A Functional Study of the Apoptosis Caspase Gene Family in the Sex Differentiation of Chinese Tongue Sole (Cynoglossus semilaevis)

Chinese tongue sole (Cynoglossus semilaevis) is an important aquaculture species in China. Under high-temperature conditions, genetically female fish can undergo sex reversal and develop into phenotypic males (pseudomale fish). Previous studies have demonstrated that apoptosis might function in sex differentiation. Based on this, we identified and characterized the functions of six caspase genes (caspase1-like, caspase3a, caspase6, caspase8, caspase8-like, caspase9) in Chinese tongue sole. These six caspase genes were expressed in all analyzed tissues of both males and females. They were detected to be expressed in the gonads at various developmental stages, with expression levels peaking between 7 months and 2 years of age. In situ hybridization (ISH) analysis showed that the caspase genes were mainly localized in spermatocytes and oocytes. Promoter activity analysis indicated that with the exception of caspase3a, the remaining five caspase genes exhibited promoter activity and were regulated by transcription factors, including sp1 and gata4. High-temperature stimulation can significantly affect the expression of caspases in the gonads of both male and female fish, with female fish showing a more pronounced response. An siRNA-mediated knockdown experiment revealed that following caspase knockdown, the expression of sex differentiation-related genes, heat shock transcription factors (hsf), and heat shock proteins (hsp) in Chinese tongue sole was significantly altered. Based on these findings, we speculate that caspases play an important role in the sex differentiation process by responding to temperature stimuli.