Effects of 17α-Ethinylestradiol (EE2) on Reproductive Endocrine Status in Mummichog (Fundulus Heteroclitus) Under Differing Salinity and Temperature Conditions

Waterborne exposure to 17α-ethinylestradiol (EE2), a synthetic estrogen, has previously been shown to decrease reproductive endocrine status in the estuarine killifish or mummichog (Fundulus heteroclitus macrolepidotus; northern subspecies). To evaluate if variations in salinity or temperature holding conditions modify the effects of EE2 on gonad size, plasma reproductive steroid levels, and gonadal steroidogenesis, mummichog were exposed in vivo for 14 days to 0, 50 and 250 ng/L EE2 in 0, 16 and 32 ppt salinity at 18°C and to 0 and 250 ng/L EE2 at 10, 18 and 26°C at 16 ppt salinity. Effects due to salinity were limited; however, 250 ng/L EE2 decreased plasma 17β-estradiol (E2) levels and in vitro gonadal E2 production and plasma 11-ketotestosterone (11-KT) across all salinities. Higher temperatures triggered gonadal growth in both sexes as well as increased plasma E2 and gonadal E2 production in females, while 11-KT production was decreased in males. EE2 counteracted the effect of temperature as determined by depressed gonadal growth in males. In both exposures, the effects of EE2 on testosterone (T) production were variable. The use of steroidogenic precursors (25-OH cholesterol, and/or pregnenolone and/or testosterone) in the in vitro gonadal incubations indicated decreased E2 production in females and 11-KT production in males were predominately due to suppression of the terminal conversion step between T and E2 or 11-KT. Ovarian cyp19a gene expression was not affected by 250 ng/L EE2 compared to controls at 16 ppt and 18oC (the only treatment combinations tested). The lack of effects of salinity could be protective for a species spawning in such a variable environment. Gonadal growth at higher temperatures confirms previous work on northern mummichog while EE2 effects on gonadal growth could be due to temperature-related increases in EE2 uptake and/or increased susceptibility during gonadal maturation. In conclusion, the present work demonstrated that environmental conditions impact effects of EE2, including terminal steroid production in the gonads. These results should be considered in designing standardized estuarine fish reproductive bioassays and in understanding the potential effects of reproductive contaminants in estuarine environments.