Skp1 Proteins Function In Early Mouse Embryonic Development Process

To screen some proteins that function in reprogramming and the development of preimplantation embryos,our laboratory studied the protein expression of mouse MII oocytes and parthenogenetically activated oocytes by 2D gel electrophoresis,through mass spectrum identification,bioinformatical analysis and reading pertinent literature, we selected the Skp1 protein for further investigation.According previous researches,in yeast Skp1 interacts with Bub1 to activate the spindle checkpoint in response to defects in microtubule's attachment to the kinetochore and in kinetochore tension,the spindle checkpoint ensures the fidelity of chromosome segregation by preventing cell-cycle progression until all the chromosomes make proper bipolar attachments to the mitotic spindle and come under tension.Skp1 is also a multifunctional protein involved in many critical cellular pathways,taken together,these reports suggest that Skp1 is important for the process of preimplantation embryonic development.In the present study,first of all,we studied the protein expression of mouse MII oocytes and parthenogenetically activated oocytes by 2D gel electrophoresis,and obtained information from 2D gels as below:Skp1 is a protein with high expression level,and its expression level doesn't change as MII oocytes are parthenogenetically activated;Skp1 is a small (M_r=21,000)protein and it has two M_r positions in 2D gels,we speculate that the higher band represent a glycosylated form of skp1 proteins.We then purchased rabbit polyclonal antibody of Skp1 and confirmed its existence in MII oocytes and parthenogenetically activated oocytes by western blot,its expression level and M_r are similar to that from 2D gels. Immunohistochemistry of ovary sections indicate that skp1 distribute diffusely in the cytoplasm of oocytes.Immunofluorescence show the same result,Skp1 protein localize to the cytoplasm of MII-stage oocytes and embryos,and its distribution and expression level don't change as one-cell embryos develop until the blastocyst stage.To investigate the role of Skp1 protein during the development of preimplantation embryos in mice,we constructed plasmid encoding fusion proteins of pEGFP with full-length Skp1,and injected them into the pronulceus of one-cell zygotes.It is observed that overexpression of Skp1 proteins will lead to the arrest of preimplantation embryos,and the results were confirmed through western blot.Inversely,after injection with Skp1 antibody into one-cell zygotes,we found that the development of these embryos were promoted,the results suggest that block of Skp1 protein would promote the development of preimplantation embryos in mice,which is in coincidence with result of overexpression.With above phaenotypes,we then studied the mechanism that how Skp1 influence the development of preimplantation embryos in mice. Previous study shows that in yeast Skp1 interacts with spindle checkpoint protein Bub1 to activate the spindle checkpoint in response to defects in microtubule's attachment to the kinetochore and in kinetochore tension, so the block of Skp1 protein would abolish the checkpoint and cells with errors could fulfill cell cycles.Our results support the mechanism,so we further studied these treated embryos.To further investigate the effect of skp1 deletion on embryos, immunofluorescence was performed to examine the morphology of the spindles of MⅡoocytes after the treatment.We observed that skp1 antibody-injected oocytes had a higher percentage of spindle abnormity than control group,the abnormities include multi-pole spindle,deranged spindle and even collapse spindle.Furthermore,some skp1 antibody-injected MⅡstage oocytes showed aberrant chromosome alignment,some chromosomes were scattered apart from the metaphase plate in the cytoplasm.The same results were taken from one-cell embryos.Taken together,these results demonstrate that the deletion of skp1 in MⅡoocytes and one-cell zygotes induced spindle abnormality and aberrantly aligned chromosome.Also karyotypic status was analyzed in one-cell zygotes that had completed oocyte meiosis.Aneuploid rate is much higher in skp1 antibody-injected one-cell zygotes than that in control zygotes.These results demonstrate that the inhibition of skp1 proteins induced aneuploidy,which might led to embryo death after implantation.We then examined the growth of these treated embryos after implantation,the result revealed that recipient females transplanted with antibody-treated embryos exhibited a sharp reduction in litter size,and more pups died after birth.With these results,we found that the block of Skp1 protein would promote the development of embryos,however,with a higher rate of abnormal spindle and aneuploidy,which might be the cause of embryo loss after implantation and death of pups.So,we presume that Skp1 protein might play a key role in activation and maintenance of the spindle checkpoint pathway in mammalians.Our research studied the distribution and expression level of Skp1 proteins in oocytes and embryos,showed that overexpression of Skp1 proteins would arrest the development of embryos,while block of Skp1 proteins would promote the process in contrast.And the promoted embryos showed more errors including spindle abnormity,aberrantly aligned chromosome and aneuploid,which led to a sharp reduction in litter size and death of pups.Our work proves that Skp1 plays an important role during the development of preimplantation embryos in mouse.We also speculate that Skp1 is critical in activation and maintenance of the spindle checkpoint pathway.