OOCYTE MATURATION IN CLARIAS-BATRACHUS .3. PURIFICATION AND CHARACTERIZATION OF MATURATION-INDUCING STEROID

Maturation-inducing steroid (MIS) in the Indian female catfish, Clarias batrachus, was purified and characterized from the incubation medium in which fully grown but immature folliculated oocytes were incubated with salmon gonadotropin (SG-G100) for 36 h. Maturation-inducing (MI) activity of residues obtained at various steps of extraction and purification was assessed by in vitro germinal vesicle breakdown (GVBD) assay using folliculated oocytes of C. batrachus. The post incubation medium was extracted with diethyl ether. The ether phase was partitioned using 50% methanol plus n-hexane. The methanol phase which had MI activity was fractionated into 7 fractions using reverse-phase high-performance liquid chromatography. Of these 7 fractions, fraction 3 was found to be active in having MI ability and identified as 17-alpha,20-beta-dihydroxy-4-pregnen-3-one (17-alpha,20-beta-diOHprog). The authenticity of 17-alpha,20-beta-diOHprog as the major follicular mediator of gonadotropin-induced oocyte maturation was further confirmed by thin-layer chromatography (TLC) in which fraction 3 was run along with authentic 17-alpha,20-beta-diOHprog standard. This investigation gives a direct evidence that 17-alpha,20-beta-diOHprog is the major naturally occurring MIS in Indian female catfish, C. batrachus.