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(18-46) Biological validation of the transgenic zebrafish assay: effects of (xeno-)estrogens on sexual differentiation and male reproduction. Legler, Juliette*,1,2, Van der Zee, Margriet1, Lanser, Peter2, Jonkers, Niels3, Van der Ven, Leo4, Vethaak, Dick5, Van der Burg, Bart2, Murk, Tinka1, 1 Department of Toxicology, Wageningen, Netherlands2 Netherlands Institute for Developmental Biology, Utrecht, Netherlands3 Department of Environmental Toxicology and Chemistry, Amsterdam, Netherlands4 National Institute of Public Health and the Environment, Bilthoven, Netherlands5 National Institute for Coastal and Marine Management, Middelburg, Netherlands ABSTRACT- We recently developed an in vivo test system using transgenic zebrafish to rapidly determine the effects of estrogenic chemicals on critical life stages and sensitive target organs in the fish. In the transgenic zebrafish, an estrogen binding sequence linked to a TATA box and luciferase reporter gene was stably introduced. Binding of a substance to endogenous estrogen receptors (ER) and the subsequent transactivation of the ER result in luciferase gene induction that is easily measured in tissue lysates. In order to determine if reporter gene induction correlates with effects on sexual differentiation and male reproductive fitness, transgenic zebrafish were exposed to 0.1 and 10 nM E2, as well as 500 nM nonylphenol (NP), for three weeks during the period of gonad differentiation (3 to 6 weeks of age). Exposure to E2 and NP induced luciferase induction as well as vitellogenin mRNA, as measured with quantitative RT-PCR. An increase in the number of fish with undifferentiated gonads was found in fish exposed to 0.1 nM E2 and 500 nM NP, whereas 100% females were found in the 10 nM E2 group. Interestingly, in fish returned to clean water following exposure and cultured to sexual maturity (4 months of age), no effect of the treatments was found on phenotypic sex ratios. However, reduced fecundity of male fish was found in the 10 nM E2 and 500 nM NP groups. Further research will determine critical levels of reporter gene induction for predicting reproductive effects. Key words: zebrafish, transgenic, estrogen, reproduction |
