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 Gsdf Y (gonadal soma derived growth factor on the Y chromosome) has replaced Dmy as the master sex-determining gene in this species. We found that Gsdf Y showed high expression specifically in males during sex differentiation. Furthermore, the presence of a genomic fragment that included Gsdf Y converts XX individuals into fertile XX males. Luciferase assays demonstrated that the upstream sequence of Gsdf Y 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 sexdetermining gene in O. luzonensis. IN most vertebrates, sex is determined genetically. Mammals and birds with cytogenetically well-differentiated sex chromosomes have sex determination systems that differ between the taxonomic classes but not within them (Solari 1994). In mammals, for example, the sex-determining (SD) gene SRY/Sry on the Y chromosome has a universal role in sex determination (Gubbay et al. 1990;Sinclair et al. 1990;Koopman et al. 1991;Foster et al. 1992). By contrast, some fish groups, such as salmonids, sticklebacks, and Oryzias fishes, have sex chromosomes that differ among closely related species (Devlin and Nagahama 2002;Woram et al. 2003;Takehana et al. 2007a;Ross et al. 2009).A DM-domain gene, Dmy, was the first SD gene identified in a nonmammalian vertebrate, the fish medaka Oryzias latipes (Matsuda et al. 2002(Matsuda et al. , 2007. In this species, the term Y chromosome is employed to refer to a recombining chromosome that carries the male-determining gene Dmy, and X is used for the homologous chromosome; these are not a heteromorphic pair. This gene is conserved among all wild populations of O. latipes examined to date . The closely related species O. curvinotus also has Dmy on its Y chromosome, which is orthologous to the O. latipes Y chromosome (Matsuda et al. 2003). However, Dmy has not been detected in any other type of fish, including other Oryzias fishes (Kondo et al. 2003). Analysis of the Y-specific region of the O. latipes sex chromosome has demonstrated that Dmy arose from duplication of the autosomal Dmrt1 gene (Nanda et al. 2002;Kondo et al. 2006). This Dmrt1 duplication is estimated to have occurred within the last 10 million years in a common ancestor of O. latipes, O. curvinotus, and O. luzonensis. In O. luzonensis, however, no functional duplicated copy of Dmrt1 has been detected (Kondo et al. 2003) (Figure 5A).O. luzonensis possesses an XX-XY system, which is homologous to an autosomal linkage group (LG 12) and Uwa 1985). In the d-rR strain, the wild-type alle...

Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3 Y transgenic fish, and Sox3 Y loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.

Sex chromosomes and the sex-determining (SD) gene are variable in vertebrates. In particular, medaka fishes in the genus Oryzias show an extremely large diversity in sex chromosomes and the SD gene, providing a good model to study the evolutionary process by which they turnover. Here, we investigated the sex determination system and sex chromosomes in six celebensis group species. Our sex-linkage analysis demonstrated that all species had an XX-XY sex determination system, and that the Oryzias marmoratus and O. profundicola sex chromosomes were homologous to O. latipes linkage group (LG) 10, while those of the other four species, O. celebensis, O. matanensis, O. wolasi, and O. woworae, were homologous to O. latipes LG 24. The phylogenetic relationship suggested a turnover of the sex chromosomes from O. latipes LG 24 to LG 10 within this group. Six sex-linkage maps showed that the former two and the latter four species shared a common SD locus, respectively, suggesting that the LG 24 acquired the SD function in a common ancestor of the celebensis group, and that the LG 10 SD function appeared in a common ancestor of O. marmoratus and O. profundicola after the divergence of O. matanensis. Additionally, fine mapping and association analysis in the former two species revealed that Sox3 on the Y chromosome is a prime candidate for the SD gene, and that the Y-specific 430-bp insertion might be involved in its SD function.