Müllerian-inhibiting substance (MIS) is both necessary and sufficient for testicular differentiation in Chinese soft-shelled turtle Pelodiscus sinensis

Mullerian-inhibiting substance ( Mis , or anti-mullerian hormone, Amh ), a member of TGF-β superfamily, as initiator or key regulator in sexual development has been well documented in some vertebrates, especially in fish. However, its functional role has not been identified yet in reptiles. Here we characterized the Mis gene in Chinese soft-shelled turtle Pelodiscus sinensis ( P. sinensis ), a typical reptilian species exhibiting ZZ/ZW sex chromosomes. The mRNA of Mis was initially expressed in male embryonic gonads by stage 15, preceding gonadal sex differentiation, and exhibited male-specific expression pattern throughout embryogenesis. Moreover, Mis was rapidly up-regulated during female-to-male sex reversal induced by aromatase inhibitor letrozole. Most importantly, Mis loss of function by RNA interference led to complete feminization of genetic male (ZZ) gonads, suppression of the testicular marker Sox9 , and upregulation of the ovarian regulator Cyp19a1 . Conversely, overexpression of Mis in ZW embryos resulted in female-to-male sex reversal, characterized by the formation of testis structure, ectopic activation of Sox9 , and a remarkable decline in Cyp19a1 . Collectively, these findings provide the first solid evidence that Mis is both necessary and sufficient to drive testicular development in a reptilian species, P. sinensis , highlighting the significance of the TGF-β pathway in reptilian sex determination.