Evidence for different origins of sex chromosomes in closely related Oryzias fishes: substitution of the master sex-determining gene

The medaka Oryzias latipes and its two sister species, O. curvinotus and O. luzonensis, possess an XX-XY sex-determination system. The medaka sex-determining gene DMY has been identified on the orthologous Y chromosome [O. latipes linkage group 1 (LG1)] of O. curvinotus. However, DMY has not been discovered in other Oryzias species. These results and molecular phylogeny suggest that DMY was generated recently [approximately 10 million years ago (MYA)] by gene duplication of DMRT1 in a common ancestor of O. latipes and O. curvinotus. We identified seven sex-linked markers from O. luzonensis (sister species of O. curvinotus) and constructed a sex-linkage map. Surprisingly, all seven sex-linked markers were located on an autosomal linkage group (LG12) of O. latipes. As suggested by the phylogenetic tree, the sex chromosomes of O. luzonensis should be "younger" than those of O. latipes. In the lineage leading to O. luzonensis after separation from O. curvinotus approximately 5 MYA, a novel sex-determining gene may have arisen and substituted for DMY. Oryzias species should provide a useful model for evolution of the master sex-determining gene and differentiation of sex chromosomes from autosomes.     

Oryzias curvinotus has DMY,a gene that is required for male development in the medaka,O. latipes

DMY is a Y-specific DM-domain gene required for male development and appears to be the sex-determining gene in the teleost fish medaka, Oryzias latipes. Although the genomic region containing DMY appears to have originated through duplication of the DMRT1 region, it is unknown when the duplication occurred. Here we show that O. curvinotus also has the DMY gene on the Y chromosome, which is homologous to the Y chromosome of medaka, and that DMY is expressed in XY embryos. A phylogenetic tree based on the amino acid sequence including the DM-domain shows that DMY was derived from DMRT1 immediately before speciation of O. latipes and O. curvinotus.     

Evolution of different Y chromosomes in two medaka species, Oryzias dancena and O. latipes

Although the sex-determining gene DMY has been identified on the Y chromosome in the medaka (Oryzias latipes), this gene is absent in most Oryzias species, suggesting that closely related species have different sex-determining genes. Here, we investigated the sex-determination mechanism in O. dancena, which does not possess the DMY gene. Since heteromorphic sex chromosomes have not been reported in this species, a progeny test of sex-reversed individuals produced by hormone treatment was performed. Sex-reversed males yielded all-female progeny, indicating that O. dancena has an XX/XY sex-determination system. To uncover the cryptic sex chromosomes, sex-linked DNA markers were screened using expressed sequence tags (ESTs) established in O. latipes. Linkage analysis of isolated sex-linked ESTs showed a conserved synteny between the sex chromosomes in O. dancena and an autosome in O. latipes. Fluorescence in situ hybridization (FISH) analysis of these markers confirmed that sex chromosomes of these species are not homologous. These findings strongly suggest an independent origin of sex chromosomes in O. dancena and O. latipes. Further analysis of the sex-determining region in O. dancena should provide crucial insights into the evolution of sex-determination mechanisms in vertebrates.     

Identification of the sex-determining locus in the Thai medaka, Oryzias minutillus

A sex-determining gene, DMY, which is comparable to the SRY gene in mammals, has been identified in the medaka, Oryzias latipes. Although Oryzias curvinotus, a closely related species to O. latipes also has DMY, this gene has not been found in other Oryzias fishes. It has recently been demonstrated that the sex chromosomes of Oryzias dancena and Oryzias hubbsi differ from those of O. latipes and these species have XX/XY and ZZ/ZW systems, respectively. This may suggest that Oryzias species have evolved different sex-determining genes on different sex chromosomes. In the present study, we investigated the sex determination mechanism in Oryzias minutillus, which is closely related to O. dancena and O. hubbsi. Linkage analysis using 14 isolated sex-linked DNA markers showed that this species has an XX/XY sex determination system. These sex-linked markers were located on linkage group 8 of O. latipes, suggesting that the sex chromosomes of O. minutillus are homologous to the autosomes of other Oryzias species. Furthermore, fluorescence in situ hybridization using a tightly sex-linked marker demonstrated that the XY sex chromosomes of O. minutillus and O. dancena were not homologous. These findings provide additional evidence for independent origins of sex chromosomes and sex-determining genes in these closely related species.     

