Kamimura Satoshi, Hatanaka Yuki, Hirasawa Ryutaro, Matsumoto Kazuya, Oikawa Mami, Lee Jiyoung, Matoba Shogo, Mizutani Eiji, Ogonuki Narumi, Inoue Kimiko, Kohda Takashi, Ishino Fumitoshi, Ogura Atsuo
RIKEN BioResource Center, Tsukuba, Ibaraki, Japan Graduate School of Life and Environmental Science, University of Tsukuba, Ibaraki, Japan.
RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
Biol Reprod. 2014 Nov;91(5):120. doi: 10.1095/biolreprod.114.120451. Epub 2014 Sep 17.
In mice, the establishment of paternal genomic imprinting in male germ cells starts at midgestation, as suggested by DNA methylation analyses of differentially methylated regions (DMRs). However, this information is based on averages from mixed populations of germ cells, and the DNA methylation pattern might not always provide a full representation of imprinting status. To obtain more detailed information on the establishment of paternal imprinting, single prospermatogonia at Embryonic Days 15.5 (E15.5), E16.5, and E17.5 and at Day 0.5 after birth were cloned using nuclear transfer; previous reports suggested that cloned embryos reflected the donor's genomic imprinting status. Then, the resultant fetuses (E9.5) were analyzed for the DNA methylation pattern of three paternal DMRs (IG-DMR, H19 DMR, and Rasgrf1 DMR) and the expression pattern of imprinted genes therein. The overall data indicated that establishment of genomic imprinting in all paternally imprinted regions was completed by E17.5, following a short intermediate period at E16.5. Furthermore, comparison between the methylation status of DMRs and the expression profiles of imprinted genes suggested that methylation of the IG-DMR, but not the H19 DMR, solely governed the control of its imprinted gene cluster. The Rasgrf1 DMR seemed to be imprinted later than the other two genes. We also found that the methylation status of the Gtl2 DMR, the secondary DMR that acquires DNA methylation after fertilization, was likely to follow the methylation status of the upstream IG-DMR. Thus, the systematic analyses of prospermatogonium-derived embryos provided additional important information on the establishment of paternal imprinting.
在小鼠中，正如对差异甲基化区域（DMRs）进行的DNA甲基化分析所表明的那样，雄性生殖细胞中父本基因组印记的建立始于妊娠中期。然而，该信息基于生殖细胞混合群体的平均值，并且DNA甲基化模式可能并不总是能完全代表印记状态。为了获得关于父本印记建立的更详细信息，利用核移植技术克隆了胚胎第15.5天（E15.5）、E16.5、E17.5以及出生后第0.5天的单个精原细胞；先前的报告表明克隆胚胎反映了供体的基因组印记状态。然后，对由此产生的胎儿（E9.5）进行了三个父本DMR（IG-DMR、H19 DMR和Rasgrf1 DMR）的DNA甲基化模式以及其中印记基因表达模式的分析。总体数据表明，所有父本印记区域的基因组印记建立在E16.5的短暂中间期之后于E17.5完成。此外，DMRs的甲基化状态与印记基因表达谱之间的比较表明，IG-DMR的甲基化而非H19 DMR的甲基化单独控制其印记基因簇。Rasgrf1 DMR似乎比其他两个基因的印记形成得更晚。我们还发现，受精后获得DNA甲基化的二级DMR即Gtl2 DMR的甲基化状态可能遵循上游IG-DMR的甲基化状态。因此，对源自精原细胞的胚胎进行的系统分析为父本印记的建立提供了额外的重要信息。
