The recent discovery of the DMY gene (DM domain gene on Y chromosome and one of the DMRT1 family genes) as a key determinant of male development in the medaka (Oryzias latipes) has led to its designation as the prime candidate gene for sex-determination in this species. This study focused on the sites and pattern of expression of DMY and DMRT1 genes during gonadal differentiation of medaka to further determine their roles in testis development. DMY mRNA and protein are expressed specifically in the somatic cells surrounding primordial germ cells (PGCs) in the early gonadal primordium, before morphological sex differences are seen. However, somatic cells surrounding PGCs never express DMY during the early migratory period. Expression of DMY persists in Sertoli cell lineage cells, from PGC-supporting cells to Sertoli cells, indicating that only DMY-positive cells enclose PGCs during mitotic arrest after hatching. DMRT1 is expressed in spermatogonium-supporting cells after testicular differentiation (20 -30 days after hatching), and its expression is much higher than that of DMY in mature testes. In XX sex-reversed testes, DMRT1 is expressed in the Sertoli cell lineage, similar to the expression of DMY in XY testes. These results suggest strongly that DMY regulates PGC proliferation and differentiation sex-specifically during early gonadal differentiation of XY individuals and that DMRT1 regulates spermatogonial differentiation. Developmental Dynamics 231:518 -526, 2004.

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Highlights d A trans-species SNP in a steroidogenic Hsd17b1 determines sex in genus Seriola d The Z-linked allele of HSD17B1 attenuates E1 to E2 conversion activity d The data support a long-standing hypothesis on steroidinduced sex determination

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The sexual plasticity of the gonads is not retained after the completion of sex differentiation in vertebrates, except in some hermaphroditic species. Here, we report that the depletion of estradiol-17β (E2) by aromatase inhibitors (AI) for up to six months resulted in a functional female-to-male sex reversal in sexually-mature adults of two gonochoristic fish species, Nile tilapia and medaka. The sex-reversed fish showed a typical male pattern of E2 and androgen levels, secondary sexual characteristics, and male-like sex behavior, producing fertile sperm. Conversely, co-treatment of E2 inhibited AI-induced sex reversal. In situ hybridization of medaka gonads during AI-induced sex reversal indicated that cysts on the dorsal side of the adult ovaries are the origin of germ cells and Sertoli cells in the newly formed testicular tissue. Gonochoristic fish maintain their sexual plasticity until adulthood and E2 plays a critical role in maintaining the female phenotype.

The study of sex determination and sex chromosome organisation in non-model species has long been technically challenging, but new sequencing methodologies are now enabling precise and high-throughput identification of sex-specific genomic sequences. In particular, Restriction Site-Associated DNA Sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software designed to specifically search for sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analyzed a published dataset of Japanese medaka, Oryzias latipes , where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyze new RAD-Seq datasets from 15 fish species spanning multiple systematic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in non-model species and outperforms the commonly used RAD-Seq analysis software STACKS.RADSex in speed, resource usage, ease of application, and visualization options. Furthermore, our analysis of new datasets from 15 species provides new insights on sex determination in fish.

The study of sex determination and sex chromosome organisation in non-model species has long been technically challenging, but new sequencing methodologies are now enabling precise and high-throughput identification of sex-specific genomic sequences. In particular, Restriction Site-Associated DNA Sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software designed to specifically search for sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analyzed a published dataset of Japanese medaka, Oryzias latipes , where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyze new RAD-Seq datasets from 15 fish species spanning multiple systematic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in non-model species and outperforms the commonly used RAD-Seq analysis software STACKS.RADSex in speed, resource usage, ease of application, and visualization options. Furthermore, our analysis of new datasets from 15 species provides new insights on sex determination in fish.