Steroidogenesis in Fundulus heteroclitus 5.: Purification, characterization, and metabolism of 17 alpha ,20 beta -dihydroxy-4-pregnen-3-one by intact follicles and its role in oocyte maturation

In vitro steroidogenesis of ovarian follicles incubated with radioactive precursors or a Fundulus heteroclitus pituitary extract (FPE) was investigated. Steroids were extracted from both the medium and follicular tissue and fractionated by liquid or thin-layer chromatography. A similar pattern of steroid metabolites was obtained with either [ super(14)C]pregnenolone or [ super(14)C]progesterone as exogenous precursor. Several metabolites comigrated with known reference steroids and thus were tentatively identified. Some have been previously reported to induce germinal vesicle breakdown (GVBD) in this and other species, particularly 17 alpha ,20 beta -dihydroxy-4-pregnen-3-one (DHP). [ super(3)H]DHP added to intact follicles or denuded oocytes was also extensively metabolized. All the DHP metabolites produced by the intact follicle were tested for biological activity. Three of the metabolites were almost as effective inducers of GVBD as DHP itself, and two were tentatively identified as 5 alpha -pregnan-3 alpha ,17 alpha ,20 beta -triol and 5 alpha -pregnan-3 beta ,17 alpha ,20 beta -triol. However, DHP was the most potent and the quickest inducer of GVBD, indicating that its maturational action is not due to metabolic conversion to a more active form. In addition, we found two very active fractions after HPLC analysis of steroid extracts from FPE-stimulated follicles: one that corresponded to and was further identified (mass spectroscopy) as DHP and a second tentatively identified as the DHP metabolite 5 alpha -pregnan-3 beta ,17 alpha ,20 beta -triol. This study provides strong evidence that DHP plays the major role as a maturation inducing-steroid in F. heteroclitus, even though DHP is not the only active steroid produced by maturing follicles.