An in vitro floating agarose method for evaluating the effects of endocrine disrupting chemicals on steroidogenesis of Atlantic salmon (Salmo salar) previtellogenic oocytes

Endocrine disruptor chemicals (EDCs) may mediate their effects through direct receptor and/or non-receptor mechanisms and can cause reproductive disturbances on vertebrate and invertebrate organisms. Despite the potential effects of EDCs on steroidogenesis, this endocrine pathway is not well studied. It is generally believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and subsequent conversion to pregnenolone by cytochrome P450-mediated side-chain cleavage enzyme (P450scc). In our research group, we recently showed that the synthetic pharmaceutical estrogen, ethynylestradiol (EE2) induced concentration specific modulation of CYP19 gene isoforms and its enzymatic activity and modulated the StAR, P450scc and CYP11b gene expressions in Atlantic salmon. The aim of this study is to investigate the effect of EE2 and nonylphenol (NP) on key oocyte steroidogenic pathways using recently developed floating agarose in vitro culture system in our laboratory and state-of-art molecular approaches. Immature oocyte (previtellogenic) samples from salmon were collected and cultured in vitro. The samples were cultured in media containing essential nutrients and test chemicals at different concentrations - EE2 (0.01, 0.1 and 1.0 M) and NP (1, 10, 50 M). Samples were collected at 0 (control), 3, 7 and 14 days after exposure. Hormone analyses (estradiol (E2), 11-ketotestosterone (11-KT) and testosterone (T)) were performed using enzyme immune assay (EIA). Our data show that EE2 and NP produced effects on E2, 11-KT and T levels that were dependent on individual chemical concentration and time of exposure. Gene expression patterns for StAR, 3b-HSD, P450scc, CYP19 isoforms and activity levels are currently under analysis. In addition, histological evaluation of oocyte structure and size are also under analysis. We hypothesize that differences in the profile of these key steroidogenic genes and protein responses, including over or down expression in exposed oocytes, will be novel, indicative, prognostic and predictive of chemical susceptibility and adverse endocrinological, and reproductive effects in exposed fish.