The overall objective of this proposal is to determine how the mitochondria of Leydig cells are altered during the course of normal development and in response to LH stimulation. Leydig cells have been shown to be the major steroid producing cells in the testis, with testosterone being the steroid synthesized in greatest amount. The production of testosterone is essential for normal spermatogenesis and the maintenance of secondary sex characteristics. The rate limiting step by which LH controls Leydig cell steroidogenesis is the side chain cleavage of cholesterol, a reaction which occurs exclusively in the mitochondria. I propose to study changes which occur in Leydig cell mitochondria during the course of normal development and in response to LH stimulation. Leydig cells from rats 10-60 days of age will be isolated on Metrizamide gradients. Mitochondria will be isolated from these cells and analyzed for cholesterol side chain cleavage activity and protein content. The latter analysis will be performed using 2-dimensional gel electrophoresis, followed by a very sensitive silver staining technique. In the next series of experiments, animals 10-20 days of age will be injected with LH followed by isolation of Leydig cells and their mitochondria. Cholesterol side chain cleavage activity and protein content will be analyzed as in the developmental studies. I then plan to stimulate freshly isolated Leydig cells in vitro with LH and determine the effects of this gonadotropin on cleavage activity, protein content and, through the use of inhibitors and radiolabeled methionine, cytoplasmic and mitochondrial protein synthesis. Lastly, I propose to perform the studies just outlined on Leydig cells grown in primary culture for up to three days. In all studies, changes in the levels of the CSCC components will be determined by immunoblotting techniques in which mitochondrial proteins will be transblotted onto APT sheets and immunostained with antibodies raised to these proteins. I also plan to perform a preliminary characterization of any other mitochondrial proteins which are synthesized in response to LH treatment. These studies are designed to further our knowledge of the role of Leydig cell mitochondria in the regulation of steroidogenesis by LH.
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