【作者】 刘文强；

【导师】 高绍荣；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 体细胞重编程是指分化细胞的特异性表观遗传修饰被擦除,细胞基因组重新回到多能性状态。核移植是目前体细胞重编程最有效的方法之一。之前研究证明处于第二次减数分裂中期即MⅡ期的卵母细胞胞质中的重编程因子可以快速高效地重编程体细胞。以受精卵为受体的核移植实验进一步表明重编程因子在受精后集中到合子的雌雄原核中并在第一次有丝分裂中期重新释放到胞质中。因此,只有中期的去核受精卵能支持克隆胚胎的发育。虽然来源于母本的雌原核与来源于父本的雄原核在受精卵中经历了迥然不同的重编程过程以形成全能性的合子。但是,雌雄原核对受精卵重编程能力的影响仍然是一个未知的问题。在本研究中,我们对受精卵进行了三次显微操作来解决这个发育生物学的基本问题。首先,我们在小鼠受精卵的间期移除一个雄原核或雌原核。然后,将单倍体的受精卵继续培养并阻滞在有丝分裂的中期并去除染色体。最后,将处于中期的供体细胞染色体注入去核的受精卵。我们的研究表明,去除雌原核(FPD)的小鼠受精卵可以获得由胚胎干细胞(ESCs)作为核供体的克隆小鼠,并且能支持体细胞核移植克隆胚胎发育至囊胚并建立具有多能性的核移植胚胎干细胞(ntESCs)系；而去除雄原核(MPD)的受精卵则无法支持以ESCs为供核细胞的克隆胚胎发育到期,并且也不能支持体细胞克隆胚胎发育到囊胚。对这两种克隆胚胎的表观遗传标记检测表明去除雌原核的受精卵对体细胞组蛋白乙酰化和DNA去甲基化的能力明显强于去除雄原核的受精卵。去除雄原核的受精卵不能使克隆胚胎中多能性基因Oct4的启动子成功地发生去甲基化。进一步的研究表明,受精卵中融合入一个额外的雄原核能明显提高核移植的效率。以ESCs和体细胞为供核细胞的核移植试验表明融合额外雄原核的受精卵能够显著提高克隆胚胎的体外发育率。克隆胚胎的体内发育试验表明增加雄原核可以提高合子获得以ESCs为核供体的克隆小鼠的能力。总之,这项研究证明了小鼠合子雌雄原核具有不对称的重编程能力,重编程因子在受精后有选择性地集中在雄原核中,且合子的重编程能力主要由雄原核决定。这些发现为进一步在雄原核中寻找关键的重编程因子奠定了坚实的实验基础。更多还原

【Abstract】 During nuclear reprogramming, the epigenome of a differentiated cell reversed to the undifferentiated embryonic state. Somatic cell nuclear transfer (SCNT) is one of the most effective methods of nuclear reprogramming. Oocytes enucleated at the metaphase Ⅱ stage of meiosis are traditionally used as the recipients for SCNT. Following fertilization, the reprogramming factors are believed to translocate into the pronuclei of zygotes because zygotes enucleated at M-phase instead of interphase retain the ability to reprogram somatic cells. The parental pronucleus derived either from the sperm or the oocytes in a fertilized egg undergo widely different reprogramming process to form a totipotent zygote, however the contribution of the pronuclei in repromgramming capacity remains elusive.In the experiments, three times of micromanipulations were performed to address this fundamental question of developmental biology. First, we removed either male or female pronucleus from a mouse zygote at interphase stage. Then, the haploid zygote was cultured to arrest at M-phase stage of mitosis and the metaphase spindle was subsequently removed by second time micromanipulation. Finally, the M-phase somatic cell chromosomes were microinjected into the enucleated zygote. The results demonstrated that only the female pronucleus-depleted (FPD) zygotes enucleated at M-phase of mitosis can support somatic cell reprogramming, the derivation of chromosome transfer embryonic stem (ctES) cells with full pluripotency and the full-term development of cloned embryos. In striking contrast, male pronucleus-depleted (MPD) zygotes fail to support the pre-implantation development of somatic cell cloned embryos and no cloned pups were got after embryos transfer. Furthermore, the epigenetic markers analysis of cloned embryos reconstructed from MPD or FPD zygotes showed that the ablity to histone acetylation and DNA demethylation of FPD zygotes was obviously stronger than MPD zygotes. Furthermore, bisulfite sequencing analysis indicated that successful demethylation occurred in the promoter of Oct4in cloned embryos reconstructed using FPD zygotes. In contrast, the promoter of Oct4remained highly methylated in the cloned embryos reconstructed using MPD zygotes. Our further studies demonstrate that fusion of an additional male pronucleus into zygote greatly enhances reprogramming efficiency. The results of nuclear transfer using ESCs or somatic cells showed that fusion an additional male pronucleus can significantly improve in vitro development of cloned embryos. In addition, adding a male pronuclear to zygotes can improve the efficiency of the full-term development of cloned embryos in vivo.Therefore, our present study provides solid evidence demonstrating that the parental pronuclei of the mouse zygote have asymmetric reprogramming capacities and that the reprogramming factors preferentially translocate into the male pronucleus following fertilization. Our data provide a clue to further identify critical reprogramming factors in the male pronucleus.更多还原