. J. ( 1965). Meiotic drive i n /lro\op/ii/~rinvolving chromosome breakage. Gerirtrc~,s51, 557-571.

I-ield, C. M.. and Alherts, B. M. (1995). Anillin. :I contractile ring protein that cycles from the nucleus to the cell cortex. J . Cell B i d 131, I 6 5 - 178. I-ishkind, D. J., and Wang, Y. L. (1995) New h o w o n s for cytokincsis. Curr: Opiu. Cell Biol. 7, 23-31. Ford, S. K., and Pringle, J. R. ( 1991 ). Cellular morphogenesi\ in the Sticcharoiiqces c.ertwi.siae cell cycle: Locallration of the CDC'I I gene product and the timing of events at the budding site /lei,. Grner. 12, 28 1-29?,

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