Molecular cloning and expression pattern of <italic>Amh </italic>and its preliminary functional analysis in male sexual differentiation in <italic>Pelodiscus sinensis</italic>

Abstract

To investigate the regulatory role of Amh in male sexual differentiation in Pelodiscus sinensis ( P. sinensis ), we performed the full-length cDNA cloning of Amh and its differential expression analysis, and subsequently conducted the loss-of-function of Amh through the lentivirus-mediated RNA interference approach in this study. RACE results showed that the complete cDNA sequence of P. sinensis Amh is 3233 bp, including 997 bp of 5′ un-translated region, 834 bp of 3′ un-translated region and 1401 bp of open reading frame, which encodes 466 amino acids. Real-time PCR analysis showed the testis-specific expression of Amh in P. sinensis . The male-specific expression of Amh in embryonic gonads initiated early at stage 16, preceding the onset of gonadal sex differentiation. However, the female embryonic gonads exhibited extremely low expression level of Amh throughout the embryogenesis. In addition, the expression of Amh was induced in ZW embryonic gonads that were masculinized by aromatase inhibitor treatment, whereas, Amh expression was dramatically decreased in feminized ZZ embryonic gonads induced by estrogen. Most importantly, RNA interference analysis showed that ZZ embryos with Amh knockdown exhibited male to female sex reversal, characterized by obviously feminized gonads with developed cortical regions and degenerated medulla, a decline in the testicular marker Sox9 , and up-regulation of ovarian marker Cyp19a1 . These data indicate that Amh is essential for primary male sexual differentiation and might functions as an upstream regulator in male pathway cascade in P. sinensis . This study provides a foundation for understanding the mechanism of sex determination in P. sinensis .