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Keywords:

  • sex determination;
  • teleost fish;
  • TGF-β;
  • turnover of sex chromosomes

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

Although the molecular mechanisms underlying many developmental events are conserved across vertebrate taxa, the lability at the top of the sex-determining (SD) cascade has been evident from the fact that four master SD genes have been identified: mammalian Sry; chicken DMRT1; medaka Dmy; and Xenopus laevis DM-W. This diversity is thought to be associated with the turnover of sex chromosomes, which is likely to be more frequent in fishes and other poikilotherms than in therian mammals and birds. Recently, four novel candidates for vertebrate SD genes were reported, all of them in fishes. These include amhy in the Patagonian pejerrey, Gsdf in Oryzias luzonensis, Amhr2 in fugu and sdY in rainbow trout. These studies provide a good opportunity to infer patterns from the seemingly chaotic picture of sex determination systems. Here, we review recent advances in our understanding of the master SD genes in fishes. Developmental Dynamics 242:339–353, 2013. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

The existence of female and male sexes is a conserved feature in diverse vertebrate taxa. However, there is conservation as well as diversity in the developmental mechanisms that control this determination, ranging from genetic to environmental triggers (Ospina-Álvarez and Piferrer, 2008). Even in the case of animals where sex is determined by genetic factors, the molecular processes that lead to the formation of either testis or ovary are evolutionary labile (Capel, 2000; True and Haag, 2001; Volff et al., 2007). For example, while sex determination in most therian mammals is triggered by the testis-determining gene, Sry, this role is played by Dmy/dmrt1bY and DMRT1 in medaka (Oryzias latipes) and chicken, respectively (Fig. 1) (Sinclair et al., 1990; Matsuda et al., 2002; Smith et al., 2009). In addition, sex determination in Xenopus laevis is triggered by the ovary-determining gene, DM-W (Yoshimoto et al., 2008). The identification of these master sex-determining (SD) genes in the past 2 decades has provided valuable insights into our understanding of the mechanisms of sex determination and their evolution.

image

Figure 1. Known sex-determining genes in tetrapods and teleost fishes and the phylogeny of the species where they are found. Different sex-determining genes have been identified in different lineages. Note that the master sex-determining genes have not been identified in fishes regarded as model species in developmental biology: zebrafish (Bradley et al., 2011; Anderson et al., 2012) and evolutionary biology: stickleback (Ross et al., 2009) and tilapia (cichlid) (Roberts et al., 2009; Eshel et al., 2012). The phylogeny of tetrapods and teleost is based on Kumar and Hedges (1998) and Setiamarga et al. (2009), respectively.

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Teleost fishes represent nearly half of all extant vertebrates (Nelson, 2006), and show a wide variety of sex determination mechanisms. Their sex can be determined by genetic factors, environmental factors, or both (Devlin and Nagahama, 2002; Ospina-Álvarez and Piferrer, 2008; Penman and Piferrer, 2008). The genetic sex determination includes monofactorial systems involving a single master SD gene such as Dmy in medaka and polyfactorial systems involving several genes on multiple chromosomes (Vandeputte et al., 2007; Penman and Piferrer, 2008; Bradley et al., 2011; Anderson et al., 2012; Liew et al., 2012). Fish also show different sexual reproductive strategies, ranging from hermaphroditism to gonochorism (Helfman et al., 1997). Therefore, they form an attractive group of organisms to study the evolution of sex determination systems and sex chromosomes. Recently, four novel SD genes (or strong candidates) in vertebrates were reported, all of them in fishes: amhy in the Patagonian pejerrey (Odontesthes hatcheri), Gsdf in Oryzias luzonensis (a relative of medaka), Amhr2 in fugu (Takifugu rubripes), and sdY in rainbow trout (Oncorhynchus mykiss) (Hattori et al., 2012; Kamiya et al., 2012; Myosho et al., 2012; Yano et al., 2012).

Fish sex determination and differentiation have been extensively reviewed (Nakamura et al., 1998; Devlin and Nagahama, 2002; Schartl, 2004a; Volff et al., 2007; Penman and Piferrer, 2008; Siegfried, 2010). Therefore, this short review focuses on the recent advances in our understanding of the master SD genes in fish species in which sex is mainly determined by monogenic factors. We first provide a brief overview of sex determination in medaka and sex chromosome evolution among closely related species, because medaka was the first nonmammalian vertebrate in which the master SD gene was identified (Matsuda et al., 2002; Nanda et al., 2002), and this species has provided a framework for understanding genetic sex determination in fish species. Next, we summarize four novel vertebrate SD genes, or strong candidates, except for the gene in O. luzonensis, a congeneric of medaka, in the order of publication. We then discuss how these findings have formulated our views of sex determination and sex chromosome evolution in vertebrates. Finally, we comment on some theoretical models that describe transitions among SD mechanisms and the turnover of sex chromosomes.

SEX DETERMINATION IN MEDAKA

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

Medaka is a small freshwater fish species that lives in inland waters of East Asia (Takehana et al., 2003, 2004). It is perhaps the best studied species among nonmammalian vertebrates with respect to sex determination. Because sex determination in medaka has been well reviewed elsewhere (Matsuda, 2003, 2005; Schartl, 2004a; Kondo et al., 2009; Herpin and Schartl, 2009), we provide only a brief overview here.

Medaka has an XX/XY sex determination system in which the Y chromosome harbors the master SD gene, Dmy/dmrt1bY (DM domain gene on the Y chromosome/doublesex and mab-3 related transcription factor 1b on the Y chromosome) (Matsuda et al., 2002; Nanda et al., 2002). This gene is a duplicated copy of the Dmrt1 gene (Nanda et al., 2002). Approximately 10–18 million years ago (Mya), the duplicated fragment containing Dmrt1 was inserted into another chromosome in an ancestral species of medaka, and this chromosome became the Y chromosome, while its homologous chromosome became the X (Kondo et al., 2004, 2006; Herpin and Schartl, 2009). Henceforth, this duplicated gene in the Y chromosome will be referred to as Dmy in this review.

Dmrt1 belongs to the Dmrt gene family, which is characterized through the presence of a DM-domain (doublesex (dsx) and male abnormality-3 (mab-3) domain). It is expressed in the embryonic gonads of many vertebrates (Herpin and Schartl, 2011) and, although it is not the primary sex determination gene in all species, it has been shown to be essential for testicular differentiation in mammals, chicken, and medaka (Raymond et al., 2000; Smith et al., 2009; Masuyama et al., 2012).

