Effects of 17α‐Ethinylestradiol on Development and Gonadal Differentiation in the Estuarine Mummichog, Fundulus heteroclitus

In fish, sex determination and gonadal development are controlled by various genetic and environmental factors. In this study, experiments were conducted on the estuarine mummichog ( Fundulus heteroclitus ) to investigate gonadal differentiation following exposure to the synthetic estrogen, 17α‐ethinylestradiol (EE 2 ). The period of sensitivity to EE 2 was explored by initiating the exposures at different times post fertilization. In Experiment 1, mummichog embryos were collected within 8 h of fertilization and then continually exposed to increasing concentrations of EE 2 (up to 229 ng/L) for up to 10 weeks. For controls, complete differentiation of the gonad to a testis or ovary in mummichog occurred by 3 weeks post hatch (wph) and there was an equal distribution of males and females. Exposure to all concentrations of EE 2 accelerated female gonadal differentiation as early as 1 wph and contributed to a highly female skewed sex ratio with 80% to 100% of the fish displaying ovaries. Exposure to EE 2 also resulted in a concentration‐dependent increase in skeletal abnormalities and mortalities whereas larval lengths were not affected. In Experiment 2, 24 h post‐hatch larvae were exposed for 5 weeks to three treatments with measured concentrations (ng/L) of 0.4 (Control), 2.5 ng/L, and 18.0 ng/L). The sex ratio was approximately 50/50 in controls and did not change with EE 2 treatment. EE 2 did not alter the proportion of oocyte stages (chromatin nucleolus, perinucleolar, cortical alveolar) but spermatogenesis was impeded as male fish had greater proportions of spermatogonia, and spermatids were only observed in controls. EE 2 treatment did not change the expression of genes in the ovary implicated in gonadal development including cyp19a1a , foxl2 , gdf9 , bmp15 , dmrt1 , and amh . In contrast, testis expression of dmrt1 was decreased and cyp19a1a , foxl2 , gdf9 , and amh were increased following EE 2 treatment. Overall, by the time of hatching (about 3 weeks post fertilization), sex differentiation was complete, and the sex determination mechanisms were only sensitive to EE 2 during a window of embryonic development before hatch. Early stages of testis differentiation may be more sensitive to exogenous EE 2 exposure than ovarian tissue in the mummichog.