Molecular mechanisms of the induction of spermatogenic cell apoptosis through mitochondria

在哺乳动物精子发生过程中，生殖细胞凋亡是非常普遍的现象。从细胞凋亡中拯救出来的机制似乎是理解生命程序重置的必要条件。由于线粒体衍生的凋亡相关蛋白，Bax和细胞色素C（cytC），在细胞质中的再分配，在生殖细胞凋亡的诱导有牵连，在本研究中，我们调查了线粒体的分布与Bax和cytC在凋亡的成年雄性小鼠生殖细胞的再分配。本研究还尝试建立了电穿孔法基因转染睾丸的方法和睾丸生殖细胞的原代培养条件。当凋亡的生殖细胞中的线粒体及其蛋白质的分布进行了检查，通过免疫电镜，Bax和cytC的重新分布并不伴随着线粒体分布的变化。事实上，在电穿孔转染pEYFP-Mito DNA的睾丸中，YFP的荧光信号与Bax和cytC共定位，而不与SP-22蛋白共定位，SP-22蛋白是线粒体的另一个标志物。这些结果表明，从线粒体衍生的阿尔茨海默病相关蛋白质的重新分布可能是由其锚这些线粒体蛋白质的能力的丧失引发的。此外，我们检查了成年小鼠睾丸分离生殖细胞长期原代培养的各种培养条件，现在可以在体外维持细胞数周，但批次间的总体特征取决于支持细胞饲养层的质量。

In mammalian spermatogenesis, germ cell apoptosis is very common. The mechanism to be rescued from the apoptosis seems to be essential to understand the reset of life program. Since redistribution of mitochondria derived apoptosis-related proteins, Bax and cytochrome C (cytC), in the cytoplasm was implicated in the induction of germ cell apoptosis, in the present study we investigated the distribution of mitochondria in relation with the redistribution of Bax and cytC in apoptotic germ cells in adult male mice. During the study, we also attempted to establish the method of gene transfection by electroporation in to testis in vivo and the conditions of primary culture of testicular germ cells. When the distribution of mitochondria and their proteins in apoptotic germ cells was examined by immuno-electron microscopy, the redistribution of Bax and cytC was not accompanied with a change in mitochondrial distribution. In fact, in the testis transfected with pEYFP-Mito DNA by electroporation, the fluorescence signal of YFP was co-localized with Bax and cytC, but not with SP-22 protein, which is another marker of mitochondria. These results indicate that the redistribution of apoptosis-related proteins derived from mitochondria may be triggered by loss of their ability to anchor those mitochondrial proteins. In addition, we examined various culture conditions for a long term primary culture of isolated germ cells from adult mouse testes and now can maintain the cells for several weeks in vitro, but the overall characteristics were variable among batches depending upon the quality of Sertoli cell feeder layer.