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.     

The XX-XY sex-determination system in Oryzias luzonensis and O. mekongensis revealed by the sex ratio of the progeny of sex-reversed fish

The sex-determining gene in Oryzias latipes and O. curvinotus has been proved to be DMY. Although O. curvinotus has the DMY gene on the Y chromosome which is homologous to the Y chromosome of O. latipes, the sex-determining mechanism of other Oryzias fishes has not been identified. In order to uncover the sex-determining mechanism of O. luzonensis and O. mekongensis, which are most closely related species to O. latipes and O. curvinotus, we analyzed the sex ratio of the progeny of sex-reversed fish. We were able to obtain sex-reversed males by the administration of methyltestosterone, and found that these yielded all-female offspring in both species. These results indicate that O. luzonensis and O. mekongensis have the XX-XY sex-determination system.     

Common spontaneous sex-reversed XX males of the medaka Oryzias latipes

In the medaka, a duplicated version of the dmrt1 gene, dmrt1bY, has been identified as a candidate for the master male sex-determining gene on the Y chromosome. By screening several strains of Northern and Southern medaka we identified a considerable number of males with normal phenotype and uncompromised fertility, but lacking dmrt1bY. The frequency of such males was >10% in some strains and zero in others. Analysis for the presence of other Y-linked markers by FISH analysis, PCR, and phenotype indicated that their genotype is XX. Crossing such males with XX females led to a strong female bias in the offspring and also to a reappearance of XX males in the following generations. This indicated that the candidate male sex-determining gene dmrt1bY may not be necessary for male development in every case, but that its function can be taken over by so far unidentified autosomal modifiers.     

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.     

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.     

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.     

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.     

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.     

Genetic risk involved in stock enhancement of fish having environmental sex determination

Environmental sex determination is reported in various fish species, including some fishes subject to stock enhancement. We studied the influence of stock enhancement on the sex-determining system of fish with both genotypic and environmental sex determination. We constructed and analyzed recurrence formula models for the dynamics of gene frequency in the male heterogametic (XX female and XY male) sex-determining system of the Japanese flounder (Paralichthys olivaceus). In this species, an XX individual can develop as a phenotypic male (sex-reversed male) depending on the conditions experienced by a juvenile. We show that the release of sex-reversed males may result in the extinction of the Y gene that determines sex. We also studied how the risk depends on the kind of hatchery broodstock used (e.g., fish collected from the wild or fish from a lineage established in a hatchery), sex-reversal rates in the hatchery and in the wild, and the relative contribution of released fish to reproduction in the wild. We propose some ways to reduce the risk. Received: October 10, 2001 / Accepted: December 17, 2001     

An Immune-Related Gene Evolved into the Master Sex-Determining Gene in Rainbow Trout, Oncorhynchus mykiss

Since the discovery of Sry in mammals [1, 2], few other master sex-determining genes have been identified in vertebrates [3-7]. To date, all of these genes have been characterized as well-known factors in the sex differentiation pathway, suggesting that the same subset of genes have been repeatedly and independently selected throughout evolution as master sex determinants [8, 9]. Here, we characterized in rainbow trout an unknown gene expressed only in the testis, with a predominant expression during testicular differentiation. This gene is a male-specific genomic sequence that is colocalized along with the sex-determining locus. This gene, named sdY for sexually dimorphic on the Y?chromosome, encodes a protein that displays similarity to the C-terminal domain of interferon regulatory factor 9. The targeted inactivation of sdY in males using zinc-finger nuclease induces ovarian differentiation, and the overexpression of sdY in females using additive transgenesis induces testicular differentiation. Together, these results demonstrate that sdY is a novel vertebrate master sex-determining gene not related to any known sex-differentiating gene. These findings highlight an unexpected evolutionary plasticity in vertebrate sex determination through the demonstration that master sex determinants can arise from the de novo evolution of genes that have not been previously implicated in sex differentiation.     

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.     

Genome assembly,sex-biased gene expression and dosage compensation in the damselfly Ischnura elegans

The evolution of sex chromosomes, and patterns of sex-biased gene expression and dosage compensation, are poorly known among early winged insects such as odonates. We assembled and annotated the genome of Ischnura elegans (blue-tailed damselfly), which, like other odonates, has a male-hemigametic sex-determining system (X0 males, XX females). By identifying X-linked genes in I. elegans and their orthologs in other insect genomes, we found homologies between the X chromosome in odonates and chromosomes of other orders, including the X chromosome in Coleoptera. Next, we showed balanced expression of X-linked genes between sexes in adult I. elegans, i.e. evidence of dosage compensation. Finally, among the genes in the sex-determining pathway only fruitless was found to be X-linked, while only doublesex showed sex-biased expression. This study reveals partly conserved sex chromosome synteny and independent evolution of dosage compensation among insect orders separated by several hundred million years of evolutionary history.     