It is now known that medaka Dmy is expressed in the somatic cells surrounding germ cells in males, and its expression is a necessary and sufficient condition for triggering testicular development in bipotential gonads (Matsuda et al., 2002, 2007). After the onset of Dmy expression, the first sign of morphological differences between male and female gonads is seen in the number of germ cells 1 or 2 days before hatching (Fig. 2) (reviewed in Matsuda, 2005; Kondo et al., 2009; Herpin and Schartl, 2011). From this stage, the proliferation of germ cells in female embryos becomes more active than that in male embryos (Saito et al., 2007). The first sex-specific features of somatic cells are the acinous structures in males and ovarian follicles in females (Matsuda, 2005). At the gene expression level, Dmrt1 is expressed in the differentiating testis, whereas Foxl2 and aromatase are expressed in the differentiating ovary (Fig. 2) (Matsuda, 2005; Nakamoto et al., 2006; Siegfried, 2010).

image

Figure 2. Illustration showing the development of gonads in medaka. The black horizontal line at the top represents a time line of developmental age, given in days post hatching. Testis-specific or enriched gene expression is represented by the blue bars (summarized in Matsuda, 2005), whereas ovary-specific or enriched expression is denoted by the red bars (Nakamoto et al., 2006). Genes expressed in both sexes (Sox9b and Amh, summarized in Nakamura et al., 2012a) are shown by gray bars. Landmarks of morphological events in gonadal sex differentiation (reviewed in Matsuda, 2005) are shown below the corresponding gene expression bars.

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Although the sex-specific regulation of morphogenesis and gene expression in the gonads obviously lie downstream of Dmy, there are still many unanswered questions about the detailed molecular processes connecting these events to Dmy function. For example, although Dmy regulates germ cell proliferation negatively in males (Paul-Prasanth et al., 2006; Herpin et al., 2007), the molecular mechanisms by which the Dmy-expressing supporting cells interact with Dmy-negative germ cells remain elusive. Indeed, despite the considerable attention given to Dmy, its direct downstream targets have yet to be characterized, except that Dmy itself is a target (Herpin et al., 2010). Recently, strong candidates for the direct downstream targets of DMRT1 in mice have been reported (Matson et al., 2011). The list of these genes help identify the targets of Dmy in medaka and thus understand the regulation of sex determination directed by the Dmy gene.

RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

Sex chromosomes and the SD gene they harbor have been maintained in therian mammals and birds, respectively, for over one hundred million years (Veyrunes et al., 2008). However, there is accumulating evidence of turnover in these chromosomes in diverse vertebrate taxa such as fishes, reptiles, and amphibians (Ezaz et al., 2009; Charlesworth and Mank, 2010; Graves and Peichel, 2010; Bewick et al., 2011). A series of papers on medaka and its closely related congenerics has provided solid evidence indicating that the turnover in the sex chromosomes and the SD gene can occur in a range of just a few million years (Kondo et al., 2004; Takehana et al., 2005, 2007a, 2007b, 2008; Tanaka et al., 2007).

Sex chromosomes have been identified in eight Oryzias species by means of genome-wide linkage analysis using expressed sequence tag (EST) markers from medaka (Fig. 3A) (summarized in Takehana et al., 2008). Among these species, Dmy acts as the SD gene only in medaka and O. curvinotus (Matsuda et al., 2003; Kondo et al., 2004; Takehana et al., 2008). Therefore, a master SD gene other than Dmy apparently triggers the SD pathway in the other Oryzias species. Moreover, these comparative analyses also identified seven non-orthologous sex chromosomes among the species studied (Fig. 3A). While six species have an XY system, two possess a ZW system (Fig. 3A). Therefore, medaka and its closely related species provide an excellent model group for investigating the mechanisms that led to the rapid turnover of sex chromosomes.

image

Figure 3. Turnover of sex chromosomes and sex-determining genes in Oryzias species and pufferfishes. A: Phylogeny of selected members of Oryzias species. Next to the phylogeny are shown the sex-determining (SD) gene involved, the chromosome in medaka (O. latipes) homologous to the sex chromosome of each species, and the corresponding mode of heterogametic sex-determination. The downward facing arrow in the tree indicates the origin of Dmy, while the upward facing arrow denotes the replacement of the sex-determining gene Dmy by Gsdf (Myosho et al., 2012). The phylogenetic position and data on sex-determining loci were derived from Takehana et al. (2008). The divergence time between medaka and the common ancestor of O. luzonesis and O. curvinotus was obtained from Kondo et al. (2004). B: Phylogeny of selected members of Takifugu species and the spotted green pufferfish (Tetraodon nigroviridis). The fugu chromosome homologous to the sex chromosome of each species is shown in the middle column (Kamiya et al., 2012). The sex-determining SNP in Amhr2 was not found in Tetrodon (Kamiya et al., 2012). The phylogenetic position and estimated divergence time are obtained from Yamanoue et al. (2009).

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Gsdf in a Closely Related Species of Medaka, O. luzonensis

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

The identification of the master SD genes in two different genetic sex determination systems should contribute to a mechanistic understanding of the evolutionary transition between the two systems, if the corresponding species are part of a well-resolved phylogeny. Medaka and a closely related species, O. luzonensis, provide an excellent opportunity for this type of study (Fig. 3A). The common ancestor of O. luzonensis and O. curvinotus was estimated to have diverged from an ancestor of medaka approximately 10 Mya (Kondo et al., 2004). While O. curvinotus retains Dmy as its SD gene, O. luzonensis lost Dmy, and yet, sex determination behaves as a simple mendelian trait in this fish (Kondo et al., 2004; Tanaka et al., 2007). Thus, Dmy is most likely to have been replaced by a new SD gene in O. luzonensis after its divergence from the lineage of O. curvinotus.

By using a genetic mapping approach, Myosho and colleagues identified Gsdf (gonadal soma derived growth factor) as a strong candidate for the master SD gene in this species (Myosho et al., 2012). Gsdf encodes a secretory protein belonging to the transforming growth factor-beta (TGF-β) superfamily (Sawatari et al., 2007) (Fig. 4). This gene product was originally found as a somatic factor controlling the proliferation of primordial germ cells and spermatogonia in rainbow trout (Sawatari et al., 2007), and its expression in gonads was also observed in medaka and zebrafish (Shibata et al., 2010; Gautier et al., 2011). A survey of the genome databases suggests that Gsdf is likely unique to teleosts (Sawatari et al., 2007; Gautier et al., 2011). Currently, the receptors for Gsdf remain to be elucidated.

