MAPK Signaling Pathway Affects Trophectoderm Specification And Blastocoele Formation

MAPK signaling pathway plays an important role in embryonic development,wholebody tissue homeostasis and various pathological processes.There are three major signal pathway members that have been thoroughly studied on MAPK signaling pathway:p38 MAPK signaling pathway,ERK MAPK signaling pathway,and JNK MAPK signaling pathway.For the activation of MAPK signaling pathway,some or more extracellular stimuli are required to activate intracellular activated proteins such as Ras,Rac1,Cdc42 and Rho in a receptor-dependent or non-dependent way.Then,upstream cascade signals(also known as effector)are transmitted phosphokinase from MAPKKK to MAPKK,phosphorylation and activation of MAPK signaling pathway further exercise regulatory functions.In human preimplantation embryo development,how polarity protein transfer the signal of the lineage differentiation down and go into the internal and external cleavage cell lineage specific transcription factor in the different expression patterns is still a matter of discussion,this research analysis the regulation of MAPK signaling pathways downstream transcription factors and the lineage specific expression of the orientation and the influence of the embryonic lineages diverged,combing analysis signal polarity into lineages diverged command signal transmission way.Illustrates the MAPK signaling pathways decide the fate of human preimplantation embryo ICM/TE lineage molecular mechanisms.The main purpose of this experiment is to explore the regulatory effects of three MAPK signaling pathways,p38,ERK and JNK,on trophoblast lineage differentiation and cyst formation of mouse preimplantation embryos,and to provide experimental basis and theoretical basis for the subsequent study on the effect of MAPK on embryo development.The main findings are follows:1.Three inhibitors(PD98059,SB203580,SP600125)were simultaneously inhibited and added to the corresponding microdroplets in the culture medium.The formation rate and cavity formation of blastocyst after inhibition by three inhibitors were observed under the microscope,and the phosphorylation of downstream transcription factors when inhibiting p38,JNK and ERK at the same time was studied.The results showed that the mouse blastocyst could be formed normally under the condition of any single inhibition of one signaling pathway,which indicated that the compensatory mechanism existed in the MAPK signaling pathway,and the effect of single inhibition of one of the signaling pathways was not obvious.In the case of inhibition of two signaling pathways,the embryonic part cannot form a blastocyst.Some embryos have small or even multiple cavities.However,most embryos in the experimental group that inhibited three signaling pathways at the same time were unable to form blastocysts.Phosphorylation of MAPK downstream transcription factor SP1 in blastocysts was also detected.It was found that phosphorylation of SP1 could still be detected in TE cell nucleus in blastocysts with two inhibitors,while phospho SP1 could not be detected in TE cell nucleus in blastocysts with the addition of three inhibitors.In addition,we also detected the expression and localization of TFAP2c(the expression of TFAP2 c is positively regulated by MAPK pathway)in the blastocyst of embryo.The results showed that TFAP2 c in the embryos of the control group was located in the nucleus of the outer blastomere,while the inhibition of three MAPK pathways resulted in the down-regulation of TFAP2 c in the nucleus of the outer blastomere.It indicates that the activation or expression of downstream transcription factors are affected after inhibiting the ERK/p38/JNK MAPK pathway.Therefore,in the following experiments,we further analyzed the expression and localization of lineage-specific transcription factors CDX2 and GATA3 regulated by these transcription factors in embryos.It was found that CDX2 and GATA3 expressions were significantly down-regulated when the ERK pathway,p38 pathway and JNK pathway were simultaneously inhibited.It indicates that MAPK signaling pathway is play an important role in TE/ICM lineage differentiation,and TE lineage differentiation.2.On the basis of previous research,the effects of simultaneous inhibition of ERK pathway,p38 pathway and JNK pathway on trophoblastic lineage differentiation and expansion were further discussed.The results showed that trophoblastic lineages could not differentiate and expand when three pathways were inhibited simultaneously.Moreover,the expression of trophoblastic lineage-specific transcription factors was significantly down-regulated.The expression of the trophoblast giant cell marker Hand1 and pl-1 was detected,and it was found that the expression of these marker genes was significantly down-regulated.It indicates that when the ERK pathway,p38 pathway and JNK pathway are inhibited at the same time,the differentiation of trophoblastic lineages is abnormal,which affects the differentiation of trophoblastic lineages in the early embryonic development of mice.3.Then,on the basis of inhibition,we use Pholliodin staining cytoskeleton.Under the laser confocal microscope,we found that when ERK,p38 and JNK pathways were inhibited at the same time,the blastocyst development rate was significantly reduced,and the embryo was abnormally extraneous,with multiple or small cavities.We examined the embryonic cytoskeleton with an abnormal exit chamber and found that the cytoskeleton was destroyed.Most cytoskeletons in the three inhibitor groups were dispersed.We believe that the inhibition of MAPK signaling pathway causing abnormal cytoskeletal formation,resulting in the abnormal development of blastocyst.In conclusion,since there is a compensatory relationship among the three parallel pathways of MAPK signaling pathway,this study simultaneously inhibited the three parallel pathways and found that MAPK signaling pathway regulates trophoblastic lineage differentiation of embryo and blastocyst cavitation by regulating cytoskeleton.