去SUMO化蛋白酶SENP7对小鼠卵子减数分裂和胚胎发育的调控及其机制研究

发布时间：2018-06-08 00:27

本文选题：SENP7 + 减数分裂恢复　；参考：《华中农业大学》2017年博士论文

【摘要】：随着社会进步,人类生殖障碍已成为全世界令人堪忧的医学和社会学难题。其中一个重要因素就是由各种已知或未知原因导致的女性卵子数量和/或质量恶化。恶化的卵子往往无法完成减数分裂或在减数分裂过程中染色体更易发生异常分离产生非整倍体配子,进而导致女性不孕、流产和新生儿先天缺陷。因此,从分子水平阐明调控卵子减数分裂和胚胎发育的机制对于提高和改善人类生殖能力具有重要的医学和社会学意义。SENP7是一种去SUMO化蛋白酶,作用于多聚SUMO链的剪切,从而维持SUMO化修饰的动态平衡。体细胞中,SENP7参与调控染色质稳态和DNA损伤修复。然而,SENP7在哺乳动物卵子和胚胎发育过程中的作用仍有待研究。本研究以小鼠为模型,通过敲低卵子和早期胚胎中内源性SENP7的表达以研究其在哺乳动物卵子成熟和早期胚胎发育过程中的作用及其分子机制。主要研究结果如下:(1)敲低GV期(减数第一次分裂前期)卵子内SENP7的表达后卵子减数分裂进程受到明显抑制。表现为卵子恢复减数分裂(GVBD)和第一极体排放(PBE)受阻,导致成熟卵子的产生显著减少;(2)敲低SENP7表达后,组蛋白H3K9和H3K27表观修饰状态的改变(乙酰化上调和三甲基化下调),以及Cdc14B/C表达水平上调伴随着的CyclinB1和CyclinB2降解导致卵子恢复减数分裂受阻以及卵子内纺锤体组装异常;(3)γ-tubulin定位紊乱以及泛素化修饰介导的γ-tubulin表达量下调也直接引起SENP7缺失型卵子内纺锤体组装异常;(4)纺锤体组装异常使恢复减数分裂的SENP7缺失型卵子后续发育受阻;同时持续活化的纺锤体组装检验点将这些卵子的发育进程阻滞在MI期之前,导致这些卵子无法进入AI期,进而无法排出第一极体;(5)敲低受精卵内SENP7表达后,胚胎发育同样出现明显缺陷。表现为胚胎卵裂受阻,大部分胚胎甚至无法发育到2细胞时期,导致囊胚发育率趋于零水平。同时,伴随着时间的延长,胚胎会发生凋亡;(6)SENP7缺失型胚胎内组蛋白H3K9和H3K27表观修饰的紊乱(乙酰化水平上调和三甲基化水平下调)以及DNA 5hmC修饰水平的下调导致DNA损伤位点邻近的染色质无法维持DNA修复所需的理想空间结构;HP1α在常染色质上的分布导致DNA修复蛋白Rad51C的表达量下调;胚胎有丝分裂过程中纺锤体组装同样出现异常。这些因素共同导致缺失母源性SENP7的胚胎无法修复其基因组中存在的DNA损伤,因而胚胎发育出现缺陷甚至胚胎发生凋亡;综上,本研究揭示了母源性SENP7在哺乳动物卵子和胚胎发育过程中的关键作用及其分子机制。另外,本研究将SUMO化修饰与染色质上的其他修饰在生殖生物学的一些事件中联系起来,有助于认识生殖发育过程中蛋白质水平的复杂调控网络,为提高动物繁殖和人类生殖健康提供重要理论依据。
[Abstract]:With the progress of society, human reproductive disorders have become a serious medical and sociological problem all over the world. An important factor is the deterioration in the number and / or quality of female eggs due to various known or unknown causes. Worsening eggs often fail to complete meiosis or are more prone to abnormal segregation of chromosomes during meiosis to produce aneuploidy gamete, leading to female infertility, abortion and birth defects. Therefore, elucidating the mechanism of regulating oocyte meiosis and embryonic development at the molecular level is of great medical and sociological significance for improving human reproductive ability. SENP7 is a demo protease that acts on the shearing of polymeric sumo chains. Thus maintaining the dynamic equilibrium of SUMO-modification. SENP7 in somatic cells is involved in the regulation of chromatin homeostasis and DNA damage repair. However, the role of SENP7 in the development of mammalian eggs and embryos remains to be studied. In this study, the mouse model was used to study the role and molecular mechanism of endogenous SENP7 in mammalian egg maturation and early embryonic development by knocking down the expression of endogenous SENP7 in oocytes and early embryos. The main results were as follows: 1) the expression of SENP7 in GV stage (premeiesis) was significantly inhibited after the first meiosis was knocked down. The results showed that GVBDD and PBE of the first polar body were blocked, which resulted in a significant decrease in the production of mature eggs and reduced the expression of SENP7. Changes in the apparent modification of histone H3K9 and H3K27 (upregulation of acetylation and downregulation of trimethylation, and up-regulation of CDc14B / C expression) accompanied by degradation of Cyclin B1 and Cyclin B2, resulting in delayed recovery of meiosis and abnormal assembly of egg spindle The abnormal localization of 纬 -tubulin and the down-regulation of 纬 -tubulin expression mediated by Ubiquitin modification also caused the abnormal spindle assembly of SENP7 oocytes. The abnormal spindle assembly caused the subsequent development of SENP7 deletional eggs which resumed meiosis was blocked. 3) the abnormal localization of 纬 -tubulin and the down-regulation of 纬 -tubulin expression mediated by Ubiquitin modification were also directly related to the abnormal spindle assembly of SENP7 deletions. At the same time, the continuously activated spindle assembly test point blocked the development of these eggs before MI stage, leading to the inability of these eggs to enter AI stage, and thus to expel the first polar body, 5) to lower the expression of SENP7 in fertilized eggs. The development of embryos also showed obvious defects. The results showed that the cleavage of embryos was blocked, and most embryos could not even reach 2 cell stage, which led to the development rate of blastocyst reaching zero level. At the same time, with the extension of time, Apoptosis occurs in embryos with the loss of histone H3K9 and H3K27 (up-regulation of acetylation and down-regulation of trimethylation) and down-regulation of 5hmC modification of DNA leading to the absence of chromatin in the vicinity of DNA damage sites. The distribution of HP1 伪 in chromatin resulted in the down-regulation of the expression of DNA repair protein Rad51C. The spindle assembly is also abnormal during the process of embryonic mitosis. These factors together lead to the failure of embryos that lack maternal SENP7 to repair DNA damage in their genomes, resulting in defects in embryonic development and even embryo apoptosis. This study revealed the key role of maternal SENP7 in the development of mammalian eggs and embryos and its molecular mechanism. In addition, SUMO-modification is associated with other chromatin modifications in reproductive biology events, which is helpful to understand the complex regulatory network of protein level during reproductive development. It provides important theoretical basis for improving animal reproduction and human reproductive health.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S814

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