Gonadotropin receptors in the plasma membrabes of rat luteal cells

Abstract 1. 1. In in vivo studies, human chorionic gonadotropin (HCG) labeled with 131I was concentrated by the ovaries of the normal and superovulated rats, whereas 131I-labeled bovine serum albumin and 131I were not incorporated. The distribution of 131I-labeled HCG was examined in the subcellular fractions of the ovaries. Injection of unlabeled HCG or HCG-antisera decreased the uptake of 131I-labeled HCG by mitochondrial and microsomal fractions suggested that the HCG entered the luteal cells. 2. 2. In in vitro studies, the 125I-labeled HCG bound to plasma membranes decreased proportionately to the increasing amounts of unlabeled HCG. Unlabeled human luteinizing hormone also decreased the uptake of 125I-labeled HCG by the plasma membranes, however to a lesser degree than the unlabeled HCG. The plasma membranes did not bind 131I-labeled bovine serum albumin or 131I. The uptake of 125I-labeled HCG by the plasma membranes was not decreased by human follicle-stimulating hormone, subunits of human follicle-stimulating hormone, HCG and human luteinizing hormone, ovine prolactin and human growth hormone, suggesting the presence of specific receptors for HCG in the plasma membranes of rat luteal cells. The mitochondrial and microsomal fractions bound both labeled HCG as well as labeled bovine serum albumin. The presence of unlabeled HCG did not decrease the uptake of 131I-labeled HCG by mitochondrial and microsomal fractions. 3. 3. In in vitro studies, the binding of 125I-labeled HCG to plasma membranes was maximum at pH 7.0. Binding of labeled HCG to the plasma membranes was rapid at both 4 and 38 °C. Approximately 1.8 · 10−14 moles of 125I-labeled HCG saturated receptor sites in 1 mg of plasma membrane protein. Incubation of plasma membranes saturated with 125I-labeled HCG in a hormone-free medium showed a dissociation of 125I-labeled HCG from the plasma membranes which was significantly greater at 38 °C than at 4 °C. At 38 °C the rate of association was 1.0 · 108 moles−1 · s−1 and the rate of dissociation was 1.7 · 10−4 · s−1. The association and dissociation constants obtained from the ratio of rate constants were 6.5 · 1011 M−1 and 1.5 · 10−12, respectively. Increasing salt concentration to 0.5 M NaCl and treatment with phospholipase C resulted in an increase in binding of 125I-labeled HCG to the plasma membranes. Temperature of 95 °C and treatment of plasma membranes with chloroform-methanol (2:1, v/v) abolished hormone-receptor binding due to denaturation of proteinsas well as delipidization. 4. 4. In in vivo experiments, HCG and human luteinizing hormone injected into superovulated rats stimulated adenylate cyclase activity in ovarian homogenates. The subunits of HCG and human luteinizing hormone, on the other hand, were significantly less potent in stimulating adenylate cyclase activity. Injection of HCG, human luteinizing hormone and their α- and β-subunits in superovulated rats did not show increase in the progesterone levels of the ovarian homogenates; however, the progesterone levels in peripheral plasma were significantly elevated. These observations indicate that the action of native hormones on the ovaries was mediated via a specific receptor in the plasma membranes and membrane-bound adenylate cyclase of the luteal cells, whereas subunits act via a different receptor or pathway, without mediation via membrane-bound adenylate cyclase.