Fujino Ryu-Suke, Ishikawa Yu-Ichiro, Tanaka Kiyoko, Kanatsu-Shinohara Mito, Tamura Kazuhiro, Kogo Hiroshi, Shinohara Takashi, Hara Takahiko
Stem cell project group, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Tokyo, Japan.
Mol Reprod Dev. 2006 Aug;73(8):955-66. doi: 10.1002/mrd.20504.
We recently established a technique to expand male germ line stem (GS) cells in long-term culture without losing their spermatogenic capacity. To gain insight into the genetic program of these cells, we compared the mRNA expression profile of GS cells with that of embryonic stem (ES) cells using DNA microarrays. We found 79 genes that were upregulated in GS cells compared to ES cells, including synaptonemal complex protein-1, deleted in azoospermia-like, ubiquitin-conjugating enzyme E2B, and ubiquitin carboxy-terminal hydrolase L1, all of which are functionally important for spermatogenesis. In addition, we identified a cDNA encoding the mouse ortholog of capillary morphogenesis gene (CMG)-1. CMG-1 transcripts were predominantly produced in spermatogonia and spermatocytes in mouse testis. When CMG-1 expression was attenuated in a mouse spermatocyte-derived cell line, GC-2spd(ts), by a target-specific short interfering RNA, the morphology of the cells was changed and the expression of cyclin D2 was abrogated. A reporter assay using a genomic region upstream of the mouse cyclin D2 gene revealed that this downmodulation occurs at the transcriptional level. We detected FLAG-tagged CMG-1 protein in the nuclei of transfected COS7 cells, suggesting that CMG-1 may play a unique role in the transcriptional regulation of the cyclin D2 gene. The upregulated GS genes identified in this study will provide useful information for the future investigation of spermatogonial stem cells and the early phase of male germ cell differentiation.
我们最近建立了一种技术，可在长期培养中扩增雄性生殖系干细胞（GS细胞），同时不丧失其生精能力。为深入了解这些细胞的遗传程序，我们使用DNA微阵列比较了GS细胞与胚胎干细胞（ES细胞）的mRNA表达谱。我们发现与ES细胞相比，GS细胞中有79个基因上调，包括联会复合体蛋白-1、无精子症样缺失基因、泛素结合酶E2B和泛素羧基末端水解酶L1，所有这些基因在精子发生中都具有重要功能。此外，我们鉴定出一个编码小鼠毛细血管形态发生基因（CMG）-1直系同源物的cDNA。CMG-1转录本主要在小鼠睾丸的精原细胞和精母细胞中产生。当通过靶向特异性小干扰RNA使小鼠精母细胞衍生的细胞系GC-2spd(ts)中的CMG-1表达减弱时，细胞形态发生改变，细胞周期蛋白D2的表达被消除。使用小鼠细胞周期蛋白D2基因上游基因组区域进行的报告基因检测表明，这种下调发生在转录水平。我们在转染的COS7细胞核中检测到FLAG标签的CMG-1蛋白，这表明CMG-1可能在细胞周期蛋白D2基因的转录调控中发挥独特作用。本研究中鉴定出的上调的GS基因将为未来精原干细胞和雄性生殖细胞分化早期阶段的研究提供有用信息。
