The Function And Mechanism Of Nucleoporin Nup35 And Rae1 During Mouse Oocyte Meiotic Maturation

During the interphase of eukaryotic cells,NPC is embedded in the nuclear envelope and participates in nucleocytoplasmic traffic of diverse molecules.One functional NPC structure is normally composed of 30 kinds of proteins termed Nups.When vertebrate cells enter the open mitotic phase,NPCs and nuclear membrane disassemble during prophase and Nups are distributed within the cytoplasm.In the past years,Nups were thought to be dormant from the nuclear membrane breaking down to the nuclear membrane reformation.However,in recent years,more and more research has showed that Nups are also involved in mitotic progress,including spindle assemble,chromosome alignment,K-MT attachments and SAC activity.Compared with mitosis,oocyte meiosis is more cline to defects of spindle assembly and chromosome segregation,while the molecular mechanism of oocyte meiosis is not deeply explored,this study investigated the role and mechanism of Nups during meiotic maturation.Nup35 and Rae1 are key components of Nups,participating in spindle architecture,chromosome alignment and SAC activity during mitosis.In this study,in vitro maturation of mouse oocytes was used as the research model,small RNA interference technology was used to knock down Nup35 or Rae1,immunofluorescence and confocal,immunoblotting,Real-time PCR and chromosome spread were used to explore the sublocation and protein expression pattern,the function and mechanism of Nup35 and Rae1 during meiotic maturation process of mouse oocyte.1.The function and mechanism of Nup35 in oocyte meiotic maturation process(1)The immunofluorescent staining of Nup35 was concentrated in the rim of nuclear membrane at GV stage,and at pro-MI,MI and MII stages,Nup35 is located at spindle architecture,but transferred to the spindle poles at AI and TI stages.Moreover,the immunofluorescent signal of Nup35 was almost disappeared when the microtubules were completely depolymerized.The immunoblotting result showed that Nup35 was evenly expressed at GV,GVBD,MI and MII stage;however,Nup35 existed as a modified form after GVBD stage.(2)Nup35 was knocked down by microinjection of Nup35 specific si RNA into GVstage oocytes and following in vitro maturation.Knockdown of Nup35 significantly compromised the extrusion of first polar body(P < 0.01)but not GVBD(P > 0.05).Nup35 interference led to abnormal spindle assembly(P < 0.01),including no spindle,no-pole spindle and small spindle.Moreover,MI plate width was enormously elevated(P< 0.001),accompanying with high rate of misaligned chromosome(P < 0.001)in the Nup35 RNAi oocytes.In addition,knockdown of Nup35 impaired K-MT attachments(P< 0.001).In conclusion,during meiotic maturation process of mouse oocyte,Nup35 was involved in PBE,spindle assembly and chromosome alignment process.(3)This study examined the ?-tubulin and p-ERK1/2 localization patterns by immunofluorescent staining.The results showed that knockdown of Nup35 resulted in no significant abnormality in ?-tubulin signaling;however,it caused abnormal localization of p-ERK1/2 signaling,disconnected from the spindle pole to the cytoplasm.This study also found that SAC was activated in Nup35 knockdown oocytes.In brief,Nup35 regulated the localization pattern of p-ERK1/2 and SAC activity.2.The function and mechanism of Rae1 in oocyte meiotic maturation process(1)The immunofluorescent staining result showed that Rae1 was concentrated in the nuclear membrane at GV stage,and then translocated to kinetochore structure after resumption of meiosis.The immunoblotting result verified that Rae1 had a comparable expression level at GV,MI and MI stage(P > 0.05),and a higher expression level at MII stage(P < 0.01).(2)The data showed that interference of Rae1 by specific si RNA impaired the progress of GVBD(P < 0.05)and PBE(P < 0.05).But unexpectedly,after GVBD,compared to the control oocytes,the PBE rate had no significant difference in the Rae1-knockdown oocytes(P > 0.05).Moreover,the misaligned chromosomes(P < 0.01),weakened K-MT attachments(P < 0.01)and high rate of aneuploidy(P < 0.01)were detected in Rae1-knockdown oocytes.In brief,Rae1 participated in GVBD,chromosome alignment and aneuploidy process.(3)The protein levels of Cyclin B1,Cyclin B2 and CDC25 B at GV stage oocytes were investigated by immunoblotting.The data displayed that there was without significant difference in the protein level of Cyclin B1,Cyclin B2 and CDC25B(P > 0.05)between control and Rae1 knockdown groups.The protein levels of Cyclin B1,Cyclin B2 and Securin in MI oocytes were examined by immunoblotting.The result showed that knockdown of Rae1 did not alter Cyclin B1 and Cyclin B2 protein levels(P > 0.05),but Securin was significantly reduced(P < 0.01).Moreover,Securin was not altered at the m RNA level in the Rae1 knockdown oocytes(P > 0.05).In addition,localization patterns of Bub R1 and Mad1 were not altered after Rae1 knockdown.Conclusion: Nup35 regulated p-ERK1/2 localization patterns and SAC activity,thus involving in spindle assembly,chromosome alignment and PBE process.Rae1 participated GVBD progression,moreover,Rae1 was involved in chromosome alignment and aneuploidy by modulating protein level of Securin.