Expression of Ad4-BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long-term cultures of human granulosa cells

Freshly isolated granulosa cells obtained from women undergoing in-vitro fertilization (IVF) become refractory to further gonadotrophin stimulation in culture due to their previous hormonal treatment. However, when precurtured for 7 days in gonadotrophin-free medium they regain their response to both human chorionic gonadotrophin (HCG) and follicle stimulating hormone (FSH) with a 10-fold and 5-fold increase in progesterone production respectively, within an additional 7 days of culture. Forskolin, a direct activator of adenylate cyclase, increased progesterone levels 12-fold compared with non-stimulated cultures. Oestradiol formation was also significantly elevated (P < 0.005) following 48 h stimulation with luteinizing hormone (LH), FSH or forskolin. Intracellular cAMP levels rose 1.5-fold, 10-fold and 15-fold after 1 h of FSH, HCG or forskolin treatment. Expression of both cytochrome P450 side chain cleavage enzyme (SCC) and the steroidogenic transcription factor SF1/Ad4BP could be demonstrated by Western blotting. However, elevation of P450 SCC alone was evident following FSH and HCG stimulation. In the presence of serum, the ultrastructure of these cultured cells displayed numerous lipid droplets and well-developed mitochondria, characteristic of highly steroidogenic cells. The proportion of apoptotic nuclei in these cultures was < 30%. Removal of the serum increased apoptotic incidence to 40%, whereas addition of FSH prevented cell death significantly (P < 0.01). HCG and forskolin increased apoptosis to similar to 50%, while treatment with 8Br-cAMP led to 80% cell death. Our data suggest that, after prolonged culture, human granulosa cells can regain cAMP and steroidogenic response to gonadotrophin stimulation. Moreover, our experiments indicate that apoptosis and steroidogenesis can coexist in the same cell population while the interrelationship between these processes can be determined by the intracellular levels of cAMP.