Evaluation of testicular oocytes and gonadal development in smallmouth bass (Micropterus dolomieu) under presumed and known exposure to estrogenic compounds

University of Minnesota Ph.D. dissertation. April 2017. Major: Integrated Biosciences. Advisors: David Mount, Patrick Schoff. 1 computer file (PDF); x, 118 pages.Testicular oocytes (TOs), a form of gonadal intersex, have been found in black basses (Micropterus spp.) from many locations in North America. The presence of TOs is often assumed to imply exposure to estrogenic endocrine disrupting compounds (EDCs), however, a definitive causal relationship has yet to be established, and TO prevalence is not consistently low in fish from areas lacking evident EDC sources. This could suggest unknown or unidentified EDC sources, or that TOs can be stimulated by other stressors, or that they arise spontaneously during normal development. The goal of this dissertation research was to improve our ability to interpret observations of TOs in smallmouth bass (M. dolomieu) in particular, with approaches that are applicable to any other species. These approaches included statistical analysis of TO occurrence in wild populations where estrogenic exposure is more and less likely, and laboratory studies to characterize the early gonadal development in smallmouth bass, both in the presence and absence of exogenous estrogen. TO occurrence in smallmouth bass was assessed in eight watersheds in Northeastern Minnesota watersheds with differing degrees of human development, and hence, likelihood of EDC exposure. Testicular tissues from mature fish were evaluated using a semi-quantitative method that estimated TO density, normalized by cross-sectional area. TO prevalence and density among populations from more developed watersheds were higher than in populations from less developed watersheds. However, TO prevalence was unexpectedly high and variable (7-43%) in some populations from less developed watersheds, and only weak evidence was found for a relationship between TO density and watershed development, suggesting alternative or more complex explanations for TO presence in smallmouth bass. To characterize early gonadal development in smallmouth bass, both in the presence and absence of estrogen, swim-up fry were exposed to control water or the potent model estrogen, 17α-ethinylestradiol (EE2), for 90 days, and reared for another 90 days in clean water. Development of gonadal tissue was assessed at 11 time points using standard histology methods. Ovarian differentiation was observed in 50% of control fish by test day 49, and testicular differentiation occurred by day 92. Among EE2-exposed groups, ovarian phenotypes were observed in 83% of fish by day 49, and in 98% of fish between days 92 and 180, indicating that EE2 can cause complete, and likely permanent, sex reversal in smallmouth bass. Among the EE2-exposed fish that developed ovaries between days 92 and 180, 38% had abnormal characteristics, suggesting that ovaries in sex reversed males can be distinguished from ovaries in genetic females. In a second laboratory study, smallmouth bass gonadal development was assessed during early life stage exposure across a range of concentrations of EE2, including environmentally realistic concentrations. 97% of fish exposed to 4.3 ng/L EE2 (mean measured concentration) had ovaries on test days 90 and 120, and 35% of ovaries in those groups had abnormal characteristics. Among control groups and fish exposed to lower concentrations (0.1 and 0.4 ng/L nominally, and 1.6 ng/L, mean measured concentration), only histologically normal ovaries and testes developed, in ratios that were not significantly different than 50:50. TOs were not observed in any control or EE2-exposed fish during these studies. This suggests that early life stage exposure to low concentrations of EE2 may not cause TOs in wild smallmouth bass, although the long-term outcomes of EE2 exposure in adult bass are unknown. The results of these studies underscore the importance of understanding gonadal development in fish reared in both the absence and presence of exogenous estrogen for interpreting the occurrence of TOs in wild populations. Overall, the evidence produced by this research does not support the hypothesis that estrogenic compounds are the unique cause of TOs in wild bass. With current understanding, the occurrence of TOs in wild populations of smallmouth bass alone is not an appropriate biomarker for estrogenic exposure