Molecular mechanisms of the induction of spermatogenic cell apoptosis through mitochondria

在哺乳动物的精子发生中，生殖细胞的凋亡是非常常见的。从细胞凋亡中解救出来的机制似乎是理解生命程序重置的关键。由于线粒体衍生的凋亡相关蛋白Bax和细胞色素C(CytC)在细胞质中的重新分布与生殖细胞的凋亡有关，本研究探讨了线粒体的分布与Bax和细胞色素C在成年雄性小鼠生殖细胞中的重新分布的关系。在本研究中，我们还试图建立体内睾丸电穿孔基因转移的方法和睾丸生殖细胞原代培养的条件。用免疫电子显微镜观察凋亡生殖细胞内线粒体及其蛋白的分布时，Bax和CytC的重新分布并不伴随线粒体分布的改变。事实上，在电穿孔转导pEYFP-Mito DNA的睾丸中，YFP的荧光信号与Bax和CytC共定位，而与线粒体的另一个标志SP-22蛋白不共定位。这些结果表明，线粒体来源的凋亡相关蛋白的重新分布可能是由于它们失去了锚定线粒体蛋白的能力而触发的。此外，我们还研究了从成年小鼠睾丸分离的生殖细胞长期原代培养的各种培养条件，现在这些细胞可以在体外保持几周，但总体特征在不同批次之间是不同的，这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.