Expression of Ad4-BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long-term cultures of human granulosa cells
Expression of Ad4-BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long-term cultures of human granulosa cells
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DOI：
10.1093/molehr/2.6.391
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发表时间：
1996-06-01
影响因子：
4
通讯作者：
Amsterdam, A.
中科院分区：
文献类型：
--
作者：
Breckwoldt, M.;Selvaraj, N.;Amsterdam, A.
从接受体外受精（IVF）的妇女身上获得的新分离的颗粒细胞由于先前的激素治疗，在培养中对进一步的促性腺激素刺激变得难以抵抗。然而，在不含促性腺激素的培养基中培养7天后，它们对人绒毛膜促性腺激素（HCG）和促卵泡激素（FSH）的反应又恢复了，黄体酮的产量分别增加了10倍和5倍。福斯克林是腺苷酸环化酶的直接激活剂，与未受刺激的培养物相比，孕酮水平增加了12倍。黄体生成素（LH）、卵泡刺激素（FSH）或福斯克林刺激48小时后，雌二醇的形成也显著升高（P < 0.005）。FSH、HCG或福斯克林处理1小时后，细胞内cAMP水平分别升高1.5倍、10倍和15倍。细胞色素P450侧链切割酶（SCC）和甾体生成转录因子SF1/Ad4BP均可通过Western blotting表达。然而，在FSH和HCG刺激后，P450 SCC的升高是明显的。在血清存在的情况下，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。细胞凋亡核比例均< 30%。去除血清可使细胞凋亡发生率增加40%，而添加FSH可显著阻止细胞死亡（P < 0.01）。HCG和forskolin可使凋亡增加50%，而8Br-cAMP可使80%的细胞死亡。我们的数据表明，经过长时间的培养，人颗粒细胞可以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，凋亡和甾体生成可以共存于同一细胞群中，而这些过程之间的相互关系可以由细胞内cAMP水平决定。
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.