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

Anti-Müllerian hormone (, or Müllerian-inhibiting substance, ), 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 gene in the Chinese soft-shelled turtle , a typical reptilian species exhibiting ZZ/ZW sex chromosomes. The messenger RNA of was initially expressed in male embryonic gonads by stage 15, preceding gonadal sex differentiation, and exhibited a male-specific expression pattern throughout embryogenesis. Moreover, was rapidly upregulated during female-to-male sex reversal induced by aromatase inhibitor letrozole. Most importantly, loss of function by RNA interference led to complete feminization of genetic male (ZZ) gonads, suppression of the testicular marker , and upregulation of the ovarian regulator Conversely, overexpression of in ZW embryos resulted in female-to-male sex reversal, characterized by the formation of a testis structure, ectopic activation of , and a remarkable decline in Collectively, these findings provide the first solid evidence that is both necessary and sufficient to drive testicular development in a reptilian species, , highlighting the significance of the TGF-β pathway in reptilian sex determination.