Activation And Influence Of Early Embryonic Development Of Sheep Phospholipase C Zeta(PLCζ)of Sheep Oocytes

Objective: In our country the sheep meat wool is widely used in livestock, resulting in a large number of ovarian tissue in the slaughter process. However, the utilization rate of the ovarian tissue in the real production is not high, and the technology of producing high quality embryos using sheep ovarian tissue is slow, so it is a problem to improve the quality of the embryos. The production of high quality embryos in ovarian tissue may be mainly influenced by the efficiency of oocyte activation, which is activated by protein kinase inhibitors or other artificial activation methods. However, this activation method can not make us understand the mechanism of oocyte activation, and it may be a misunderstanding to us to study the mechanism of oocyte activation, so that we can not determine whether adverse effects on the development of the late embryo. Thus, we need a new biological activation preparation to activate the oocyte. The study found that there is a specific activator of sperm, phospholipase C zeta(PLCζ). It was confirmed by the increase of Ca2+ concentration in the cytoplasm of oocyte to activate the oocyte, and it can make the early embryonic development. It will help us to further study the mechanism of oocyte activation and the development of early embryos, and the technology of producing high quality embryos at the same time will also be rapid development. In this experiment, the initial exploration of sheep phospholipase C zeta(PLCζ) of sheep oocytes activation effect and influence of early embryonic development.Methods:(1)Digestion of laboratory has been constructed the constructed recombinant plasmid p EGFP-N1-PLCζ bacilli PCR and double enzyme, after the completion of the identification of extract recombinant plasmid and empty vector. Sheep ovarian tissue was collected to pick out the oocytes, and the recombinant plasmid and empty vector were injected into the M II oocytes to observe the activation of oocytes.(2)were cultured in the culture of recombinant plasmid of 22 h, and then stained with Rhod2-AM, and the dynamic changes of Ca2+during the oocyte and early embryos were scanned by laser scanning confocal microscope.(3) the whole process of the development of the early embryonic development was observed by culturing the recombinant plasmid.Result:(1)Identification of the recombinant plasmid p EGFP-N1-PLCζ, and the recombinant plasmid p EGFP-N1-PLCζ microinjected into the sheep oocyte cells managed to reactivate the oocytes to the parthenogenetic development.(2) the dynamic changes of Ca2+ during the development of oocytes and early embryos were collected by laser scanning confocal microscope.(3) micro injection of recombinant plasmid p EGFP-N1-PLCζ of ovine oocytes after activation, sheep oocytes normal development formed early embryos.Conclusion:(1) sheep PLCζ plasmid injection M II oocytes parthenogenetic activation of sheep oocytes,and can develop into normal embryos.(2) Ca2+ dynamic changes during the development of the oocyte and early embryonic development of the sheep.