Effects Of Cytochalasin B On Asymmetric Division In Mouse Oocytes

The mammalian oocyte maturation process consists of two consecutive asymmetric divisions,and produces three daughter cells of vastly different sizes: one larger egg cell and two smaller polar bodies.Asymmetric division is a typical feature of mammalian oocyte meiosis that results in a highly polar egg cell.The mitosis of the cell after fertilization exhibits restored symmetric division,but the polarity characteristics formed during meiosis of oocytes are preserved and affect the polarity of early embryos.The first meiotic process of mouse oocytes includes oocyte meiotic recovery,spindle formation,spindle migration,and discharge of the first polar body.These processes require the coordinated and orderly function of chromosomes,cytoskeletal proteins,and various protein molecules to ensure precise control of this process.Cytochalasin B(CB)is an actin polymerization inhibitor that binds to the ends of microfilaments to block the polymerization of actin at this site and it is also a cytoskeletal reversible stabilizer.In this experiment,mouse oocytes were treated with CB to make modles of symmetric division and cytokinesis failure.This model is uesd to detect the characteristics and mechanisms of asymmetrical division process and to explore the polar complexes.Role in the establishment of mammalian oocyte polarity.The main results obtained in this study were as follows: 1.CB effected the maturation of mouse oocytesThe mouse oocytes were treated with CB at three concentration gradients(1 ?g/mL,2.5 ?g/mL,and 5 ?g/mL,respectively).It was found by statistical data that the low concentration of CB treatment did not affect the mouse oocytes GVBD.The occurrence of high concentration of CB will affect the incidence of GVBD.CB significantly decreases the rate of discharge of the first polar body and presents a dose-dependent reduction,oocyte equivalence splits,or cytokinesis failure.In addition,the longer the CB treatment,the lower the ability of the cells to restore cytokinesis.2.CB disrupted typical characteristics of asymmetric divisionAfter CB treatment,by immunofluorescence staining and fluorescence gray value analysis,it was found that CB disrupted spindle morphology and migration,and affected the distribution and expression of p-MAPK compared with the normal group;CB disrupted the formation of actin caps,CGFD and the distribution of mitochondria.3.CB disrupted the distribution of major polarity proteinsAfter CB treatment,through the cell immunofluorescence staining experiment,it was found that CB disturbs the polar distribution of the polar proteins aPKC,Par6 a,Crb3,DLG1 compared with the normal group,and destroyed the polarity of the oocyte as a result..4.CB influenced the expression of cytokinesis-related factorsAfter CB treatment,the expression levels of cytokinesis-related factors such as Arp2/3,Cdc42,Fyn,Anillin,?Actinin4,Septin2 and Septin7 in oocytes by real-time fluorescence quantitative PCR.By comparison,Arp2/3,Cdc42,Fyn,?Actinin4 and Septin7 were significantly up-regulated,Anillin was significantly down-regulated,and Septin2 did not change significantly.In conclusion,this experiment shows that CB disrupts the maturation of mouse oocytes and disrupts the typical features of asymmetric division and the distribution of major polarity proteins and the expression of factors related to cytokinesis.