The Function And Regulation Of Microtubule Severing Protein And ZP3 In Meiosis And Fertilization Of Mouse Oocytes

Microtubule-severing proteins(MTSPs)play important roles in mitosis and interphase;however,no studies have assessed the role of MTSPs in meiosis in mammalian females.Thus,we examined the expression and localization of all seven MTSPs in mouse oocytes obtained using in vitro maturation.Notably,KL2 is the only MTSP that localized at chromosomes,suggesting that it may be important for chromosome-based spindle organization and meiosis.To test our hypothesis,we showed that KL2 knockdown with siRNA significantly diminished microtubule(MT)nucleation,increased aberrant kinetochore-microtubule(K-MT)attachment,significantly delayed meiosis,and severely affected normal fertility.To further investigate KL2 functions,we completed three key experiments.First,inhibition of aurora B,the key kinase for correcting aberrant K-MT attachment,completely abolished KL2 from chromosomes,indicating that active aurora B may be important for the unique chromosomal localization of KL2.Second,we characterized phosphorylated eukaryotic elongation factor 2 kinase(p-EEF2K).P-EEF2K and KL2 interact with each other and a reduction in either of these two significantly increased the expression of the other by approximately 50%,indicating that KL2 may compete with p-EEF2K for chromosome binding.Indeed,KL2 and p-EEF2K co-immunoprecipitated with H3.Third,we made a phospho-specific antibody to characterize a phosphorylation site at the N-terminus of KL2.We found that phosphorylated KL2 was mainly localized at spindle poles,indicating that phosphorylation can dislocate a small portion of KL2 from chromosomes.This study is the first to characterize the unique functions of KL2 in female meiosis and establish that multiple kinases coordinate to regulate KL2 levels at chromosomes.It was also found that another microtubule severing protein,Fidgetin,was strongly clustered on the zona pellucida of oocytes,which was also significantly different from that of other members of the same family.When the specific siRNA was used to knock down Fidgetin,there was a significant abnormal fertilization of oocytes,which showed that the proportion of multiple nuclei in fertilized eggs was significantly higher.According to the fertilization mode,when the first sperm penetration of zona pellucida and contact the oocyte membrane,will stimulate the cortical reaction and zona reaction,followed by egg membrane and the zona pellucida hardening,thus preventing the second sperm penetration of zona pellucida.Since the first sperm penetration process takes a certain amount of time(more than 30 minutes),so the model can not explain in the period the first sperm penetration through the zona pellucida,no the second sperm penetration into the zona pellucida.Since Fidgetin is clustered in the zona pellucida,the absence of Fidgetin in oocytes is likely to result in multipronuclear zygotes,and we speculate that Fidgetin may play a role in preventing the penetration of multiple sperm.We detected the interaction of Fidgetin with several zona pellucida proteins by mass spectrometry.Moreover,it was found that in the initial stage of fertilization,the hexamer of Fidgetin in zona pellucida increased obviously.Because the microtubule severing protein need to form hexamer to function,so the results are consistent with our speculation.That is,when the sperm penetrates the zona pellucida,it activates the Fidgetin monomer in the region,which makes it form a six polymer and prevents the penetration of the second sperm.In addition,we found that inactivation of Fidgetin(phosphorylation)requires Erk1/2 regulation.While Erkl/2 is the key enzyme in sperm acrosome reaction,we speculate that Fidgetin may prevent the second sperm from penetrating the zona pellucida by competing the key enzyme with sperm.This study is the first to investigate the role of Fidgetin in oocyte fertilization,and may provide important supplements and modifications to existing fertilization patterns.ZP3 is a principal component of the zona pellucida(ZP)of mammalian oocytes and is essential for normal fertility,and knockout of ZP3 causes complete infertility.ZP3 promotes fertilization by recognizing sperm binding and activating the acrosome reaction;however,additional cellular roles for ZP3 in mammalian oocytes have not been yet reported.In the current study,we found that ZP3 was strongly expressed in the nucleus during prophase and gradually translocated to the ZP.Knockdown of ZP3 by a specific siRNA dramatically inhibited germinal vesicle breakdown(GVBD)(marking the beginning of meiosis),significantly reducing the percentage of M?oocytes.To investigate the ZP3-mediated mechanisms governing GVBD,we identified potential ZP3-interacting proteins by immunoprecipitation and mass spectrometry.We identified Protein tyrosine phosphatase,receptor type K(Ptprk),Aryl hydrocarbon receptor-interacting protein-like 1(Aipl1),and Diaphanous related formin 2(Diaph2)as potential candidates,and established a working model to explain how ZP3 affects GVBD.Finally,we provided preliminary evidence that ZP3 regulates Akt phosphorylation,lamin binding to the nuclear membrane via Aipl1,and organization of the actin cytoskeleton via Diaph2.These findings contribute to our understanding of a novel role played by ZP3 in GVBD.