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.

Use of a radioimmunoassay which detects C21 steroids with a 17, 20beta-dihydroxyl configuration to identify and measure steroids involved in final oocyte maturation in female plaice (Pleuronectes platessa).

A radioimmunoassay (RIA) has been developed to detect a range of C21 (pregnane) steroids with a 17,20beta-dihydroxyl (17,20beta) configuration. In conjunction with reverse-phase high-performance liquid chromatography (HPLC), it identifies and quantifies the metabolites of 17,20beta-dihydroxy-4-pregnen-3-one, the putative "maturation-inducing steroid" in female plaice Pleuronectes platessa. Total levels of 17,20beta metabolites which can be extracted from plasma or urine with diethyl ether (i.e., free steroids) are very low (<3 ng/ml). However, total levels of 17,20beta metabolites which can be released by solvolysis (i.e., sulphated steroids) are very high (up to 1 microg/ml in plasma and 10 microg/ml in urine). On HPLC, these sulphated metabolites have been identified (in order of abundance in plasma) as: 5beta-pregnane-3alpha,17,20beta-triol, 5beta-pregnane-3beta,17,20beta-triol, 17, 20beta-dihydroxy-4-pregnen-3-one, and 17, 20beta-dihydroxy-5beta-pregnan-3-one. These steroids are absent from plasmas of fish which have not yet begun final oocyte maturation. The results support the hypothesis that 17, 20beta-dihydroxy-4-pregnen-3-one is the maturation-inducing steroid in plaice but that it is rapidly metabolised to render it inactive. The results also show that the '17,20beta'-RIA, in combination with an overnight acid solvolysis procedure, is a useful procedure for monitoring the effects of exogenous factors (such as gonadotrophin injections) on final oocyte maturation in female plaice.

A novel M phase-specific H1 kinase recognized by the mitosis-specific monoclonal antibody MPM-2.

At the onset of mitosis, eukaryotic cells display an abrupt increase in a Ca2(+)- and cyclic nucleotide-independent histone H1 kinase activity, referred to as growth-associated or M phase-specific H1 kinase. The molecular basis for this activity is generally attributed to a kinase complex that consists of the p34cdc2 protein and cyclin, and exhibits maturation-promoting factor (MPF) activity. In the present study, we show that more than one kinase contributes to M phase-specific H1 kinase activity. When mature Xenopus oocyte extract prepared with ATP gamma S and NaF was fractionated by gel filtration, two prominent peaks of H1 kinase activity were detected, with apparent molecular masses of 600 and 150 kDa. The 150-kDa kinase copurified with the p34cdc2 protein and was immobilized by the suc 1 gene product p13 and anti-cyclin B2, which are specific for the cdc2 kinase complex. However, the 600-kDa kinase did not satisfy any of these criteria, thus identifying it as a novel M phase-specific H1 kinase. Only the 600-kDa kinase was recognized by the mitosis-specific monoclonal antibody, MPM-2, which inhibits Xenopus oocyte maturation and immunodepletes MPF activity. Furthermore, not only did the full activation of this kinase (MPM-2 kinase) coincide with the activation of MPF during the cell cycle, but also MPM-2 kinase-positive fractions obtained by gel filtration accelerated progesterone-induced oocyte maturation. It is, therefore, likely that MPM-2 kinase is a positive regulator in the M phase induction pathway.