Use of a Radioimmunoassay Which Detects C 21 Steroids with a 5 b-Reduced, 3 a-Hydroxylated Configuration to Identify and Measure Steroids Involved in Final Oocyte Maturation in Female Plaice ( Pleuronectes platessa) R. Moses Inbaraj, 1 A. P. Scott, and E. L. M. Vermeirssen Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory, Lowestoft, Suffolk, NR33 OHT, United Kingdom Accepted July 25, 1996 Two radioimmunoassays (RIAs) have been developed which detect C 21 (pregnane) steroids with a 5 b-reduced, 3 a-hydroxyl (5 b,3 a) configuration. One RIA only detects 3 a,17,21-trihydroxy-5 b-pregnan-20-one and 3 a,17-dihy- droxy-5 b-pregnan-20-one, whilst the other detects a range of 5 b,3 a steroids, including 5 b-pregnane-3 a,17,20 b-triol, a major metabolite of 17,20 b-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 5 b,3 a metabolites which can be extracted with diethyl ether (i.e., free steroids) are relatively low ( F10 ng/ml). However, total levels of 5 b,3 a metabolites released by solvolysis (i.e., sulphated steroids) are very high (up to 1000 ng/ml in plasma and 20 mg/ml in urine). On HPLC, these metabolites have been identified (in order of their abundance in plasma) as: 3 a,17,21-tri- hydroxy-5 b-pregnan-20-one, 5 b-pregnane-3 a,17,20b-triol, 5b-pregnane-3 a,17,20 a-triol, 3 a,11 b,17,21-tetrahydroxy- 5 b-pregnane-20-one, and 3 a,17-dihydroxy-5 b-pregnan- 20-one. Levels of the first three steroids are significantly elevated in female plaice undergoing natural or gonadotro- phin-induced final oocyte maturation. r 1997 Academic Press In most teleosts studied, oocyte final maturation is mediated by either 17,20b-P 2 or 17,20b,21-P. However, in North Sea plaice, Pleuronectes platessa, it has proved difficult to establish which, if either, of these steroids is the ‘‘maturation-inducing steroid’’ (MIS). This is be- cause the plaice is a ‘‘batch spawner’’; at the start of the spawning season, the size frequency distribution of oocytes in the ovaries shows a clear distinction be- tween primary and secondary (vitellogenic) oocytes (Witthames and Greer Walker, 1987). The vitellogenic oocytes form a distinct bell-shaped mode, but only a 1 Present address: Department of Zoology, Madras Christian College, Madras 600 059, India. 2 Steroid abbreviations: 17,20b-P, 17,20b-dihydroxy-4-pregnen- 3-one; 17,20b,21-P, 17,20b,21-trihydroxy-4-pregnen-3-one; 17-P, 17- hydroxy-4-pregnene-3,20-dione (17-hydroxyprogesterone); 11-deoxy- cortisol, 17,21-dihydroxy-4-pregnene-3,20-dione; 3a,17-P-5b, 3a, 17-dihydroxy-5b-pregnan-20-one; 3a,17,21-P-5b,3a,17,21-trihydroxy- 5b-pregnan-20-one; 3a,17,20a-P-5b, 5b-pregnane-3a,17,20a-triol; 3a,17,20b-P-5b,5b-pregnane-3a,17,20b-triol; 3a,17,20b,21-P-5b,5b- pregnane-3a,17,20b,21-tetrol; 3a,11b,17,21-P-5b,3a,11b,17,21-tetra- hydroxy-5b-pregnan-20-one; 3a,17,20b-P-5a, 5a-pregnane-3a,17, 20b-triol; 3b,17,20b-P-5a,5a-pregnane-3b,17,20b-triol; 17,20b-P-5b, 17,20b-dihydroxy-5b-pregnan-3-one; 17,20b-preg, 5-pregnene-3b,17, 20b-triol; 17,20b-P-sulphate, 17,20b-dihydroxy-4-pregnen-3-one 20- sulphate; 17,20b-P-glucuronide, 17,20b-dihydroxy-4-pregnen-3-one 20-glucuronide; 3a,17,20b-P-5b 20-sulphate, 5b-pregnane-3a,17,20b- triol 20-sulphate; 3a,17,20a-P-5b 20-sulphate, 5b-pregnane-3a,17,20a- triol 20-sulphate; 3a,17,21-P-5b 21-sulphate, 3a,17,21-trihydroxy-5b- pregnan-20-one 21-sulphate. For further explanations of steroid nomenclature, see Kime (1995). General and Comparative Endocrinology 105, 50–61 (1997) Article No. GC966797 50 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.