Hormones and Reproduction of Vertebrates, Volume 1: Fishes

By David Norris and Kristin Lopez

This series of volumes represents a comprehensive and integrated treatment of reproduction in vertebrates from fishes of all sorts through mammals. It is designed to provide a readable, coordinated description of reproductive basics in each group of vertebrates as well as an introduction to the latest trends in reproductive research and our understanding of reproductive events. Whereas each chapter and each volume is intended to stand alone as a review of that topic or vertebrate group, respectively, the volumes are prepared so as to provide a thorough topical treatment across the vertebrates. Terminology has been standardized across the volumes to reduce confusion where multiple names exist in the literature, and a comprehensive glossary of these terms and their alternative names is provided.

• A complete, essential and up to date reference for research scientists working on vertebrate hormones and reproduction - and on animlals as models in human reproductive research
• Covers the endocrinology, neuroendocrinology, physiology, behaviour and anatomy of vertebrate reproduction
• Structured coverage of the major themes for all five vertebrate groups allows a consistent treatment for all
• Special chapters elaborate on features specific to individual vertebrate groups and to comparative aspects, similarities and differences between them

• Language: English
• Publisher: Academic Press
• Release date: May 4, 2011
• ISBN: 9780080962290

Book preview

Table of Contents

Cover image

Front Matter

Copyright

Dedication

Preface

Preface

Contributors

Chapter 1. Sex Determination in Fishes

1. Introduction

2. Sex Determination

3. Sexual Differentiation

4. Environmental Effects on Sex Determination and Differentiation

5. Conclusions and Future Directions

Chapter 2. Conserved and Divergent Features of Reproductive Neuroendocrinology in Teleost Fishes

1. Introduction

2. The Unique Hypothalamic–Pituitary–Gonadal (HPG) Axis of Teleosts

3. Gonadotropin-Releasing Hormone (GnRH)

4. Other Brain Factors Stimulating GTH Release

5. Dopamine (DA), A Brain Inhibitor of Reproduction

6. KISS, A New Actor in the Brain’s Control of Reproduction

7. Sex Steroids in the Brain of Fishes

8. Conclusions and Perspectives

Chapter 3. Testicular Function and Hormonal Regulation in Fishes

1. Introduction

2. Testis Structure and Spermatogenesis: An Overview

3. Testicular Hormones

4. Endocrine Regulation of Testis Structure and Function

5. Temporal Aspects of Testicular Function

6. Accessory Gonadal Structures

7. Intraspecific Variation in Sperm Characteristics and Testicular Function

8. Conclusions

Chapter 4. Regulation of Ovarian Development and Function in Teleosts

1. Introduction: Fish Models of Reproductive Strategies

2. Morphological Aspects of the Teleost Ovary and Stages Of Oocyte Development

3. Differentiation of Primordial Germ Cells into Oogonia

4. Oogenesis, Oocyte Growth, and Development

6. Final Considerations

Chapter 5. Thyroid Hormones and Reproduction in Fishes

1. Introduction

2. Thyroid Hormone Delivery

3. The Thyroid Tissue of Fishes

4. Thyroid Hormone (TH) and Reproduction in Fishes

5. Conclusions

Chapter 6. Stress and Reproduction

1. Introduction

2. Effects of Stress on the Hypothalamic–Pituitary–Gonadal (HPG) Axis

3. Life Stage-Specific Effects of Stress on Reproduction

4. Effects of Sex and Reproduction on the Hypothalamic–Pituitary–Interrenal (HPI) Axis

5. Reproduction and Resistance to Stress

6. Conclusions

Chapter 7. Hormones and Sexual Behavior of Teleost Fishes

1. Theoretical Constructs: Appetitive and Consummatory Phases

2. Patterns of Sexual Behavior

3. Endocrine Mechanisms Regulating Sexual Behavior

4. Brain Circuits Underlying Sexual Behavior in Fishes

5. Prospects for Future Research

Chapter 8. Neuroendocrine Regulation in Sex-changing Fishes

1. Introduction

2. Hermaphroditism in Fishes

3. Hypotheses of Natural Sex Reversal

4. Social Factors Affecting Sex Reversal

5. Neuroendocrine Factors Affecting Sex Reversal

6. Studies on the Saddleback Wrasse

7. Future Research

Chapter 9. Hormonally Derived Sex Pheromones in Fishes

1. Introduction

2. Hormonal Pheromones in Fishes

3. Hormonal Pheromones and the Issue of Species Specificity

Chapter 10. Reproduction in Agnathan Fishes: Lampreys and Hagfishes

1. Introduction

2. Hagfish Reproduction

3. Lamprey Reproduction

4. Summary

Chapter 11. Hormones and Reproduction in Chondrichthyan Fishes

1. Reproduction in Chondrichthyan Fishes

2. The Hypothalamic–Pituitary–Gonadal (HPG) Axis

3. Hormonal Regulation in Females

4. Hormonal Regulation in Males

5. Other Hormones Involved in Reproduction in Males and Females

6. Hormones, Sexual Differentiation, and Sexual Maturation

7. Hormones, Reproductive Behaviors, and Sensory Function

8. Environmental Influences on Circulating Hormone Levels and Reproduction

9. Conclusions and Future Directions

Chapter 12. Hormones and Reproduction of Sarcopterygian Fishes

1. Introduction

2. Coelacanths

3. Lungfishes

4. Concluding Remarks

Chapter 13. Endocrine-active Chemicals (EACs) in Fishes

1. Introduction

2. Mechanisms of Endocrine-Active Chemical (EAC) Signaling

3. Multidimensional Mixture Complexity

4. Consequences of Specific Life-Stage Exposures

5. Organizational and Activational Effects of Endocrine-Active Chemicals (EACs)

6. Evidence of Reproductive Disruption in Free-Living Fishes

7. Conclusions

Color Plates

Species Index

Index

Front Matter

Hormones and Reproduction of Vertebrates, Volume 1—Fishes

Hormones and Reproduction of Vertebrates

Volume 1: Fishes

David O. Norris

Department of Integrative Physiology, University of Colorado, Boulder, Colorado

Kristin H. Lopez

Department of Integrative Physiology, University of Colorado, Boulder, Colorado

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Academic Press is an imprint of Elsevier

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Copyright

Academic Press is an imprint of Elsevier

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First edition 2011

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Cover images

Front cover image: Amphiprion percula, the orange clownfish. Courtesy of iStockphoto: Image 6571184.

Back cover image: Atlantic hagfish (Myxine glutinosa) eggs. Courtesy of Stacia A. Sower, University of New Hampshire, Durham, NH, USA and Scott I. Kavanaugh, University of Colorado, Boulder, CO, USA.

