Floating agarose method: an in vitro organ culture system for evaluating endocrine disruptor biomarker responses on fish steroidogenic pathways

Steroid hormone (estrogens and androgens) synthesis and regulation involve a large number of enzymes and potential biochemical pathways. In the context of these biochemical pathways, it is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein, and the subsequent conversion to pregnenolone by cytochrome P450-mediated side-chain cleavage enzyme (P450scc). Oocyte maturation is a complex process that is triggered by the maturation-promoting factor (MPF) and during this process, cyclin-B functions as a regulatory factor. Thus, the accumulation of cyclin-B in oocytes controls the timing of early embryonic cell cycle. Herein, we present an in vitro previtellogenic oocyte culture technique that was based on an agarose floating method for evaluating endocrine-disruption of fish steroidogenic pathways using the Atlantic cod (Gadus morhua) as model species. Despite its high economic significance, the Atlantic cod is not a well-studied species neither from an endocrinological or toxicological standpoint. Tissue was cultured in a humidified incubator at 10 °C for 1, 5, 10 and 20 d with different concentrations of the non-aromatizable androgen - methyltestosterone (MT at 0, 1, 10, 100 and 1000 mM) dissolved in ethanol (0.3%). Gene expressions were detected using validated real-time PCR with specific primers. Immunohistochemistry and in-situ hybridization of the StAR protein and P450scc were performed using antisera prepared against synthetic peptide for both proteins and specific gene probes, respectively. Tissue levels of E2 and T were estimated using enzyme immunoassay. Our data showed significant concentration-specific increase (at day 1 and 5) and decrease (day 10 and 20) of StAR mRNA expression after exposure to MT. P450scc expression showed an MT concentration-specific decrease during the exposure periods and cyclin-B mRNA expression were decreased in MT concentration-dependent manner at days 10 and 20 (reaching almost total inhibition after exposure to highest MT concentration (1000 mM). MT exposure produced variable effect on the aromatase mRNA expression that can be described as concentration-specific increase (at day 1 and 20) and decrease (at days 5 and 10). For hormone levels, MT produced an apparent concentration- and time-dependent increase of E2 and T levels. Thus, the present study reveals some novel aspects of hormonal effects on maturation and oocyte growth in teleosts. The impaired steroidogenesis and hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleost.