Effects Of LPA On The Early Embryo Development And Blastocyst Cells Culture Of Ovine In Vitro Fertilizated Embryos

As an important technology in embryo engineering,in-vitro fertilization technology is widely used in domestic animals and provides technical supports in domestic genetic breeding.The ovine is one of the most important domestic animals in China and also the first domestic animal has been performed in-vitro fertilization technology,while the efficiency of in-vitro fertilization was significantly lower than other domestic animals.In addition,stem cells have great application potentials in domestics.The in-vitro culture of ovine stem cells can be used not only as a research model of mammalian development,but also in treatment of disease and breeding.However,the study of ovine stem cells is still at the primary stage.Lysophosphatidic acid(LPA)is a small glycerol phospholipid which has growth-factor and hormone-like activities in varieties of animal cells.Combining with G Protein-Coupled Receptors,LPA can activate multiple pathways and biological effects,such as cell proliferation,differentiation,migration,invasion,morphogenesis occurrence,etc.Studies have shown that LPA can promote oocyte maturation and embryo development,and has a positive effect on cell proliferation.By adding LPA in the process of in-vitro fertilization and blastocyset cell culture,we explored the effect of LPA in the in-vitro fertilization and blastocyset cell culture.When different concentration of LPA(0.1?M,1?M and 10?M)is added into the culture,the maturation rate,cleavage rate and blastocyst rate gradually increased with the increase of LPA concentration,and the blastocyst has normal morphology.But when the LPA concentration reached 15?M,the maturation rate,cleavage rate and blastocyst rate decreased and the blastocysts has unnormal morphology,which indicated by abnormal inner cell mass distribution.In order to detect LPARs and and the stem gene markers OCT-4 and CDX-2 expression in the early embryo under the different concentrations of LPA,the quantitative real-time PCR of LPAR1,LPAR2,LPAR3,LPAR4,LPARS?LPAR6?OCT-4 and CDX-2 was perfomed on the 2-cell,4-cell,8-cell embryos,morulas and blastocysts.The results showed that,in different concentration of LPA,LPARs have mRNA expression in early embryonic stages.The mRNA expression of LPAR2 was significantly higher than other 5 receptors,the mRNA expression of LPAR4 was only lower than LPAR2,and the expression of LPAR1,LPAR3,LPAR5,and LPAR6 is very low.The mRNA expression of LPAR2 and LPAR4 in the 2-cell stage to blastocyst stage showed an increasing trend in the 2-cell stage to blastocyst stage and increase with LPA concentration in 0-10?M LPA culture systems while decrease in 1?M LPA culture system.In order to explore the effects of LPA on blastocyst cell culture,different concentration of LPA was added into the culture system to observe the growth of blastocysts.By immunofluorescence staining and the quantitative real-time PCR,we detect the expression of stem cell pluripotency markers OCT4 and trophoblast stem cell marker CDX2.We choose GFF as the feeder layers,2i as the basic culture system combined with hLIF,Wnt-3a,Forskolin and LPA to form 8 culture systems.The results showed that the outgrowth of blastocysts grew slowly and differentiation appeared in the 2i sysytem.When adding Wnt-3a in 2i culture system,the outgrowth of blastocysts formed dome structure and was OCT4 and CDX2 positive.When human Lif was added,the outgrowth of blastocyst was three-dimensional,which was similar to the mouse embryonic stem cells and OCT4 positive and CDX2 negitive.When adding hLiif and Forskolin in culture system,the outgrowth of blastocyst was like trophoblast stem cells structure and OCT4 and CDX2 positive.Adding hLif,Forskolin and different concentrations of LPA in culture system,the outgrowth of blastocyst was also like trophoblast stem cells,which showed monolayer,clear outline of the outgrowth,thickened cell colony edge and dome structure.The immunofluorescence staining of OCT4 and CDX2 was positive,the fluorescence intensity,to some extent,was enhanced with the increase of LPA concentration.The quantitative real-time PCR showed that the expression of OCT4 and CDX2 enhanced with the increase of LPA concentration,but the high concentration of LPA could inhibit the expression of OCT4 and CDX2.These results indicated that LPARs are expressed in the ovine early embryos,LPA may play an important role in ovine embryo development,and LPA has effects on the culture of stem cell from the blastocysts.This study lay the foundation for the IVF and stem cell research of domestics.