Molecular Mechanisms of Gonadal Differentiation Induced by 17β-Estradiol and Testosterone Propionate in Rana dybowskii.

Rana dybowskii is an economically important amphibian in Northeast China, valued for its medicinal oviduct (Oviductus Ranae). However, the low proportion of females in artificial culture severely restricts industrial productivity. To elucidate the molecular mechanisms of hormone-induced sex reversal, based on our previous research results, we exposed tadpoles to 17β-estradiol (E2) or testosterone propionate (TP). We then monitored gonadal histology, conducted multi-stage transcriptome sequencing, and validated key genes via qRT-PCR. The results show that induction treatment with 40 μg·L-1 E2 and 80 μg·L-1 TP resulted in 93.33% female and 100% male populations, respectively. Both hormones promoted germ cell proliferation at the undifferentiated stage and directed stable ovarian or testicular development without intersex abnormalities. Ovarian differentiation involved progressive multi-wave transcriptional reprogramming, whereas testicular differentiation exhibited a concentrated gene expression burst during gonadal maturation. Steroid hormone biosynthesis, Wnt, and PPAR pathways formed the core regulatory network. Three female-biased genes (3α-hsd, Adcy3, Rspo1) and three male-biased genes (Ptgs2, Sox9, Shbg) were identified, with their expression dynamics aligned with histological progression. This study defines the optimal parameters and molecular signatures of bidirectional sex reversal in R. dybowskii. It provides a practical foundation for sex-control breeding and offers amphibian-based evidence for the conservation and plasticity of vertebrate sex determination.