| It is well acknowledged that the wide applications of pig embryo engineering technology in animal husbandry and human medicine is severely limited by low efficiency and poor quality of early embryos cultured in vitro.As known,early embryonic development is regulated by series of molecular events,among which regulation of gene networks and epigenetic modifications are two major pathways.WD repeat-containing protein(WDR5)is a core component of mixed lineage leukemia(MLL)protein family,playing important roles in transcriptional regulation,epigenetic modifications,stem cell pluripotency and self-renewal in human and mouse cells.However,effect of WDR5 in early embryonic development in mammals is still unknown.Therefore,the current study aims to explore dynamic expression profiles of WDR5 and determine its function and mechanism involved during preimplantational development of embryos in pigs,which will not only better our understanding of molecular regulation in early embryos,but also provide theoretical basis for improving in vitro production of pig embryos.Firstly,the coding sequence of pig WDR5 was cloned,and the sequence has 99.9% similarity to the predicted sequence on NCBI.Regarding to amino acid homology,porcine WDR5 has 99.1% similarity between pigs and mice or humans and has 98.8% similarity between pigs and cattle.The structure area of WD40 repeat protein highly conserved between pig and other three species and its amino acid sequences are consistent completely.Secondly,we found that WDR5 was express in all analyzed pig tissues using RT-q PCR,and the relative expression was highest in testis.WDR5 was expressed in porcine embryonic fibroblasts(PEF),adipose stem cells and induced pluripotent stem cells,and the relative expression of WDR5 is negatively correlated with cell differentiated potency.The expression of WDR5 displays dynamic pattern in porcine oocytes and preimplantation embryos,and the relative expression was highest in GV stage oocytes and lowest in 8-cell stage embryos.Indirect immunofluorescence staining results showed that WDR5 proteins were localized in the nucleus and cytoplasm of PEF,while only localized in cytoplasm of GV and MII oocytes and only in nucleus of blastocyst stage.WDR5 proteins were expressed in the nucleus and cytoplasm from pronucleus to morula stage.Thirdly,WDR5 was knocked down using microinjection combination with RNA interfering and inhibited by using specific protein inhibitor in porcine parthenogenetic activation or in vitro fertilization embryos.The results showed that the blastocyst developmental rate and total cell number per blastocyst decreased obviously when WDR5 was knocked down.Meanwhile,knockdown of WDR5 induces apoptosis index increased sharply and causes pluripotency genes disturbedexpression in blastocyst.Epigenetic modifications were changed in WDR5-knockdown(WDR5-KD)4-cell embryos,as evidenced by great increasing of H3K4me3 levels and severe declining of H4K16 ac levels mediated by MOF.DNA strand break was significantly increased in WDR5-KD blastocyst.The major regulator ATR of DNA damage response was upregulated,and the expression of BRCA1 and MRE11A in HR repair pathway was downregulated obviously in WDR5-KD blastocyst.The negative regulated factor P21 of cell cycle was increased notably in WDR5-KD blastocyst.The abnormal development of blastocyst caused by WDR5-KD was rescued when the P21 was interfered after RNAi.In conclusion,the present study reveals the effect of WDR5 on preimplantation in vitro development of porcine embryos' viability for the first time,to our knowledge.WDR5 could maintain the normal development of porcine preimplantation embryos through regulation of cell apoptosis,epigenetic modifications,pluripotency genes and maintaining of genomic stability and cell cycle progression.The results not only fullfil our understanding of the molecular regulation mechanism in early development of embryos in pigs,but also provide theoretical reference for improving in vitro production of pig embryos. |
PDF Full Text Request