The Role of Anti-Müllerian Hormone in Testis Differentiation Reveals the Significance of the TGF-β Pathway in Reptilian Sex Determination

Anti-Müllerian hormone (Amh, or Müllerian-inhibiting substance, Mis), a member of TGF-β superfamily, has been well documented in some vertebrates as initiator or key regulator in sexual development, and particularly in fish. However, its functional role has not yet been identified in reptiles. Here, we characterized the Amh gene in the Chinese soft-shelled turtle Pelodiscus sinensis, a typical reptilian species exhibiting ZZ/ZW sex chromosomes. The messenger RNA of Amh was initially expressed in male embryonic gonads by stage 15, preceding gonadal sex differentiation, and exhibited a male-specific expression pattern throughout embryogenesis. Moreover, Amh was rapidly upregulated during female-to-male sex reversal induced by aromatase inhibitor letrozole. Most importantly, Amh 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 Amh in ZW embryos resulted in female-to-male sex reversal, characterized by the formation of a testis structure, ectopic activation of Sox9, and a remarkable decline in Cyp19a1. Collectively, these findings provide the first solid evidence that Amh 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.