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

Abstract

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.

Figure 1

(A) Genetic and physical map of the sex-determining regions on the X and Y chromosomes. 1, normal XY male; 2, XX female; 3 and 4, recombinant male. Blue column, regions derived from the Y chromosome; red, from the X chromosome. Red bar, BAC clones; blue bar, fosmid clones. Black arrows indicate predicted genes. (B) RT–PCR products of PGs (PG1–9) in the XX and XY body trunk at 0 days after hatching. M, size marker.

Figure 2

(A) Genomic structure of Gsdf^Y and Gsdf^X. Two synonymous substitutions are present in exon 3. Open boxes, exons. Horizontal bars, introns. Numbers represent nucleotide sequence length (bp). Open arrowheads indicate the translation start (ATG) and stop (TGA) sites. (B) Amino acid sequences of Gsdf. Gsdf^Y and Gsdf^X are the same. Signal peptide is indicated in italics. The six conserved cysteine residues are underlined. (C) Neighbor-joining (NJ) tree for the TGF-β superfamily using the amino acid sequence of the mature domain. The tree was rooted by using BDNF.

Figure 3

Expression of Gsdf. (A) Real-time PCR of Gsdf in XX and XY fry from 2 days before hatching (dbh) to 10 days after hatching (dah). Blue bars, expression of PG5 on the Y chromosome (Gsdf^Y); red, the X chromosome (Gsdf^X). Significant differences were analyzed by using two-way analysis of variance (ANOVA) followed by Bonferroni post-tests. Columns and error bars represent mean ± SEM (n = 4 per developmental stage). (b–d) In situ hybridization of Gsdf in the gonad. (B) Expression of Gsdf in 5-dah XY fry. Gsdf was detected only in somatic cells surrounding germ cells. (C) Expression of Gsdf in 5-dah XX fry. Weak signals were detected. (D) Gsdf expression in adult testis. Strong signals were detected in Sertoli cells (arrowheads) surrounding spermatogonia. Modest signals were found around the efferent duct (ed). (E) Gsdf expression in adult ovary. Signals were detected in the granulosa cells (arrows) surrounding well-developed oocytes (oc). nd, nephric duct; go, gonad; gu, gut.

Figure 4

Mutations in the Gsdf cis-regulatory element contribute to Gsdf^Y-specific high expression. (A) Illustration of Gsdf^Y-specific mutations in the 1.8-kb upstream and 2-kb downstream regions of Gsdf^Y, comparing with Gsdf^X and Gsdf (O. latipes). Numbers represent positions of Gsdf^Y-specific mutations. (B) Gsdf^Y-specific sequences in the upstream region of Gsdf^Y. (C) Luciferase assay analysis of sequences responsible for Gsdf^Y-specific high expression. *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA, post hoc comparisons, Turkey’s test. Columns and error bars represent mean ± SEM (n = 12).

Figure 5

Changes in the sex-determining gene of O. luzonensis. (A) Evolutionary history up to the appearance of Gsdf^Y. Dmy appeared as a common ancestor of O. latipes, O. curvinotus, and O. luzonensis. Gsdf^Y appeared in, and Dmy disappeared from, the ancestor of O. luzonenesis. (B) Change in the sex-determining cascade. (Top) Sex-determining cascade in an ancestor of O. luzonensis. (Bottom) Current sex-determining cascade in O. luzonensis. Gsdf was downstream of Dmy. A mutation then occurred in Gsdf, allowing its expression without Dmy. Gsdf^Y then became the new sex-determining gene.