Expression of GFP in zebrafish embryos in the transient expression assay using the mixture of the plasmid pERE-Gal4ff containing theTol2transposase mRNA and the plasmid pUAS-GFP. Fertilised zebrafish eggs were microinjected at the 1–2 cell stages with the mixture of two plasmids. Injected embryos were then exposed for up to 72 hour post-fertilisation (hpf) to 17α-ethinyloestradiol (EE₂). These embryos showed specific mosaic expression of the reporter. The injected embryos exposed to 1000, 100 and 10 ng/L of EE₂ expressed GFP in cells most strongly in the somite muscle, heart and liver (72 hpf). This experiment was run in duplicate and was repeated at least seven times.

Comparison of detectable response concentrations for different oestrogen compounds in the zebrafish embryo transient assay. Injected embryos were exposed for up to 72 h post-fertilisation to 17β-oestradiol (E₂) and nonylphenol (NP) and fluorescence detected using fluorescence microscopy (Leica DMI 4000 B). No GFP expression was observed in controls (A1-A3), but low level GFP expression occurred in the liver for exposure to 1000 ng E₂/L (C2) and high level GFP expression was detected in the somite muscle and heart at exposure concentrations of 100 and 1000 ng E₂/L. Weak GFP expression was also detected in the heart and somite muscle of embryos for exposure to 10 μg/L NP (D1-D3). Embryos exposed to the higher concentrations of NP (100 μg/L) died. This experiment was run in duplicate and was repeated at least seven times.

Analysis of transient expression of GFP in response to oestrogens in medaka embryos. The plasmid pERE-Gal4ff containing the Tol2 transposase mRNA and the plasmid pUAS-GFP were injected into 1–2 cell stage medaka embryos and the embryos were exposed to 17α-ethynyloestradiol (100 ng EE₂/L) via the water. No GFP expression was detected in controls. The injected medaka embryos exposed to EE₂ showed strong GFP expression in the skin in the anterior region and in the body trunk (B). At 11 dpf, fish exposed to EE₂ showed GFP expression in the somite muscle, heart, liver and gall bladder (D, F, H and J).

GFP expression at 4 dpf embryos in the transient expression assay. Injected embryos without chemical exposure (A) or exposed to the oestrogenic chemicals 17α-ethinyloestradiol (1000 ngEE₂/L) (B), 17β-oestradiol (1000 ng E₂/L) (C) and nonylphonol (10 μgNP/L) (D) for 4 days. Head with lateral (L) and ventral (V) views (i and ii) and trunk with lateral view (iii). The shape of the liver is outlined with a white line. No GFP expression was observed in the unexposed control (A). EE₂ and E₂ induced GFP expression in the heart (h), liver (li), neuromasts (n) and somite muscles (sm) (B,C). In NP exposed larvae, GFP expression was observed mainly in the muscle and heart (D).