Bovine ovarian non-genomic progesterone binding sites: presence in follicular and luteal cell membranes

We have shown recently that the bovine corpus luteum (CL) possesses specifi c luteal cell surface membrane binding sites for progesterone. We have now confirmed and extended these observations to compare the subcellular distri bution of these binding sites in developing, mature and regressing CL. The median buoyant densities of luteal progesterone binding sites from early-, mid- and late-luteal phase CL were similar (though three of five density pr ofiles for late-luteal phase CL showed association of steroid binding with a fraction with a lower density), and clearly resolved from nuclear, mitoch ondrial, lysosomal, peroxisomal, Golgi-endoplasmic reticulum-lysosomal and smooth endoplasmic reticulum markers. Specific binding of [H-3]progesterone overlapped with the distributions of 5'-nucleotidase and luteinizing hormo ne receptor (luteal cell surface membrane markers) in both control and digi tonin-treated gradients at all stages of the luteal phase. Since steroidogenic 'large luteal' and 'small luteal' cells of the CL are d erived from the granulosa cells (GC) and theca of the preovulatory follicle , we also investigated whether similar receptors were present in the follic le, and describe for the first time specific membrane binding sites for pro gesterone in purified GC and thecal membranes from healthy bovine follicles of different sizes. Specific binding increased linearly with GC and thecal membrane protein concentration; however, it was detectable only when digit onin was included in the binding incubation. Binding sites were specific fo r progesterone; unlabelled progesterone competed for [H-3]progesterone bind ing at low concentrations (IC50, 35 and 31 nmol/l) compared with testostero ne (IC50, 905 and 870 nmol/l) and Delta(4)-androstenedione (IC50, 1050 and 660 nmol/l) for GC and thecal receptors respectively. In contrast, oestradi ol, oestrone, pregnenolone, cortisol, cholesterol, and genomic progesterone receptor antagonist, RU486, competed poorly. Steroid binding was present in GC and thecal membranes of follicles of all sizes, but [H-3]progesterone binding to GC membranes decreased significantl y with increasing follicle size (P<0.02), perhaps indicating developmental regulation of GC membrane non-genomic progesterone receptors in the preovul atory bovine follicle. We suggest that these membrane steroid receptors may be involved in the autocrine/paracrine regulation of follicular function b y progesterone.