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Abstract: The purpose of this study was to investigate the physiological maturation-inducing steroid (MIS) in the marine protandrous yellowfin porgy (Acanthopagrus latus). Female fish were injected with 2 doses of LHRH analog (10 and 40μg per kg). Ovarian tissue was obtained at 6h intervals for in vitro analysis of oocyte maturation. The most effective steroids for inducing in vitro maturation (germinal vesicle breakdown and GVBD) in cultured oocytes were 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) and 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S). 17,20βP was less potent than 20βS in inducing oocyte maturation. At higher concentrations, 11-deoxycortisol, 17α-hydroxy-progesterone, and 20β-21-dihydroxy-4-pregnen-3-one also significantly induced oocyte maturation. A tritiated precursor [3H]-pregnenolone, was cultured in vitro together with the maturing ovarian tissue. The tritiated metabolites were purified and identified by solvent extraction, HPLC, TLC, acetylation reaction and recrystallization. HPLC, TLC and recrystallization analysis showed that significant levels of tritiated 11-deoxycortisol (a precursor of 20β-S) and 20β-S, but not 17,20βP, were biosynthesized from [3H]-pregnenolone. Similar TLC profiles were obtained from the tritiated products that were isolated from the HPLC/TLC 20β-S fraction and standard 20β-S after the acetylation reaction. Constant specific radioactivity of tritiated 11-deoxycortisol and 20β-S but not 17,20βP by recrystallization was obtained in the tritiated metabolites isolated from HPLC and TLC fractions. The expression of 20β-hydroxysteroid dehydrogenase (20β−HSD) mRNA (a key enzyme that converts 11-deoxycortisol to 20β-S) was significantly increased in maturing ovarian tissue. This study provides the first evidence that 20β-S is converted from 11-deoxycortisol and is the possible MIS in yellowfin porgy. [Copyright &y& Elsevier]