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Dedication

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Richard Evan Jones

This series of five volumes on the hormones and reproduction of vertebrates is appropriately dedicated to our friend and colleague of many years, Professor Emeritus Richard Evan Jones, who inspired us to undertake this project. Dick spent his professional life as a truly comparative reproductive endocrinologist who published many papers on hormones and reproduction in fishes, amphibians, reptiles, birds, and mammals. Additionally, he published a number of important books including The Ovary (Jones, 1975, Plenum Press), Hormones and Reproduction in Fishes, Amphibians, and Reptiles (Norris and Jones, 1987, Plenum Press), and a textbook, Human Reproductive Biology (Jones & Lopez, 3rd edition 2006, Academic Press). Throughout his productive career he consistently stressed the importance of an evolutionary perspective to understanding reproduction and reproductive endocrinology. His enthusiasm for these subjects inspired all with whom he interacted, especially the many graduate students he fostered, including a number of those who have contributed to these volumes.

Preface

Hormones and Reproduction of Vertebrates Preface to the Series

Every aspect of our physiology and behavior is either regulated directly by hormones or modified by their actions, as exemplified by the essential and diverse roles of hormones in reproductive processes. Central to the evolutionary success of all vertebrates are the regulatory chemicals secreted by cells that control sexual determination, sexual differentiation, sexual maturation, reproductive physiology, and reproductive behavior. To understand these processes and their evolution in vertebrates, it is necessary to employ an integrated approach that combines our knowledge of endocrine systems, genetics and evolution, and environmental factors in a comparative manner. In addition to providing insight into the evolution and physiology of vertebrates, the study of comparative vertebrate reproduction has had a considerable impact on the biomedical sciences and has provided a useful array of model systems for investigations that are of fundamental importance to human health. The purpose of this series on the hormones and reproduction of vertebrates is to bring together our current knowledge of comparative reproductive endocrinology in one place as a resource for scientists involved in reproductive endocrinology and for students who are just becoming interested in this field.

In this series of five volumes, we have selected authors with broad perspectives on reproductive endocrinology from a dozen countries. These authors are especially knowledgeable in their specific areas of interest and are familiar with both the historical aspects of their fields and the cutting edge of today’s research. We have intentionally included many younger scientists in an effort to bring in fresh viewpoints. Topics in each volume include sex determination, neuroendocrine regulation of the hypothalamus–pituitary–gonadal (HPG) axis, separate discussions of testicular and ovarian functions and control, stress and reproductive function, hormones and reproductive behaviors, and comparisons of reproductive patterns. Emphasis on the use of model species is balanced throughout the series with comparative treatments of reproductive cycles in major taxa.

Chemical pollution and climate change pose serious challenges to the conservation and reproductive health of wildlife populations and humans in the twenty-first century, and these issues must be part of our modern perspective on reproduction. Consequently, we have included chapters that specifically deal with the accumulation of endocrine-disrupting chemicals (EDCs) in the environment at very low concentrations that mimic or block the critical functions of our reproductive hormones. Many authors throughout the series also have provided information connecting reproductive endocrinology to species conservation.

The series consists of five volumes, each of which deals with a major traditional grouping of vertebrates: in volume order, fishes, amphibians, reptiles, birds, and mammals. Each volume is organized in a similar manner so that themes can be easily followed across volumes. Terminology and abbreviations have been standardized by the editors to reflect the more common usage by scientists working with this diverse assembly of organisms we identify as vertebrates. Additionally, we have provided indices that allow readers to locate terms of interest, chemicals of interest, and particular species. A glossary of abbreviations used is provided with each chapter.

Finally, we must thank the many contributors to this work for their willingness to share their expertise, for their timely and thoughtful submissions, and for their patience with our interventions and requests for revisions. Their chapters cite the work of innumerable reproductive biologists and endocrinologists whose efforts have contributed to this rich and rewarding literature. And, of course, our special thanks go to our editor, Patricia Gonzalez of Academic Press, for her help with keeping us all on track and overseeing the incorporation of these valuable contributions into the work.

David O. Norris and Kristin H. Lopez

Preface

Preface to Volume 1 Fishes

Fishes represent the most diverse and abundant paraphyletic category of vertebrates. They also have experienced the longest evolutionary history. The endocrine system of fishes is the prototype vertebrate system; it has undergone extensive evolution within the fishes and has provided the basic foundation for the reproductive endocrinology of all vertebrates. Additionally, fishes are of great economic importance for humans as they provide billions of tons of food annually.

Unfortunately, in spite of their seeming abundance, the numbers and diversity of fishes are decreasing in the face of persistent human impacts, not only because of their commercial uses but as a result of extensive chemical and thermal pollution of freshwater and marine habitats. Our understanding of fish reproductive endocrinology is basic not only to understanding the reproductive endocrinology of other vertebrates but also to the continued survival of fishes.

In this volume, we focus on the basic components of the reproductive endocrinology of fishes, setting the pattern we have followed in all subsequent volumes of this series. We begin by looking at sex determination, neuroendocrine regulation of the hypothalamic–pituitary–gonadal axis, and basic aspects of ovarian and testicular function. Additional chapters deal with the roles of pheromones and stress, and the effects of thyroid function on reproduction of fishes, as well as the endocrine control of reproductive behaviors, including sex reversal, exhibited by certain teleosts. Special chapters deal with hormones during the reproductive cycles of agnathan fishes (lampreys, hagfishes), sarcopterygian fishes (lungfishes), and chondrichthyan fishes (sharks and their relatives). The teleost fishes, which represent the largest segment of living fishes by far, are also the best studied. Consequently, they are thoroughly covered throughout the basic chapters in this volume. The final chapter in this volume addresses the growing problem of disruption of the fish reproductive system by hormone mimics and antagonists, which are appearing in the aquatic environment as a consequence of widespread chemical pollution and hence are capable of reducing the reproductive potential of fishes on a worldwide basis.

Contributors

Nicholas J. Bernier

University of Guelph, Guelph, ON, Canada

Sharon L. Carlisle

University of Oklahoma, Norman, OK, USA

Sylvie Dufour

Muséum National d'Histoire Naturelle, Paris, France

Meghan L.M. Fuzzen

University of Guelph, Guelph, ON, Canada

James Gelsleichter

University of North Florida, Jacksonville, FL, USA

David M. Gonçalves

Instituto Superior de Psicologia Aplicada, Lisboa, Portugal

Universidade do Algarve, Faro, Portugal

Jean M.P. Joss

Macquarie University, Sydney, NSW, Australia

Olivier Kah

Université de Rennes, Rennes, France

Hiroshi Kawauchi

Laboratory of Molecular Endocrinology, Sendai, Miyagi, Japan

Rosemary Knapp

University of Oklahoma, Norman, OK, USA

Earl T. Larson

Northeastern University, Boston, MA, USA

Karen P. Maruska

Stanford University, Stanford, CA, USA

Yoshitaka Nagahama

Solution-Oriented Research for Science and Technology (SORST)

Kawaguchi, Saitama, Japan;

National Institute for Basic Biology, Okazaki, Japan

Masaru Nakamura

Solution-Oriented Research for Science and Technology (SORST), Kawaguchi, Saitama, Japan;

University of the Ryukyus, Motobu, Okinawa, Japan

David O. Norris

University of Colorado at Boulder, Boulder, CO, USA

Rui F. Oliveira

Instituto Superior de Psicologia Aplicada, Lisboa, Portugal

Instituto Gulbenkian de Ciência, Oeiras, Portugal

Bindhu Paul-Prasanth

Solution-Oriented Research for Science and Technology (SORST), Kawaguchi, Saitama, Japan;

