DEMONSTRATION OF PUTATIVE MEMBRANE AND CYTOSOL STEROID-RECEPTORS FOR 17-ALPHA,20-BETA-DIHYDROXY-4-PREGNEN-3-ONE IN BROOK TROUT SALVELINUS-FONTINALIS OOCYTES BY PHOTOAFFINITY-LABELING USING SYNTHETIC PROGESTIN 17,21-DIMETHYL-19-NOR-PREGN-4,9-DIENE-3,20-DIONE (R5020)

Cytosol from brook trout ovarian follicles (stages 1-3) was photoaffinity (PA) labelled using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione ([H-3]R5020). The covalently bound cytosol protein had a relative mass of 501,000 M(r) following Sephacryl S-300 column chromatography. The zona radiata membrane fraction from brook trout oocytes which had gone through the first phase of meiotic maturation (stages 6-7) was isolated by ultracentrifugation of the whole oocytes. The zona radiata solubilized protein presumably from the oocyte membrane was also PA labelled and found to give a peak at 355,000 M(r). The SDS PAGE of the cytosol and zona radiata PA labelled protein gave very similar subunits indicating that the membrane protein and the cytosol protein, both of which bind the maturation inducing steroid (MIS) 17-alpha,20-beta-dihydroxy-4-pregnen-3-one (17-alpha,20-beta-DHP), have similar subunit structures. The isolated zona radiata protein showed cooperativity of binding to [H-3]17-alpha,20-beta-DHP and PA labelling to [H-3]R5020. The association constant (K(a)) was 2.0 x 10(7)M-1 and maximum binding capacity (N(max)) 427 fmoles/mg protein with MIS [H-3]17-alpha,20-beta-DHP. No evidence for nuclear binding of MIS [H-3]17-alpha,20-beta-DHP or PA labelling of [H-3]R5020 to nuclei was observed. The nuclei were isolated from stages 1 and 3 fresh ovarian follicles of brook trout. The experimental evidence presented demonstrates the presence of MIS 17-alpha,20-beta-DHP receptor-like protein from the zona radiata membranes by PA labelling in brook trout oocytes during final stages of maturation.