Reprogramming Capacity Of Parental Pronuclei In Mouse Zygotes

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.