Endogenous progestogens are important regulators of vertebrate reproduction. Synthetic progestins are components of human contraceptive and hormone replacement pharmaceuticals. Both progestogens and progestins enter the environment through a number of sources, and have been shown to cause profound effects on reproductive health in various aquatic vertebrates. Progestins are designed to bind human progesterone receptors, but they also have been shown to strongly activate androgen receptors in fish. Levonorgestrel (LNG) activates fish androgen receptors and induces development of male secondary sex characteristics in females of other species. Although behavior has been postulated to be a sensitive early indicator of exposure to certain environmental contaminants, no such research on the reproductive behavior of gestagen-exposed fish has been conducted to date. The goal of our study was to examine the exposure effects of a human contraceptive progestin, LNG, on the reproductive development and behavior of the viviparous eastern mosquitofish (Gambusia holbrooki). Internal fertilization is a requisite characteristic of viviparous species, and is enabled by an androgen driven elongation of the anal fin into the male gonopodium (i.e., phallus). In this study, we exposed adult mosquitofish to ethanol (EtOH control), 10ng/L, and 100ng/L LNG for 8d using a static replacement exposure design. After 8d, a subset of males and females from each treatment were examined for differences in the 4:6 anal fin ratio. In addition, paired social interaction trials were performed using individual control males and control females or females treated 10ng/L or 100ng/L LNG. Female mosquitofish exposed to LNG were masculinized as evidenced by the elongation of the anal fin rays, a feature normal to males and abnormal to females. LNG caused significant increases in the 4:6 anal fin ratios of female mosquitofish in both the 10ng/L and 100ng/L treatments, although these differences were not significant between the two treatments. LNG caused significant increases in the 4:6 anal fin ratio of males exposed to 100ng/L, with no effects observed in the 10ng/L treatment. In addition, the reproductive behavior of control males paired with female mosquitofish exposed to 100ng/L LNG was also altered, for these males spent more time exhibiting no reproductive behavior, had decreased attending behavior, and a lower number of gonopodial thrusts compared to control males paired to control female mosquitofish. Given the rapid effects on both anal fin morphology and behavior observed in this study, the mosquitofish is an excellent sentinel species for the detection of exposure to LNG and likely other 19-nortestosterone derived contraceptive progestins in the environment.

Endogenous progestogens and pharmaceutical progestins enter the environment through wastewater treatment plant effluent and agricultural field runoff. Lab studies demonstrate strong, negative exposure effects of these chemicals on aquatic vertebrate reproduction. Behavior can be a sensitive, early indicator of exposure to environmental contaminants associated with altered reproduction yet is rarely examined in ecotoxicology studies. Gestodene is a human contraceptive progestin and a potent activator of fish androgen receptors. Our objective was to test the effects of gestodene on reproductive behavior and associated egg deposition in the fathead minnow. After only 1 day, males exposed to ng/L of gestodene were more aggressive and less interested in courtship and mating, and exposed females displayed less female courtship behavior. Interestingly, 25% of the gestodene tanks contained a female that drove the male out of the breeding tile and displayed male-typical courtship behaviors toward the other female. Gestodene decreased or arrested egg deposition with no observed gonadal histopathology. Together, these results suggest that effects on egg deposition are primarily due to altered reproductive behavior. The mechanisms by which gestodene disrupts behavior are unknown. Nonetheless, the rapid and profound alterations of the reproductive biology of gestodene-exposed fish suggest that wild populations could be similarly affected.

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The synthetic progestin levonorgestrel is commonly utilized in human oral contraceptives. It enters the environment as a component of wastewater treatment plant effluent, and has been measured at low ng/L concentrations in surface waters. It has been shown to activate fish androgen receptors, causing the physical masculinization of females, changes in reproductive behavior, and decreases in fecundity. In the present study, the effects of levonorgestrel exposure on early-stage oogenesis in the fathead minnow (Pimephales promelas) was examined. Adult females were exposed to 0, 10, or 100 ng/L levonorgestrel for 14 d using a flow-through exposure system. The ovaries from each female were then removed via dissection and weighed for gonadosomatic index (GSI) calculations, and oocytes from one lobe preserved in Serra's fixative. Total numbers of late-stage vitellogenic oocytes exhibiting a germinal vesicle were then quantified. In a second exposure, blood plasma samples were collected from adult females and analyzed for vitellogenin concentrations using enzyme-linked immunosorbent assay. Females exposed to both concentrations of levonorgestrel developed male secondary sexual characteristics in a dose-dependent manner, and ovaries contained significantly fewer late stage oocytes. Exposure to 100 ng/L of levonorgestrel resulted in decreased GSI and blood plasma vitellogenin concentrations. The results suggest that female exposure to levonorgestrel alone may have profound effects on reproduction in progestin-contaminated environments. Environ Toxicol Chem 2017;36:3299-3304. © 2017 SETAC.

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