National Institute for Basic Biology, Okazaki, Japan

Jason C. Raine

University of Saskatchewan, Saskatoon, SK, Canada

E. Rocha

University of Porto, Porto, Portugal

M.J. Rocha

University of Porto, Porto, Portugal

Superior Institute of Health Sciences-North (ISCS-N), Paredes, Portugal

Stacia A. Sower

University of New Hampshire, Durham, NH, USA

Norm Stacey

University of Alberta, Edmonton, AB, Canada

R. Urbatzka

University of Porto, Porto, Portugal

Alan Milan Vajda

University of Colorado at Denver, Denver, CO, USA

Glen Van Der Kraak

University of Guelph, Guelph, ON, Canada

Chapter 1. Sex Determination in Fishes

Bindhu Paul-Prasanth∗†, Masaru Nakamura∗∗∗ and Yoshitaka Nagahama∗†

∗Solution-Oriented Research for Science and Technology (SORST), Kawaguchi, Saitama, Japan

†National Institute for Basic Biology, Okazaki, Japan

∗∗University of the Ryukyus, Motobu, Okinawa, Japan

SUMMARY

Among the vertebrates, teleost fishes display the greatest diversity of sexual phenotypes, thus providing an excellent model to investigate molecular mechanisms of sex determination/differentiation. Sex in fishes is determined by genetically or environmentally based signals. The second vertebrate sex-determining gene, dmy/dmrt1by, was discovered in medaka (Oryzias latipes) but was found to be present only in two Oryzias species, illustrating the vast diversity of sex-determining genes in fishes. By contrast, molecular mechanisms involved in sexual differentiation appear to be conserved among fishes. Several factors have been identified: Gsdf/Dmrt1 for testicular differentiation and Foxl2/Cyp19a1a/estrogens for ovarian differentiation. Among these factors, Gsdf is specific to the fish lineage and a new player in the field of sexual differentiation of fishes. These factors may play major roles in both genetic and environmental modes of gonadal sex differentiation.

1. Introduction

Most vertebrates adopt a sexual mode of reproduction. Sexually reproducing organisms generally exist in one of two anatomical and physiological states, namely male or female. Males develop testes, while females develop ovaries for production of gametes: sperm and ova, respectively. Development of testes or ovaries is the outcome of differential morphogenesis of the gonadal primordium. Essentially, differential morphogenesis is established through repression of the manifestation of one sex and promotion of the other sex by sex determination (SD). Vertebrates exhibit a wide array of sex-determining mechanisms, sometimes even at the species level, ranging from environmental sex determination (ESD) to genetic sex determination (GSD). Among vertebrates, fishes are the only group that possesses all the various mechanisms of sex determination known for vertebrates. Therefore, study of sex determination using various fishes as animal models constitutes an essential and exciting part of the field of sex determination.

Sex determination kicks off the program for sexual development in the undifferentiated gonadal primordium and sex differentiation orchestrates further structural development of the gonads according to their designation at the SD step. In general, mechanisms underlying sex differentiation are more or less conserved across genera. During sexual differentiation, the somatic as well as the germ cells in the gonads differentiate and the gonads undergo testis- or ovary-specific morphogenesis. In fishes, these processes are controlled largely by genetic and hormonal factors. However, most of the earlier studies describing the involvement of steroid hormones in sexual differentiation are based on the exogenous administration of sex steroids. Since numerous reviews are available on this subject, we have not dealt much with this topic in the present chapter. Instead, we will discuss the role of endogenous sex steroids as revealed by studies on the expression patterns of steroidogenic enzymes during early sexual differentiation stages.

A comprehensive state-of-the-art analysis of the field concerning sex determination and differentiation among fishes was done by Devlin and Nagahama in 2002, the year in which the first male sex-determining gene among fishes was identified from a small teleost fish, the Japanese medaka (Oryzias latipes) (Matsuda et al., 2002 and Nanda et al., 2002). This review extensively covered about three decades of research prior to 2002 and gave elaborate descriptions of almost all aspects of fish sex determination/differentiation; e.g., various modes of sex determination, gonadal differentiation and development, endocrine regulation of gonadal differentiation and morphogenesis, etc. More recently, several prominent studies in the field of sex determination among fishes have unraveled more facts, factors, and mechanisms involved at the molecular, cellular, and physiological levels, filling in the gaps in the evolution of sex-determining mechanisms. Use of modern molecular tools such as transgenesis and gene knock-down have made it possible to study the cellular and molecular processes involved in the early stages of gonadal development among fishes so as to reveal the evolutionary relationships among different vertebrate classes. Some of the genes found in fishes have similar roles in higher vertebrates, while some possess reversed roles. Here, we have reviewed mainly more prominent studies that have come out within and after the year 2002. Though fishes include innumerable species, emphasis will be given mostly to model organisms such as medaka, tilapia (Oreochromis niloticus), zebrafish (Danio rerio), and rainbow trout (Oncorhynchus mykiss), as most of the studies on sex determination/differentiation are centered on these species.

2. Sex Determination

Fishes have both XX/XY and ZZ/ZW heterogametic modes of sex determination. In the XX/XY system, males carry the heteromorphic sex chromosomes and the presence of the Y-chromosome triggers gonadogenesis into the testicular pathway. Although a ZZ/ZW system, with females carrying the heteromorphic chromosomes, has been reported in several fishes belonging to the Teleostei (Anguilliformes, etc.), only a limited number of studies dealing with this topic are available. Therefore, for practical reasons, the ZZ/ZW system is not analyzed in detail in this review.

In vertebrates with GSD, undifferentiated gonads develop strictly under the control of the programs within their genomes. According to the master regulator positioned at the top of the sex differentiation cascade, gonads develop along one of two alternate pathways, either testis or ovary. In mammals, the SD gene, SRY/Sry (sex-determining region of the Y chromosome), initiates testicular differentiation through the sex-specific upregulation of Sox9, which in turn promotes the testis-specific differentiation of the somatic cell lineages and, thus, the male-specific gonadal architecture (Sekido et al., 2004, Kidokoro et al., 2005 and Sekido and Lovell-Badge, 2008). Concomitant to the promotion of the testicular cascade, Sox9 represses the alternate cascade of development by suppressing key genes involved in ovarian differentiation (Wilhelm et al., 2009). Hence, the role of the SD gene is to promote the expression of those factors required for the establishment of the sex-specific gonadal architecture, which will in turn support the differentiation, development, and maturation of the gamete that the gonad produces.

