The Comparative Study Of Different Factors On Oocyte Maturation And The Effect Of Donor Cell Passages On Porcine SCNT

Somatic cell nuclear transplantation(SCNT)which include several process such as thematuration of oocyte in vitro(IVM),the synchronization of donor cell cycle,the process of oocyte de-nucleation,cell fusion andthe culture and development embryonic in vitro,is a complicate mammalian cloning techniques.The technology of IVM not only provided high-quality matured oocytes for SCNT,but it also played significance roles in the process of in vitro fertilization(IVF),intracytoplasmic sperm injection(ICSI)and so on.Matured Oocytesact as a receptor cell provided a strict microenvironment for the reprogramming of nuclear-donor cells,thus the oocyte quality(the characteristics of oocytes are the same as those of natural maturation in vivo or not)is very important.In addition,nuclear-donor cells provide the genetic information for oocytes with free nucleus,so the chromatin state directly affect to the nuclear reprogramming.Furthermore,itis also necessary that the synchronization of donor cells and oocytes in the development of re-constructed embryos.In this study,we began our work around the two aspects described above.In summary,on the one hand,we added different hormones(RosA,pNPPB/E2,FGF2,LIF,IGF1,LH)to the pig oocyte culture systemto study whether it improve the quality of pig oocytes and the effect on development ability of parthenogenetic embryo.On the other hand,we investigated the effect of thecell passages and the diameter size of donor cells,and the synchronization of donor and oocyte cycles on preimplantation embryo development which formed after SCNT.Finally,we demonstrated that the optimal treatment time of 100nMpNPPB/100nME2optimal treatment time and the optimal treatment concentration ofRosA,FGF2,LIF,IGF1 for oocytes.The result shows that,1 Treatment of oocyte with the 100nMpNPPB/100nME2 for 2h,the maturation2The optimal concentration of LH is 5ng/ml,nevertheless under this concentration,the rate of maturity and development of oocytes have no obvious difference compared with control group.3The optimal concentration ofIGF1,LIF,FGF2are respectively 15ng/ml,15ng/ml,25ng/ml,under this condition the rate of oocyte maturation and blastocyst was promoted.4The optimal concentration of RosA is 5?M,under this condition,the maturation rate and blastocyst rate of parthenogenetic embryos were improved.On this basis,weculture the oocytes using different combinations of hormones(pNPPB/E2+FLI+RosA,pNPPB/E2+FLI,pNPPB/E2+RosA,FLI+RosA),and observation the development of parthenogenetic embryos.The result shows thatthere was no significant difference in maturation rate and cleavage rate between the control group when blastocyst rate is rised.Other combinations(pNPPB/E2+FLI+RosA,pNPPB/E2+RosA,FLI+RosA)have no obvious effect on oocyte maturation and development,and may even also be adverse effectors.After that,the effect of donor cells on the porcine SCNT was studied.When using different passages donor cells in the process of SCNT,we found that the development of preimplantation embryos injected with F7-F8 passages is better.And that,the diameter of porcine fetal fibroblasts have no a significant effect on embryo development after SCNT,but it has some influence on the number of cells and apoptosis in the blastocyst stage of reconstructed embryo.All in all,this experiment explores that the effects of different stimulating hormones on oocyte IVM and the effect of donor cell state on embryonic development after SCNT.It provides a reference for the study of oocyte maturation in vitro and the efficiency of porcine SCNT in the future.