Particulate binding sites for steroid hormones in subcellular fractions of the ovine corpus luteum: properties and hormone specificity

Abstract

We have described previously the presence of binding sites in particulate fractions of the porcine corpus luteum (CL) which were specific for progesterone. We now demonstrate the presence of similar progesterone-specific binding sites in particulate fractions of the ovine CL. Preincubation of ovine luteal membranes with radiolabelled steroids demonstrated binding of progesterone and pregnenolone to a low-density particulate fraction (1.07–1.09 g/cm 3). Preincubation with digitonin perturbed the buoyant density of this fraction (to1.10–1.14 g/cm 3) without causing release of steroid. Androgens and oestrogens did not bind appreciably to control luteal membranes, but were bound when preincubated with digitonin. In contrast, steroid conjugates (oestrone sulfate, pregnanediol glucuronide), cortisol, fatty acids (arachidonic acid, prostaglandin F 2α) 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 steroid binding to ovine luteal membrane fractions in vitro. Specific progesterone binding was absent in the absence of digitonin, even at very high membrane concentrations. However, binding of 3H-labelled progesterone was stimulated 5–15-fold in a dose-dependent fashion by increasing digitonin concentrations, reaching a plateau at about 100 μM. In the presence of digitonin, [ 3H]progesterone binding increased linearly with luteal membrane concentration. Other detergents, saponins and cardiotonic steroids tested did not stimulate progesterone binding to ovine luteal membranes. [ 3H]Progesterone binding was dependent on the pH, duration and temperature of incubation. Unlabelled progesterone decreased binding of [ 3H]progesterone (half-maximal displacement of specific binding (IC 50) at about 60 nM) whereas androgens were less potent (IC 50, 500–3300 nM), whilst a wide range of other steroids and inhibitors of steroidogenic enzymes were neffective, except at very high concentrations. Similarly, a number of progesterone receptor agonist and antagonist analogues failed to compete for progesterone binding to luteal 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 of the A-ring decreased binding potency dramatically, whereas changes to the 17 or 20 positions had relatively minor effects. Our results indicate the presence of a low density particulate fraction in ovine corpora lutea which contains specific binding sites for endogenous and exogenous progesterone.