Effects Of Lpa On Early Development And The Expression Of Pluripotency Markers Ganes Of Porcine Parthenogenetic Embroys

Pigs,as an important livestock in China,play an important role in Chinese agricultural economy.At present,the main methods of obtaining pig pluripotent stem cells were generated through in vitro isolation from early blastcysts and induced pluripotency techonical,however,currently,the "naive" state of porcine pluripotent stem cells were not able to captured in vitro.In order to add more chances to solve this problem,in this study,we investigated the effects of Lysophosphatidic acid(LPA)on the early development of porcine parthenogenetic embryos and naive pluripotency transformation.The results are summarized as follows:1.Effects of LPA(Lysophosphatidic acid)on early development of porcine parthenogenetic embryos and related germ layers.First of all,according to cleavage rate and blastocyst number,the concentration of 50μM LPA treatment was significantly better than other treatment groups,and the quality of blastocyst was better than that of the control group.In order to further confirm the previous test results,we selected OCT4 antibody from the protein level for immunofluorescence staining,the results show that 50μM LPA treatment concentration of immunofluorescence intensity is better than other experimental groups and control group,so to determine the 50μM LPA treatment Concentration was followed by experiments.The results of QRT-PCR showed that LPA had no significant effect on primordial ectoderm,but LPA could significantly upregulate the expression of trophoblast marker gene.The results showed that LPA could significantly increase the expression of trophoblast marker Genes and promote the development of early porcine parthenogenetic blastocysts.2.The Role of LPA in the Signal Pathways of Parthenogenetic Embryos in Pigs.The effect of FGF signaling pathway on the original endoderm of porcine parthenogenetic embryos was explored.QRT-PCR results showed that the addition of 30ng/mL bFGF could significantly up-regulate the expression of GATA4 at porcine blastocysts.The addition of LPA and bFGF together decreased the expression of original endodermal marker gene GATA4.Then we explored whether the LPA acts through the ROCK signaling pathway.The use of ROCK signaling pathway inhibitor Y27632 inhibits the expression of ROCK signaling pathway with addition of LPA,however the original blastocyst germ cell marker GATA4 did not change significantly.The above results indicated that,as discovered at mouse early embryos,the FGF signaling pathway can regulate the primary endoderm development of the pig embryos.At the same time,the results indicated LPA acts as on the porcine primordial endoderm through the ROCK signaling pathway which may have more influence than that of the FGF signaling pathway.3..In order to further study the effects of LPA on the expresson of pluripotency genes in early swine blastocyst,the candidate genes of"naive" and "primed" were quantitatively determined.The results showed that LPA had no significant effect on "naive" gene,but significantly down-regulate the expression of "primed" pluripotent gene NODAL and Activin-A.At the same time LPA also increased the expression of ES cell self-renewal factors,OCT4 and c-Myc.The results showed that LPA could promote the early development of porcine parthenogenetic embryos and may promote the "naive" pluripotency by inhibiting the expression ofprimed pluripotent genes.The above results is expected to promote theisolation and culture of naive pig ES cell line.