PARTICULATE BINDING-SITES FOR STEROID-HORMONES IN SUBCELLULAR-FRACTIONS OF THE OVINE CORPUS-LUTEUM - PROPERTIES AND HORMONE SPECIFICITY

We have described previously the presence of binding sites in particul ate fractions of the porcine corpus luteum (CL) which were specific fo r progesterone. We now demonstrate the presence of similar progesteron e-specific binding sites in particulate fractions of the ovine CL. Pre incubation of ovine luteal membranes with radiolabelled steroids demon strated binding of progesterone and pregnenolone to a law-density part iculate fraction (1.07-1.09 g/cm(3)). Preincubation with digitonin per turbed the buoyant density of this fraction (to 1.10-1.14 g/cm(3)) wit hout causing release of steroid. Androgens and oestrogens did not bind appreciably to control luteal membranes, but were bound when preincub ated with digitonin. In contrast, steroid conjugates (oestrone sulfate , pregnanediol glucuronide), cortisol, fatty acids (arachidonic acid, prostaglandin F-2 alpha) and cholesterol ester failed to bind to ovine luteal membranes, with or without digitonin pretreatment. The effects of digitonin on steroid binding led us to examine its effects on ster oid binding to ovine luteal membrane fractions in vitro. Specific prog esterone binding was absent in the absence of digitonin, even at very high membrane concentrations. However, binding of H-3-labelIed progest erone was stimulated 5-15-fold in a dose-dependent fashion by increasi ng digitonin concentrations, reaching a plateau at about 100 mu M. In the presence of digitonin, [H-3]progesterone binding increased linearl y with luteal membrane concentration. Other detergents, saponins and c ardiotonic steroids tested did not stimulate progesterone binding to o vine luteal membranes. [H-3]Progesterone binding was dependent on the pH, duration and temperature of incubation. Unlabelled progesterone de creased binding of [H-3]progesterone (half-maximal displacement of spe cific binding (IC50) at about 60 nM) whereas androgens were less poten t (IC50, 500-3300 nM), whilst a wide range of other steroids and inhib itors of steroidogenic enzymes were ineffective, except at very high c oncentrations. Similarly, a number of progesterone receptor agonist an d antagonist analogues failed to compete for progesterone binding to l uteal membranes, suggesting that these binding sites were unrelated to progesterone receptors. Modifications to the 3, 4, 5 and 11 positions of progesterone, removal of the steroid side-chain or aromatization o f the A-ring decreased binding potency dramatically, whereas changes t o the 17 or 20 positions had relatively minor effects. Our results ind icate the presence of a low density particulate fraction in ovine corp ora lutea which contains specific binding sites for endogenous and exo genous progesterone.