We will investigate the biochemical mechanism by which gontropin regulate luteal cell function. Luteal cells obtained from pregnant mare serum gonadotropin and human chorionic gonadotropin primed rats will be used as a model system. These cells respond to gonadotropins and cyclic nucleotides with increase in progesterone production. Additionally, plasma lipoprotein fractions LDL and HDL are specifically taken up by these cells and their cholesterol is used for progesterone production. It is proposed to examine the following aspects of the problem during the project period. 1) Identification and characterization rat luteal cell HDL receptor, 2) to define the mechanism by which HDL delivers cholesterol for luteal cell steroidogenesis and control of this process by gonadotropins, 3) regulation of luteal cell mitochondrial cholesterol side chain cleavage enzyme by gonadotropin as determined by EPR spectroscopy and to determine the mechanism of hCG/LH-induced refractoriness of luteal cell steroidogenesis, and 4) the role of luteal cell cytoskeleton in hCG/LH-induced steroidogenesis. Objective 1) will be accomplished by determining the biochemical characteristics of rat luteal cell HDL receptor with the help of HDL receptor antibody and by attempting to purify the receptor using conventional biochemical techniques. The regulation of the receptor by gonadotropin and the turnover of the receptor will be then determined. Objective 2) will be accomplished by examining the fate of (125I) labeled apolipoproteins of HDL after binding to rat luteal cell HDL receptor. Apolipoprotein DMPC vesicles will be prepared with differentially labeled cholesterol, apolipoprotein and the ability of apolipoprotein DMPC vesicles to deliver cholesterol for luteal cell progesterone production will be determined. Objective 3) will be accomplished by measuring cytochrome P450 of rat luteal cell mitochondria and the effect of hCG/LH on the binding of Cyt P450 with cholesterol will be determined. Objective 4) will be examined by determining the regulation of cytoskeletal function by gonadotropins as it relates to luteal cell progesterone production.
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