Understanding the genetic sex-determining mechanism in Hyla eximia treefrog inferred from H-Y antigen

Genetic sex-determining mechanisms have been extensively elucidated in mammals; however, the sex chromosomes, sex-determining genes, and gene regulatory networks involved in sex differentiation remain poorly understood in amphibians. In this study, we investigated the sex-determining mechanism in the Hyla eximia treefrog based on karyotypic analysis and identification of H-Y antigen, a sex-linked peptide that is present in the gonads of the heterogametic sex (XY or ZW) in all vertebrates. Results show a diploid chromosome number 2n = 24 with homomorphic sex chromosomes. The heterogametic sex, ZW-female, were hypothesized based on H-Y antigen mRNA expression in female gonads (24,ZZ/24,ZW). The treefrog H-Y peptide exhibited a high percentage of identity with other vertebrate sequences uploaded to GenBank database. To obtain gene expression profiles, we also obtained the coding sequence of the housekeeping Actb gene. High H-Y antigen expression levels were further confirmed in ovaries using real-time polymerase chain reaction (RT-PCR) during non-breeding season, we noted a decrease in the expression of the H-Y antigen during breeding season. This study provides evidence that sex hormones might suppress H-Y antigen expression in the gonads of heterogametic females 24,ZW during the breeding season. These findings suggest that H-Y gene expression is a well-suited model for studying heterogametic sex by comparing the male and female gonads.