The first decade of the twenty-first century has witnessed a major breakthrough in the field of fish sex determination. Matsuda et al. (2002) and Nanda et al. (2002) independently identified the first male sex-determining gene among fishes, dmy (DM-domain gene on the Y chromosome)/dmrt1bY from medaka. It was named so because dmy/dmrt1bY was found to be a duplicate copy of the well-conserved downstream gene, Dmrt1 (doublesex and mab-3 related transcription factor 1), of vertebrate sexual differentiation on the Y-chromosome. dmrt1 carries the DM domain, which is characterized as the zinc finger-like DNA-binding motif, and the DM domain was originally found in two separate phyla, insects and nematodes: D for doublesex in Drosophila melanogaster and M for male abnormal-3 in Caenorhabditis elegans. These two genes control sexual development in these invertebrates and, more interestingly, DM domain genes have a conserved role in sex differentiation across various phyla.

dmy was the second sex-determining gene to be identified among vertebrates, almost a decade after the discovery of the mammalian SRY/Sry (Sinclair et al., 1990 and Koopman et al., 1991). Though both dmy/dmrt1bY and SRY/Sry are functionally analogous, no structural similarities have been noticed between them. Unlike SRY/Sry, which appears to be unique to mammals, the involvement of DM-domain genes in sex determination appears to be more highly conserved, as evidenced by two recent discoveries. In Xenopus laevis, DM-W has been identified as the sex-determining gene (Yoshimoto et al., 2008). In contrast to dmy, DM-W is involved in ovarian determination. More recently, dmrt1 has been revealed as the male sex-determining gene of chickens (Smith et al., 2009), in which the male carries homomorphic chromosomes, ZZ, and two copies of dmrt1 likely are required for testicular differentiation, unlike in medaka. Thus, the DM domain is involved in the sex determination of animals belonging to three out of five classes that constitute the phylum chordata: fishes, amphibians, and birds.

Dmy has been found in only two species of Oryzias: Oryzias latipes (Matsuda et al., 2002 and Nanda et al., 2002) and O. curvinotus (Matsuda et al., 2003 and Kondo et al., 2004) and not in any other genus of fishes. Though the medaka follows an XX/XY heterogametic mode of sex determination, the X and Y chromosomes are homomorphic, unlike in mammals. To date, the only difference noted between X and Y chromosomes of O. latipes is the exclusive presence of dmy on the Y chromosome. Accordingly, a natural mutant XY fish in which there was a single insertion in exon 3 of dmy resulting in truncation of its protein showed a female phenotype (Matsuda et al., 2002). Similarly, another XY mutant female showed reduced expression of dmy and a high proportion of XY females were found among the offspring of this mutant female. In addition to these observations, which gave important clues about the sex-determining role of Dmy, collection of new data in recent years has provided further insights about the functions of Dmy protein.

In order to understand the role of Dmy during initiation of testicular differentiation in medaka, a functional analysis was carried out by knocking it down with engineered peptide nucleic acid (gripNA) (Paul-Prasanth et al., 2006). dmy-gripNA and (human cAMP response element–binding) hCREB-gripNA were injected into one-cell stage medaka embryos. Simultaneously, we treated another batch of the fertilized eggs with 17β-estradiol (E2) to compare the process of female sexual differentiation in these larvae with that of the knock-down larvae, as the mechanisms behind ovarian differentiation could be different. Ovarian differentiation in E2-treated XY larvae was found to be initiated in the presence of an unaltered Dmy (Suzuki, Nakamoto, Kato, & Shibata, 2005); however, the gonads of dmy-gripNA-XY larvae were expected to undergo ovarian differentiation like the normal genetic females as Dmy was not supposed to be present in knock-down embryos. The fry collected on the day of hatching were subjected to both histological (for germ cell counting) and whole-mount in situ hybridization analyses. The sex of the fry was assessed by genomic polymerase chain reaction (PCR) of the DNA extracted from the head or tail region of each fry, using common primers for dmrt1 and dmy. Some of the 0 days after hatching (dah) dmy knock-down XY fry had germ cell numbers comparable to those of the normal XX fry. However, the germ cell numbers in the E2-treated XY gonads at 0dah were found to be similar to those of the hCREB (control) gripNA-treated and normal XY gonads of the same stage (Figure 1.1). Whole-mount in-situ hybridization with the meiosis-specific marker gene, synaptonemal complex protein 3 (scp3), revealed that meiosis was induced in the gonads of the 0dah dmy knock-down (Figure 1.1) and E2-treated XY fry, but not in the control XY fry. These findings revealed that the mitotic and meiotic activities of the germ cells in the 0dah dmy knock-down XY larvae were similar to those of the normal XX larvae, suggesting that the microenvironment of these XY gonads is similar to that of the normal XX gonad, where Dmy is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active Dmy, even though it could initiate meiosis. This study showed that germ cells in the XY gonad can resume mitotic activity if dmy is knocked down. Corroborating this data, Herpin et al. (2007) found that injection of dmy/dmrt1bY morpholino resulted in increased rate of germ cell proliferation in XY embryos of medaka. These results demonstrate that Dmy is sufficient for male development in medaka, and suggest that the functional difference related to sex differentiation between the X and Y chromosomes in medaka is a single gene.

Over-expression of the 117-kb bacterial artificial chromosome (BAC) clone harboring the dmy genomic region in medaka embryos initiated testicular differentiation in the gonads of genetic females (Matsuda et al., 2007). Over-expression of dmy led germ cells in XX gonads into mitotic as well as meiotic arrests. In the dmy over-expression experiments, the total number of germ cells at 0dah was significantly reduced in both the XX and XY fry. In the 5dah XX fry injected with dmy, not only was the total germ cell number reduced but the number of germ cells at various stages of development was also reduced. As the total number of germ cells reflects the outcome of active mitosis, the reduced number of germ cells in the transgenic fry may be due to a signal or signals from the surrounding somatic cells that express dmy. The over-expression of dmy using the dmy genomic region induced sex reversal in XX gonads. However, since the genomic region used was large (117-kb), it could not be ruled out that a region within this 117-kb DNA segment but outside the dmy open reading frame (ORF) might be involved in the induction of sex reversal. Therefore, to investigate the ability of the dmyORF to induce sex reversal, we constructed over-expression vectors in which cytomegalovirus (CMV) promoter controlled the dmy ordmrt1 cDNA. These constructs were injected into one-cell-stage medaka embryos of the Hd-rR strain, and the injected embryos were reared until the secondary sexual characteristics became apparent. Female-to-male sex-reversed fish were obtained from the embryos that over-expressed dmy, whereas no sex-reversed fish were obtained from the embryos that over-expressed dmrt1. The sex-reversed medaka were fertile.

