The Change And Associated Mechanism Of Kinetochore During Oocyte Aging

Oocyte meiosis is an extremely complicated cell division process,including the formation of acentrosomal spindle microtubule,homologous chromosome segregation during meiosis I and sister chromatids segregation during meiosis II.In particular,the occurrence of chromosome segregation error during meiotic division increases with advanced maternal age,and may lead to the generation of egg with an incorrect number of chromosomes,a condition termed as aneuploidy.When an aneuploid egg is fertilized by a sperm,it will give birth to an aneuploidy embryo and may eventually end in a spontaneous abortion,which is the predominant cause of age-associated decline in mammalian female fertility.Several lines of evidence indicate that age-related changes that contribute to aneuploidy are multifactorial.Most well characterized age-associated changes that predispose chromosome mis-segregation are the progressive loss of chromosomal cohesion during maternal aging,age-related defects in spindle microtubule dynamics and mitochondrial dysfunction.Kinetochore is the proteinaceous complex that assembles on centromeric nucleosome,and transfer the force from microtubule dynamic by interacting with spindle microtubule to direct chromosome movement.However,remarkably little is known about the effect of kinetochore on chromosome segregation during oocyte aging.Therefore,in this study,we try to investigate the assembly dynamic of kinetochore during CD1 mouse oocyte meiosis I and the underlying effect of kinetochore on chromosome segregation during oocyte aging using immunofluorescence,microinjection,oocyte enucleation and cell fusion.The major results of this study were as follows:1.The assembly dynamic of kinetochore during oocyte meiosis IImmunofluorescence revealed that CENP-A,CENP-C and CENP-T stably localized on the centromere in oocyte from GV-stage to the completion of meiosis I.In contrast,at GVstage,Ndc80 was excluded from centromere.Once GVBD,Ndc80 was rapidly recruited on the centromere and consistently localized during the subsequent divison process.In addition,the expression of four proteins at the centromere all exhibited a dynamic trend.2.Aurora kinase regualted the assembly dynamic and maintenance of kinetochore during meiosis IFirst,treated GV-stage oocyte with different concentration of ZM447439(Ctrl、2.5and 5 μM).The results revealed that ZM447439 treatment strongly suppressed Ndc80recruitment(p < 0.0001)in a dose-dependent manner.ZM447439 addition had no impact on the expression of CENP-A.However,ZM447439 treatment significantly promoted the expression of CENP-C and CENP-T(p < 0.01).Secondly,treated oocyte which already went GVBD with higher concentration of ZM447439(Ctrl、5 and 10 μM),the results showed that 10 μM ZM447439 stimulation could remarkably inhibit the expression of Ndc80(p <0.0001)and CENP-A(p < 0.05).However,it showed no effect on the expression of CENP-C and CENP-T.Moreover,Monastrol(200 n M)and Nocodazole(1 μM)treated oocyte,respectively.The results showed that both treatment had no effect on the expression of Ndc80.Finally,Roscovitine treatment(Ctrl、12.5 、25 and 50 μM)displayed no impact on recruitment and maintenance of Ndc80.3.Oocyte aging could destroy kinetochore architectureComparing to young oocyte,the expression of CENP-A,CENP-C,CENP-T and Ndc80 all reduced remarkably in aged oocyte(p < 0.0001,p < 0.0005,p < 0.01 and p < 0.0005).Moreover,similar results were obtained after treatment with APCin(100 μM)synchronizing cell cycle.In addition,relative to the young oocyte,INCENP phosphorylation level in aged oocytes slightly decreased without significant difference.Finally,comparing the four proteins expression,respectively,between fused sister kinetochores and separated sister kinetochores in aged oocytes,the results revealed that they were identical in different sister kinetochores geometry.4.Centromere weakness in aged oocytes led to kinetochore assembly defectAnalysing by Pearson method,there existed strong correlation between Ndc80 and CENP-A in young oocyte(r = 0.416).Using oocyte enucleation and cell fusion,reconstructed three types of hybrid oocytes,that are YN-YC,YN-AC and AN-YC.The results displayed that CENP-A was identical between hybrid oocytes(YN-YC and YN-AC),but showed strongly reduction in hybrid oocyte(AN-YC)(p < 0.0001).Similar to CENP-A,Ndc80 also showed significantly decrease in hybrid oocyte(AN-YC)(p < 0.0001),relative to YN-YC and YN-AC hybrid oocytes.Moreover,we generated another two types of hybrid oocytes,that are single oocytes containing two GVs,one from a young oocyte and one from an aged oocyte,both within the same cytoplasm,either in young cytoplasm or aged cytoplasm.Consistent with previous data,remarkably reduced recruitment of Ndc80 on chromosomes from the aged oocytes was observed,when compared to chromosomes from young oocytes.Importantly this effect was the same regardless of whether the cytoplasm of the reconstructed oocyte was young(p < 0.0005)or aged(p < 0.01).5.Kinetochore function was impaired in aged oocyteAfter APCin(100 μM)pre-treatment,Nocodazole(1 μM)and Monastrol(200 n M)treatment shortened the distance between CENP-A protein and Ndc80 protein,or CENP-C protein and Ndc80 protein,indicating there exists stretch function in kinetochore architecture.Then the result displayed that the distance between CENP-A protein and Ndc80 protein increased gradually along the progression of meiosis I.The distance between CENP-C protein and Ndc80 protein increased dramatically when closely entering anaphase.The result showed that there was a strong reduction in the distance between CENP-A protein and Ndc80 protein(p < 0.0001),or CENP-C protein and Ndc80 protein(p < 0.05)in aged oocytes,relative to young oocytes.In addition,the results showed that the profile of correct kinetochore-microtubule(KT-MT)attachments was substantially decreased in aged oocytes.Moreover,kinetochore-fibers(K-fibers)in aged oocytes were significantly thinner than those in young oocytes(p < 0.01).With APCin(100 μM)treatment synchronizing cell cycle,same results were also obtained.Finally,created three types of hybrid oocytes,that are YNYC,YN-AC and AN-YC,and performed cold-shock,the resultes revealed that nuclear aging is the primary cause leading to the aberrant KT-MT attachments in aged oocytes.Altogether,the assembly of kinetochore in oocyte begins upon meiosis I resumption,and kinetochore assembly and its maintenance rely on the activity of Aurora kinase.Moreover,kinetochore structure undergoes deterioration with advanced female age,and this phenomenon is a result of a nuclear aging that leads to a weakened centromere.Additionally,in aged oocytes,the stretch of kinetochore architecture attenuates,and the capability of KTMT interaction decreased,and the nuclear aging is the primary cause for the alteration of KT-MT attachment status.This study comprehensively and systematically investigates the assembly dynamics of kinetochore during oocyte meiosis I,as well as the deterioration in kinetochore architecture and function with oocyte aging,providing a novel theoretical basis for clarifying the associated chromosome mis-segregation and aneuploidy with advanced female mammal age and ensuring an instructive reference significance for extending the breeding and reproduction years of livestock in the animal husbandry industry.