De Felici Massimo, Lobascio Anna Maria, Klinger Francesca Gioia
Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Rome, Italy.
Autophagy. 2008 Feb;4(2):240-2. doi: 10.4161/auto.5410. Epub 2007 Dec 12.
We devised a short-term culture system allowing us to define novel characteristics of programmed cell death (PCD) of fetal oocytes and to underscore new aspects of this process. Mouse fetal oocytes cultured in conditions allowing meiotic progression underwent apoptotic degeneration as revealed by TUNEL staining, DNA ladder, Annexin V binding, PARP cleavage and, usually, caspase activation. TEM observations show, however, recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features are also observed. Moreover, under the fluorescence microscope a subpopulation of TUNEL(+) oocytes appear morphologically healthy and do not show detectable caspase activity. Finally, caspase inhibitors are able to slow down, but not to abolish, oocyte cell death, whereas calpain inhibitor I significantly reduces the number of TUNEL(+) oocytes after 4 days of culture, and rapamycin (mTOR inhibitor) increases such numbers both at day 3 and 4. These observations together with results showing expression in cultured oocytes undergoing cell death of apoptosis inducing factor and Beclin 1, two important players of caspase independent and autophagic cell death, respectively, demonstrate that fetal oocytes possess and are able to activate several players of various forms of cell death. However, causal correlation among different cell death pathways in such oocytes remains to be determined and stimuli causing the activation of these pathways in vitro and in vivo also clarified.
我们设计了一种短期培养系统，使我们能够定义胎儿卵母细胞程序性细胞死亡（PCD）的新特征，并强调这一过程的新方面。在允许减数分裂进程的条件下培养的小鼠胎儿卵母细胞发生凋亡性退化，这通过TUNEL染色、DNA梯状条带、膜联蛋白V结合、PARP裂解以及通常的半胱天冬酶激活得以揭示。然而，透射电镜观察显示，存在反复出现的非典型凋亡形态，其特征是缺乏染色质边缘化和核碎片化；还观察到具有自噬和坏死特征的卵母细胞。此外，在荧光显微镜下，一部分TUNEL（+）卵母细胞在形态上看起来健康，并且未显示出可检测到的半胱天冬酶活性。最后，半胱天冬酶抑制剂能够减缓但不能消除卵母细胞死亡，而钙蛋白酶抑制剂I在培养4天后显著减少TUNEL（+）卵母细胞的数量，雷帕霉素（mTOR抑制剂）在第3天和第4天均增加此类细胞数量。这些观察结果以及显示凋亡诱导因子和Beclin 1在经历细胞死亡的培养卵母细胞中表达的结果，这两个分别是半胱天冬酶非依赖性和自噬性细胞死亡的重要参与者，表明胎儿卵母细胞拥有并能够激活多种形式细胞死亡的多个参与者。然而，此类卵母细胞中不同细胞死亡途径之间的因果关系仍有待确定，并且在体外和体内导致这些途径激活的刺激因素也有待阐明。
