Aims: In ovarian periovulatory follicles, granulosa cells in response to gonadotropins and intraovarian factors produce ample progesterone essential to promote oocyte maturation and ovulation (1-3). This study was to explore the capability of granulosa cells under FSH and TGFβ1 induction to de novo synthesize cholesterol for producing progesterone when external cholesterol supply is limited, and the associated cellular metabolic reform.
Methods: Ovarian granulosa cells from mid-to-large antral follicles of gonadotropin-primed immature rats were cultured in serum-and-lipoprotein-free medium. To induce progesterone synthesis, cells were given FSH±TGFβ1; 24h later, simvastatin (HMGCR inhibitor) was added to block de novo cholesterol synthesis. At the end of 48-h hormonal treatment period, progesterone secretion, cellular content of cholesterol, steroidogenic and cholesterogenic proteins, and crucially associated metabolic proteins were determined using enzyme-immunoassay, immunoblotting and qPCR analyses.
Results: This study provides interesting original findings. First, FSH+TGFβ1-induced progesterone production was suppressed by simvastatin cotreatment, while simvastatin had no effect on steroidogenic protein levels (StAR, P450scc-FDX1-FDXR complex, 3βHSD). Second, FSH+TGFβ1 treatment decreased cellular cholesterol level, which was further reduced by simvastatin cotreatment. Consistent with our earlier study (4), FSH+TGFβ1 upregulated cholesterogenic proteins (HMGCR, LDLR, SR-B1) and key regulator SREBP2. Third, we demonstrated that simvastatin cotreatment further increased HMGCR, LDLR, and SREBP2 without affecting SR-B1. The above results together support that cellular cholesterol homeostatic control is functional, and SR-B1 is insensitive to such control. Fourth, FSH+TGFβ1 upregulated key metabolic proteins that support cholesterol biosynthesis, involving mitochondrial anaplerotic process providing citrate (PC, FASN, CPT1A), and cataplerotic process providing cytosolic citrate conversion to acetyl-CoA (CiC, ACLY); interestingly, simvastatin cotreatment further increased FASN and ACLY.
Conclusion: Our work discloses that to assure maturation of enclosed oocyte and ovulatory process, granulosa cells display amazing capability to de novo synthesize cholesterol for progesterone production when external cholesterol resource is limited, and this involves effecting mitochondrial anaplerosis-and-cataplerosis.