Interspecific hybridization between Oryzias latipes and Oryzias curvinotus causes XY sex reversal

The teleost fish, Oryzias curvinotus, is a closely related species to the medaka, Oryzias latipes, and both species have the DMY gene, which is required for male development in O. latipes. It suggests that the molecular function of the DMY gene and the following molecular events of sex differentiation are conserved between these two species. In the present study, we obtained interspecific hybrids between O. curvinotus and O. latipes and demonstrated sex-reversed XY females in the hybrids. The incidence of sex-reversed females in F1 XY hybrids between O. curvinotus females and O. latipes males, and hybrids between O. latipes females and O. curvinotus males were 21% and 100%, respectively. These results indicate that DMY does not always determine maleness in hybrid fish even though it is able to specify normal male development on its native genetic background and suggest that there are some differences between DMY(latipes) and DMY(curvinotus) alleles. Appearance of XY females in F1 hybrids also suggests that an autosomal or X-liked gene(s) from the maternal species interferes in the function of the paternal DMY gene in the male-determining process of the hybrid fish. These hybrid fish would supply a new experimental approach for investigating the genetic and molecular pathway of testis determination and differentiation.     

Tracing the emergence of a novel sex-determining gene in medaka, Oryzias luzonensis

Three sex-determining (SD) genes, SRY (mammals), Dmy (medaka), and DM-W (Xenopus laevis), have been identified to date in vertebrates. However, how and why a new sex-determining gene appears remains unknown, as do the switching mechanisms of the master sex-determining gene. Here, we used positional cloning to search for the sex-determining gene in Oryzias luzonensis and found that GsdfY (gonadal soma derived growth factor on the Y chromosome) has replaced Dmy as the master sex-determining gene in this species. We found that GsdfY showed high expression specifically in males during sex differentiation. Furthermore, the presence of a genomic fragment that included GsdfY converts XX individuals into fertile XX males. Luciferase assays demonstrated that the upstream sequence of GsdfY contributes to the male-specific high expression. Gsdf is downstream of Dmy in the sex-determining cascade of O. latipes, suggesting that emergence of the Dmy-independent Gsdf allele led to the appearance of this novel sex-determining gene in O. luzonensis.     

The medaka sex-determining gene DMY acquired a novel temporal expression pattern after duplication of DMRT1

The male sex-determining gene, DMY, of the medaka is considered to have arisen via gene duplication of DMRT1. In the medaka, both genes are expressed in Sertoli cell lineage cells, but their temporal expression patterns are quite different. DMY expression starts just before the sex-determining period, whereas DMRT1 expression occurs during the testicular differentiation period. To evaluate the alterations to the expression patterns of the DMRT1 genes after duplication, we analyzed the morphological gonadal sex differentiation processes and expression patterns of DMRT1 in Oryzias luzonensis and Oryzias mekongensis, which are closely related to the medaka but do not have DMY. Male-specific upregulation of DMRT1 in these two species occurred during the testicular differentiation period, similar to the case for DMRT1 in the medaka. These findings suggest that DMY acquired a novel temporal expression pattern after duplication and that this event played a critical role in the evolutionary process of this gene.     

