Wild type adult male and female zebrafish, Danio rerio, were obtained from a commercial supplier (Ekkwill, Gibsonton, FL) and maintained in 30 gal aquaria at 28°C on a 14:10 light-dark cycle. Fertilized eggs were collected after natural spawning, washed, and distributed into 20 × 100 mm culture plates (Fisher Scientific). Embryos (150 embryos/50 ml egg water) were allowed to develop at 28°C on a 14L:10D cycle [36 ]. For developmental expression analysis embryos were collected after timed intervals: 2, 6, 12, 24, 48, 72, and 120 hours post-fertilization (hpf), quick-frozen on dry ice, and stored at -70°C until analysis (3 independent embryo pools, 50 embryos per pool, per time point from the same spawning group). For treatment expression analysis embryos were left untreated until 24 hpf and then exposed to 17β-estradiol (E2; 0.1 μM), testosterone (T; 1 μM), ICI 182,780 (ICI; 10 μM; Tocris Bioscience, Ellisville, MO), β-napthaflavone (BNF; 10 nM), or 2,3,7,8, tetrachlodibenzo-p-dioxin (TCDD; 1 nM; Ultra Scientific, N. Kingstown, RI) dissolved in dimethyl sulfoxide (DMSO). All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Stock solutions of chemicals were added directly to egg water and replaced daily. In addition, embryos were treated with DMSO alone (final concentration, 0.0006%), EtOH alone (final concentration 0.0005%), or left untreated as a control. Embryos were collected at 96 hpf, quick-frozen on dry ice, and stored at -70°C until analysis (3 independent embryo pools per treatment). Treated embryo RNAs were used for both housekeeping gene expression analysis (Table 3 ) and gene of interest normalization (Figure 2 ). Tissues (brain, eye, heart, liver, muscle, gonad) were collected from adult male and female zebrafish, pooled by sex (3 pools per tissue type/sex, 5 fish per pool), quick-frozen on dry ice, and stored at -70°C. Adult fish were reproductively active stock from our breeding colony.
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