BIOCHEMICAL-IDENTIFICATION OF APOPTOSIS (PROGRAMMED CELL-DEATH) IN GRANULOSA-CELLS - EVIDENCE FOR A POTENTIAL MECHANISM UNDERLYING FOLLICULAR ATRESIA
BIOCHEMICAL-IDENTIFICATION OF APOPTOSIS (PROGRAMMED CELL-DEATH) IN GRANULOSA-CELLS - EVIDENCE FOR A POTENTIAL MECHANISM UNDERLYING FOLLICULAR ATRESIA
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DOI：
10.1210/endo-129-5-2415
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发表时间：
1991-11-01
期刊：
影响因子：
4.8
通讯作者：
GOROSPE, WC
中科院分区：
文献类型：
--
作者：
HUGHES, FM;GOROSPE, WC
在本研究中，我们研究了卵泡闭锁过程中颗粒细胞死亡通过细胞凋亡(程序性细胞死亡)发生的可能性。为了探讨这种可能性，通过注射15IU PMSG诱导未成熟雌性大鼠闭锁。对照组要么接受车辆治疗，要么不接受治疗。注射PMSG后第1~5天处死动物，对照组在注射后第1天或第5天处死动物。通过光镜观察5 mU/m组织切片，并通过定量血清孕酮和雌激素水平来评估闭锁的发生。细胞凋亡的特征是基因组DNA被依赖于Ca~(2+)/Mg~(2+)的内源内切酶切割成寡核小体长度的片段。这些片段在电泳法分离时形成独特的梯形图案。因此，通过琼脂糖凝胶电泳法检测细胞裂解物中DNA片段化的模式来评估颗粒细胞中是否发生了细胞凋亡。凝胶用溴化乙锭染色，DNA用紫外透射法显示，注射PMSG后4d出现最早的闭锁征象，表现为颗粒细胞从基底板变性和脱落。在PMSG治疗后第3天，血清雌激素水平从基础水平上升到对照组的7倍，到第4天下降到对照组的水平，此后下降到对照组的水平。相比之下，孕酮在最初的3天内保持在基础水平，在PMSG治疗后4天和5天分别上升到对照组的3倍和8倍。雌孕激素分泌比例的这种变化是卵泡闭锁的特征。最后，在注射PMSG后的第4天和第5天，颗粒细胞裂解物中的低分子DNA的电泳图显示出明显的梯形条带(以细胞凋亡为特征)。在治疗后第1天和第2天检测不到这种模式。在PMSG处理3天后获得的裂解产物显示出微弱的DNA片段的凋亡样图案；这一结果与其他系统的结果一致，在其他系统中，DNA裂解在任何形态死亡迹象之前就开始了。有趣的是，对照裂解液中DNA片段呈梯形分布，提示未成熟大鼠在正常(与诱导)闭锁条件下颗粒细胞死亡是通过细胞凋亡发生的。这些数据表明，凋亡样事件与颗粒细胞死亡密切相关，从而支持了细胞凋亡参与卵泡闭锁诱导的可能性。
In the present study, we examined the possibility that granulosa cell death during ovarian follicular atresia occurs by apoptosis (programmed cell death). To investigate this possibility, atresia was induced in immature female rats by injecting 15 IU PMSG. Controls received either vehicle or no treatment. PMSG-treated animals were killed on days 1-5 post-injection while controls were killed on days 1 or 5. The onset of atresia was assessed histologically by light microscopic inspection of 5-mu-m tissue sections and functionally by quantification of serum progesterone and estrogen levels. Apoptosis is characterized by the cleavage of genomic DNA into oligonucleosomal length fragments by a Ca2+/Mg2+-dependent endogenous endonuclease. Such fragments form a distinctive ladder pattern when separated electrophoretically. Accordingly, the occurrence of apoptosis in granulosa cells was assessed by examining the pattern of fragmented DNA in cell lysates after agarose gel electrophoresis. Gels were stained with ethidium bromide and DNA visualized by UV transillumination.The earliest morphological signs of atresia were detected 4 days after PMSG injection as evidenced by degeneration and detachment of granulosa cells from the basal lamina. Serum estrogen increased from basal to levels 7-fold over controls by day 3 after PMSG treatment, falling to control values by day 4 and thereafter. In contrast, progesterone remained basal for the first 3 days, rising to levels 3-fold and 8-fold above controls 4 and 5 days after PMSG treatment, respectively. Such shifts in the ratio of estrogen to progesterone production are known to be characteristic of follicular atresia. Finally, electrophoretic analysis of low mol wt DNA in granulosa cell lysates revealed a definitive ladder pattern of oligonucleosomal length DNA fragments (characteristic of apoptosis) on days 4 and 5 after PMSG injection. This pattern was not detectable on days 1 and 2 after treatment. Lysates obtained 3 days after PMSG treatment showed a faint apoptotic-like pattern of DNA fragments; a result consistent with other systems in which DNA cleavage begins before any morphological signs of death. Interestingly, a ladder pattern of DNA fragments was present in control lysates suggesting that granulosa cell death under normal (vs. induced) conditions of atresia in immature rats occurs by apoptosis.These data demonstrate an intimate association between apoptotic-like events and dying granulosa cells and thus support the possibility that apoptosis is involved in the induction of follicular atresia.