Wild-derived XY sex-reversal mutants in the Medaka, Oryzias latipes

The medaka, Oryzias latipes, has an XX/XY sex-determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a sex-determining gene in this species. Previously, we found 23 XY sex-reversed females from 11 localities by examining the genotypic sex of wild-caught medaka. Genetic analyses revealed that all these females had Y-linked gene mutations. Here, we aimed to clarify the cause of this sex reversal. To achieve this, we screened for mutations in the amino acid coding sequence of DMY and examined DMY expression at 0 days after hatching (dah) using densitometric semiquantitative RT-PCR. We found that the mutants could be classified into two groups. One contained mutations in the amino acid coding sequence of DMY, while the other had reduced DMY expression at 0 dah although the DMY coding sequence was normal. For the latter, histological analyses indicated that YwOurYwOur (YwOur, Y chromosome derived from an Oura XY female) individuals with the lowest DMY expression among the tested mutants were expected to develop into females at 0 dah. These results suggest that early testis development requires DMY expression above a threshold level. Mutants with reduced DMY expression may prove valuable for identifying DMY regulatory elements.     

Sex determination in fish: Lessons from the sex-determining gene of the teleost medaka, Oryzias latipes

Although sex determination systems in animals are diverse, sex-determining genes have been identified only in mammals and some invertebrates. Recently, DMY (DM domain gene on the Y chromosome) has been found in the sex-determining region on the Y chromosome of the teleost medaka fish, Oryzias latipes. Functional and expression analyses of DMY show it to be the leading candidate for the male-determining master gene of the medaka. Although some work is required to define DMY as the master sex-determining gene, medaka is expected to be a good experimental animal for investigating the precise mechanisms involved in primary sex determination in non-mammalian vertebrates. In this article, the process of identification of DMY and is summarized and the origins of DMY and sexual development of the medaka's gonads are reviewed. In addition, putative functions of DMY are discussed.     

Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations

The medaka, Oryzias latipes, has an XX/XY sex determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a prime candidate for the sex-determining gene. Furthermore, the crucial role of DMY during male development was established by studying two wild-derived XY female mutants. In this study, to find new DMY and sex-determination related gene mutations, we conducted a broad survey of the genotypic sex (DMY-negative or DMY-positive) of wild fish. We examined 2274 wild-caught fish from 40 localities throughout Japan, and 730 fish from 69 wild stocks from Japan, Korea, China, and Taiwan. The phenotypic sex type agreed with the genotypic sex of most fish, while 26 DMY-positive (XY) females and 15 DMY-negative (XX) males were found from 13 and 8 localities, respectively. Sixteen XY sex-reversals from 11 localities were mated with XY males of inbred strains, and the genotypic and phenotypic sexes of the F(1) progeny were analyzed. All these XY sex-reversals produced XY females in the F(1) generation, and all F(1) XY females had the maternal Y chromosome. These results show that DMY is a common sex-determining gene in wild populations of O. latipes and that all XY sex-reversals investigated had a DMY or DMY-linked gene mutation.     

The Y chromosome that lost the male-determining function behaves as an X chromosome in the medaka fish, Oryzias latipes

The medaka, Oryzias latipes, has an XX/XY sex-determination system, and a Y-linked DM-domain gene, DMY, is the sex-determining gene in this species. Since DMY appears to have arisen from a duplicated copy of the autosomal DMRT1 gene approximately 10 million years ago, the medaka Y chromosome is considered to be one of the youngest male-determining chromosomes in vertebrates. In the screening process of sex-reversal mutants from wild populations, we found a population that contained a number of XY females. PCR, direct sequencing, and RT-PCR analyses revealed two different null DMY mutations in this population. One mutation caused loss of expression during the sex-determining period, while the other comprised a large deletion in putative functional domains. YY females with the mutant-type DMY genes on their Y chromosomes were fully fertile, indicating that the X and Y chromosomes were functionally the same except for the male-determining function. In addition, we investigated the frequencies of the sex chromosome types in this population over four successive generations. The Y chromosomes bearing the mutant-type DMY genes were detected every year with no significant differences in their frequencies. These results demonstrate that aberrant Y chromosomes behaving as X chromosomes have been maintained in this population.     

