Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17？-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10？ng/L, E2; 100？ng/L, after 72？h exposures). For the NP exposures, GFP expression was observed at 10？μg NP/L after 72？h (100？μg NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally.Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos.