Breckwoldt M, Selvaraj N, Aharoni D, Barash A, Segal I, Insler V, Amsterdam A
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
Mol Hum Reprod. 1996 Jun;2(6):391-400. doi: 10.1093/molehr/2.6.391.
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 precultured 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 approximately 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.
从接受体外受精（IVF）的女性体内新鲜分离得到的颗粒细胞，由于其先前接受的激素治疗，在培养过程中对进一步的促性腺激素刺激变得不敏感。然而，当在无促性腺激素的培养基中预培养7天时，它们在额外7天的培养期内恢复了对人绒毛膜促性腺激素（HCG）和促卵泡激素（FSH）的反应，孕酮分泌分别增加了10倍和5倍。福斯可林是腺苷酸环化酶的直接激活剂，与未刺激的培养物相比，其使孕酮水平增加了12倍。在用促黄体生成素（LH）、FSH或福斯可林刺激48小时后，雌二醇的生成也显著升高（P < 0.005）。在FSH、HCG或福斯可林处理1小时后，细胞内cAMP水平分别升高了1.5倍、10倍和15倍。细胞色素P450侧链裂解酶（SCC）和类固醇生成转录因子SF1/Ad4BP的表达均可通过蛋白质免疫印迹法检测到。然而，仅在FSH和HCG刺激后，P450 SCC的水平才明显升高。在有血清存在的情况下，这些培养细胞的超微结构显示出大量脂滴和发育良好的线粒体，这是高度类固醇生成细胞的特征。这些培养物中凋亡细胞核的比例<30%。去除血清会使凋亡发生率增加到40%，而添加FSH可显著防止细胞死亡（P < 0.01）。HCG和福斯可林使凋亡率增加到约50%，而用8-溴-cAMP处理则导致80%的细胞死亡。我们的数据表明，经过长时间培养后，人颗粒细胞能够恢复对促性腺激素刺激的cAMP和类固醇生成反应。此外，我们的实验表明，凋亡和类固醇生成可以在同一细胞群体中共存，而这些过程之间的相互关系可以由细胞内cAMP水平决定。
