The xenoestrogen, 4-nonylphenol, impaired steroidogenesis in previtellogenic oocyte culture of Atlantic cod (Gadus morhua) by targeting the StAR protein and P450scc expressions

The steroidogenic acute regulatory (StAR) protein and cytochrome P450-mediated cholesterol side-chain cleavage (P450scc) have been localized in most steroidogenic organs and are rapidly synthesized in response to acute tropic hormone stimulation. In this study, we present the development of cod previtellogenic oocyte in vitro culture system, histological and molecular methods for evaluating the effects of endocrine disruptors such as nonylphenol (NP) on steroid hormone levels, the StAR protein and P450scc. In addition, expression pattern of cyclin-B was studied, because of cyclin B’s role as an indicator of oocyte growth in fish. The in vitro previtellogenic oocyte culture technique was based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 °C for 4, 7, 14 and 21 d with different concentrations of nonylphenol (0 (control), 1, 10, 50 and 100 μM) dissolved in ethanol (0.3%). Gene expressions were detected using validated real-time polymerase chain reaction (PCR) with specific primers. Immunohistochemistry of the StAR protein and P450scc were performed using antisera prepared against synthetic peptide for both proteins. Estradiol-17β (E2) and 11-ketotestosterone (11-KT) tissue levels were estimated using enzyme immunoassay. Our data show that nonylphenol produced a unique and consistent concentration-specific pattern of modulation for the StAR protein, P450scc and cyclin-B gene expression at day 14 after exposure. This pattern is generally described as increasing from 0 (control) to 1 and 10 μM, and decreased at 50 and 100 μM. The observed changes in the StAR protein, P450scc and cyclin-B levels showed a direct relationship with changes in 11-KT levels at day 14 after exposure. Cellular localization of StAR and P450scc were specific to the follicular cells of previtellogenic oocytes, but with no differences in staining intensities. No significant change in oocyte diameter was observed between the exposure groups. Our data reveal some novel aspects of nonylphenol effects on maturation and oocyte growth in teleosts, suggesting impaired steroidogenesis and hormonal imbalance with potential consequences for the vitellogenic process and overt fecundity.