Control of sterol metabolism in cultured rat granulosa cells

Rat granulosa cells in culture secreted 15-35 microgram 20 alpha-hydroxypregn-4-en-3-one/mg protein . 24 h when grown in medium containing 20% calf serum. Within 24 h of exposure to lipoprotein-deficient medium, steroid production was reduced by 80%. The addition of human low density lipoprotein (LDL), human high density lipoprotein (HDL), or the polar sterol, 5-cholesten-3 beta,25-diol, rapidly restored steroidogenesis to control values. The effects of human lipoproteins were dose dependent, with maximum stimulation of steroid production observed at sterol concentrations of 150 and 200 microgram/ml for human LDL and HDL, respectively. Stimulation of steroidogenesis by lipoproteins was prevented by antiserum against the specific lipoproteins. Lipoproteins, in particular human LDL, increased the incorporation of [14C]oleate into cellular sterol esters in a dose-related manner. In the case of human LDL, sterol concentrations of 150 microgram/ml maximally stimulated sterol esterification. When cells were grown in medium containing lipoproteins, endogenous sterol production, as measured by the incorporation of [14C]acetate into nonsaponifiable lipids, was low, as was the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Moreover, steroid production was unimpaired upon the addition of ML-236B, a potent inhibitor of HMG-CoA reductase. Cells cultured in lipoprotein-deficient medium displayed significantly increased rates of [14C]acetate incorporation into nonsaponifiable lipids and increased HMG-CoA reductase activity. Under these conditions, the incorporation of [14C]acetate into sterol was markedly reduced, and steroid secretion was also lowered by ML-236B. When aminoglutethimide was added to cell cultures for a 24-h period, steroid secretion was blocked, and [14C]acetate labeling of nonsaponifiable lipids was decreased. Furthermore, sterol esterification was increased by cells incubated with aminoglutethimide. The latter effect of aminoglutethimide appeared to be to an increased availability of cellular free sterol for esterification and, to a lesser extent, to the relief of inhibition of sterol ester synthetase by progestins. We conclude that 1) an exogenous source of cholesterol (e.g. lipoproteins) is necessary for maximal steroidogenesis and sterol ester storage in cultured rat granulosa cells, because their capacity to generate substrate de novo is limited; and 2) de novo sterol synthesis and sterol esterification in these cells are regulated by sterol balance, which is determined by the availability of exogenous cholesterol on the one hand, and the rate of cholesterol utilization for hormone synthesis on the other.