Evolutionary origin of the medaka Y chromosome

Genetic sex determination in an XX-XY chromosome system can be realized through a locus on the Y chromosome that makes the undifferentiated gonad develop into a testis. Although this mechanism is widespread, only in two cases so far have the corresponding master male sex-determining genes been identified. One is Sry, which initiates testes determination in most mammals. The other is dmrt1bY (syn. dmy), from the fish medaka, Oryzias latipes. The mammalian Y is roughly estimated to be over 200 million years old. The medaka Y may be considerably younger. A comparative analysis of the genus Oryzias revealed that one sister species of the medaka has dmrt1bY on a homologous Y chromosome, whereas in another closely related species only a non-sex-linked pseudogene is present. In all other species, dmrt1bY was not detected. The divergence time for the different species was determined with mitochondrial DNA sequences. The timing was confirmed by independent calculations based on dmrt1 sequences. We show that the medaka sex-determining gene originated approximately 10 million years ago. This makes dmrt1bY and the corresponding Y chromosome the youngest male sex-determining system, at least in vertebrates, known so far.     

A highly repetitive interspersed sequence isolated from genomic DNA of the Medaka, Oryzias latipes, is conserved in three other related species within the genus Oryzias.

A highly repeated interspersed sequence (OLR1) was isolated from a genomic DNA library of the Medaka, Oryzias latipes. The OLR1 was about 160 base pairs (bp) in length. As judged from the results of colony hybridization experiments, OLR1 is one of the major repeated DNA sequences in the Medaka genome and is present in every 136 kb on average. Results of Southern and colony-hybridization analyses indicate that OLR1 is a small interspersed repetitive element (SINE). OLR1-related sequences were conserved in other three species (O. luzonensis, O. curvinotus, and O. mekongnensis) within the genus Oryzias as a repetitive sequence. These results lend support at the DNA level to the hypothesis that these four species form one group in the genus Oryzias, as has been suggested from an analysis of their karyotypes (Magtoon and Uwa, '85, Proc. Jpn. Acad., Ser. B, 61:157-160).     

Induction of female-to-male sex reversal by high temperature treatment in Medaka, Oryzias latipes

Medaka, Oryzias latipes, has a firm XX-XY sex-determining system with the sex-determining gene, DMY, on the Y chromosome. However, previous studies have suggested that high water temperature might affect sex determination in Medaka. In the present study, the influence of high water temperature on sex reversal was examined. Fertilized eggs of two inbred strains of Medaka were developed at high water temperature (32 degrees C) until hatching. The hatched fry were kept at normal water temperatures (27 degrees C) until adulthood, and the phenotypic and genotypic sex was examined. As a result, 24% (N=105) and 50% (N=36) of XX fish developed a male phenotype in the Hd-rR and HNI inbred strains, respectively. These XX sex-reversed males had a normal testis and were fully fertile. On the other hand, all XY fish were male in the both strains. These results demonstrate that high water temperatures can induce XX sex reversal and that elevated water temperatures during the embryonic stage is a simple and useful method for getting XX males in Medaka.     

Differences in recombination frequencies during female and male meioses of the sex chromosomes of the medaka, Oryzias latipes

In the medaka, Oryzias latipes, sex is determined chromosomally. The sex chromosomes differ from those of mammals in that the X and Y chromosomes are highly homologous. Using backcross panels for linkage analysis, we mapped 21 sequence tagged site (STS) markers on the sex chromosomes (linkage group 1). The genetic map of the sex chromosome was established using male and female meioses. The genetic length of the sex chromosome was shorter in male than in female meioses. The region where male recombination is suppressed is the region close to the sex-determining gene y, while female recombination was suppressed in both the telomeric regions. The restriction in recombination does not occur uniformly on the sex chromosome, as the genetic map distances of the markers are not proportional in male and female recombination. Thus, this observation seems to support the hypothesis that the heterogeneous sex chromosomes were derived from suppression of recombination between autosomal chromosomes. In two of the markers, Yc-2 and Casp6, which were expressed sequence-tagged (EST) sites, polymorphisms of both X and Y chromosomes were detected. The alleles of the X and Y chromosomes were also detected in O. curvinotus, a species related to the medaka. These markers could be used for genotyping the sex chromosomes in the medaka and other species, and could be used in other studies on sex chromosomes.     

