| The initial totipotent blastomeres will undergo the first cell fate segregation with the formation of trophoblast(TE)and inner cell mass(ICM),which develop into the extra embryonic and embryonic lineages,respectively.TE will form the foetal portion of the placenta and trophoblast giant cells,whereas the ICM segregates in to the pluripotent epiblast and the primitive endoderm.The trophoblast cells in early embryo are the precursors of the differentiated cells of the placenta;it plays a key role in the development of the placenta.More and more research is aimed to trophoblast stem cells(TSC)which is the most appropriate candidate in vitro model for studying placental development and function.The current researches on the buffalo showed that suppressing FGF signaling pathway could increase the number of trophoblast cells and also reported that inhibition of the FGF signaling promoted porcine parthenogenetic embryo development and pluripotency-related and trophoblast lineage-specific gene expression,as well as the expression of trophoblast maker genes CDX2.However,there is little research on trophoblast in early embryos of pigs.Based on the important position of the trophoblast in the positioning,attachment and invasion of the embryo in the uterus,the study on improving the number of trophoblast cells and the expression of related markers and functional genes provides a very promising way to improve the efficiency of embryo transfer.In this study,we use different concentration of FGFR inhibitor BGJ398 to interrupt the FGF signaling pathway during the porcine parthenogenetic embryo for 168h.The main results are as below:1.Porcine parthenogenetic embryos supplying with 5?M BGJ398 shows significant difference of the rate of blastocyst compared to vehicle controls(29.64%vs 22.97%,P<0.05).The addition of 10?M BJG398 slightly increased in the rate of blastocyst but it was statistically significant(22.97%vs 24.68%,P>0.05).However,the addition of 15pM and 20pM BJG398 did not improve the blastocyst rate.The total cell numbers of blastocysts were increased under in addition of 5?M(54.2±7.92 vs 45.16±8.75,P<0.05)and 10?M BJG398(48.2±4.32 vs 45.16±8.75,P>0.05),respectively.Then 5pM BGJ398 was used for quantitative PCR to investigate the expression of several pluripotency-related and lineage-specific genes in blastocysts.The results showed the pxpression of the pluritptency-relatead genes KLF4 and SOX2 was significantly increased(P<0.05),OCT4 was increase(P>0.05).Expression of trophoblast maker genes CDX2 was up-regulated in blastocyst treated with 5?M BGJ398(P<0.05)compared to that of vehicle controls.2.BGJ398 promoted the expression of trophoblast-related gene expression.The result shows that GATA3,TEAD4,CDX2,OCT4,TERT,OEMES,FGFR2 were detectable on the porcine parthenogenetic embryos.The blastocyst treated with 5?M and lOpM BGJ398 were used for quantitative PCR to investigate the expression of trophoblast identificate marker genes.The results shows that the expression of GATA3,TEAD4,TERT and EOMES was up-regulated significantly(P<0.05)in blastocyst treated with the concentration of 5?M BGJ398,but the addition of 10?M BGJ398 group did not show a specific trend.Over all,the addition of 5?M BGJ938 significantly promoted the rate and cell number of blastocyst,and the expression of pluripotency related genes as well as trophoblast specific genes,which may indicate the develpment potentials of embryo and trophoblast.The results of this study will add to the foundation for increasing the efficiency of embryo transfer at next step. |
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