CXCL12?VEGFA And WNT5A Optimize Porcine Oocytes And Mechanisms In Vitro

The combination of gene editing technology and animal cloning technology makes gene manipulation easier and more efficient in molecular breeding of pigs and the construction of animal models,but this technology system requires a large number of oocytes to produce cloned embryos.At present,in large animals,waste ovarian tissue is mainly obtained from the slaughterhouse,and immature oocytes are obtained from them for in vitro maturation.In pigs,mature oocytes obtained and subsequent developmental capacity by in vitro maturation(IVM)still have a low maturation rate,low embryonic development rate,low Inner cell mass and total cell numbers and low birth efficiency compared with those produced in the in vivo environment.In vivo,maternal regulatory factors confer strong developmental capacity on oocytes and embryos,and the current in vitro maturation culture system is difficult to provide sufficient and effective maternal regulatory factors,thereby reducing the quality of oocytes and early embryos.Transcriptome analysis confirmed that CXCL12,VEGFA and WNT5A(referred to as CVW)are important maternal cytokines during the maturation process of porcine oocytes.Therefore,in this study,the CVW maternal cytokines were added to the porcine oocyte maturation system alone or in combination,and the biological effects of the CVW on both oocyte maturation and early embryo development were explored.The main research results are as follows:(1)CVW added to the porcine oocyte maturation medium can effectively improve the maturation quality of oocytes,that is,the nuclear maturation rate increased from 57.2% in the control group to 75.9%,and reduce the early apoptosis rate.(2)After CVW was added to porcine oocyte maturation solution in vitro,the proportion of cumulus expansion diameter greater than 600 ?m was 53.0% in the control group and 64.2% in the CVW group;the number of TZPs in the MII oocyte control group was 56.0 ± 15.5 and the CVW group was 45.0 ± 18.5 and P <0.05;after in vitro fertilization,the proportion of polyspermation in the control group was 59.5% and that in the CVW group was 43.0%.Therefore,CVW can expand the gap between the oocytes spaces and promote the expansion of cumulus cells,resulting in a more complete transregional projection retraction(TZPs)in the zona pellucida,thereby reducing the incidence of polyspermy after In vitro fertilization(IVF).(3)Adding the corresponding CVW receptor inhibitor to the in vitro maturation medium of porcine oocytes not only inhibits the CVW receptor-mediated signaling pathway but also seriously affects the meiosis recovery of oocytes and the expansion of cumulus cells.(4)CVW added to the porcine oocyte maturation medium improve the maturation is achieved by advanced activating the MAPK pathway and adjusting the WNT pathway before the end of the IVM cycle.(5)Mature oocytes obtained by adding CVW to the porcine oocyte maturation medium in vitro will increase the blastocyst rate by more than 10% compared with the control group,regardless of parthenogenetic activation or in vitro fertilization.CVW also increases the total number of blastocysts and the number of inner cell mass and reduces the proportion of apoptotic cells in the blastocyst.The above results indicate that the maternal cytokine CVW can improve the quality and subsequent development potential of mature oocytes in vitro.These findings indicate that CVW optimizes the IVM system of porcine oocytes,thereby providing a large number of high-quality oocytes for pig cloning and other technologies,and ultimately supporting the improvement for pig cloning efficiency and reproductive medicine research.