小鼠排卵后卵子老化过程中及ICSI囊胚的甲基化印迹研究

发布时间：2018-06-05 02:51

本文选题：小鼠 + 卵子　；参考：《广西大学》2008年博士论文

【摘要】： 基因组印迹在调节胎儿生长、发育、胎盘功能和出生后的行为中发挥着重要的作用。一旦甲基化印迹建立起来,它们甚至在受精之后发生表观遗传重编程过程中当基因组DNA发生整体去甲基化的时候都会得到维持,从而使得亲本特异性甲基化印迹可以在基因组重编程过程中发挥重要的作用,这也是决定发育潜能的一个重要因素。正常的印迹一旦被破坏,将会导致发育异常和印迹缺陷相关的疾病。排卵后卵子老化和ICSI操作都有可能干预正常的甲基化印迹。我们进行下面的试验探讨这些问题。试验一:延长排卵后卵子停留在输卵管或者是体外培养到相对长的时间而不进行受精的情况下会导致卵子的老化。这些已老化的卵子受精后会影响着床前和着床后胚胎/胎儿的发育。本研究中,我们使用重亚硫酸盐测序法和COBRA法检测两个母本印迹基因,Snrpn和Peg1/Mest甲基化差异区在排卵后卵子的体内和体外老化过程中的甲基化状况。结果显示,卵子排放后在体内和裸卵在体外培养至注射HCG之后29 h,Snrpn的差异甲基化区域发生了去甲基化。然而,Peg1/Mest无论在体内或是体外的环境下从HCG注射之后21-29 h都没有发生去甲基化。这些数据表明,Snrpn差异甲基化区域在小鼠排卵后卵子的老化过程中会发生时间依赖性的去甲基化。试验二:胞质内单精子注射(ICSI)在十九世纪九十年代初首次获得成功之后,一直被广泛应用于治疗男性不育症。但是人们对包括IVF和ICSI在内的辅助生殖技术(ART)的安全性仍存有顾虑,尤其是近来有报导指出ART可能与基因组印迹缺陷有关。然而,目前我们还不了解ICSI对囊胚的印迹基因甲基化是否会造成影响。为了评价ICSI是否影响囊胚印迹基因的正常甲基化,我们使用基因组测序法检测了来自体内囊胚,体内受精-体外培养囊胚,体内MII卵子ICSI囊胚以及IVM-ICSI囊胚H19差异甲基化区域的DNA甲基化情况。结果显示,体内囊胚“忠实”遗传了配子的甲基化模式;KSOM培养液没有造成囊胚H19甲基化异常;ICSI体内MII卵子所得囊胚发生H19甲基化的丢失和甲基化模式的异常;ICSI联合IVM同样导致了H19甲基化的丢失和甲基化模式的异常。与以往使用多囊胚进行的研究相比较,我们的研究强调了个体之间存在的高度差异,这对临床来说更具有实用意义。从我们的实验结果可以看出,外界环境或者是ART的操作都会导致配子和胚胎印迹基因甲基化的异常,临床上有必要充分考虑这些问题。
[Abstract]:Genomic imprinting plays an important role in regulating fetal growth, development, placental function and postnatal behavior. Once methylated imprinting is established, they are maintained even during epigenetic reprogramming after fertilization, when global demethylation of genomic DNA occurs. Therefore, parental specific methylation imprinting can play an important role in genome reprogramming, which is also an important determinant of developmental potential. Normal imprinting, once destroyed, can lead to dysplasia and imprinting related diseases. Egg aging after ovulation and ICSI manipulation may interfere with normal methylation imprinting. We are going through the following experiments to explore these problems. Experiment 1: prolonged ovulation after the egg stays in the fallopian tube or in vitro culture for a relatively long time without fertilization will lead to egg aging. Fertilization of these aged eggs affects the development of preimplantation and postimplantation embryos / fetuses. In this study, we used the method of bisulfite sequencing and COBRA to detect the methylation of two female imprinted genes, SNPN and Peg1/Mest, during the in vivo and in vitro aging of ovulation eggs. The results showed that demethylation occurred in the differential methylation region of Snrpn 29 h after HCG injection in vivo and naked eggs. However, Peggy / Mest had no demethylation 21-29 h after injection of HCG in vivo or in vitro. These data suggest that the differential methylation region of Snrpn occurs time-dependent demethylation during the aging of mouse oocytes after ovulation. Experiment 2: ICSII has been widely used in the treatment of male infertility since its first success in the early 1890s. But there are still concerns about the safety of assistive reproductive technologies, including IVF and ICSI, especially recent reports that ART may be related to genomic imprinting defects. However, we do not know whether ICSI affects blastocyst imprinting gene methylation. To evaluate whether ICSI affects the normal methylation of blastocyst imprinted genes, we used genomic sequencing to detect blastocysts from in vivo, fertilization and in vitro culture of blastocysts. In vivo DNA methylation of ICSI blastocyst and IVM-ICSI blastocyst H 19 in MII eggs. The results show that In vivo blastocyst "faithfully" inherited the methylation pattern of gametes. KSOM culture medium did not cause abnormal H19 methylation of blastocyst. Loss of H19 methylation and abnormal methylation pattern of blastocyst derived from MII eggs in ICSI The loss of H 19 methylation and the abnormality of methylation pattern were induced. Compared with previous studies using polyblastocysts, our study emphasizes the high level of individual differences, which is more practical for clinical use. From our experimental results, we can see that the outside environment or the operation of ART can lead to abnormal methylation of gametes and embryo imprinting genes, and it is necessary to consider these problems in clinic.
【学位授予单位】：广西大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R321

【引证文献】