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Figure 4. A model of AMH signaling by SMAD protein in mammals. Two AMH ligands form a dimer and bind to AMHR2 (reviewed in Drummond, 2005; Fan et al., 2011), which then recruits and activates a type I receptor (ACVR1, BMPR1A, or BMPR1B) (Jamin et al., 2003; Belville et al., 2005) by phosphorylating its intercellular domain. This facilitates the formation of the ligand–receptor complex that consists of a ligand dimer and four receptor molecules. This complex then phosphorylates the SMAD proteins (SMAD 1/5/8). The fugu sex-determining SNP is located in the kinase domain of Amhr2 (Kamiya et al., 2012). Note that binding between Amh and Amhr2 has not been reported in any fish species. Receptors for Gsdf have also not been identified.

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Unlike medaka Dmy or other known vertebrate SD genes (e.g., mammalian Sry, Xenopus DM-W, and chicken DMRT1) that reside on only one of the two sex chromosomes, Gsdf is located on both chromosomes (X and Y) in O. luzonensis (Myosho et al., 2012). Transgenic experiments have demonstrated that the allele of this gene residing on the Y chromosome (GsdfY) was sufficient to cause female-to-male sex-reversal in 94% of XX O. luzonensis (15 of 16 fish in G1 generation) (Myosho et al., 2012). Moreover, analysis of this gene's promoter showed that evolutionary changes in the upstream sequence of GsdfY contributed to male-specific higher expression of the Y allele, which is associated with later testicular development (Myosho et al., 2012).

Of interest, the interspecific transgenic experiment between medaka and O. luzonensis indicated that GsdfY from O. luzonensis was able to produce a male phenotype in XX medaka in the absence of Dmy (Myosho et al., 2012). Given that the spatial and temporal expression pattern of Gsdf is closely correlated to that of Dmy in medaka (Shibata et al., 2010), and that expression patterns of more downstream genes in gonadal differentiation, such as Sox9a2, Dmrt1 and Foxl2, are similar between medaka and O. luzonensis (Nakamoto et al., 2009), the transition of the SD system in the lineage leading to O. luzonensis can be explained by the following scenario: a downstream gene became independent of an existing SD gene, and usurped control of the downstream cascade (Fig. 3A) (Myosho et al., 2012). In the previous studies of the identification of master SD genes of vertebrates (Sinclair et al., 1990; Matsuda et al., 2002; Yoshimoto et al., 2008; Smith et al., 2009), the ancestral mechanisms overthrown by these SD genes were unclear. However, this study (Myosho et al., 2012) elucidates genomic changes associated with the evolutionary transition between the ancestral and derived SD systems at the DNA level.

The situation in O. luzonensis may provide an opportunity to see a degeneration process of the old SD gene. Indeed, O. luzonensis possesses a pseudogene of Dmrt1 named Oludmrt1p, in addition to the autosomal Dmrt1 gene designated as Oludmrt1 (Kondo et al., 2004). However, the genomic position of the pseudogene in O. luzonensis makes the interpretation of the degeneration process difficult (Kondo et al., 2004; Tanaka et al., 2007). O. luzonensis Dmrt1/Oludmrt1 is located on the chromosome homologous to the medaka chromosome 9 containing Dmrt1, whereas the pseudogene (Oludmrt1p) is on the chromosome corresponding to the medaka chromosome 18 (Tanaka et al., 2007). Because the pseudogene (Oludmrt1p) is not located on the chromosome corresponding to that of medaka containing Dmy (chromosome 1), the origin of the pseudogene (Oludmrt1p) cannot be easily speculated upon (Tanaka et al., 2007). It is possible that the past Y chromosome harboring Dmy has been lost in the O. luzonensis lineage, and the pseudogene is the result of another independent duplication of Dmrt1 (Kondo et al., 2004; Tanaka et al., 2007). Alternatively, the pseudogene (Oludmrt1p) may have been derived from Dmy through a transposition event.

DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

After the identification of medaka Dmy (Matsuda et al., 2002; Nanda et al., 2002), no other SD gene or strong candidate has been reported in fish species until recently. However, Hattori et al. (2012) reported a male-specific, duplicated copy of the anti-Müllerian hormone gene (amh), designated amhy, as a strong candidate for the master SD gene in the Patagonian pejerrey, a teleost species that lives in inland waters of southern South America. AMH is a secretory protein belonging to the TGF-β superfamily (Fig. 4), and is important for the development and maintenance of reproductive organs in mammals (Mishina et al., 1996; Drummond, 2005; Fan et al., 2011), where it is also responsible for the regression of the Müllerian duct in males (Mishina et al., 1996). Loss-of-function of this gene in the male mouse leads to partial hermaphroditism, with the uterus and oviduct present along with the testis, but no ovaries. Although teleost fish lack the Müllerian duct, the Amh protein has been implicated in the regulation of germ cell proliferation in medaka (Shiraishi et al., 2008) and spermatogenesis in Japanese eel (Miura et al., 2002).

The Patagonian pejerrey possesses an XX/XY sex determination system (Strüssmann et al., 1997). This fish and its closely related species, Odontesthes bonariensis, have shown promise for aquaculture (Inazawa et al., 2011). These two species have also been of interest for studies on sex determination as O. bonariensis presents marked temperature-dependent sex determination, whereas the rearing temperature affects sex determination only near the extremes of the thermal range in Odontesthes hatcheri (Strüssmann et al., 1996, 1998).

Hattori et al. (2012) used molecular cloning and fluorescent in situ hybridization (FISH) on metaphase chromosomes and showed that male Patagonian pejerrey carry amhy in a sex-specific manner. Their study indicated that the expression of amhy preceded the first signs of morphological differentiation of ovaries and testes in genotypic male fish, at which time the expression of autosomal amh was not detected in the gonads of either sex (Hattori et al., 2012). Morpholino-mediated knock-down of amhy resulted in male to female sex reversal in 22% (11 of 50 XY larvae) of the fish carrying the amhy gene (Hattori et al., 2012). Collectively, these results suggest that amhy is likely to be the testis-determining gene in the Patagonian pejerrey. This study was published earlier than the study of Gsdf in O. luzonensis and provided the first evidence suggesting that a nontranscription factor can also become a master SD gene in vertebrates. We discuss this issue later together with other examples.

