Sox9 is indispensable for testis differentiation in the red-eared slider turtle, a reptile with temperature-dependent sex determination

SOX9 (SRY-related HMG box gene 9) is an essential regulator of male sex determination and testis differentiation in many vertebrate species. However, the functional role of Sox9 in testis differentiation has not yet been identified in any reptilian species. Herein, Sox9 knockdown in the red-eared slider turtle (Trachemys scripta) embryos at a male-producing temperature led to complete male-to-female sex reversal, characterized by the formation of an ovary-like structure, disappearance of male marker AMH, and ectopic expression of ovarian regulator FOXL2, as well as a female distribution pattern of germ cells. Conversely, in-ovo overexpression of Sox9 at a female-producing temperature resulted in partial masculinization of putative female embryos, with the co-existence of AMH and FOXL2. Our study provides the first direct evidence that Sox9 is indispensable for testicular differentiation in a reptilian species, further confirming the conserved role of Sox9 in vertebrate sexual development.