Foghi A, Teerds K J, van der Donk H, Moore N C, Dorrington J
Banting and Best Department of Medical Research, University of Toronto, Canada.
J Endocrinol. 1998 Jun;157(3):489-94. doi: 10.1677/joe.0.1570489.
Follicular atresia is characterized by the initial rapid loss of granulosa cells by apoptosis, followed by the loss of thecal cells at a slower rate. We have previously shown that treatment of subconfluent cultures of thecal/interstitial cells (T/I) with transforming growth factor (TGF) alpha plus TGF beta caused chromatin condensation and internucleosomal fragmentation characteristic of apoptosis, whereas in the presence of either TGF alpha or TGF beta alone the cells remained healthy. In this study we have examined the effect of TGF alpha and TGF beta alone and in combination on the levels of mRNA encoding bcl-2 and interleukin-1 beta-converting enzyme (ICE) in T/I cells using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Bcl-2, a cell survival gene, has been implicated in regulating the balance between cell proliferation and cell death in physiological processes. ICE, the homolog of the C. elegans cell death gene, ced-3, is also involved in apoptotic signal transduction. The levels of mRNA encoding specific PCR products for bcl-2 (430 bp) and ICE (453 bp) were amplified from T/I cell cDNA. Untreated T/I cells and TGF alpha- or TGF beta-treated cells contained comparable levels of bcl-2 mRNA. Treatment of T/I cells with TGF alpha plus TGF beta significantly decreased the levels of bcl-2 mRNA expression. TGF alpha plus TGF beta caused a significant decrease in bcl-2 mRNA levels within 3 h of treatment of T/I cells, followed by a progressive decline to 10% of control levels after 24 h of treatment. In contrast, in control T/I cells, the levels of ICE mRNA were low. TGF alpha plus TGF beta caused a progressive increase in ICE mRNA, reaching levels 2- and 3-fold higher than control cells after 5 and 7 h respectively. DNA analysis showed that DNA fragmentation, indicative of apoptosis, occurred after 10 h of treatment with TGF alpha plus TGF beta. These studies demonstrated that treatment of T/I cells with TGF alpha plus TGF beta influenced gene expression of bcl-2 and ICE prior to the time at which DNA fragmentation was observed. We propose that the gene products of bcl-2 and ICE are involved in the apoptotic signal transduction pathway induced by TGF alpha plus TGF beta in T/I cells.
卵泡闭锁的特征是颗粒细胞最初通过凋亡迅速丢失，随后卵泡膜细胞以较慢的速率丢失。我们之前已经表明，用转化生长因子（TGF）α加TGFβ处理卵泡膜/间质细胞（T/I）的亚汇合培养物会导致染色质浓缩和凋亡特有的核小体间断裂，而单独存在TGFα或TGFβ时细胞保持健康。在本研究中，我们使用半定量逆转录 - 聚合酶链反应（RT-PCR）分析检测了单独及联合使用TGFα和TGFβ对T/I细胞中编码bcl-2和白细胞介素-1β转换酶（ICE）的mRNA水平的影响。Bcl-2是一种细胞存活基因，已被证明在生理过程中调节细胞增殖和细胞死亡之间的平衡。ICE是秀丽隐杆线虫细胞死亡基因ced-3的同源物，也参与凋亡信号转导。从T/I细胞cDNA中扩增出编码bcl-2（430 bp）和ICE（453 bp）特异性PCR产物的mRNA水平。未处理的T/I细胞以及经TGFα或TGFβ处理的细胞含有相当水平的bcl-2 mRNA。用TGFα加TGFβ处理T/I细胞会显著降低bcl-2 mRNA表达水平。TGFα加TGFβ在处理T/I细胞3小时内导致bcl-2 mRNA水平显著下降，随后在处理24小时后逐渐降至对照水平的10%。相反，在对照T/I细胞中，ICE mRNA水平较低。TGFα加TGFβ导致ICE mRNA逐渐增加，分别在5小时和7小时后达到比对照细胞高2倍和3倍的水平。DNA分析表明，在用TGFα加TGFβ处理10小时后出现了指示凋亡的DNA片段化。这些研究表明，在用TGFα加TGFβ处理T/I细胞后，在观察到DNA片段化之前就影响了bcl-2和ICE的基因表达。我们提出，bcl-2和ICE的基因产物参与了TGFα加TGFβ在T/I细胞中诱导的凋亡信号转导途径。
