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Molecular mechanisms of the induction of spermatogenic cell apoptosis through mitochondria

通过线粒体诱导生精细胞凋亡的分子机制

基本信息

批准号：
15390058
负责人：
KOJI Takehiko
金额：
$ 9.6万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15390058/
关键词：
spermatogenic cells apoptosis mitochondria redistribution of Bax cytochrome C endocrine disruptor electroporation primary culture Bax TUNEL 遺伝子導入

项目摘要

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.

在哺乳动物精子发生过程中，生殖细胞凋亡是很常见的。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。由于细胞质中线粒体衍生的凋亡相关蛋白Bax和细胞色素C （cytC）的重新分布与生殖细胞凋亡的诱导有关，因此本研究研究了成年雄性小鼠凋亡生殖细胞中线粒体分布与Bax和cytC重新分布的关系。在研究过程中，我们还尝试建立睾丸电穿孔基因转染的方法和睾丸生殖细胞原代培养的条件。免疫电镜观察凋亡生殖细胞中线粒体及其蛋白的分布，发现Bax和cytC的重新分布不伴有线粒体分布的变化。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，而不与另一种线粒体标志物SP-22蛋白共定位。这些结果表明，来自线粒体的凋亡相关蛋白的重新分配可能是由它们锚定这些线粒体蛋白的能力丧失引起的。此外，我们研究了从成年小鼠睾丸中分离的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，但根据Sertoli细胞饲养层的质量，批次之间的总体特征是可变的。