Progesterone production in ovarian granulosa cells of large antral follicle is crucial to ovulation induction. We and others have demonstrated in granulosa cells that transforming growth factor TGFβ1 enhances follicle-stimulating hormone (FSH)-induced progesterone production, which is associated with the increased expression of cholesterol-side-chain-cleavage enzyme (P450scc/Cyp11a1) localized mainly in inner mitochondrial membrane. We are intrigued to understand whether and how FSH and TGFβ1 regulate mitochondrial function in close relation to the increase of P450scc activity using primary culture of ovarian granulosa cells from gonadotropin-primed immature rats.
P450scc functions as a complex together with adrenodoxin reductase (AdxR) and adrenodoxin (Adx), which are elemental to P450scc activity. Here, we disclosed that treatment with FSH and TGFβ1 also increase the protein level of AdxR and Adx. Also, immunofluorescent analysis indicates that P450scc localized mainly in mitochondria. We then further examined the mitochondrial NAD(P)H-generating enzymes as NADPH is an essential cofactor for AdxR, and found that FSH and TGFβ1 increase the protein level of TCA cycle constituents, NADPH-generating isocitrate dehydrogenase IDH2, and NADH-generating IDH3 and α-ketoglutarate dehydrogenase (αKGDH). Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial function. PGC-1α activity is importantly stimulated by sirtuin Sirt1, and is involved in facilitating estrogen-related receptor alpha (ERRα)-transactivated expression of Sirt3, which modulates the activity of mitochondrial molecules including IDH2. We previously revealed that PGC-1α is involved in FSH and TGFβ1-upregulated expression of P450scc. Here, we further demonstrated that FSH and TGFβ1 increase the protein level of Sirt1, PGC-1α, ERRα and Sirt3, and that pretreatment with a selective inhibitor of Sirt1 attenuates FSH and TGFβ1-induced progesterone production. In all, this study indicates that FSH and TGFβ1 induction of progesterone synthesis is closely associated with upregulating Sirt1–PGC-1α–Sirt3 mediation of mitochondrial function including NADPH–NADH balance in ovarian granulosa cells.