Endocrine disrupting compounds in the environment, their impact on reproduction of Xenopus laevis, and basic mechanisms underlying sexual differentiation in amphibians

In the last decades major concern evolved about the potential of endocrine disrupting compounds (EDC) to interfere with the endocrine system of animals and to cause adverse effects on reproductive biology of vertebrates and invertebrates including reduced fertility, decreased hatching success, behavioural and developmental abnormalities or altered sex ratios. In the present study, different aspects of this topic were addressed. In the first part, the presence of EDC with (anti)estrogenic and (anti)androgenic modes of action was investigated in water and sediment samples of the river Lambro, a polluted river from Northern Italy, using yeast-based in vitro bioassays coupled with a fractionation procedure. Moderate estrogenic and high anti-androgenic activities were found presumably present in biologically effective concentrations. In the second part, the effects of EDC on aquatic living organisms were evaluated by exposing adult South African clawed toads (Xenopus laevis) in vivo to Lambro river water and to EDC model compounds with (anti)estrogenic and (anti)androgenic modes of action. Feedback mechanisms on the hypothalamus-pituitary-gonad axis (mRNA expression of luteinising hormone, follicle stimulating hormone, gonadotropin releasing hormone), mRNA expression of potential biomarker genes in the liver (vitellogenin, transferrin, transthyrethin, retinol-binding protein), plasma sex steroids and histology of gonads were investigated as endpoints indicating potentially adverse effects on the reproductive biology of X. laevis. Response of the molecular biomarkers was observed mainly in (anti)estrogenic treatments. In contrast, histology of gonads was the most sensitive tool clearly demonstrating effects of exposures to model compounds and a feminization effect in adult male X. laevis following exposure to Lambro river water. In the third part, basic mechanisms of sexual differentiation, those processes regulating the development of organisms into males or females, were analysed in X. laevis to broaden our knowledge how gonadal development might be influenced by exogenous steroids or EDC. In particular, the mRNA expressions of steroidogenic enzymes (aromatase, 5α-reductase type 1 and type 2) were investigated during the ontogeny of X. laevis tadpoles. Aromatase and 5α-reductase type 2 seemed to be important for the maturation of gametes since sex-specific mRNA expression of both genes was found. Exposure of X. laevis tadpoles during ontogeny to an inhibitor of 5α-reductase activity (finasteride) revealed disruption of sperm maturation highlighting the fundamental role of 5α-reductases and whose product dihydrotestosterone, respectively, for spermatogenesis.