Induction of masculinization in mosquitofish (Gambusia affinis) by the synthetic progestin etonogestrel exposure: Insights from morphology, ovarian histology, and transcriptomic analyses.

The demand for synthetic progesterone has surged due to its widespread use in contraceptives and veterinary drugs. However, these compounds are not fully removed by wastewater treatment plants, leading to their release into aquatic environments and potential disruption of fish reproduction and development. Etonogestrel (ETO), a third-generation synthetic progestogen, is widely used in oral contraceptives. In this study, adult female mosquitofish were exposed to ETO at measured concentrations of 0 ng/L, 3.88 ng/L, 39.5 ng/L, and 385.18 ng/L for 36 days. Morphology, ovarian histology, and transcriptomic analyses were conducted to assess the effects of ETO. Results showed that ETO exposure reduced the body weight-to-total length ratio and promoted gonopodia and skeletal modifications. Particularly notable was the finding of decreased ovarian weight and the induction of testicular structures in the 385.18 ng/L ETO group, indicating a significant masculinization effect. Transcriptomic analysis revealed that genes related to sexual differentiation (cyp17a1, hsd17b3 and sox9), which were enriched in the GnRH signaling pathway and steroid hormone biosynthesis, may drive masculinization by promoting androgen production and inhibiting estrogen synthesis. Additionally, ETO may promote morphological masculinization by altering lipid metabolism patterns. These findings indicate that ETO could induce masculinization and negatively impact reproductive health in mosquitofish, an effect potentially exacerbated by changes in lipid metabolism.