Molecular pathway of germ cell apoptosis following ischemia/reperfusion of the rat testis
Molecular pathway of germ cell apoptosis following ischemia/reperfusion of the rat testis
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DOI：
10.1095/biolreprod63.5.1465
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发表时间：
2000-11-01
影响因子：
3.6
通讯作者：
Turner, TT
中科院分区：
文献类型：
--
作者：
Lysiak, JJ;Turner, SD;Turner, TT
本研究探讨了大鼠睾丸急性缺血后生殖细胞的分子凋亡途径，对大鼠进行缺血诱导的扭转，并在再灌注后收获睾丸。用单链 DNA 抗体鉴定凋亡细胞。通过 RNase 保护测定或逆转录酶聚合酶链式反应 (RT-PCR) 检查生精小管 RNA 中凋亡分子的存在和调节，来自生精小管的蛋白质用于细胞色素 c 的蛋白质印迹分析。扭转修复后24小时生殖细胞凋亡达到最大。生精上皮周期II-III阶段的生殖细胞是主要的早期反应者。 RNase 保护测定表明 Bcl-X-L 是主要的 mRNA 种类。 Caspases 1,2, 3 和 Bar mRNA 持续上调；然而，扭转后上调的时间是可变的。 Bcl-X-L 和 Bcl-X-S mRNA 的上调不太一致，并且没有证据表明 Fas 或 Bcl-2 上调。 RNase保护实验未检测到Fas配体（FasL），但RT-PCR显示扭转修复后4小时FasL表达显着增加。细胞色素 c 释放的蛋白质印迹分析表明，扭转修复后 4 小时显着增加。结果表明，大鼠睾丸缺血/再灌注后的生殖细胞凋亡是通过线粒体相关分子 Bar 以及 Fas-FasL 相互作用启动的。
The present study investigates the molecular apoptotic pathway in germ cells following acute ischemia of the rat testis, Rats were subjected to ischemia-inducing torsion and testes were harvested after reperfusion. Apoptotic cells were identified with an antibody to single-stranded DNA. Seminiferous tubule RNA was examined by RNase protection assay or by reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence and regulation of apoptotic molecules, Proteins from seminiferous tubules were used for Western blot analysis of cytochrome c. Germ cell apoptosis was maximal at 24 h after repair of torsion. Germ cells in stages II-III of the seminiferous epithelium cycle were the predominant early responders. The RNase protection assays revealed that Bcl-X-L was the prominent mRNA species. Caspases 1,2, 3, and Bar mRNA were consistently upregulated; however, the time of upregulation after torsion was variable. The Bcl-X-L and Bcl-X-S mRNAs were less consistently upregulated and there was no evidence for upregulation of Fas or Bcl-2. Fas ligand (FasL) was not detected by RNase protection assay, but RT-PCR revealed a significant increase in FasL expression 4 h after the repair of torsion. Western blot analysis for cytochrome c release demonstrated a significant increase 4 h after the repair of torsion. Results suggest that germ cell apoptosis following ischemia/reperfusion of the rat testis is initiated through the mitochondria-associated molecule Bar as well as Fas-FasL interactions.