Very recent evidence (Otake et al., 2009) indicated that the exogenous dmy in the above experiments was located on linkage group (LG) 23 in one strain and 5 in another strain, whereas the endogenous dmy was originally located on LG 1. Male development in those fish carrying the heritable artificial chromosomes with exogenous dmy demonstrates that this gene is indeed involved in initiation of testicular differentiation. Localization of the signals for Dmy exclusively in the somatic cells sequestering the primordial germ cells (PGCs) of stage 36 XY embryos suggests that it may regulate further differentiation of PGCs directly or indirectly in these embryos to prevent female sex differentiation (T. Kobayashi, H. Kobayashi, & Nagahama, 2004). In mammals, the role of SRY/Sry is considered to be critical for the upregulation of Sox9 and thus Sertoli cell differentiation (Sekido et al., 2004, Kidokoro et al., 2005 and Sekido and Lovell-Badge, 2008). Recent evidence from mice has elucidated the molecular mechanism involved in this process. Sry along with steroidogenic factor-1 (SF-1) bind to multiple elements within the Sox9 gonad-specific enhancer region on its promoter in order to upregulate Sox9 expression in a male-specific manner (Sekido & Lovell-Badge, 2008). Loss- and gain-of-function experiments have shown that Dmy is necessary and sufficient for initiating testicular differentiation in the primordial gonads of genetically male medaka (Paul-Prasanth et al., 2006 and Matsuda et al., 2007). However, the molecular mechanisms underlying the action of Dmy remain largely unknown, even after more than half a decade since its discovery, mainly because of lack of knowledge regarding its target genes. Ample evidence collected hitherto indicates that this gene is involved directly or indirectly in the regulation of germ cell proliferation. No other clues have revealed the precise function of Dmy.

Other species in which intensive searches for an SD gene are being carried out include tilapia, zebrafish, fugu (Takifugu rubripes), three-spined stickleback (Gasterosteus aculeatus), and rainbow trout; however, to date studies have not pinpointed a single factor as the SD gene in these species, although several candidate genes were identified. Studies have been able to locate only the SD region, but the genes are not yet deciphered (fugu: Kikuchi et al. (2007); rainbow trout: Woram et al. (2003) and Alfaqih, Brunelli, Drew, and Thorgaard (2009); three-spined stickleback: Peichel et al. (2004), Ross and Peichel (2008), Ross et al., 2009 and Kitano et al., 2009 and Leder et al. (2010)). Thus, the status of dmy as the only SD gene among fishes continues.

3. Sexual Differentiation

Gonadal morphogenesis is initiated soon after the PGCs are translocated to the gonadal primordium. The process of morphogenesis is initiated by species-specific sex-determining mechanisms. In gonochoristic teleosts such as tilapia, medaka, etc., PGCs start to proliferate (medaka: stage 38 (Kobayashi et al., 2004); tilapia: 8dah (Kobayashi, Kajiura-Kobayashi, Guan, & Nagahama, 2008)) female-specifically and enter into meiosis, giving a distinct morphology to the gonads of genetic females. On the other hand, genetic males of these species carry gonads at this time that appear undifferentiated at the morphological level as the PGCs remain mitotically and meiotically silent.

3.1. Testicular Differentiation

In most fish species, testicular differentiation remains morphologically inconspicuous during initial stages of gonadal development. In the case of medaka, the gonads of genetic males look undifferentiated up to 10dah, because the germ cells remain mitotically and meiotically quiescent. At 10dah, the gonads in this species show the first morphologically differentiated testis-specific structure, the acinus, which is the precursor of the seminiferous tubule. This process is comparable to the process of testicular cord formation in mammals. In general, morphologically discernable testicular differentiation is delayed among fishes but there exists much diversity in the developmental stage at which the presumptive testes exhibit the first sign of morphological sex differentiation. However, accumulating data from medaka indicate that the first event during the course of gonadal differentiation in fishes is the differentiation of Sertoli cells.

Numerous studies on fishes have shown that treatment of XX larvae with androgens induces sex reversal toward testicular development. However, no direct experimental evidence has been provided to prove whether endogenous androgens have an essential role in initial testicular differentiation in fishes. In tilapia, mRNAs of most steroidogenic enzymes such as cholesterol side-chain cleaving enzyme (P450scc = CYP11A1), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxylase (P45017α = CYP17a1), and 17β-hydroxysteroid dehydrogenase type 1(17β-HSD1) were constantly detected in XY gonads at 5–7dah (Ijiri et al., 2008). However, unlike in XX gonads, in XY gonads there were no further increases in these levels at 10–25dah. Another important observation in this study is that the expression of cytochrome P450 11β-hydroxylase (P45011β = CYP11B2)—which contributes to the synthesis of 11-ketotestosterone (11-KT), the most potent androgen in teleost fishes, from testosterone (T)—is not detected in either XX or XY gonads at 5–25dah. These findings may indicate that 11-KT is not produced in tilapia gonads until 25dah and, thus, androgens do not appear to play a major role in testicular differentiation of this species. In contrast, at 70dah, the mRNA levels of four steroidogenic enzymes including P45011β are higher in XY gonads than in XX gonads (Ijiri et al., 2008). As spermatogenesis is initiated during this period, increased steroidogenic activity of XY gonads may be involved. A recent investigation in rainbow trout confirmed that initiation of testicular differentiation does not require androgen production (Vizziano, Randuineau, Baron, Cauty, & Guiguen, 2007). Further studies are necessary to confirm whether androgen is involved in testicular differentiation in fishes or not.

Our recent studies revealed a new factor, gonadal–soma-derived factor (Gsdf), associated with testicular differentiation in medaka (Shibata et al., 2010) (Figure 1.2). Originally identified from rainbow trout as a unique member of the transformation growth factor superfamily, Gsdf is a comparatively new player in the field of sexual differentiation of fishes (Sawatari, Shikina, Takeuchi, & Yoshizaki, 2007). Phylogenetic analyses encompassing Gsdfs from several other fish species such as fugu, zebrafish, three-spined stickleback, and medaka, along with rainbow trout, have revealed that this gene is specific to the fish lineage. There are two forms of Gsdf in rainbow trout—Gsdf1 and -2—while only one has been found in medaka (Lareyre et al., 2008).

Unlike dmy, expression of the gsdf gene is found in the somatic cells surrounding the germ cells, not only in the XY gonads but also in the XX gonads, especially in granulosa cells around previtellogenic oocytes. However, prior to the onset of early signs of testicular differentiation, a rise is noticed in expression levels of gsdf in XY gonads at around six days post-fertilization (dpf) (Shibata et al., unpublished). Such a rise is not observed in XX gonads; rather, the expression of gsdf becomes clearly visible in XX gonads only from 10dah. Colocalization studies have demarcated two types of somatic cells in the XY gonads: one type that expresses only dmy protein and another type showing expression of both dmy and gsdf. The cells expressing both dmy and gsdf could be undergoing differentiation into Sertoli cells, and it is possible that this process precedes all the other morphological changes that have been recorded so far. Therefore, higher levels of gsdf mRNA in somatic cells of XY gonads than that of the XX gonads could be critical for the masculinization of XY somatic cells. Gsdf seems to play a role equally important to Dmy in driving the XY gonads towards testicular differentiation. Expression of dmy is linked to the genotype of the gonads, not to the phenotype. Accordingly, in XY females generated by exogenous E2 treatment, dmy expression has been detected in granulosa cells, suggesting that the presence of dmy is not detrimental to ovarian development (Suzuki et al., 2005). In contrast to dmy, expression of gsdf is closely linked to the gonadal phenotype, because E2 administration downregulated the expression levels of gsdf mRNA in exposed XY embryos (Shibata et al., unpublished). It is possible that Gsdf is the first target of estrogenic chemicals in the male sex-differentiation cascade in medaka.

