Functional characterization of <italic>Cyp19a1</italic> in female sexual differentiation in <italic>Pelodiscus sinensis</italic>

Abstract

Aromatase is the key enzyme for estrogen synthesis, and functions as a key regulator in early female sexual differentiation in non-mammalian vertebrates. In this study, the expression pattern of Cyp19a1 was examined in tissues and its functional role in ovarian differentiation was investigated through lentivirus-mediated RNA interference and over-expression systems. Results showed that Cyp19a1 was robustly expressed in adult ovary, and was not or weakly detected in testis, heart, liver, spleen, lung, kidney, muscle and intestine. Moreover, Cyp19a1 exhibited female sexual dimorphic expression pattern in embryonic gonads early at stage 18, preceding the onset time of gonadal differentiation (stage 19). Loss-of-function analysis revealed that ZW embryos with AI treatment or Cyp19a1 knockdown exhibited female to male sex reversal, characterized by obviously masculinized gonads with well-organized testis cord structure in the medulla and a degenerated cortex, an upregulation of testicular marker Sox9 and Amh , and a decline in ovarian marker Foxl2 . Gain-of-function analysis showed that ZZ gonads overexpressing Cyp19a1 were feminized, determined by a thickened outer cortex with a number of germ cells and a vacuolated medulla, as well as upregulation of Foxl2 and downregulation of Sox9 and Amh . In summary, Cyp19a1 is both necessary and sufficient to initiate ovarian development, thereby functioning as a key regulator of female sexual differentiation. This study provides a foundation for understanding the mechanism of sex determination in Pelodiscus sinensis.