Knockdown of R-spondin1 leads to partial sex reversal in genetic female Chinese soft-shelled turtle Pelodiscus sinensis

Chinese soft-shelled turtle Pelodiscus sinensis is an important aquaculture species in China, the male individual being more valuable in aquaculture because of its larger body size, higher growth rate and less fat compared with females. Understanding the mechanism of ovarian differentiation and development is crucial for the production of mono-sex male offspring. However, little is known about the molecular mechanism underlying turtle ovarian differentiation. Here, we characterized the Rspo1 gene, an upstream regulator of vertebrate female sexual differentiation, in P. sinensis. The messenger RNA of Rspo1 was initially expressed at stage 14, preceding gonadal sex differentiation, and exhibited a sexually dimorphic expression pattern throughout the sex determination and gonadal differentiation periods. Rspo1 was rapidly downregulated during aromatase inhibitor-induced female-to-male sex reversal, which occurred prior to gonadal differentiation. Rspo1 loss of function by RNA interference led to partial female-to-male sex reversal, with masculinized changes in the phenotype of gonads, the distribution of germ cells and the expression of testicular regulators. Collectively, these findings suggest that Rspo1 is necessary for primary female sexual differentiation in P. sinensis. This study demonstrates for the first time the functional role of Rspo1 in reptilian sex determination, and is of fundamental significance for the production of fertile pseudo-female parents and mono-sex male offspring of P.sinensis.