Among fish species, dmrt1 shows a consistent pattern of expression with an exception in medaka. In tilapia (Guan et al., 2002 and Ijiri et al., 2008), rainbow trout (Vizziano et al., 2007), and zebrafish (Jørgensen, Morthorst, Anderson, Rasmussen, & Bjerregaard, 2008), expression of dmrt1 coincides with initiation of testicular differentiation (Figure 1.3). Conversely, in medaka, dmrt1 expression becomes testis-specific only at around 20dah, which is well down the pathway of the initiation of testicular differentiation (Kobayashi et al., 2004). More interestingly, during temperature-induced masculinization of genetic females with no dmy/dmrt1bY in their genomes, dmrt1 expression is upregulated in 50% of the exposed embryos from stage 36, which is much earlier than its usual temporal expression pattern, and, concordantly, 40% of embryos developed testes, suggesting that Dmrt1 in this case acts as the master regulator to initiate testicular differentiation in the absence of Dmy (Hattori et al., 2007).

Most recently, in-vitro and in-vivo functional analyses of Dmrt1 in tilapia revealed the possible mechanism by which this gene orchestrates testicular differentiation in genetic males of this species and probably other vertebrates (Wang et al., 2010). Overexpression of dmrt1 driven by CMV promoter in genetically female tilapia embryos caused retardation of ovarian cavity formation, follicular degeneration, and partial to complete sex reversal. Transcription of the ovarian aromatase gene, P450aro (= CYP19A1), was repressed in these embryos, resulting in reduced serum E2 levels. In-vitro analysis revealed that Dmrt1 protein could directly bind to the palindrome sequence, ACATATGT, on the promoter region of P450aro, suggesting further that Dmrt1 probably induces the male pathway through its direct blocking action on estrogen production.

Medaka and tilapia have two sox9 genes, sox9a1 and sox9a2. Of these, only sox9a2 is linked with testicular differentiation in these fishes (Nakamoto et al., 2005 and Ijiri et al., 2008). Although sox9a2 is expressed in the somatic cells in the gonads of both XY and XX fishes from stage 33, which is before the onset of Dmy expression, its expression becomes male-specific only from 10dah. This occurs at around the same stage as acinus formation, leading to the hypothesis that Sox9a2 is involved in the process of acinus formation (Nakamoto et al., 2005 and Nakamura et al., 2008). Expression of the sex-determining gene, SRY/Sry, and the upregulation of SOX9/Sox9 in mammals are closely associated with testicular cord formation, while timings of the initiation of expression of the sex-determining gene of medaka, dmy, and acinus formation are not synchronized, leading to the assumption that these two processes are not directly correlated. However, unlike Sry, dmy expression does not decline in XY gonads until at least 10dah, suggesting that dmy and sox9a2 might be involved with Sertoli cell differentiation and first male-specific structure formation, which are very prolonged processes in medaka. Hence, it is still valid to consider sox9a2 as having a role during early stages of male gonadogenesis in fishes. However, the mechanisms or other cofactors involved remain largely unexplored.

In addition to this purported role, sox9a2 is useful in tracing the lineage of somatic cells in both the testis and ovary of medaka (Nakamura et al., 2008). In sox9a2-EGFP (enhanced green fluorescent protein), transgenic medaka testes sox9a2-expressing cells develop as Sertoli cells, while in ovaries the same cells develop as granulosa cells. In other species of fishes such as rainbow trout (Vizziano et al., 2007) and the rice field eel (Monopterus albus) (Lu et al., 2003 and Zhou et al., 2003), sox9a2 is associated with testicular differentiation.

Anti-Müllerian hormone (AMH) is a glycoprotein belonging to the transforming growth factor-β superfamily. A major function of AMH in mammals is to mediate regression of Müllerian ducts in males. Targeted mutagenesis has shown that AMH is not required for testicular determination in mice (Behringer, Finegold, & Cate, 1994). Importantly, unlike other vertebrates, fishes do not have Müllerian ducts, indicating a different role for this gene in fishes. In tilapia, amh mRNA starts to be expressed in indifferent gonads of both sexes, and the expression is upregulated in male gonads but downregulated in female gonads (Ijiri et al., 2008). The sexually dimorphic expression of amh in fish gonads during sex differentiation also has been reported in the Japanese flounder (Paralichthys olivaceus) (Yoshinaga et al., 2004). In these studies, amh expression starts in supporting cells of indifferent gonads of both sexes. In medaka, a mutation in the receptor for Amh, AmhrII, resulted in sex reversal of the gonads in half of the XY fish that had the mutant gene (Morinaga et al., 2007).

3.2. Ovarian Differentiation

Ovaries in adult fishes possess oocytes at various stages of development, granulosa cells, theca cells, and blood vessels. During embryogenesis in most fish species, the primordial gonads of genetic males and females are morphologically indistinguishable. In zebrafish, all the hatched fry, regardless of genetic sex, possess gonads with primary oocytes. Later in presumptive testes, the primary oocytes undergo apoptosis, giving way to the development of testicular tissue. On the other hand, in medaka, germ cells start to proliferate and increase in number first in the presumptive ovaries. Germ cell proliferation is followed by entry into a clonal mode of proliferation where dividing germ cells form clusters. These germ cells then enter into meiotic prophase and start to develop as oocytes. Thus, the ovaries in medaka acquire a distinct morphology at very early stages of development. An earlier study postulated that, in mammals, initiation of ovarian formation occurred by default because germ cells that migrated to areas other than gonadal anlagen developed as oocytes (McLaren & Southee, 1997). However, recent evidence has proved that ovarian formation in mammals also is regulated actively at the genetic level. Genes such as Wnt4 (wingless-type MMTV integration site family, member 4) (Kim et al., 2006), Foxl2 (Forkhead box L2) (Ottolenghi et al., 2007 and Garcia-Ortiz et al., 2009), and Rspo1 (Roof plate-specific Spondin 1,R-Spondin 1) (Parma et al., 2006) were found to be critical for ovarian differentiation and morphogenesis in mammals. In fishes, there have not been many studies revealing the molecular mechanisms underlying ovarian formation.

