Pcbp1 Protein In The Mouse Egg Activation And Function In Early Embryonic Development

In mammals,a mass of maternal factors accumulate during oocyte maturation,which are extremely important in the early events of embryonic development,such as oocyte activation,zygote genome activation,ect.Oocyte activation of particular concern,including the completion of meiosis,the formation of pronuclei and cytoskeletal rearrangements,is totally dependent on the remarkable post-transcriptional regulatory mechanisms that control maternal mRNA stability and translation.For the sake of a group of differentially expressed maternal proteins,our lab constructed the pre-activated and post-activated mouse oocyte protein profile by means of two-dimensional PAGE and MALDI-TOF techniques.PCBP1(poly(rC)binding protein 1), one of differentially expressed proteins,dispalys dephosphorylation post activation.PCBP1 belongs to the poly(rC)-binding protein family which contains three KH domains.On the one hand,it functions as a molecular chaperone to bind to CU-rich element((CyU)CCANxCCC(UyA) PyxUC(CyU)CC)of mRNA 3'-UTR by KH domains,thus taking part in mRNA stabilization and translation.Dephosphorylation of PCBP1 can boost its ability to bind to mRNAs;on the other,it participates in some protein-protein interactions.In our research we focus on its roles in mouse oocyte activation and early embryonic development for the first time.A series of mRNAs whose stability and translation might be regulated by PCBP1 and proteins which will interact with PCBP1 are accessible in our early analysis by literature search and bioinformatics according to the characteristics of CU-rich element.Thereinto,H2A.X mRNA and protein Lamin A/C aroused our intense interest.H2A.X,a variant histone H2A and with a CU-rich element in its 3'-UTR,palys an impotant role in nucleosome assembly,chromatin organization,DNA repair and so on.Phosphorylated H2A.X is closely related with sperm chromatin decondensation and male pronucleus formation after fertilization in xenopus oocyte research.Lamin A/C,located in the nuclear envelope and caryoplasm of nucleated cells,is a potential marker for nuclear remodeling since its close relation with nuclear envelope assembly and chromatin organization.Furthermore,it has been reported that Lamin A/C can interact with PCBP1 in Hutchinson-Gilford progeria syndrome.A hundred to one that PCBP1 might play an important role in pronucleus formation by controlling H2A.X stablity and translation or interacting with protein Lamin A/C.Western blot validated the result of two-dimensional PAGE and MALDI-TOF. Immunohistochemistry and immunofluorescence showed that PCBP1 was located in the cytoplasm of MII oocyte,whereas it mostly transfered to the pronucleus of zygote.PCBP1 antibody microinjection and RNAi of MII oocytes,blocking the function and expressing of PCBP1,retarded the rate of pronucleus formation obviously.H2A.X antibody microinjection, as we expected,dramatically retarded the rate of pronucleus formation. Moreover,the level of H2A.X mRNA was significantly reduced following PCBP1 antibody microinjection indicating that stability of H2A.X was controlled by PCBP1 in mouse oocyte.In addition,double immunofluorescence manifested that PCBP1 was co-localized with Lamin A/C in pronucleus which gave us a hint that PCBP1 could also interact with Lamin A/C in mouse oocyte.Thus,to sum up above points, PCBP1 might play an important role in pronucleus formation through two ways at least:1.Dephosphorylated PCBP1 enhances the ability to bind to H2A.X mRNA after oocyte activation whose stability was strengthened consequently,thus facilitating the pronucleus formation;2.PCBP1 takes part in nuclear and chromatin remodeling by interaction with Lamin A/C. So exploring the functions of maternal proteins,such as PCBP1,may help us to understand the potential reasons for early embryonic development failure and enhance the development of reconstructed embryos.