Use of a radioimmunoassay which detects C21 steroids with a 5beta-reduced, 3alpha-hydroxylated configuration to identify and measure steroids involved in final oocyte maturation in female plaice (Pleuronectes platessa).

Two radioimmunoassays (RIAs) have been developed which detect C21 (pregnane) steroids with a 5beta-reduced, 3alpha-hydroxyl (5beta, 3alpha) configuration. One RIA only detects 3alpha,17, 21-trihydroxy-5beta-pregnan-20-one and 3alpha, 17-dihydroxy-5beta-pregnan-20-one, whilst the other detects a range of 5beta,3alpha steroids, including 5beta-pregnane-3alpha,17,20 beta-triol, a major metabolite of 17, 20beta-dihydroxy-4-pregnen-3-one, the putative oocyte maturation-inducing steroid in plaice Pleuronectes platessa. The RIAs, in conjunction with reverse-phase high-performance liquid chromatography (HPLC), have identified and quantified the steroids in plasma and urine of reproductively mature females. Total levels of 5beta,3alpha metabolites which can be extracted with diethyl ether (i.e., free steroids) are relatively low (<10 ng/ml). However, total levels of 5beta,3alpha metabolites released by solvolysis (i.e. , sulphated steroids) are very high (up to 1000 ng/ml in plasma and 20 microg/ml in urine). On HPLC, these metabolites have been identified (in order of their abundance in plasma) as: 3alpha,17, 21-trihydroxy-5beta-pregnan-20-one, 5beta-pregnane-3alpha,17, 20beta-triol, 5beta-pregnane-3alpha,17,20alpha-triol, 3alpha,11beta, 17,21-tetrahydroxy-5beta-pregnane-20-one, and 3alpha, 17-dihydroxy-5beta-pregnan-20-one. Levels of the first three steroids are significantly elevated in female plaice undergoing natural or gonadotrophin-induced final oocyte maturation.