Fish are exposed to steroids of different classes in contaminated waters, but their effects are not sufficiently understood. Here we employed an anti-sense technique using morpholino oligonucleotides to knockdown the glucocorticoid receptors (GRs, GRα and GRβ) and androgen receptor (AR) to investigate their role in physiological and transcriptional responses. To this end, zebrafish embryos were exposed to clobetasol propionate (CLO), androstenedione (A4) and mixtures containing different classes of steroids. CLO caused a decrease of spontaneous muscle contraction and increase of heart rate, as well as transcriptional induction of pepck1, fkbp5, sult2st3 and vitellogenin (vtg1) at 24 and/or 48 h post fertilization (hpf). Knockdown of GRs eliminated these effects, while knockdown of AR decreased the ar transcript but caused no expressional changes, except induction of sult2st3 after exposure to A4 at 24 hpf. Exposure to a mixture of 6 steroids comprising progesterone (P4) and three progestins, cyproterone acetate, dienogest, drospirenone, 17β-estradiol (E2) and CLO caused a significant induction of pepck1, sult2st3, vtg1 and per1a. Knockdown of GRs eliminated the physiological effects and the up-regulation of vtg1, sult2st3, pepck1, fkbp5 and per1a. Thus, as with CLO, responses in mixtures were regulated by GRs independently from the presence of other steroids. Exposure to a mixture comprising A4, CLO, E2 and P4 caused induction of vtg1, cyp19b, sult2st3 and fkbp5. Knockdown of AR had no effect, indicating that regulation of these genes occurred by the GRs and estrogen receptor (ER). Our findings show that in early embryos GRs cause vtg1 and sult2st3 induction in addition to known glucocorticoid target genes. Each steroid receptor regulated its own target genes in steroid mixtures independently from other steroids. However, enhanced expressional induction occurred for vtg1 and fkbp5 in steroid mixtures, indicating an interaction/cross-talk between GRs and ER. These findings have importance for the understanding of molecular effects of steroid mixtures.

鱼在被污染的水中暴露于不同类别的类固醇，但其作用尚不充分。在这里我们采用了一种反义技术，使用吗啉代寡核苷酸敲低糖皮质激素受体（GRs，GRα和GRβ）和雄激素受体（AR），以研究它们在生理和转录反应中的作用。为此，将斑马鱼的胚胎暴露于丙酸氯倍他索（CLO），雄烯二酮（A4）和含有不同类固醇的混合物。CLO引起自发性肌肉收缩的减少和心率的增加，以及pepck1，fkbp5，sult2st3和卵黄蛋白原（vtg1）的转录诱导）在受精后24和/或48小时（hpf）。GRs的敲除消除了这些影响，而AR的敲除降低了ar转录本，但没有引起表达变化，除了在24 hpf暴露于A4后诱导sult2st3。暴露于包含黄体酮（P4）和三种孕激素的6种类固醇的混合物中，醋酸环丙孕酮，二诺孕酮，屈螺酮，17β-雌二醇（E2）和CLO显着诱导了pepck1，sult2st3，vtg1和per1a的诱导。减少GRs消除了生理效应和vtg1，sult2st3，pepck1，fkbp5和per1a的上调。因此，与CLO一样，混合物中的响应均由GR调节，而与其他类固醇的存在无关。暴露于包含A4，CLO，E2和P4的混合物中会引起vtg1，cyp19b，sult2st3和fkbp5的诱导。击倒AR没有作用，表明这些基因的调节由GR和雌激素受体（ER）发生。我们的发现表明，除了已知的糖皮质激素靶基因外，GRs在早期胚胎中还引起vtg1和sult2st3诱导。每个类固醇受体在类固醇混合物中独立于其他类固醇调节自己的靶基因。但是，vtg1和fkbp5的表达诱导增强在类固醇混合物中，表明GR和ER之间存在相互作用/串扰。这些发现对于理解类固醇混合物的分子作用具有重要意义。