A Duplicated, Truncated <i>amh</i> Gene Is Involved in Male Sex Determination in an Old World Silverside

Abstract

A master sex-determining gene, the Y chromosome-linked anti-Müllerian hormone (<i>amhy</i>) gene, has been described in two New World atheriniform species but little is known on the distribution, evolution, and function(s) of this gene in other Atheriniformes. Interestingly, <i>amhy</i> has been found to coexist with temperature-dependent sex determination (TSD), providing a unique opportunity to explore the interplay between genotypic and environmental sex determination. In this study, the search for an <i>amhy</i> homolog was extended to an Old World atheriniform, the cobaltcap silverside <i>Hypoatherina tsurugae</i> (Atherinidae). The full sequences, including the coding and noncoding regions, of the autosomal <i>amh</i> (<i>amha</i>) and a putative <i>amhy</i> were obtained. The deduced Amha and Amhy proteins comprised 511 and 340 amino acids (aa), respectively. PCR analysis with genomic DNA from wild adults and from laboratory-reared juveniles revealed a high, but not complete association of ∼95% between <i>amhy</i> and maleness. The spatiotemporal expression of <i>amhy</i> and <i>amha</i> during gonadal sex differentiation was analyzed by qRT-PCR and <i>in situ</i> hybridization (ISH). <i>amhy</i> transcription (in <i>amhy</i>-positive larvae) started before and peaked during histological differentiation of the gonads whereas <i>amha</i> was negligible during the same period in both genotypes. These results demonstrate that the <i>amhy</i>, although with some structural differences in relation to the <i>amhy</i> of some New World atheriniforms, is strongly associated with maleness and probably important for testicular development in this Old World atheriniform. Thus, <i>amhy</i> is a candidate sex determination gene in cobaltcap silverside and it will be key to scrutinize the mechanism of sex determination in this species.