Hitting the mark studies of alterations in behaviour and fertility in ethinyl estradiol-exposed zebrafish and search related biomarkers

Abstract: In this thesis, we have analysed the effects of EE2 on non-reproductive behaviours and fertility. We have showed that two doses of EE2 in male adult short-term exposures evokes opposite behaviours in the novel tank test. A lower dose induced increased bottom-dwelling, a sign of increased anxiety and a higher dose increased surface-dwelling, which would likely expose themselves to predation in a natural environment. Increased shoaling was observed in both exposures, possibly affecting feeding and reproduction opportunities. Fertility analysis of these fish demonstrated a complete inhibition of spawning in the highest dose group. To investigate mechanisms behind the spawning failure, we examined expression levels of genes involved in zebrafish sex differentiation and maintenance of gonadal function. We found downregulated transcription levels of male-predominant genes, suggesting a demasculinization of the testes contributing to functional sterility in these fish. We have demonstrated that non-reproductive behaviour in zebrafish is highly sensitive to EE2 exposure during development. After exposing male and female zebrafish to low doses of EE2 followed by remediation in clean water until adulthood, the fish displayed increased anxiety and shoaling behaviour, demonstrating persistent effects of EE2. Furthermore, behavioural effects were transferred to their progeny. Decreased fertilisation success of the developmentally exposed fish was observed in both sexes when mated to untreated animals of the opposite sex. These fertility effects persisted although the fish had a long remediation period, implying likely reduced fitness of fish populations in aquatic environments. Based on our findings on non-reproductive behaviours and fertility, we performed RNAsequencing analysis of the brain and testes in order to investigate possible biological mechanisms behind the persistent effects. There is a need for biomarkers allowing detection of both reversible and irreversible effects in animals exposed to estrogenic substances, hopefully contributing to better risk assessments for EDCs. Results from RNA-sequencing would serve as a basis for continued studies in pursuit of potential biomarkers.