Genetic mapping of the autosomal region involved in XX sex-reversal and horn development in goats

Contrary to other genetic disorders, the genetic study of sex determination anomalies in humans stumbles over the difficulty in observing large pedigrees. In goats, abnormalities in sex determination are intimately linked to a dominant Mendelian gene coding for the ``polled' (hornless) character, which could render this species an interesting animal model for the rare human cases of SRY-negative XX males. In this report, we describe genetic linkage between the polled/intersex synchome (PIS) and four microsatellite markers of the distal region of goat Chromosome 1 (CHI1), quite distinct from the bovine ``polled' region. According to comparative mapping data, no sex-determining gene has been described so far in homologous regions in the human. This genetic localization constitutes a first step towards identifying a new autosomal sex-determining gene in mammals. Received: 7 July 1995 / Accepted: 6 September 1995     

The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate

The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and act as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. We propose that this repression is achieved by the Y-linked male-determining factor (M).     

Potency of testicular somatic environment to support spermatogenesis in XX/Sry transgenic male mice

The sex-determining region of Chr Y (Sry) gene is sufficient to induce testis formation and the subsequent male development of internal and external genitalia in chromosomally female mice and humans. In XX sex-reversed males, such as XX/Sry-transgenic (XX/Sry) mice, however, testicular germ cells always disappear soon after birth because of germ cell-autonomous defects. Therefore, it remains unclear whether or not Sry alone is sufficient to induce a fully functional testicular soma capable of supporting complete spermatogenesis in the XX body. Here, we demonstrate that the testicular somatic environment of XX/Sry males is defective in supporting the later phases of spermatogenesis. Spermatogonial transplantation analyses using XX/Sry male mice revealed that donor XY spermatogonia are capable of proliferating, of entering meiosis and of differentiating to the round-spermatid stage. XY-donor-derived round spermatids, however, were frequently detached from the XX/Sry seminiferous epithelia and underwent cell death, resulting in severe deficiency of elongated spermatid stages. By contrast, immature XY seminiferous tubule segments transplanted under XX/Sry testis capsules clearly displayed proper differentiation into elongated spermatids in the transplanted XY-donor tubules. Microarray analysis of seminiferous tubules isolated from XX/Sry testes confirmed the missing expression of several Y-linked genes and the alterations in the expression profile of genes associated with spermiogenesis. Therefore, our findings indicate dysfunction of the somatic tubule components, probably Sertoli cells, of XX/Sry testes, highlighting the idea that Sry alone is insufficient to induce a fully functional Sertoli cell in XX mice.     

Mutation in a sex-determining gene in rainbow trout: detection and genetic analysis

In rainbow trout (Oncorhynchus mykiss), the acknowledged sex-determining system is genetic sex determination (GSD) with female homogamety (female symbolXX-male symbolXY). Subsequently, mitotic gynogens are all expected to be females. Unexpected maleness was fortuitously observed in a mitotic gynogenetic family of rainbow trout (13 males out of 27). An equal ratio of males and females suggested the possible segregation of some Mendelian sex-influencing factor. In order to perform a comprehensive analysis of the inheritance and expression of the factor involved, the transmission of maleness was studied across the next three generations, using both conventional and/or meiotic and mitotic gynogenetic offspring. On the whole, males as well as intersexes were observed in crosses between two expected carrier parents, and in gynogenetic offspring of expected carrier females, but not in crosses between one expected carrier parent and one normal XX control. Sex ratios in the different crosses often fitted Mendelian proportions, but not always. Both excess and lack of maleness were observed. The simplest hypothesis consistent with most results is a one-locus model, assuming the existence of a mutation (termed mal) of a sex-determining gene, which is able to override the primary XX mechanism of sex determination and to induce the development of testicular tissue in the gonads of expected XX individuals. The one-locus model requires that the mal mutation usually, but not systematically, behave as a recessive mutation and have a limited penetrance, that is, heterozygous (mal/+) may be sex reversed, homozygous (mal/mal) may remain female, and carrier individuals may undergo partial masculinization alone (many intersexes were recorded). Inconsistency in sex ratios among offspring of parents expected to respond the same way was recorded, indicating that other modifier loci may also be involved. Finally, the occurrence of both males and females in clonal progenies showed that epigenetic factors also likely influence the expression of maleness. The effects of the mal mutation are compared to similar mutations recently described in other fish species. The nature and location of the mal gene (carried by heterochromosomes or an autosomal pair) is briefly discussed in view of the knowledge recently acquired on the subject.