Inventing a sex-specific gene: a conserved role of DMRT1 in teleost fishes plus a recent duplication in the medaka Oryzias latipes resulted in DMY

DMY, the first sex-determining gene to be described in a nonmammal vertebrate was recently characterized in the medaka fish (Oryzias latipes). It is homologous to DMRT1, a conserved gene of the sex determination cascade in vertebrates. We have checked the near complete genomes of two other percomorph fishes, Tetraodon nigroviridis and Takifugu rubripes, for supplementary homologs of DMRT1 and DMY. We also compared the new gene, DMY, to its homolog DMRT1 from all available vertebrates. Finally, we found evidence for sex-specific expression and alternative splicing of the homolog from T. nigroviridis. Our results show that DMY is a recent duplicate of DMRT1 in the medaka. Its role in sex determination was not acquired through an acceleration of evolutionary rates, but by translocation to the Y chromosome and possibly changes at key positions.     

Evolution of ZZ/ZW and XX/XY sex-determination systems in the closely related medaka species, <Emphasis Type="Italic">Oryzias hubbsi</Emphasis> and <Emphasis Type="Italic">O. dancena</Emphasis>

A DM-domain gene on the Y chromosome was identified as the sex-determining gene in the medaka, Oryzias latipes, and named DMY (also known as dmrt1bY). However, this gene is absent in most Oryzias fishes, suggesting that closely related species have another sex-determining gene. In fact, it has been demonstrated that the Y chromosome in O. dancena is not homologous to that in O. latipes, whereas both species have an XX/XY sex-determination system. Through a progeny test of sex-reversed fish and a linkage analysis of isolated sex-linked DNA markers, we show that O. hubbsi, which is one of the most closely related species to O. dancena, has a ZZ/ZW system. In addition, genetic and fluorescence in situ hybridization mapping of the sex-linked markers revealed that sex chromosomes in O. hubbsi and O. dancena are not homologous, indicating different origins of these ZW and XY sex chromosomes. Furthermore, we found that O. hubbsi has morphologically heteromorphic sex chromosomes, in which the W chromosome has 4,6-diamidino-2-phenylindole (DAPI)-positive heterochromatin blocks and is larger than the Z chromosome, although such differentiated sex chromosomes have not been observed in other Oryzias species. These findings suggest that a variety of sex-determining mechanisms and sex chromosomes have evolved in Oryzias.     

Cytogenetic studies on the origin and species differentiation of the Philippine medaka, Oryzias luzonensis

The origin and species differentiation of the Philippine medaka, Oryzias luzonensis , was studied by means of karyotype analyses and interspecific hybridyzations. The karyotype revealed 2n = 48 consisting of 24 pairs of biarmed chromosomes (NF = 96). Characteristic C-bands were found in three sub-metacentric pairs. Nucleolar organizer regions (NORs) were detected on the short arms of the satellited sub-metacentric pair. Estimated DNA-value was 1.9 pg per nucleus. Interspecific crosses revealed that O. luzonensis was allied most closely to O. latipes (biarmed chromosome type), less closely to O. celebensis (fused chromosome type), and least closely to O. melastigma and O.javanicus (monoarmed chromosome type), which are distributed in east Asia, the Sulawesi, and south-west Asia and west side of south-east Asia, respectively. The external appearance of O. luzonensis was also more similar to that of O. latipes . From these results, we concluded that O. luzonensis is a member of the biarmed chromosome group and the nearest relative to O. latipes . Thus, the Philippine medaka seems to be of east Asiatic origin, having migrated to northern Luzon from the China continent via Formosa.