Disrupted reproductive behavior in unexposed female zebrafish (Danio rerio) paired with males exposed to low concentrations of 17α-ethinylestradiol (EE2)

Abstract

Estrogenic substances, including the contraceptive pharmaceutical 17α-ethinylestradiol (EE2), pose a threat to aquatic wildlife causing endocrine disturbances of physiological processes and reproductive behavior. We have previously demonstrated that the reproductive behavior of male zebrafish, Danio rerio, remain intact after lifelong exposure to low concentrations of EE2 (0.05 and 0.5ngL(-1)), concentrations high enough to cause morphological alterations of the male phenotype. Despite normal courtship behavior, the reproductive output is suppressed when these males were paired with unexposed females. In this study, we include the female courtship behavior in the analyzes of EE2's effects on behavior. Groups of male zebrafish were exposed to nominally 0, 1, 3 and 10ngL(-1) of EE2 from hatching to adulthood and subsequently individually paired with an unexposed female. Eleven distinct elements of the reproductive behavior were then extracted and analyzed using an automated video tracking system. Subsequently, male brains were isolated and the expression of genes encoding estrogenic receptors ERα, ERβ1 and ERβ2, the androgen receptor AR, and the aromatase cyp19a1b were compared with the expression in brains of unexposed males. (In this article gene expression is taken synonymous to transcription, although it is acknowledged that it can also be regulated by, e.g., mRNA and protein stability, and translation). The results confirmed that the male reproductive behavior was unaffected by the EE2 treatments. Also, the expressions of genes encoding estrogen and androgen receptors were unaffected. Only the gene encoding aromatase was 0.6 fold down-regulated. In contrast, and most surprisingly, nearly all the elements in the female courtship behavior were significantly disturbed, despite the fact that these females had never been exposed to EE2; most likely elicited by differences in male morphology, pheromones or some other unrevealed mechanism.

Keywords: 17α-ethinylestradiol; EE2; Indirect estrogenic effect; RT-qPCR; Reproductive behavior; Zebrafish.