Amhr2 IN FUGU AND ITS RELATED SPECIES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

The tiger pufferfish (fugu) is a large marine fish, and its genome was sequenced to the draft level in 2002 (Aparicio et al., 2002). Since then, several genome assemblies for this species (the latest being fugu version 5) have been released (Kai et al., 2011). These assemblies have been used as a reference genome for identifying genes and other functional elements in vertebrate genomes (e.g., Woolfe et al., 2004), and for understanding the evolution of vertebrate genomes (e.g., Christoffels et al., 2004; Kai et al., 2011). This fish is economically important in East Asia (Kai et al., 2005), and has an XX/XY sex determination system (Kikuchi et al., 2007).

Kamiya et al. (2012) investigated the SD locus in fugu by means of a combination of family-based genetic mapping and association mapping in natural populations. They found that a missense single-nucleotide polymorphism (SNP) in the kinase domain of the anti-Müllerian hormone receptor type II (Amhr2) was perfectly associated with the phenotypic sex of the fish. In a natural population consisting of 58 females and 47 males, all males were heterozygous (G/C) at the SNP site, whereas all females were homozygous for the C allele. There were no other polymorphic sites or sex-specific genomic segments in the SD locus perfectly associated with the phenotypic sex. In addition, the association between the SNP and phenotypic sex was conserved in two other species closely related to fugu (Kamiya et al., 2012) (Fig. 3B). Therefore, the missense SNP in Amhr2 is likely responsible for sex determination in fugu.

Intriguingly, Amhr2 codes for a receptor for Amh that is also implicated as the likely SD factor, Amhy, in the Patagonian pejerrey (Hattori et al., 2012) (Fig. 4). Amhr2 belongs to the type II receptors for the TGF-β superfamily proteins and contains a single transmembrane domain and a kinase domain (Imbeaud et al., 1995) (Fig. 4). In mammals, the binding of AMH to AMHR2 recruits and phosphorylates the type I receptor(s), BMPR1A, ACVR1 or BMPR1B that in turn, then transduce signals by phosphorylating SMAD proteins (Jamin et al., 2003; Belville et al., 2005) (Fig. 4). The “sex-determining SNP” in fugu is located within the kinase domain of Amhr2 (His384Asp) that is responsible for the phosphorylation (Kamiya et al., 2012). Five natural mutations in the kinase domain of human AMHR2 result in a partial hermaphrodite phenotype similar to the knockout phenotype of Amh and Amhr2 in mice (Mishina et al., 1996; Belville et al., 2009). Importantly, a homozygous mutation in the kinase domain of Amhr2 (Tyr390Cys) (hotei mutant) results in sex reversal in ∼50% of XY males in medaka (Morinaga et al., 2007). The proposed model of sex determination in fugu, where the missense mutation (SNP) in Amhr2 causes a female phenotype when homozygous, fits well with this phenotype.

Kamiya et al. (2012) also showed that the fugu SD gene resides in a region of the sex chromosomes that still recombines, unlike the previously identified SD genes such as therian Sry (Graves, 2006) and medaka Dmy (Kondo et al., 2006). Given that many fish species and other poikilotherms are thought to have a pair of sex chromosomes that are indistinguishable by microscopic observation (Devlin and Nagahama, 2002; Eggert, 2004; Ezaz et al., 2009), the SD gene residing in a recombining region may be more common than previously thought. Association mapping using wild populations will be a powerful approach to pinpoint the SD gene/allele for this type of sex determination system (Kamiya et al., 2012). It should be noted that the SD system in fugu is similar to that observed in O. luzonensis (Myosho et al., 2012). In both cases, the sex is likely to be determined by an allele or a combination of alleles of a gene encoding a nontranscription factor.

EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

Until recently (2011), all four vertebrate master SD genes (or strong candidates) were known to code for transcription factors (Sinclair et al., 1990; Matsuda et al., 2002; Yoshimoto et al., 2008; Smith et al., 2009), which could have been construed as evidence that gonadal sex determination in vertebrates is always triggered by transcription factors. However, the three novel candidates for the master SD genes in the Patagonian pejerrey, O. luzonensis and fugu, code for growth factors or one of their receptors. Thus, these findings suggest alternative mechanisms of genotypic sex determination in which the main trigger is not constrained to be a transcription factor.

It is noteworthy that these three novel SD genes code for components of the TGF-β signaling pathway (Fig. 4). In mammals, this pathway has been shown to play numerous important roles in the development of ovarian (reviewed in Drummond, 2005) and testicular function (reviewed in Fan et al., 2011). For example, with respect to gonadal sex determination, AMH has been shown to inhibit oogonial proliferation and induce the formation of seminiferous cord-like structures in fetal ovaries exposed to it in organ culture (Vigier et al., 1987) and in transgenic mice (Behringer et al., 1990). Follistatin, that encodes a TGF-β superfamily binding protein, inhibits formation of the XY-specific coelomic vessel in XX gonads, a feature normally associated with testis differentiation (Yao et al., 2004). However, there were no knockout mice or known human diseases associated with components of the TGF-β pathway that exhibit complete gonadal sex reversal (Mishina et al., 1996; Massagué et al., 2000; Harradine and Akhurst, 2006; Memon et al., 2008). In contrast, the above findings in the three fish mark the critical role of TGF-β signaling in determining the path toward the development of either testicular or ovarian tissue (Fig. 5).

image

Figure 5. A model for cellular and molecular mechanisms of sex determination in fishes by TGF-β related genes. Gray round cells represent germ cells, whereas blue bean-shaped cells denote somatic steroid-producing cells. Germ cell proliferation provides a niche for the somatic steroid-producing cells resulting in higher steroid and estradiol (E2) production, which would lead to ovarian differentiation and/or maintenance of the ovarian state (Guiguen et al., 2010). Inhibition of germ cell proliferation by the TGF-β related sex-determining gene (amhy, Amhr2, and Gsdf) in somatic cells would have the opposite effect, resulting in less E2 production, which in turn would facilitate testicular differentiation. Alternatively, the sex-determining gene expressed in somatic cells may act on either germ cells to inhibit their proliferation through unidentified signaling or somatic cells to inhibit the E2 production through paracrine mechanisms. While medaka Dmrt1 is known to negatively regulate aromatase expression (Masuyama et al., 2012), medaka Dmy expressed in somatic cells also regulates germ cell proliferation negatively by means of some unknown mechanism (Paul-Prasanth et al., 2006; Herpin et al., 2007).

