The overall objective of the research in this proposal is to determine how Leydig cell mitochondrial and cytosolic proteins are altered both during the course of normal development and in response to gonadotropin. Leydig cells are the major steroid producing cells in the testis with testosterone being the steroid synthesized in greatest amount. Steroid production in the Leydig cells is under the control of LH and has as its rate limiting step the side chain cleavage of cholesterol, a reaction which occurs exclusively in the mitochondria of steroidogenic cells. In addition to the cleavage enzymes themselves, several other factors have been implicated in the overall regulation of this step in steroid production by LH. Cholesterol binding protein, sterol carrier protein, calmodulin and several other as yet unidentified proteins have all been linked to this regulatory process. I proposed to study this regulatory process in both freshly isolated Leydig cells as well as in MA-10 cells which are a permanent cell line cloned from a Leydig cell tumor. Both the mitochondrial and cytosolic proteins will be isolated from Leydig cells during the cousre of normal development and in response to gonadotropin stimulation. The protein content of these fractions will be analyzed by 2-dimensional gel electrophoresis followed by a very sensitive silver staining technique. In addition, the synthesis of proteins by Leydig cells in response to hormone stimulation will be determined by radiolabelling the cells with S35-methionine and analyzing the results with 2-D electrophoresis and fluorography. Concommitant with these studies the activity of the cholesterol side chain cleavage (CSCC) reaction will be measured. In addition, 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 characterize other proteins which are synthesized in Leydig cells in response to LH treatment. These studies are designed to further characterize both mitochondrial and cytosolic proteins which are synthesized by Leydig cells in response to gonadotropin stimulation.
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