LIPOPROTEIN SUPPORT OF STEROIDOGENESIS BY LUTEINIZING MACAQUE GRANULOSA CELLS: DIFFERENTIAL EFFECTS OF VLDL, LDL, AND HDL

Abstract

Circulating lipoproteins are hypothesized to deliver cholesterol as substrate for ovarian steroidogenesis. The present study was designed to determine which of the 3 main lipoproteins (very low density [VLDL], low density [LDL], high density [HDL]) is the preferred source of cholesterol to support human chorionic gonadotropin (hCG)-stimulated progesterone (P) production by primate granulosa cells. Following the onset of menstruation, adult female rhesus monkeys were treated with recombinant human FSH (r-hFSH) for 7 days and non-luteinized granulosa cells were aspirated by an ultra-sound guided procedure. Some animals received FSH for 7 days followed by hCG for up to 24 hr to initiate periovulatory events before aspiration. Non-luteinized cells were cultured in serum-free media with hFSH (25 ng/ml) or hCG (20 IU/ml) + hFSH for up to 48 hrs. The expression of VLDL receptor mRNA decreased (p<0.05) within 3 hr of hCG administration in vivo, while LDL receptor mRNA increased (p<0.05) by 3–12 hr post-hCG and returned to pre-hCG levels at 24 hr. The expression of scavenger receptor-BI (SR-BI) mRNA increased by 6 hr and was maintained throughout 24 hr. Similar results were obtained from non-luteinized granulosa cells treated with FSH+hCG in vitro. VLDL, LDL, or HDL were added to cultures based either on molar concentration of lipoproteins (5, 25, 50 nM) or cholesterol content (1, 5, 10 microg/ml) for 24 hr (0–24 hr) in the presence of FSH or FSH+hCG. Alternatively, cells were cultured in the presence of only FSH or FSH+hCG for 24 hr to deplete stored cholesterol, and treated subsequently with lipoproteins for the next 24 hr (24–48 hr). During the initial 24 hr culture interval (0–24 hr), levels of P in lipoprotein-free media were significantly higher (p<0.05) in FSH+hCG treated cultures versus FSH only, suggesting the use of stored cholesterol. Molar equivalents of VLDL and LDL dose-dependently increased (p<0.05) hCG-stimulated P 0–24 hr, while HDL did not alter P synthesis. When lipoproteins were balanced to cholesterol content, similar results were obtained. In the absence of lipoproteins, P levels were not increased significantly by FSH+hCG vs FSH alone after 48 hr (24–48 hr), suggesting depletion of stored cholesterol esters between 24 and 48 hr after hCG treatment. The addition of VLDL and LDL during the 24–48 hr interval increased P levels in hCG-stimulated cells whether balanced to molar concentrations of lipoproteins or to cholesterol. Lipases involved in lipid metabolism (monoacylglycerol lipase [MGL]) and hydrolysis of estrified cholesterol (hormone sensitive lipase [HSL]) were examined to determine their role in P synthesis in vitro. Inhibitors of both MGL (0–100 microM) and HSL (0–400 mM) dose-dependently decreased (p<0.05) hCG-induced P synthesis after 6 hr. It is concluded that steroidogenesis by primate granulosa cells is supported primarily by stored cholesterol during the first 24 hr after an ovulatory stimulus, and thereafter by LDL and VLDL, but not HDL. The use of cholesterol stores during early luteinization is most likely mediated by hCG-activated lipases such as monoacylglycerol lipase and hormone sensitive lipase. The increased expression of LDL receptor and SR-BI after hCG suggest that either or both of these lipoprotein receptors mediate the use of LDL and VLDL during luteinization of primate granulosa cells. Supported in part by NIH HD043358 (CLC), RR13439 (CAV), RR00169 (CNPRC), HL075646 (AR). (poster)