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It has been hypothesized that this pathway may play a more central role in gonadal sex determination in nonmammalian vertebrates than in mammals (Smith and Sinclair, 2004). For example, while Amh in mammals clearly lies far downstream in the SD pathway, being controlled by a mammalian key gene, Sox9, the expression of chicken Amh precedes Sox9 expression in males (Oreal et al., 1998; Smith et al., 2007). Similar expression patterns of the two genes were observed in the American alligator (Alligator mississippiensis) (Western et al., 1999a). In addition, AMH is suspected to be a factor responsible for female-to-male sex reversal in the chicken, as determined by grafting undifferentiated testes into ZW female embryos (Maraud et al., 1990). The above-mentioned findings in fish species corroborate this hypothesis.

One possible mechanism of sex determination by the TGF-β signaling is that it decreases the number of germ cells, which facilitates the development of testes (Fig. 5) (Saito and Tanaka, 2009). This assumption is based on the observation that male-to-female sex-reversal in Amhr2 deficient medaka is associated with over-proliferation of germ cells (Morinaga et al., 2007). In addition, experimental germ cell depletion during the early steps of gonadal differentiation resulted in masculinization of gonads in medaka and zebrafish (Kurokawa et al., 2007; Siegfried and Nüsslein-Volhard, 2008). This hypothetical mechanism is consistent with the observation that female gonads contain a greater number of germ cells than male gonads in early development of O. luzonensis (Nakamoto et al., 2009). However, in apparent contradiction with this hypothesis, Amh and Gsdf have also been found to be positive regulators in the proliferation of germ cells in medaka and rainbow trout, respectively (Sawatari et al., 2007; Shiraishi et al., 2008). Thus, the effect of these TGF-β members on germ cell proliferation appears to depend on additional signaling partners (di Clemente et al., 2003; Guiguen et al., 2010; Nakamura et al., 2012a). Moreover, it is currently unclear whether the germ cell number is critical in sexual fate determination in species other than medaka and zebrafish. For example, the depletion of germ cells did not appear to affect the sexual fate of gonadal somatic cells in gold fish Carassius auratus (Goto et al., 2012), loach Misgurnus anguillicaudatus (Fujimoto et al., 2010) and the red-eared slider turtle Trachemys scripta (Dinapoli and Capel, 2007).

Another possible mechanism of sex determination by TGF-β signaling is that this signaling could inhibit aromatase activity as reported for mammalian Amh and Gdf9 (Vigier et al., 1989; di Clemente et al., 1992; Kim et al., 1992; Yamamoto et al., 2002) (Fig. 5). High levels of estrogen synthesized by aromatase are known to function as a strong inducer of ovarian differentiation in fish, amphibians, reptiles and birds (Devlin and Nagahama, 2002; Smith and Sinclair, 2004). Therefore, suppression of aromatase activity by TGF-β signaling could lead to the formation of testes.

Recently, a suppressive role of Amh on steroidogenesis was also reported in fish (Skaar et al., 2011). By using zebrafish testis tissue culture, Skaar et al. (2011) showed that recombinant Amh down-regulated gonadotropin-stimulated Leydig cell gene expression (star, insl3, and cyp17a1) and reduced androgen release. Among the genes suppressed, cyp17a1/P450c17-I is of interest in the context of this review as this gene encodes a steroidogenic enzyme required for the production of both estrogen and androgen (Zhou et al., 2007). In female medaka, a subset of supporting cells surrounding oocytes expresses Cyp17a1/P450c17-I (Nakamura et al., 2009). Moreover, in Cyp17a1/P450c17-I-deficient medaka, XX mature fish appeared to fail to maintain ovarian differentiation and contained gonads with many spermatozoa (Sato et al., 2008). This phenotype suggests that down-regulation of Cyp17a1/P450c17-I expression facilitates the development of testis at the expense of maintaining the ovarian fate. Now that functional recombinant Amh has been obtained from eel and zebrafish (Miura et al., 2002; Skaar et al., 2011), it should be possible to test the role of Amh on steroidogenesis, including aromatase activity in undifferentiated gonads and differentiating ovaries in fish species.

The relative importance of the two hypothetical roles of the TGF-β signaling, the control of germ cell number and the regulation of steroidogenic enzymes in undifferentiated and differentiating gonads during gonadal sex determination, may vary among species as well as between developmental stages within a species. However, the two mechanisms may act cooperatively in the determination and maintenance of gonadal sex. Indeed, the two mechanisms may not be mutually exclusive, as inhibition of germ cell proliferation would reduce the number of somatic steroid-producing cells surrounding the germ cells, which in turn would decrease estrogen production and promote testicular differentiation (Fig. 5) (Guiguen et al., 2010). Saito and Tanaka (2009) also proposed a model suggesting that the masculinization effects of germ cell depletion in zebrafish and medaka (Kurokawa et al., 2007; Siegfried and Nüsslein-Volhard, 2008), and the feminization phenotype in Amhr2 deficient medaka (Morinaga et al., 2007) are potentially mediated by the regulation of aromatase expression.

TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

The previously characterized master SD genes are related to genes that are believed to be common key actors in the gonadal differentiation pathway across vertebrates. For example, the master SD gene in medaka is a transposed copy of Dmrt1 whose expression in the differentiating testis is well conserved across vertebrates (Herpin and Schartl, 2011) and shown to be necessary for testicular development in mice and medaka (Raymond et al., 2000; Masuyama et al., 2012). In Xenopus laevis, a truncated copy of DMRT1, DM-W, triggers ovary development presumably through inhibition of the function of DMRT1 (Yoshimoto et al., 2010). Another example is Sry. A member of the Sry-like high mobility group domain protein family, SOX9 acts downstream of Sry and is necessary and sufficient for testis specification in mice (Wagner et al., 1994; Vidal et al., 2001). The expression of Sox9 in the differentiating testis has been observed in many vertebrate species (da Silva et al., 1996; Western et al., 1999b; Barske and Capel, 2010). However, the role of Sox9 in the sex determination of non-mammalian vertebrates is less clear. In fact, in medaka Sox9b is not necessary for testicular development but is required for maintenance of germ cells (Nakamura et al., 2012b).