In several fish species (e.g., tilapia), ovarian differentiation is primarily under the control of E2 (Yamamoto, 1969, Piferrer et al., 1991, Nakamura et al., 1998, Kobayashi et al., 2003 and Guiguen et al., 2010) (Figure 1.4). The gene Cyp19 codes for the steroidogenic enzyme, P450aro, responsible for estrogen production in steroidogenic tissues such as the brain, gonads, adrenal gland, etc. In fishes, there are two forms of P450aro, the ovarian form and the brain form, encoded by two different genes, cyp19a1a and cyp19a1b; cyp19a1a is expressed in the ovaries, while cyp19a1b is found predominantly in the brain. Cyp19a1a-expressing cells were apparent near blood vessels in the primordial gonads of tilapia at 7dah (Sakai, Kobayashi, Matsuda, & Nagahama, 2008). Blocking production of E2 through inhibition of P450aro caused testicular differentiation in XX tilapia fry (Kobayashi, Kajiura-Kobayashi, & Nagahama, 2003), demonstrating that E2 is the natural inducer of ovarian differentiation in this species. More recent investigations in tilapia revealed that cyp19a1a was one of the first genes to exhibit a sexually dimorphic expression pattern prior to any morphological sex differentiation in the primordial gonads (Ijiri et al., 2008) (Figure 1.3). cyp19a1a transcripts were abundant in the XX females of this species at 5dah, while XY fry of the same stage did not show any such rise in the expression levels of cyp19a1a. Genes coding for other steroidogenic enzymes involved in the E2 pathway are expressed during the same time frame. As expression of cyp19a1a signifies the production of E2, all these data collectively confirm the notion that E2 is critical for female sexual differentiation in tilapia. Further, in rainbow trout, administration of 17α-ethinylestradiol during the first two months of development upregulated early female genes including cyp19a1a and foxl2, confirming that E2 has a leading role in the induction of ovarian differentiation in this species also (Vizziano-Cantonnet et al., 2008; Guigen et al., 2010). In zebrafish, cyp19a1a expresses in excess in those gonads that will be transformed into ovaries (Jørgensen et al., 2008). It has been suggested that, in presumptive testes, decrease in cyp19a1a expression leads to apoptosis of the oocytes, eventually resulting in the emergence of testicular tissue (Wang & Orban, 2007).

For sex steroids to have their effects during gonadal development, their receptors must also be present. In tilapia, all three types of estrogen receptor (ER) (ERα, ERβ1, and ERβ2) were consistently expressed at relatively high levels at 5dah in gonads of both sexes, with no sexually dimorphic expression until 35dah (Ijiri et al., 2008). Similar levels of expression of ERα in undifferentiated XX and XY gonads were reported in medaka (Kawahara, Omura, Sakai, & Yamashita, 2003) and rainbow trout (Guiguen et al., 1999). These results indicate that E2 synthesized in female gonads mediates female sexual differentiation by stimulating development of undifferentiated XX gonads through ERs. Expression of ERs in XY gonads early during differentiation explains the susceptibility of males to feminization by exogenous E2.

Nevertheless, the role of E2 in female sexual differentiation of medaka remains unresolved. Although exogenous E2 administration successfully induces ovarian differentiation in the gonads of genetic males, treatment with the P450aro inhibitor fadrozole during embryogenesis does not cause sex change in genetic females (Kawahara & Yamashita, 2000). Expression of cyp19a1a in medaka is initiated only after morphological sex differentiation has been initiated in the presumptive ovaries. However, in this experiment, the embryos were exposed to fadrozole from the day of fertilization to the day of hatching only and, in medaka, cyp19a1a transcripts are found only from 5dah (Suzuki, Tanama, Nagahama, & Shibata, 2004). Whether fadrozole remains in the hatched fry even after the withdrawal of the drug was not confirmed in this study. Therefore, it is necessary to ascertain whether extending the treatment until after the initiation of cyp19a1a expression might cause sex reversal. Alternatively, next-generation P450aro inhibitors such as letrozole, anastrozole, and exemestane also could be tried to reverse the phenotypic sex of genetic females.

Foxl2 is a transcription factor belonging to the forkhead family. In goats, Foxl2 has been implicated in ovarian differentiation, possibly by regulating the transcription of the CYP19 gene (Pailhoux et al., 2005 and Pannetier et al., 2006). Similarly, investigations using teleosts have revealed that the role of Foxl2 in ovarian differentiation is conserved among vertebrates. Initiation of foxl2 expression has been linked to ovarian differentiation in a number of teleosts, e.g. tilapia (Wang et al., 2004, Wang et al., 2007 and Ijiri et al., 2008) (Figure 1.4), medaka (Nakamoto et al., 2006 and Nakamoto et al., 2009), rainbow trout (Baron et al., 2004 and Baron et al., 2005), and Silurus meridionalis (Liu, Zhang, & Wang, 2008). foxl2 mRNA was detected specifically in the somatic cells in presumptive ovaries, and overexpression of a dominant negative mutant form of Foxl2 in tilapia demonstrated that this factor was critical for ovarian differentiation and morphogenesis (Wang et al., 2007). Overexpression of foxl2 genomic DNA in genetically male embryos of tilapia resulted in partial to complete sex reversal of the gonads into ovaries. Promoter analysis revealed that Foxl2 regulates the transcription of cyp19a1a either by directly binding to the promoter of the latter or through an interaction with Ad4bp/sf-1 (Figure 1.5). Thus, in tilapia, Foxl2 is essential for ovarian differentiation and morphogenesis.

Very recently, conditional ablation of Foxl2 in adult female mice resulted in transdifferentiation of the granulosa cells into Sertoli cells, showing the essential role of FOXL2 in maintaining the ovarian phenotype even after completion of sexual development (Uhlenhaut et al., 2009). This study demonstrated active repression of the male pathway in granulosa cells by FOXL2. Exploration of a similar role for FOXL2 in ovarian sex differentiation and maintenance of ovarian phenotype among fishes might provide insight into the labile nature of sex determination among fishes.

Ad4bp/sf-1 is involved in sexual differentiation and early gonadogenesis in vertebrates. Recently, in mice, association of Sry with SF-1 was found to be critical for testicular differentiation (Sekido & Lovell-Badge, 2008). On the other hand, studies involving the role of Ad4bp/sf-1 role in fish sexual differentiation linked it only with initiation of ovarian differentiation. During early stages of ovarian differentiation in tilapia XX gonads, interaction between Ad4bp/sf-1 and Foxl2 increases the transcription of cyp19a1a, accelerating E2 production in a female-specific manner (Wang et al., 2007) (Figure 1.4). Foxl2 interacts with the ligand-binding domain of Ad4bp/sf-1 through its forkhead domain to form a heterodimer, which further enhances cyp19a1a transcription, demonstrating that, though ad4bp/sf-1 does not exhibit a sexually dimorphic expression pattern in the gonads during early stages of sexual differentiation, it plays pivotal roles in sex-specific development of the gonads through its association with other sex-specific factors. Further research on the mechanisms involved in testicular differentiation of tilapia revealed that Dmrt1 repressed the female pathway in XY gonads by suppressing the basal as well as the Ad4bp/sf-1-activated transcription of cyp19a1a, proving that Ad4bp/sf-1 is critical for ovarian differentiation of tilapia (Wang et al., 2010).

A few studies associate Wnt4 with initiation of ovarian development in fishes. In the protandrous black porgy (Acanthopagrus schlegeli), wnt4 expression is closely linked with ovarian development (Wu and Chang, 2009 and Wu et al., 2009). In this species all the fry first develop as males and, after three years, there is male-to-female conversion, during which WNT4, along with Cyp19a1a and Foxl2, is instrumental in the initiation of ovarian tissue development. Nevertheless, the role of Wnt4 in female sex determination and ovarian development among fishes remains largely uncertain.

