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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Modified Research Career Development Award (K04)
Project #
5K04HD000685-02
Application #
3073281
Study Section
Reproductive Biology Study Section (REB)
Project Start
1985-08-01
Project End
1990-07-31
Budget Start
1986-08-01
Budget End
1987-07-31
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Texas Tech University
Department
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430
Stocco, D M; Ascoli, M (1993) The use of genetic manipulation of MA-10 Leydig tumor cells to demonstrate the role of mitochondrial proteins in the acute regulation of steroidogenesis. Endocrinology 132:959-67
Stocco, D M; Khan, S A (1992) Effects of steroidogenesis inducing protein (SIP) on steroid production in MA-10 mouse Leydig tumor cells: utilization of a non-cAMP second messenger pathway. Mol Cell Endocrinol 84:185-94
Stocco, D M (1992) Further evidence that the mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are involved in the acute regulation of steroidogenesis. J Steroid Biochem Mol Biol 43:319-33
Stocco, D M; Sodeman, T C (1991) The 30-kDa mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are processed from larger precursors. J Biol Chem 266:19731-8
Chaudhary, L R; Stocco, D M (1991) Effect of different steroidogenic stimuli on protein phosphorylation and steroidogenesis in MA-10 mouse Leydig tumor cells. Biochim Biophys Acta 1094:175-84
Stocco, D M; Chen, W (1991) Presence of identical mitochondrial proteins in unstimulated constitutive steroid-producing R2C rat Leydig tumor and stimulated nonconstitutive steroid-producing MA-10 mouse Leydig tumor cells. Endocrinology 128:1918-26
Chaudhary, L R; Stocco, D M (1990) An in vitro cell model system to study the action of retinoids on Leydig cell steroidogenesis. Biochem Int 21:1033-42
Stocco, D M; Teerds, K J; van Noort, M et al. (1990) Effects of hypophysectomy and human chorionic gonadotrophin on Leydig cell function in mature rats. J Endocrinol 126:367-75
Stocco, D M; Chaudhary, L R (1990) Evidence for the functional coupling of cyclic AMP in MA-10 mouse Leydig tumour cells. Cell Signal 2:161-70
Kilgore, M W; Rommerts, F F; Wirtz, K W et al. (1990) Regulation of steroidogenesis in subclones of the MA-10 mouse Leydig tumor cell line. Mol Cell Endocrinol 69:9-16

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