【作者】 王慧利；

【导师】 谭景和；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 生发泡期卵母细胞染色质的一个共同特点是:随卵泡生长,弥散在整个细胞核区域中的染色质逐渐发生凝集,依物种不同,或在核仁周围凝集成环或形成其它凝集结构。先前有关卵母细胞染色质的研究主要集中在不同构型与减数分裂及发育能力的关系方面,对参与染色质凝集的分子及信号通路还知之不多。最近的研究表明后成性修饰,包括DNA甲基化及组蛋白修饰在调控染色质结构及基因表达过程中发挥了重要作用。本文以小鼠卵母细胞为实验模型,利用免疫荧光技术及相关特异性抑制剂,系统研究了DNA甲基化、组蛋白磷酸化和乙酰化在卵母细胞GV期染色质及MII期染色体凝集过程中的作用。结果发现:1、MPF及MAPK抑制剂处理后,卵母细胞GV期染色质凝集正常发生,表明MPF与MAPK均不参与染色质凝集2、卵母细胞GV期染色质与MII期染色体凝集均伴随DNA甲基化水平升高,但甲基化特异性抑制剂5-aza-cytidine处理后,染色质与染色体凝集仍然发生,表明DNA甲基化不是卵母细胞染色质及染色体凝集所必需3、卵母细胞GV期染色质与MII期染色体凝集均伴随组蛋白磷酸化水平升高,但磷酸化特异性抑制剂ZM447439处理后,染色质与染色体凝集仍然发生,表明组蛋白磷酸化不是卵母细胞染色质及染色体凝集所必需4、卵母细胞GV期染色质凝集伴随着组蛋白乙酰化水平升高,但HDACs抑制剂TSA处理后,GV染色质不再继续凝集但乙酰化水平仍然升高,表明HDACs参与GV期染色质凝集,但并非通过组蛋白乙酰化/去乙酰化途径5、卵母细胞MII期染色体凝集伴随组蛋白去乙酰化,TSA处理后,组蛋白保持乙酰化状态,但染色体凝集正常发生,表明组蛋白去乙酰化不是染色体凝集所必需6、GV期及MII期卵母细胞中,HATs/HDACs保持动态平衡,协同调节组蛋白的乙酰化/去乙酰化状态受核胞质制备是核移植过程中的重要环节,作为受核胞质的卵母细胞必须除去自身遗传物质,此过程通常称之为去核。去核不完全往往会使克隆胚染色体出现多倍性或非整倍性现象,直接导致克隆胚胎发育失败。化学辅助去核是指利用药物处理卵母细胞使之形成含核物质的突起,再通过显微操作去除突起而去核。这种去核方式因其操作简便、对卵母细胞发育能力损伤较小而得到广泛应用。目前所用化学辅助去核药物一般为微管抑制剂,通过破坏纺锤体形成进而扰乱染色体分离,从而实现辅助去核。咖啡因和MG132通常用于升高MPF活性,尚未见将这两种药物应用于卵母细胞辅助去核的报道。本文首先研究了成熟培养液中添加半胱胺和胱氨酸对山羊卵母细胞孤雌胚胎及核移植胚胎发育的促进作用,然后利用此优化的成熟培养系统,首次将咖啡因和MG132这两种非微管抑制剂应用于山羊卵母细胞的化学辅助去核,结果发现:1、成熟培养液中添加100μM半胱胺和200μM胱氨酸可促进山羊孤雌胚胎的体外发育2、成熟培养液中添加100μM半胱胺和200μM胱氨酸可促进山羊体细胞核移植胚胎的体外发育3、1mM的Caffeine和5μM的MG132处理30分钟可诱导80%山羊MII期卵母细胞产生胞质突起4、与Demecolcine相比,Caffeine和MG132处理后不破坏山羊MII期卵母细胞的纺锤体结构5、Caffeine和MG132处理诱导胞质突起后不损害山羊孤雌胚胎的体外发育能力6、与盲吸去核相比,Caffeine和MG132诱导突起后去核率较高,并能提高核移植胚胎的体外发育能力7、Caffeine处理诱导产生的胞质突起的持续时间长于MG132处理,与Demecolcine处理相当8、Caffeine辅助去核的体细胞核移植胚胎可发育到期更多还原

【Abstract】 A common feature in the configuration of germinal vesicle (GV) chromatin in most species was that diffuse chromatin condenses into perinucleolar rings or other corresponding condensed configurations with or without the perinucleolar rings, depending on species. Previous investigations focused on the correlation between different chromatin configuration and the acquisition of meiotic as well as developmental competence of oocytes, little was known concerning the cellular and signaling pathways regulating chromatin condensation.Recent studies have revealed that epigenetic modifications,such as DNA methylation and histone modifications,played important roles in the regulation of chromatin structure and gene expression. In the present study, using immunofluorescence and distinct specific inhibitors, we conducted a comprehensive analysis of the DNA methylation,histone phosphorylation and acetylation during chromatin and chromosome condensation in mouse oocytes. Results as follows:1.GV oocytes treated with inhibitors of MPF and MAPK underwent chromatin condensation, suggested that chromatin condensation was not correlated with MPF and MAPK activities.2. GV chromatin and MII chromosome condensation were associated with increased levels of DNA methylation, however, condensations remained occurred after treating oocytes with 5-aza-cytidine, a specific inhibitor of DNA methylation. These results indicated that DNA methylation was not required for chromatin and chromosome condensation.3. GV chromatin and MII chromosome condensation were associated with increased levels of histone phosphorylation, however, condensations remained occurred after treating oocytes with ZM447439, a specific inhibitor of histone phosphorylation. These results indicated that histone phosphorylation was not required for chromatin and chromosome condensation.4. GV chromatin condensation