Delayed gonadal development of estrogen-induced ZZ females in Chinese soft-shelled turtles†

Exogenous estrogen induces sex reversal during embryonic development in various animal species including reptiles, yet the mechanisms underlying post-reversal gonadal development remain poorly understood. In this study, we generated ZZ female Chinese soft-shelled turtle Pelodiscus sinensis through 17β-estradiol treatment and investigated their reproductive traits and sex-specific gene expression at 1, 3, 6, 8, and 12 months post-hatching. Our results showed that by 12 months, ZZ females maintained ovarian structures and exhibited low expression levels of the male-specific genes SOX9 and DMRT1. However, at 8 months, the gonadosomatic index of ZZ female becomes significantly lower than that of ZW females, and yellow follicles were absent. Furthermore, the expression of female granulosa and theca cell markers FOXL2 and CYP19A1 in ZZ females was significantly lower than in ZW females. Our findings indicate that while sex-reversed ZZ female turtles retain female gonadal phenotypes, they experience delayed ovarian development at 8 months. The findings directly inform all-male aquaculture strategies by clarifying estrogen's long-term effects on gonadal development.