The presence of the conserved downstream key actors in the gonadal differentiation pathway can be explained by the bottom up hypothesis in which new sex determination systems arise through addition of new sex determiners independently in different lineages (Wilkins, 1995). The emergence of the new SD gene related to the downstream key actor such as Dmrt1 can be interpreted as the trimmed-down version of the SD cascade extended by the bottom up process (Schartl, 2004b). This trimmed-down model appears to be consistent with the presence of Gsdf, Amh, and Amhr2 as the SD gene in O. luzonensis, the Patagonian pejerrey and fugu, respectively. Moreover the identification of these SD genes (or strong candidates) strengthens the hypothesis that some key actors or their related genes are repeatedly recruited to the top of pathway. (Volff et al., 2007). However, the study on rainbow trout by Yano et al. (2012) suggested that there is likely an unexpected evolutionary plasticity in the mechanisms underlying vertebrate sex determination.

Rainbow trout is a species of salmonid native to tributaries of the Pacific Ocean in Asia and North America (Davidson et al., 2009). This species has been introduced as a food and sport fish in many countries (Wolf and Rumsey, 1985). Sex determination in this fish is strictly genetic, with an XX/XY system controlled by a single SD locus, although there appears to be an argument regarding the genomic position of the SD gene on the linkage map of sex chromosomes (reviewed in Davidson et al., 2009).

By characterizing genes specifically expressed in the differentiating testis of rainbow trout, Yano et al. (2012) identified a male-specific gene, sdY, that is expressed in the somatic cells surrounding germ cells. This gene encodes a novel protein that displays sequence homology with the carboxy-terminal domain of interferon regulatory factor 9 (Irf9). IRF9 is a transcription regulatory factor that mediates signaling by type I interferon in mammals (Takaoka and Yanai, 2006), and its homolog is present in several fish genomes (Yano et al., 2012). In contrast, the sdY gene seems to be highly lineage specific, because there is no homolog in the genome databases for other teleosts (Yano et al., 2012).

The sdY gene was shown to be tightly linked to the SD locus in rainbow trout (Yano et al., 2012). Microinjection of the sdY cDNA fused with its promoter region into eggs resulted in female-to-male sex reversal in 12% of XX fish (9 of 73 fish) (Yano et al., 2012). Moreover, the targeted inactivation of sdY using zinc-finger nucleases induced ovarian differentiation in F1 XY fish (Yano et al., 2012). These results demonstrated that sdY fulfills all requirements for being a master testis-determining gene in the rainbow trout: expression in the differentiating testes, genetic linkage with the SD locus, and the ability to induce testis development.

Yano et al (2012) raised a possibility that interferon signaling affects gonadal sex determination through the inhibitory role of steroidogenesis based on the fact that INFs are known to inhibit steroidogenesis in primary cultures of mammalian Leydig cells (Diemer et al., 2003), and that steroids are important inducers of fish gonadal differentiation (Devlin and Nagahama, 2002). However, there are few clues about the mechanism of action of this gene product in triggering the switch in the bipotential gonads toward the development of testes as the involvement of the interferon signaling pathway during testicular differentiation in vertebrates has not been investigated in detail (Yano et al., 2012). In addition, because SdY is clearly divergent from the Irf9 protein (47% identity in amino acids of the IRF-associated domain), it is possible that SdY has acquired a new function and lost the function of the original Irf9 (Yano et al., 2012). This study demonstrates that novel actors can be recruited at the top of the SD cascade.

EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

Sex chromosome evolution models have focused on the early stages of sex chromosome formation to explain how the reduction of recombination between the sex chromosomes may have arisen (e.g., Charlesworth et al., 2005; Rice, 1996). A well-known theory predicts that proto-sex chromosomes arose from a stepwise allelic diversification of one or two loci of a chromosome pair, where one allele became a sex determiner while another allele at the same locus retained a non-SD function or favored the development of the opposite sex (Charlesworth et al., 2005; Herpin and Schartl, 2009). Herpin and Schartl (2009) and Kondo et al. (2009) pointed out that the prediction does not hold true in the case of medaka, as the medaka SD gene is a duplicated copy of an autosomal gene, which was inserted into the future-Y chromosome during evolution (Fig. 6A). The findings in the Patagonian pejerrey (Hattori et al., 2012) and rainbow trout (Yano et al., 2012) also contradict the prediction of allelic diversification as their SD genes arose by means of duplication events. The only possible case that is consistent with the prediction in vertebrates is the Sry gene in therian mammals (Foster and Graves, 1994). Comparative sequence analyses, coupled with cytogenetic studies of sex chromosomes, have led to the hypothesis that Sry arose from mutations in the allele of Sox3 in the ancestor of therian mammals (Foster and Graves, 1994; Sutton et al., 2011). However, the genomic sequences of X and Y in extant therian mammals are so divergent (Skaletsky et al., 2003) and the genomic locations of Sry and Sox3 on the sex chromosomes are no longer comparable. For example, based on the human genome data of Ensembl (release 66), SOX3 is located at ∼139.6 Mb from the short arm telomeric end of the X chromosome, whereas SRY is located at 2.6 Mb from the short arm telomeric end of the Y chromosome. Therefore, the former allelic relationship between SRY and SOX3, if any, is not obvious at first sight. In contrast, the SD loci of O. luzonensis and fugu independently represent inceptions of allelic diversification (Fig. 6B) (Kamiya et al., 2012; Myosho et al., 2012), and have provided evidence in vertebrates to support the longstanding hypothesis in which the SD gene arose from an allelic diversification (Foster and Graves, 1994). Considering all these, the available evidence indicates that sex chromosome evolution in vertebrates is likely to begin with allelic diversification at one locus (Fig. 6B). In addition, the insertion of a genomic segment derived from a duplication event can also initiate this evolutionary process (Fig. 6A).

image

Figure 6. Origin of the sex-determining gene. A: Duplication and insertion. Sex-determining genes in medaka, the Patagonian pejerrey and rainbow trout are originally duplicated copies of autosomal genes, labeled C (medaka Dmrt1, Patagonian pejerrey amh and rainbow trout irf9) (Kondo et al., 2006; Hattori et al., 2012; Yano et al., 2012). The duplicated copy of Dmrt1 was inserted into the future Y chromosome and became the sex-determining gene in an ancestor of medaka (Kondo et al., 2006). A duplicated copy of amh appears to have been inserted into the future Y chromosome in the Patagonian pejerrey, since amhy and autosomal amh mapped to different chromosomes by fluorescent in situ hybridization (FISH) (Hattori et al., 2012). B: Allelic diversification. Sry and Sox3 have been hypothesized to be allelic in an ancestral mammals (Foster and Graves, 1994). The sex-determining loci of O. luzonensis and fugu independently represent inceptions of allelic diversification (Kamiya et al., 2012; Myosho et al., 2012).

