5-脱氧杂氮胞苷抑制小鼠附植前的胚胎发育(英文)

The Demethylating Agent 5-Aza-2′-Deoxycytidine (5-AZA-CdR) Inhibits The Development of Preimplantation Mouse Embryos

DNA甲基化在哺乳动物发育过程中有关键作用.在小鼠附植前胚胎发育过程中,DNA甲基化一直处于动态变化过程中.通过将体外受精胚在5-AZA-CdR中持续培养,研究5-AZA-CdR对小鼠附植前胚胎发育的影响,为附植前胚胎发育机理的研究及5-AZA-CdR的毒副作用研究提供试验基础.从原核期加入不同浓度的5-AZA-CdR时,胚胎不能发育到桑椹胚(0.2和1.0μmol/L)和4-细胞胚(5.0μmol/L);从2-细胞期加入时,胚胎阻滞于未致密化的8-细胞(0.2和1.0μmol/L)和3/4-细胞期(5.0μmol/L);而当从4-细胞加入时,虽然胚胎能够发育到早期桑椹胚,但发育比例同对照相比显著降低(P<0.05).进一步检测凋亡、基因组DNA甲基化和整体转录活性,结果显示,高浓度的5-AZA-CdR导致8-细胞和早期桑椹胚发生早期凋亡,而低浓度的5-AZA-CdR引起8-细胞和早期桑椹胚基因组DNA甲基化的降低和转录活性的降低,并且这种降低呈浓度依赖性.所以加入低浓度的5-AZA-CdR时,胚胎的DNA甲基化降低,引起转录活性的降低,进而导致胚胎发育的停滞.

DNA methylation is crucial for mammalian development, and DNA methylation is always in the dynamic status during preimplantation mouse embryos development. The effects of 5-AZA-CdR on the development ofpreimplantation mouse embryos were evaluated. Preimplantation mouse embryos created by in vitro fertilization were cultured continuously in 5-AZA-CdR （0.2, 1.0, or 5.0 μmol/L）. Fertilized oocytes exposed to CZB containing 5-AZA-CdR at the pronuclear stage were unable to form morulae （0.2 and 1.0 μmol/L） or 4-cell embryos （5.0 μmol/L）, while 2-cell stage embryos exposed to 5-AZA-CdR developed into uncompacted 8-cell （0.2 and 1.0 μmol/L） or 3/4-cell （5.0 μmol/L） stage embryos. The rate of morula formation was significantly lower in 4-ceU embryos cultured in 5-AZA-CdR （1.0 or 5.0 μmol/L） than that in control embryos （P 〈 0.05）. These data indicate that 5-AZA-CdR inhibits the development of mouse preimplantation embryos. Apoptosis, DNA methylation, and transcriptional activity were analyzed to determine the reason for these developmental defects. An annexin V-PI assay revealed that high doses of 5-AZA-CdR led to apoptosis. Compared to the controls, DNA methylation was significantly reduced in uncompacted 8-cell embryos and morulae （ P 〈 0.05） in a dosedependent manner, whereas no significant change was detected in 2- or 4-cell embryos （ P 〉 0.05）. The observed changes in transcriptional activity, determined by measuring the incorporation of BrUTP, were similar to the observed alterations in DNA methylation. Therefore, the developmental defects induced by 5-AZA-CdR appear to be mediated by alterations in DNA methylation and transcriptional activity in preimplantation mouse embryos.