Regulation of the Synthesis of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in the Bovine Ovaryin Vivoandin Vitro*

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Abstract
In order to investigate the pattern of ovarian cholesterol biosynthesis during the bovine estrous cycle, tissue concentrations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the synthesis of cholesterol, were determined by immunoblot techniques. Medium-sized (9-11 mm) and large (14-18 mm) follicles, after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid, late-mid, and late stages of the luteal phase were used (n = 5 per group). The specific content (per microgram of tissue homogenate protein) and total content of HMG-CoA reductase in medium-sized and large follicles were substantially lower than those of corpora lutea of the early-mid and late-mid luteal phase. The specific content was elevated in a number of the corpora lutea from the early luteal phase and was low in regressing corpora lutea. Thus during the midluteal phase, when steroid hormone production is elevated, the total and specific contents of HMG-CoA reductase are also elevated. To investigate the mechanisms whereby the levels of HMG-CoA reductase are regulated, primary monolayer cultures of bovine luteal cells (early-mid and late-mid luteal phase) were used. Cells were cultured for 24 h in Dulbecco''s modified Eagle''s medium containing lipoprotein-poor fetal calf serum (2% vol/vol). At this concentration there was no stimulation of the production of progesterone above that seen wtih no addition of serum. Under these conditions the total and specific contents, and the synthesis, of HMG-CoA reductase were stimulated by treatment with (Bu)2cAMP (1 mM). The stimulatory action of (Bu)2cAMP was inhibited by the addition of bovine high density lipoprotein and an inhibitor of cholesterol side-chain cleavage activity, clotrimazole (3.75 .mu.M). Based on these results, it is suggested that the action of (Bu)2cAMP to stimulate the synthesis of HMG-CoA reductase in bovine luteal cells is mediated primarily by changes in the intracellular content of cholesterol. It is concluded, therefore, that the substantially greater content of HMG-CoA reductase in corpora lutea over that of follicles is primarily the result of the greater use of cholesterol by corpora lutea to meet the demand for progesterone biosynthesis. This, in turn, is a consequence of the substantially greater concentration of the components of the cholesterol side-chain cleavage system in corpora lutea.