Effect Of Transcription Factor ELK1 On Developmental Potential Of Porcine Preimplantation Embryos Fertilized In Vitro And Its Mechanism

Transcription factors play essential roles for epigenetic reprogramming and zygotic gene activation during early embryonic development.In recent years,a large number of experimental studies have found the important roles of multiple transcription factors during embryonic development in different species.However,there are still few relevant studies on the transcription factors that play key regulatory roles during pig early embryonic development.To this end,we first predicted the essential role of ELK1 during pig embryo development by using bioinformatics methods,and subsequently investigated the regulation of ELK1 expression in pig in vitro fertilization(IVF)embryos by q PCR and immunofluorescence staining,and observed its effects on embryonic developmental phenotypes,apparent reprogramming,as well as pluripotency genes by silencing ELK1.The effect of ELK1 silencing on the expression profile of 4-cell embryos was finally explored by transcriptome analysis.The main results are as follows:1.We found that porcine embryo zygotic gene activation is initiated at the 4-cell stage by contrasting the transcriptome results at all stages of in vivo fertilized embryos in pigs.ELK1 was subsequently found to rank first by enrichment for transcription factors that bind upon zygotic gene activation.Enrichment of these genes revealed that most of these gene functions were focused on the process of protein synthesis,which triggered our intense interest in ELK1.To further investigate its function,we examined the regulation of ELK1 expression in preimplantation embryos undergoing IVF,and by immunofluorescence staining as well as q PCR,we found that ELK1 was significantly upregulated at the 4-cell stage and barely expressed at the blastocyst stage.Therefore,we speculate that ELK1 may play an important role during the zygotic activation phase.Simultaneous immunofluorescence staining of oocytes at the MII stage revealed the near absence of maternal expression of ELK1,which provides feasibility for subsequent experiments to silence ELK1 by si RNA.2.To further investigate the effect of ELK1 on the developmental potential of pig embryos,we silenced ELK1 of IVF embryos by cytosolic injection of ELK1 si RNA,using normal group as a control,and we found that ELK1 silencing significantly decreased cleavage and blastocyst rates of embryos,with a particularly significant decrease in blastocyst rates.Subsequently for the blastocyst cell number versus apoptosis rate statistics,we found that although there was no obvious difference in the blastocyst cell number,the apoptosis rate was significantly higher.To investigate the effect of ELK1 on epigenetic reprogramming during early embryonic development,we found by immunofluorescence staining that ELK1 silencing results in a significant decrease in H3K9 Ac modification but an increase in h3k9me3 modification in 4-cell embryos.The results suggest that abnormalities in epigenetic reprogramming result in a large number of genes that do not activate properly.Subsequently,we found by immunofluorescence staining that ELK1 silencing also significantly reduced the expression of the pluripotency gene Oct4 in blastocysts.3.To deeply investigate the effects of ELK1 on porcine embryonic development and the in-depth molecular mechanism,we contrasted the gene expression differences of IVF embryos in ELK1 silenced group versus normal group by RNA SEQ technology,and we found that a large number of protein synthesis related genes and pathways were severely affected by go with KEGG enrichment.In addition,cell cycle related genes and pathways were significantly affected,which partly explained the poor embryo development rate.Go analysis was similarly enriched for a large number of DNA damage repair related genes,which explained to some extent the large number of apoptotic cells found by TUNEL staining.The transcriptome data showed no significant differences in epigenetic modification related genes,so we hypothesized that the effect of ELK1 on epigenetic reprogramming might be due to the protein synthesis being affected which in turn leads to the inability to properly establish epigenetic modifications.Furthermore,our enrichment of upregulated genes after ELK1 silencing revealed that the function of upregulated genes was focused on the respiratory chain for aerobic respiration,which we speculate may be a " self rescue " means for cells to enlarge their energy expenditure in order to repair the damage caused by ELK1 silencing.