| Purpose:High rate of abnormal spindles assembly and chromosome segregation results in abnormal Gametogenesis、abnormal fertilization、abnormal early embryonic development and implantation;which is the main cause of low success rate of assisted reproductive technology and recurrent abortion.Kif2a,as a microtubule depolymerase,play important role in spindle integrity,proper chromosome segregation,pole coalescence in mitosis.as of now,whether Kif2a participates in meiotic spindle assembly,subsequent chromosome alignment and segregation remains unkown.In this study,we investigated the expression,localization and potential roles of Kif2a in mouse oocyte meiosis.It provide the theoretical foundation for improving oocytes quality,improving the assisted reproductive technology,preventation prevention and treatment of human reproductive diseases and abn-ormal embryonic development.Method:Immature GV oocytes were obtained from ovaries of 6-8 week old female ICR mice.Firstly,we examined the expression and subcellular localization of this protein of different stages by immunoblotting analysis and immunofluorescence.Then,we further investigate the correlation between Kif2a localization and microtubule dynamics by employing taxol and nocodazole.we employed specifc siRNA injection to deplete Kif2a in oocytes.In addition,after Kif2a depletion,we utilized immunofluorescence,Western blot,chromosome spreading and cold treatment to investigated the potential roles of Kif2a in mouse oocyte meiosis.At least three replications were performed for each treatment.All percentages were expressed as means± SEM and the number of oocytes observed(n)was put in parentheses.Data were analyzed by independent-sample t-test with SPSS 17.0 sofwere.P<0.05 was considered to be statistically signifcant.Fluorescence intensity statistics was conducted using ZEN(2012)sofware:Results:1 Kif2a may act as a microtubule depolymerase,regulating spindle assembly and chromosome congression:Expression and localization of Kif2a during mouse oocyte meiotic maturation:The expression level of Kif2a was low at GV,and gradually increased form GV to the MI stages,and then decreased slightly at the M II stage.Next,we investigated the subcellular location of Kif2a during meiotic maturation.At the GV stage,Kif2a was mainly distributed in the nucleus.Afer GVBD,Kif2a began to accumulate in the vicinity of the condensed chromosomes,displaying also faint staining around chromosomes and concentrating at centromeres of chromosomes,but the signal was just slightly above the background level.At the M I and MⅡ stage,when the chromosomes were aligned at the equatorial plate,Kif2a localized to spindle microtubules,being concentrated at poles,but also accumulated strongly around centromeres of chromosomes.Kif2a was stained at the midbody at anaphase I and telophase I.Tis localization pattern for Kif2a may implicate that it may play important role in spindle assembly and chromosome movement.In taxol-treated oocytes,the microtubule fibers were excessively polymerized,leading to notably enlarged spindles and multiple asters in the cytoplasm.After being treated with taxol,Kif2a was co-localized with a-tubulin and accumulated at abnormal spindle poles as well as in the cytoplasmic asters,but it also was localized at the centromeres.Kif2a and y-tubulin signals partially overlapped at the abnormal spindle poles as well as in the cytoplasmic asters.Knockdown of Kif2a results in abnormal spindles and misaligned chromosomes:Kif2a depletion by siRNA microinjection generated severely defective spindles(spindles with no poles,monopoles,and multipoles and the broadened spindles)and misaligned chromosomes(lagging chromosomes and irregularly scattered chromosomes),reduced spindle migration toward one side of the cortex.Depletion of Kif2a causes dissociation of y-tubulin from spindle poles:It is well known that γ-tubulin is the MTOC-specifc protein that is important for microtubule nucleation and spindle formation during mouse oocyte meiosis.y-tubulin was localized to spindle poles in the control group at the M I stage.Strikingly,after Kif2a siRNA injection,y-tubulin was no longer accumulated at spindle poles,being irregularly distributed into the cytoplasm.So,we concluded that Kif2a may play important role in spindle integrity and pole coalescence.Kif2a destabilizes the spindle microtubules in meiosis:The microtubules density,as measured by total a-tubulin immunofluorescence intensity in the M I spindles of Kif2a-knockdown oocytes was significantly higher compared to that in the control group,indicating that Kif2a destabilized the spindle microtubules in meiosis through microtubule depolymerization.2 Kif2a regulates the mouse oocyte meiosis maturation process:Kif2a depletion by siRNA microinjection led to significant pro-M I/M I arrest,failure of first polar body(PB1)extrusion and increases asymmetric division.We employed chromosome spreading to confirm whether the chromosomes could segregate correctly.Oocytes in both the Kif2a knockdown group and the control group were cultured for 12 h.Chromosomes were still bivalents in the Kif2a knockdown group;in contrast,univalent chromosomes were observed in the control group,indicating completion of frst meiosis.Kif2a-depleted oocytes were also defective in spindle pole localization of y-tubulin and showed spindle assembly checkpoint(SAC)protein Bub3 at the kinetochores even after 10 hr extended culture.Knockdown of Kif2a activates SAC and increase of cyclin B1 level:To further explore the cause for the Pro-M I/MI arrest we analyzed the localization of Bub3,an important component of SAC.Specific signal of Bub3 was detected on chromosome kinetochores in the Kif2a-knockdown oocytes at 10 h after IBMX washout,while the control oocytes entered anaphase without signals of Bub3 on kinetochores.Detection of Bub3 signal indicated that the spindle assembly checkpoint was activated in the Kif2a-depleted oocytes.Cyclin B1 degradation and MPF in activation are essential in regulating the exit from the M-phase.Western blot analysis showed that the expression level of cyclin B1 was much higher in the knockdown group compared to the control group at 10 h of culture in IBMX-free medium,suggesting the lack of degradation of cyclin B1 in the knockdown group,and thus failure of anaphase entry.Effect of Kif2a knockdown on actin cap formation in mouse oocytes meiosis:Oocytes from the Kif2a knockdown group and the control group were cultured in IBMX-free M2 medium for 8.5h,then stained with Phalloidin-FITC.We can clearly observed actin cap in the control group with peripherally localized spindle.There was no actin cap in Kif2a knockdown oocytes with centrally localized spindle.Conclusion:Kif2a may act as a microtubule depolymerase and MTOC-associated protein,regulating microtubule dynamics,spindle assembly and migration,chromosome congression,actin cap formation and frst polar body(PB1)extrusion.Kif2a-depleted oocytes were also defective in spindle pole localization of y-tubulin and showed SAC activation and increase of cyclin B1 level,which led to signifcant pro-M I/M I arrestKif2a is a member of the Kinesin-13 microtubule depolymerases.Here,we report the expression,subcellular localization and functions of Kif2a during mouse oocyte meiotic maturation.Immunoblotting analysis showed that Kif2a was gradually increased form GV to the M I stages,and then decreased slightly at the M II stage.Confocal microscopy identifed that Kif2a localized to the meiotic spindle,especially concentrated at the spindle poles and inner centromeres in metaphase and translocated to the midbody at telophase.Kif2a depletion by siRNA microinjection generated severely defective spindles and misaligned chromosomes,reduced microtubule depolymerization,which led to signifcant pro-M I/M I arrest and failure of frst polar body(PB1)extrusion.Kif2a-depleted oocytes were also defective in spindle pole localization of y-tubulin and showed spindle assembly checkpoint(SAC)protein Bub3 at the kinetochores even after 10 hr extended culture.These results demonstrate that Kif2a may act as a microtubule depolymerase,regulating microtubule dynamics,spindle assembly and chromosome congression,and thus cell cycle progression during mouse oocyte meiotic maturation. |
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