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WHY DO SEX CHROMOSOMES TURN OVER?

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

As in the cases seen above, the turnover of sex chromosomes and/or the SD genes is also suggested in many other lineages of vertebrates (Charlesworth and Mank, 2010; Graves and Peichel, 2010). For example, karyotype information suggests that lizards have evolved sex chromosomes independently many times (Ezaz et al., 2009). Genetic analysis indicates that two model amphibian species, X. laevis and Silurana tropicalis, have different master SD genes. While no DM-W has been found in S. tropicalis (Yoshimoto et al., 2008), the presence of the SD locus was shown by the tight linkage of genetic markers to its phenotypic sex (Olmstead et al., 2010).

The rapid turnover of sex chromosomes is thought to provide the advantage of avoiding the degeneration of sex chromosomes in these animals (Volff et al., 2007; Charlesworth and Mank, 2010) (Fig. 7A). It has often been suggested that the sex chromosomes are doomed to be degenerate through the evolution of suppressed recombination between sex chromosomes and sexual antagonism may be the driving force behind the suppression of recombination (Charlesworth et al., 2005). The argument is that once a male-determining gene emerged, the Y chromosome became a good place for male-beneficial genes (or alleles) to land (Rice, 1984). The suppression of recombination between the X and Y was selectively advantageous at first, as the male-determining gene and male-beneficial genes were transmitted to the next generation together (Rice, 1987). The nonrecombining segment tended to expand, but genes that were trapped in the segments began to accumulate deleterious mutations under the combined forces of genetic drift, selective sweeps, background selection, and Muller's ratchet (Charlesworth and Charlesworth, 2000). Because this degeneration would cause haploinsufficiency and reduction of fitness in males, new males with a nondegenerate Y chromosome (the turnover of sex chromosomes) would be favored in a population (Volff et al., 2007) (Fig. 7A).

image

Figure 7. Models for turnover of sex chromosomes. A: A degenerate sex chromosome facilitates the turnover (Volff et al., 2007). One autosome acquires a male-determining gene (black allele) and becomes the Y chromosome. The alleles/genes beneficial for males and disadvantageous for females (blue allele) can accumulate near the male determiner. Selection favors inhibition of recombination between the male determiner and its male beneficial alleles at first. The nonrecombining segment (yellow region) accumulates deleterious mutations and expands, and eventually becomes harmful to males. Due to the resultant decline in fitness of the males, new males carrying a nondegenerate Y chromosome that contains a new male determiner (red allele) are favored in the population. The new sex determiner (red allele) can be a pre-existing allele or one arising by de novo mutations. B: Sexually antagonistic selection facilitates the turnover (van Doorn and Kirkpatrick, 2007). The alleles beneficial to one sex and disadvantageous for the other (male beneficial (MB) allele shown in blue) may be widely distributed across the genome. The frequency of these alleles is usually restricted because of their expression in both sexes. Once a new male-determining allele (red) emerges near the MB allele, selection favors the segment containing the MB allele and male-determining allele, resulting in the segment spreading in the population.

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Sexual antagonism (sex-specific selection pressures) can also explain the establishment of a new SD locus without the assumption of the negative effects of the nonrecombining region (Fig. 7B). Theory suggests that selection will favor an increase in the frequency of any new SD allele that is linked to a sexually antagonistic allele (e.g., new male determining allele and a male beneficial allele) and will lead the new SD allele to spread to fixation, resulting in the disappearance of the previous sex determiner (van Doorn and Kirkpatrick, 2007). There is anecdotal support for this from cichlids (Roberts et al., 2009).

In addition to sexual antagonism, several models have been proposed to explain the evolution of new sex determination mechanisms, including random genetic drift (Vuilleumier et al., 2007), sex-ratio selection (Kozielska et al., 2006; Grossen et al., 2011) and pleiotropic selection favoring new SD genes (Alphen, 2001). Among them, one simple model is that the turnover in fish and other poikilotherms is the mere consequence of environmental change. Because changes in temperature will induce sex-ratio biases, the emergence of new SD genes will be favored by sex-ratio selection to reduce the biases (Bull, 1981; Caubet et al., 2000; Grossen et al., 2011).

The last model can explain why the turnovers of sex chromosomes is likely to be more frequent in fish and other poikilotherms than mammals and birds because warm-blooded animals can prevent the effect of environmental temperature on sex determination by thermoregulation (Grossen et al., 2011). In addition, the number of variations that can potentially become the SD trigger may be greater in poikilotherms than in mammals, due to the robustness of the developmental process of gonads in poikilotherms with respect to the production of functional gametes. For example, whereas Dmrt1-deficient XY medaka showed complete sex-reversal and produced fertile eggs (Masuyama et al., 2012), the gonads in Dmrt1-deficient XY mouse underwent abnormal differentiation (Raymond et al., 2000). It is known that perturbations at different steps in the production of estrogen result in the common outcome of spontaneous testicular differentiation in several fish species (Guiguen et al., 2010). These examples suggest that mutations in many genes have a potential to be a sex determiner in fish species. Alternatively, the evolutionary potential to counteract the haploinsufficiency due to sex chromosome decay may be different between poikilotherms and warm-blooded animals. Indeed different systems of dosage compensation are suggested to have evolved even between therian mammals and birds (Livernois et al., 2011).

The above-mentioned theoretical models on the transition between sex determination systems involving the turnover of sex chromosomes can be tested by means of studies on closely related species (van Doorn and Kirkpatrick, 2007). In cases where ancestral and derived sex chromosomes coexist in a species, the species will provide an excellent opportunity to test theoretical models that explain why and how sex chromosomes turn over (Roberts et al., 2009). Comparative analysis has suggested a rapid turnover of sex chromosomes among salmonids (Woram et al., 2003), sticklebacks (Ross et al., 2009), poeciliids (Schultheis et al., 2009) and cichlids (Ser et al., 2010), apart from the Oryzias species (Fig. 3A). Comparative mapping of fugu and related species (Takifugu species) (Fig. 3B) as well as the Patagonian pejerrey and related species (Hattori et al., 2012) are also ongoing. Studies on these fish groups should be promising.

