A Y-linked anti-Müllerian hormone duplication takes over a critical role in sex determination

Abstract

Gonadal sex determination in vertebrates generally follows a sequence of genetically programmed events. In what is seemingly becoming a pattern, all confirmed or current candidate "master" sex-determining genes reported in this group, e.g., SRY in eutherian mammals, DMY/dmrt1bY in medaka, DM-W in the African clawed frog, and DMRT1 in chicken encode transcription factors. In contrast, here we show that a male-specific, duplicated copy of the anti-Müllerian hormone (amh) is implicated in testicular development of the teleost fish Patagonian pejerrey (Odontesthes hatcheri). The gene, termed amhy because it is found in a single metacentric/submetacentric chromosome of XY individuals, is expressed much earlier than the autosomal amh (6 d after fertilization vs. 12 wk after fertilization) and is localized to presumptive Sertoli cells of XY males during testicular differentiation. Moreover, amhy knockdown in XY embryos resulted in the up-regulation of foxl2 and cyp19a1a mRNAs and the development of ovaries. These results are evidence of a functional amh duplication in vertebrates and suggest that amhy may be the master sex-determining gene in this species. If confirmed, this would be a unique instance of a hormone-related gene, a member of the TGF-β superfamily, in such a role.

Fig. 1.

amhy gene structure, relation with SNP marker and phenotypic sex, and physical mapping. (A) Schematic representation of amhy and amha gene structure in O. hatcheri. Exons (Ex1 to Ex7) are represented by filled boxes, and the percentage identity between corresponding exons is indicated. Open segments represent introns, and the gray-shaded area shows the location of the insertion in the amhy third intron. The location of the TGF-β domain (blue line) and of the start and stop codons (red dotted lines in the first and seventh exons, respectively) are indicated. (B) Pattern of amha (1,057 bp; Top panel, lower band) and amhy (1,614 bp; Top panel, upper band) amplification using primers flanking the third intron and of the sex-linked SNP marker in sex genotyping. Results show complete matching between genotypic sex (inferred by both amhy amplification and SNP marker) and phenotypic sex (only six animals are shown). (Middle) The SNP marker shows the amplification of the male-specific PCR fragment, and the Bottom lane the respective control. (C) Fluorescence in situ hybridization using as probe the amhy gene and its 5′ flanking region (7.3 kb) on metaphase spreads from XX and XY fish. The fragment was based on the amhy sequence but the probe hybridizes also to amha. XX individuals show two signals in a pair of acrocentric/telocentric chromosomes whereas XY individuals show, in addition to this pair of signals (presumptive amha), a strong signal (presumptive amhy) in a single metacentric/submetacentric chromosome.

Fig. 2.

Time course of gonadal sex differentiation in XX and XY fish. (A) Schematic representation of the morphological changes in female and male gonads during sex differentiation (wah: weeks after hatching). (B) Light histology of the critical developmental stages indicated by dotted boxes in A. Black (XX; 4 wah) and white (XY; 6 wah) arrowheads indicate the appearance of somatic cell outgrowths (ovarian differentiation) and rudiments of the main sperm duct (testicular differentiation), respectively.

Fig. 3.

XY-specific expression of amhy mRNA during embryogenesis and gonadal sex differentiation. (A and B) Temporal expression of amhy (1,699 bp) and amha (1,716 bp) mRNAs by RT-PCR in (A) embryos (daf: days after fertilization) and in (B) XX and XY larval trunks (wah: weeks after hatching). The β-actin (457-bp) gene was used as endogenous control for all samples. T: testis; O: ovary. (C) Localization of amhy mRNA (Right) in gonadal sections of XX and XY larvae at 3 wk after hatching (in situ hybridization with an antisense probe). (Left) H&E-stained sections of the same individuals. (Scale bars, 10 μm.)

Fig. 4.

Feminization in amhy knockdown XY fish. (A) Relative expression of foxl2 and cyp19a1a mRNAs in morpholino-injected fish at 4 weeks after hatching (wah). foxl2 and cyp19a1a expression was normalized by the respective β-actin values. The three XY individuals in the antisense morpholino group with female-like, high foxl2 values also show high values for cyp19a1a. (B) Histological appearance of the gonads in mispaired morpholino-injected XX control fish (XX-ctr), antisense morpholino-injected XY fish (XY-mo), and mispaired morpholino-injected XY fish (XY-ctr) at about 12 wah. OC: ovarian cavity. (Scale bars, 20 μm.)