Effects of exposure to 17α-ethynylestradiol during larval development on the African clawed frog (Xenopus laevis) and the wood frog (Rana sylvatica)

The exposure of aquatic vertebrates to estrogenic compounds in the environment has become a concern in recent years. These compounds are present in the environment due to their presence in agricultural runoff, wastewater, pharmaceuticals, personal care products, and industrial products. Previous studies have shown that potent estrogens have the potential to feminize or demasculinize fish and amphibians, but the responses of many species to these compounds have not been fully characterized. The overall objective of this thesis was to determine the effects of exposure to 17α-ethynylestradiol (EE2), the estrogen analog commonly utilized in oral contraceptives, during the larval period on the sexual development, growth, metamorphosis, and transcriptional profiles of the African clawed frog (Xenopus laevis) and the wood frog (Rana sylvatica). To do so, X. laevis were exposed to 0.09, 0.84, or 8.81 µg EE2/L throughout the larval period and early post-metamorphic development (89 d) and R. sylvatica were exposed to 1.08, 9.55, or 80.9 µg EE2/L throughout the larval period until the climax of metamorphosis was reached (55-100 d). In X. laevis, exposure to all concentrations of EE2 tested resulted in significant delays in time required to complete metamorphosis of 15-23 d compared to the control treatment. In addition, exposure to all concentrations of EE2 tested resulted in significantly greater proportions of X. laevis individuals with a male genotype that displayed female, abnormal male, and mixed sex phenotypes. These individuals accounted for 83-93% of all genetic males in the EE2 treatments. While the development of genetic male X. laevis was affected by exposure to EE2, complete reversal of phenotypic sex was rare at the concentrations of EE2 tested, so it was not possible to calculate an EC50 for complete feminization. The EC50 for partial feminization was determined to be approximately 8.81 µg EE2/L. At the molecular level, exposure of X. laevis to EE2 resulted in alterations in abundances of transcripts of genes involved in endocrine processes, including steroid signaling and metabolism, vitellogenesis, testicular development, and thyroid hormone signaling, both during the larval period and after the completion of metamorphosis. In general, compared to the control treatment, transcripts of genes involved in testicular development and thyroid hormone signaling were suppressed during the larval period, and transcripts of genes involved in vitellogenesis were induced after the completion of metamorphosis. In addition, both genetic male and genetic female X. laevis exposed to EE2 displayed greater production of vitellogenin protein relative to control individuals after the completion of metamorphosis. In R. sylvatica, exposure to all concentrations of EE2 tested resulted in significantly lesser proportions of phenotypic males which corresponded to greater proportions of female and mixed sex individuals. Phenotypic males accounted for 41%, 24%, 2.5%, and 0% of all individuals in the solvent control, 1.08, 9.55, and 80.9 µg EE2/L treatments, respectively. The EC50 for complete feminization for R. sylvatica was determined to be 7.7 µg EE2/L, and the EC50 for partial feminization was determined to be 2.3 µg EE2/L. At the molecular level, chronic exposure of R. sylvatica to EE2 resulted in induction of transcription of genes involved in vitellogenesis and the synthesis of cholesterol at the time of metamorphic climax. Although there were species-specific differences in effects between X. laevis and R. sylvatica after exposure to EE2, the overall response of both species was consistent with feminization or demasculinization, and both species were shown to be affected at concentrations of EE2 within the realm of environmental relevance