The Expression And Function Of BRUNOL4 On The Development Of Preimplantation Mouse Embryos

Objective:To observe the localization and expression of BRUNOL4 in mouse preimplantation embryos,and elucidate the role of BRUNOL4 in the development of mouse preimplantation embryos.Methods:1.The different stages of mouse preimplantation embryos were collected from mating mice(♀KM×♂KM),the localization and distribution of BRUNOL4 protein were observed by confocal laser microscopy.2.The expression level of Brunol4 mRNA was observed by Real-Time PCR from different stages of mouse preimplantation embryos.3.KM mouse 1-cell embryos(18 h after hCG)were collected and incubated for 3h in KSOM culture medium.1-cell embryos with female and male pronuclei were divided into three groups which were KSOM culture group(blank control group),unrelated IgG(100 μg/ml)group(negative control group)and BRUNOL4 antibody(100 μg/ml)group.Then the preimplantation embryos of three groups were further cultured in vitro.4.At 50 h after hCG,2-cell embryos were collected,the expression levels of ZGA-related marker genes MuERV-L,Zscan4 d,Hsp70.1,and eIF1 A mRNA were detected.5.At 96 h after hCG,morula embryos were collected to measured the different mRNA expression levels of stem cell transcription factors Sox2,Oct4 and the differentiation correlation factor Cdx2 by Real-time PCR.Results:1.The results of immunofluorescence showed that the expression of BRUNOL4 protein was granular form in the early stage of oocyte and preimplantation embryo.In the 8-cell embryo stage,the protein particles became significantly thicker,and the BRUNOL4 fluorescence intensity reached the strongest level in the morula stage.Then it began to decrease and falls to a minimum level in the late blastocyst stage.2.The results of Real-Time PCR showed that the expression level of Brunol4 mRNA was the highest in mature oocytes(MII eggs),and gradually decreased after fertilization.The 8-cell embryo to blastocyst stage was almost undetectable.3.After injecting of BRUNOL4 antibody,the blastocyte development rate was significantly lower than the KSOM culture group(blank control group)and negative control group(P<0.05).4 After injecting BRUNOL4 antibody,the mRNA expression levels of four ZGA-related marker genes eIF1 A,MuERV-L,Zscan4 d and Hsp70.1 in 2-cell embryos were non-significantly differences by comparing three groups(P>0.05).5.After injecting BRUNOL4 antibody,though the expression of Sox2 and Oct4 mRNA didn’t change obviously in morula embryos(P>0.05),the expression of Cdx2 mRNA was significantly decreased(P<0.05).Conclusion:The phase-specific granular expression of BRUNOL4 protein in the cytoplasm of preimplantation embryo is suggested that BRUNOL4 may plays an important role in the development of preimplantation embryo.Brunol4 mRNA is expressed at the highest level in mature oocytes(MII eggs),suggesting that it may be a maternal effector gene.After blocking the function of BRUNOL4 by specific antibody microinjection,the expression levels of four ZGA-related marker genes eIF1 A,MuERV-L,Zscan4 d,Hsp70.1 and stem cell transcription factor Sox2,Oct4 mRNA aren’t significantly differences,but the expression of Cdx2 mRNA in morula is down-regulated.It is indicated that BRUNOL4 may affect the formation of trophoblasts by changing the expression of Cdx2 gene,and further influence the development of blastocysts.