4. Environmental Effects on Sex Determination and Differentiation

Many fish species exhibit ESD. In these fishes, temperature, pH, or social factors determine the developmental fate of the bipotential gonad during the critical period of larval development (Barroiller & D’Cotta, 2001; see also Chapter 8, this volume). Temperature-dependent sex determination (TSD) was reported for the first time in fishes in the Atlantic silverside (Menidia menidia) (Conover & Kynard, 1981). Menidia peninsulae and M. menidia are the only two species that exhibit TSD in the wild (Ospina-Alvarez & Piferrer, 2008). The remaining species were tested for TSD only under laboratory conditions and it is still unclear whether they adopt TSD in their natural habitats. In general, three different patterns of sex ratio responses due to thermal influences have been observed among fishes: (1) higher incidence of males at high temperatures, (2) higher incidence of females at low temperatures, and (3) both low and high temperatures resulting in more males, whereas an intermediate temperature produces both males and females in equal proportions (Devlin and Nagahama, 2002 and Munch and Conover, 2004). Under laboratory conditions, TSD occurs in cichlids, sea bass (Dicentrarchus labrax), atherinids, and many other teleosts (Ospina-Alvarez & Piferrer, 2008). Thermal influences on sexual differentiation have been demonstrated among teleosts, even in species with GSD (Koshimizu, Strüssman, Okamoto, Fukuda, & Sakamoto, 2010). Gonads of tilapia (as reviewed in Nagahama et al., 2004 and Baroiller et al., 2009) and medaka (Hattori et al., 2007), two species with proven GSD mechanisms, were susceptible to thermal effects during critical periods of ontogenesis.

Demonstration of TSD in species with GSD mechanisms poses the question of whether fishes do possess actual TSD mechanisms like reptiles. A reinvestigation to assess the prevalence of TSD among fishes has revealed that TSD is not as widely spread as was previously thought (Ospina-Alvarez & Piferrer, 2008). In this study, 59 species with known TSD mechanisms were rechecked at a range of temperatures during development under natural conditions (range of temperature during development in the wild (RTD)); RTD is more ecologically relevant and, therefore, thermal effects on sex determination should ideally occur in a true TSD species within the range of their RTDs. Using this criterion, it was found that approximately 75% (19 out of 26) of the species reported as having TSD did not actually possess TSD. Instead, sex determination and further sexual differentiation in the gonads of these fishes were the result of thermal effects, especially extreme temperature fluctuations outside their RTDs, on their GSD. These authors concluded that there exists only one general pattern of sex ratio response to temperature, not three as described above, and suggested that fishes in general do not possess a true TSD mechanism, but sexual development in fishes is only sensitive to temperature.

Unlike reptiles, where orthologs for most of the factors involved in the mammalian sex determination and differentiation cascade have been found (as reviewed in Georges, Ezaz, Quinn, & Sarre, 2010), information on genetic factors involved in purported TSD of fishes largely remain unknown. The main factor that has been reported to be involved in TSD of fishes is the gene that encodes P450aro (cyp19a1a). At male-producing temperatures, transcripts of cyp19a1a diminish, driving the gonads toward testicular differentiation. Female-producing temperatures cause an increase in the expression levels of cyp19a1a, resulting in ovarian differentiation. In the Japanese flounder, high water temperature caused sex reversal from genetic females to phenotypic males and suppression of cyp19a1a expression. Further, follicle-stimulating hormone (FSH) signaling and Foxl2 were implicated in the regulation of cyp19a1a transcription during temperature-dependent sexual determination in this species (T. Yamaguchi, S. Yamaguchi, Hirai, & Kitano, 2007). In the atherinid Odontesthes bonariensis (pejerrey fish), with known TSD, cyp19a1a expression was closely correlated with ovarian formation in fish reared at feminizing temperatures (Fernandino et al., 2008). Conversely, masculinizing temperatures reduced the expression levels of this gene, while mixed-sex-producing temperatures showed a bimodal pattern of expression, indicating the involvement of cyp19a1a in the TSD of this species (Karube et al., 2007). In addition, a rise in dmrt1 expression was correlated with male-producing temperatures (Fernandino et al., 2008 and Lee et al., 2009). Exposure to high temperature during early gonadal development in pufferfish (T. rubripes) caused degeneration of germ cells and subsequent masculinization of the somatic cells (Lee et al., 2009). Whether this is a common mechanism during high-temperature-induced testicular differentiation is yet to be ascertained.

Regulation of the genes involved in TSD is not well understood. However, a correlation between circulating levels of insulin-like growth factor-1 (IGF-1) and changes in specific growth rate of body size at varying temperatures and different nutritional status has been demonstrated in the southern flounder (Paralichthys lethostigma). Plasma IGF-1, muscle IGF-1, and specific growth rate were suppressed at high rearing temperatures relative to low rearing temperatures (Luckenbach et al., 2007). Further analysis of this gene might reveal a possible correlation between TSD and the endocrine-growth regulatory axis.

Though pH has been shown to influence sex determination in fishes belonging to the genera Apistogramma and Pelvicachromis (Rubin, 1985; Röemer & Beisenherz, 1996), sex ratio response to pH is in general less obvious in comparison to temperature. In the molly Peocilia sphenops, a combined effect of pH and temperature was found to skew the sex ratio towards one sex (Baron, Buckle, & Espina, 2002). However, molecular mechanisms underlying pH-induced sex determination have not been studied in detail.

Socially controlled sex determination was explained for the first time in the fairy basslet (Gramma loreto) (as reviewed in Devlin & Nagahama, 2002). Social factors were found to play a major role in determining the sex in several teleost hermaphroditic species such as wrasses, damsel fishes, parrotfishes, and gobies (see also Chapter 8, this volume). Relative size and sex ratio are two prominent social factors used among fishes for sex determination and differentiation. In the midas cichlid (Cichlasoma citrinellum), sex is determined by relative size of an individual during its juvenile stage (Francis & Barlow, 1993). In the protogynous epinepheline Cephalopholis boenak, all isolated, single juveniles differentiated as males, and they clearly showed a faster growth rate than differentiating females and undifferentiated juveniles. In the bluehead wrasse (Thalassoma bifasciatum), population size during juvenile stages has an influence on the development of the primary males (Munday, Wilson, & Warner, 2006). Likewise, in false clown anemonefish (Amphiprion ocellaris) longterm social interactions promote male sexual development and growth, and 11-KT has been implicated as having a role in this process (Iwata, Nagai, Hyoudou, & Sasaki, 2008). The serial sex-changing fish Trimma okinawae possesses both testis and ovary at the same time; however, only one gonad remains fully developed (active) at a time, while the other remains in an underdeveloped state (inactive). According to their social status, these fish are capable of changing their gonadal phenotype back and forth several times. Recent evidence revealed that, soon after the change in the social status of these fish, GTH receptor genes switch their location of expression from the active gonad to the inactive gonad and initiate the development of the inactive gonad (Kobayashi et al., 2009). This fish is a good model for the study of the role of the brain in mediating gonadal sex differentiation in response to environmental stimuli.

5. Conclusions and Future Directions

Genetic and molecular approaches have been used to facilitate the studies in the field of fish sex determination/differentiation. Figure 1.6 summarizes the factors involved in sex determination/differentiation among fishes. Discovery