SPECIFIC NON-GENOMIC, MEMBRANE-LOCALIZED BINDING-SITES FOR PROGESTERONE IN THE BOVINE CORPUS-LUTEUM

Fractionation of bovine corpus luteum (CL) homogenates on continuous s ucrose density gradients with and without preincubation with H-3-proge sterone demonstrated high levels of tracer binding and high content of endogenous progesterone associated with particulate membrane fraction s. Analysis of gradient fractions for a range of luteal plasma membran e and intracellular organelle marker enzyme activities indicated that endogenous progesterone content and H-3-progesterone-binding activity were associated with fractions enriched in luteal plasma membrane mark ers. This was confirmed by pretreatment of homogenates with the saponi n, digitonin, prior to fractionation. Digitonin perturbed the buoyant density of luteal surface membrane markers and H-3-progesterone bindin g to a similar extent, but did not perturb the buoyant densities of ot her intracellular markers to the same degree. Interestingly, digitonin pretreatment also increased the proportion of progesterone tracer tha t entered the gradients. We consistently failed to demonstrate signifi cant binding of H-3-progesterone to membrane fractions incubated with progesterone tracer in vitro. However, when digitonin was included in the in vitro binding assay, we observed a dramatic, dose-dependent sti mulation of H-3-progesterone binding by digitonin. Other radiolabeled steroids tested (H-3-cortisol, H-3-testosterone) bound poorly in the p resence or absence of digitonin. H-3-Progesterone binding in the prese nce of optimal digitonin concentrations increased linearly with increa sing luteal membrane concentration; was dependent on the pH, duration, and temperature of incubation; and low levels of progesterone (68 nM) competed for tracer binding. A range of other steroids tested (androg ens, estrogens, corticosteroids, steroid precursors) competed at highe r concentrations (10- to 100-fold) or did not compete at all for H-3-p rogesterone binding. There was no correlation between the hydrophobici ty of various steroids and their ability to compete for binding. Moreo ver, a number of agonists and antagonists specific for the genomic pro gesterone receptor, agonists of peripheral benzodiazepine receptors, a nd inhibitors of a range of steroidogenic enzymes did not compete for H-3-progesterone binding. Western blots confirmed that detergent-solub ilized progesterone-binding sites could be resolved from cytochrome P4 50 side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase. Moreov er, extraction of bound steroid from the binding and HPLC demonstrated identity to progesterone, suggesting that no metabolism of the proges terone tracer had occurred during incubation. Progesterone binding to membranes of large luteal cells was higher compared with binding to sm all luteal cells, and levels were similar in membranes prepared from C L at all stages of the luteal phase. We suggest that bovine luteal pro gesterone-binding sites may play a role either in sequestration of new ly synthesized progesterone or in the mediation of autocrine and/or pa racrine actions of progesterone in the CL.