The Effect And Regulation Mechanism Of Krt8 On Mouse Blastocyst Development

There are three types of cells in the preimplantation embryo:primitive ectoderm(EPI),rophoblast(TE)and primitive endoderm(PE)cells,the EPI and PE cells were differentiated from inner cell mass(ICM)cells.The EPI cells could develop into the body and parts of the amnioticmembrance,chorioallantoic placenta and visceral yolk sac.The PE cells could develop into parts of parietal and visceral yolk sac.The TE cells could develop into parts of the chorioallantoic placenta and parietal yolk sac.It has great significance to elucidate the regulatory mechanism of the three germ layer differentiation in order to elucidate the maintenance of pluripotent embryonic stem cells and its clinical application in the future.Krt8,a kind of keratin,is composed to the intermediate fibers,and has significant difference between ICM and TE cells.The stydy focus on the differential expression of krt8 and the regulation mechanism in ICM and TE cells.In this study,the expression of multiple genes of haploid,diploid and tetraploid preimplantation embryos in different developmental stages were analyzed by single cell microfluidic high throughput qPCR technology.Krt8 was significantly different between haploid and diploid embryos at the 2-4 cell stage.In addition,the rate of blastocyst development was significantly lower than that of diploid blastocysts,but the injection of Krt8 mRNA into parthenogenetic haploid 1-cell embryos could not improve the blastocyst development rate.In diploid embryos,Krt8 was expressed from the zygote stage;krt8 protein abundance was low in the blastomere before mitosis M phase and high after the M stage of blastomeres;with the development of embryos,Krt8 gene expression level was high at morula stage,there were significant differences among different types of cells in blastocysts,the low in ICM cells and high in TE cells.In addition,the expression levels of Krt8 mRNA was low in ES cells,and high in TS cells;the expression level of Krt8 mRNA in placenta and amniotic membrane was higher than that of other organs and tissues at 13.5 dpc mouse conceptus.Injection siRNA of Krt8 gene into diploid zygote to knock down Krt8 gene,which led the mouse blastocyst rate decreased;the blastocyst hatching rate was lower;total number of blastocyst cells and TE cells decreased;the expression level of Cdx2 gene decreased significantly;at the same time,the mitochondrial membrane potential decreased;ATP content decreased;and the copy number of mtDNA decreased.It was predicted that Krt8 protein has significantly correlated with the mitochondrial autophagy,mitochondrial depolarization and ATP binding protein,which contain Hk2 protein.The results of mRNA-sequencing showed that the Hk2 expression was decreased in the Krt8-KD blastocyst.Furthermore,the Hk2 expression level also was significantly decreased in the morula and blastocyst of Krt8-KD by the qRT-PCR.The TS cells was easily lead to differentiation in TS cells with knockdown of Krt8 gene;the Cdx2 expression level decreased significantly,mitochondrial membrane potential decreased;the Hk2 expression level decreased significantly;and the Hk2 protein expression also decreased significantly.These results suggest that Krt8 affected the development and differentiation of embryos by regulating Hk2.The expression model of Krt8 in diploid and tetraploid preimplantation embryos was similar,the expression level in ICM cells was high,and the expression level in TE cells was low.Compared with the diploid blastocysts,tetraploid blastocysts of EPI cells was significantly reduced,and the outgrowth formation ability also decreased significantly.These results indicated that the defect of tetraploid EPI cells was the reason for the decrease of outgrowth formation ability of EPI cells.Tetraploid ES cells also can contribute to the ICM cells of the diploid blastocyst.The tetraploid and diploid ES cells can takepart in the similar chimeric sites in the chimeras at the period from 6.5 dpc to 10.5 dpc,but the chimeric and survival capacities were significantly reduced.In conclusion,Krt8 plays an important role in the development of TE cells,which regulate the mitochondria through Hk2.