was associated with increased levels of histone acetylation, however,chromatin condensation did not occur while levels of acetylation remained increased after treatment with TSA,a inhibitor of HDACs. These evidences provided for a role of HDACs in the control of GV chromatin condensation,but not take place via histone acetylation/deacetylation-dependent mechanism.5. MII chromosome condensation was associated with histone deacetylation, chromatin condensation remained continued while levels of acetylation increased after treatment with TSA. These results indicated that histone deacetylation was not required for chromosome condensation.6. Dynamic equilibrium between HATs and HDACs activitie were presented in GV and MII oocytes, by which, to regulate the acetylation/deacetylation state. The preparation of recipient cytoplasm was one of the key steps of somatic cell nuclear transfer,the removal of chromosomes from recipient oocytes was named enucleation. Improper measures used in enucleation resulted in problems as aneuploidy or polyploidy with subsequent detrimental effects on development.Chemically assisted enucleation was an approach of choice, oocytes were exposed to the drug, and as a result of treatment, a cortical protrusion formed on the surface of the oocyte containing all the oocyte chromosomes, this protrusion can be easily removed by micromanipulation techniques.Chemically assisted enucleation seemed to be a very attractive procedure that could simplify conventional enucleation methods with a minimal decrease of the cytoplasmic volume and without reducing the viability of the resulting cytoplast. Chemically assisted enucleation previously reported mainly focused on anti-microtubule drugs.Exposured to these drugs impaired spindle rotation, altered chromosome migration, and thereby, resulted in the generation of enucleated oocytes.Caffeine and MG132 were used to increase MPF activity, however,no study about these drugs has been reported in chemically assisted enucleation procedure. In the present study, effects of supplementation of cysteamine and cystine on the developmental capacity for parthenogenetic and SCNT embryos were investigated, and then, using the improved system of IVM, the protocol for caffeine and MG132 assisted enucleation of goat oocytes was first applied. Results as follows:1. Simultaneous supplementation of 100μM cysteamine and 200μM cystine to maturation medium improved parthenogenetic blastulation of goat oocytes.2. Simultaneous supplementation of 100μM cysteamine and 200μM cystine to maturation medium improved SCNT blastulation of goat oocytes. 3. A 30-min treatment with 1mM caffeine or 5μM MG132-induced cytoplasmic protrusions in over 80% of the MII oocytes.4. Treatment with caffeine or MG132 did not impair the structure of oocyte spindle compared with demecolcine.5. Treatment with caffeine or MG132 did not compromise the capacity of parthenogenetic development.6. Rates of enucleation and blastocyst formation were significantly higher after caffeine or MG132-assisted than after blind aspiration enucleation.7. After withdraw exposured to drugs, caffeine had longer persistence of the cytoplasmic protrusions than MG132, and equivalent to demecolcine.and so,caffeine was more suitable to application.8. Production of live cloned goat from SCNT embryos using caffeine-enucleated cytoplasts demonstrated the potential of this new agent for nuclear transfer practice.更多还原