PERSPECTIVES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES

Given the evidence from all the fish species mentioned, together with previous studies of other nonmammalian species, it seems reasonable to imagine that many other teleosts, reptiles, and amphibians also have experienced a turnover of sex chromosomes accompanied with the substitution of the SD systems and their master regulators. This large-scale natural experiment provides a resource that geneticists can use to search for genetic variants that control sex determination. Accumulation of knowledge on such variants will allow us to distinguish conserved and diversified SD pathways among vertebrates and lead us into a deeper understanding of the vertebrate SD cascade. For example, it may be worth reconsidering the role of AMH signaling in gonadal sex determination even in mammals based on the results of recent fish studies. In addition, the identification of different SD genes among closely related species enables us to understand the evolutionary forces facilitating sex chromosome turnover.

Indeed, efforts to identify the SD genes in nonmammalian vertebrates (e.g., Quinn et al., 2009; Tsend-Ayush et al., 2012), including many fish other than those mentioned above (e.g., Table 1), are in progress. Nevertheless, successful identification of such variants has been very limited because of the lack of genomic resources for genetic mapping and the difficulty in performing functional tests for the candidate SD gene/variant in nonmodel species. However, the recent advent of new technologies including high-throughput DNA sequencing and nuclease-based gene targeting approaches is making it easier to perform genetic experiments in nonmodel vertebrates (e.g., Bruneaux et al., 2012; Yano et al., 2012). The undifferentiated nature of sex chromosomes observed in many nonmammalian species (Devlin and Nagahama, 2002; Eggert, 2004; Ezaz et al., 2009) will greatly facilitate the pinpointing of the SD gene by standard linkage mapping (Myosho et al., 2012), population genomics, comparative genomics or a combination of these methods (Kamiya et al., 2012; Yano et al., 2012). We have witnessed the identification of four novel SD genes, or SD genes candidates, in 2012. The pace of such identification is unlikely to slow down. Several ongoing studies in fish and other nonmammalian species should provide further fundamental insights into our understanding of the mechanisms of sex determination and their evolution.

Table 1. Examples of genetic mapping experiments on the sex-determining locus in fish speciesa
  1. a

    Charlesworth and Mank (2010) has compiled an extensive list of fishes and other species reported until 2010. The above list shows more recent examples of fish species. In addition, fishes considered to be models in developmental biology and evolutionary biology are included. 1(Bradley et al., 2011; Anderson et al., 2012); 2(Eshel et al., 2012); 3(Ser et al., 2010); 4(Viñas et al., 2012); 5(Galindo et al., 2011); 6(Tripathi et al., 2009); 7(Schultheis et al., 2009); 8(Takehana et al., 2008); 9(Myosho et al., 2012); 10 (Kamiya et al., 2012); 11(Loukovitis et al., 2011);12(Hattori et al., 2012);13(Ross et al., 2009); 14(Yano et al., 2012); 15(Davidson et al., 2009);16(Fuji et al., 2010).

Carps (Cypriniformes)
 Danio rerio (zebrafish)1
Cichlids (Perciformes)
 Oreochromis niloticus (Nile tilapia)2, Aulonocara baenschi3, Labeotropheus trewavasae3, Metriaclima barlowi3, M. benetos3, M. callainos3, M. “daktari”3, M. fainzilberi3, M. “kompakt”3, M. lombardoi3, M. mbenjii3, M. phaeos3, M. pyrsonotus3, M. zebra3, Pseudotropheus polit3
Flatfishes (Pleuronectiforms)
 Scophthalmus maximus (turbot)4, Hippoglossus stenolepis (Pacific halibut)5
Killifish and live bearers (Cyprinodontiformes)
 Poecilia reticulata (guppy)6, Xiphophorus spp.7
Medakas (Beloniformes)
 Oryzias latipes (medaka)8, O. luzonensis9, O. curvinotus8, O. mekogenesis8, O. hubbsi8, O. javanicus8, O. dancena8, O. minutillus8
Pufferfishes (Tetraodontiformes)
 Takifugu rubripes (fugu, tiger pufferfish)10, T. pardalis10, T. poecilonotus10
Sea breams (Perciformes)
 Sparus aurata (gilthead sea bream)11
Silversides (Atheriniform)
 Odontesthes hatcherei (Patagonian pejerrey)12
Sticklebacks (Gasterosteiformes)
 Pungitius pungitius (ninespine stickleback)13, Gasterosteus aculeatus (threespine stickleback)13, G. wheatlandi (black-spotted stickleback)13
Salmons (Salmoniformes)
 Oncorhynchus mykiss (rainbow trout)14, O. kisutsch15, O. tshawytscha15, O. clarki15, Salmo salar (Atlantic salmon)15, S. trutta (brown trout)15, Salvelinus alpinus (Arctic charr)15
Yellow tails (Perciformes)
 Seriola quinqueradiata (Japanese amberjack)16

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. SEX DETERMINATION IN MEDAKA
  5. RAPID TURNOVER OF SEX CHROMOSOMES IN CLOSELY RELATED SPECIES OF MEDAKA
  6. Gsdf in a Closely Related Species of Medaka, O. luzonensis
  7. DUPLICATED COPY OF THE amh GENE IN THE PATAGONIAN PEJERREY
  8. Amhr2 IN FUGU AND ITS RELATED SPECIES
  9. EMERGING PATTERNS FROM THE NOVEL SD GENES IN THE PATAGONIAN PEJERREY, O. luzonensis AND FUGU: SEX DETERMINATION BY NONTRANSCRIPTION FACTORS AND IMPORTANCE OF THE TGF-β SIGNALING PATHWAY
  10. TRUNCATED COPY OF AN IMMUNE-RELATED GENE, sdY, IN RAINBOW TROUT: A NOVEL ACTOR
  11. EMERGING PATTERNS FROM FISH STUDIES: ORIGINS OF SEX CHROMOSOMES
  12. WHY DO SEX CHROMOSOMES TURN OVER?
  13. PERSPECTIVES
  14. ACKNOWLEDGMENTS
  15. REFERENCES