Role And Mechanisim Of Aurora-A During Meiosis In Porcine Oocytes

Mammalian oocyte maturation should undergo certain multi-stage and accurate events,which molecular regulation is one of the important themes of life science research.In recent years,it gained great advances in the aspect of protein relating to regulate oocyte maturation.Aurora kinase is widely distributed in eukaryotic organisms,involved in regulation of mitosis.In mammals,three Aurora kinase homologous proteins have been identified:Aurora-A,-B and-C respectively.They have similar sequence structure,with a highly conservative C-terminal and quite different length sequence of N-terminal.The rest of the sequence is not conservative,so these three homologous proteins have different subeellular localization,activation and substrate.Aurora-A mainly accumulate around centrosome and spindle.Aurora-A kinase is mainly regulates centrosome maturation and segregation,entry into mitosis,chromosomes alignment,formation and function of the bipolar spindle,cytokinesis and so on.It is generally known that meiotic spindle assembly,ehromosome alignment and separation are different with mitosis.Therefore,it has great theory significance and utility value to clarify the regulatory mechanisms of Aurdra-A in oocyte maturation for deepening the cognitive of regulation of meiosis,reavling the reproductive regularity of mammalian and even improving the rate of oocytes maturation.Although there have been achievements in the study of Aurora-A in regulation of oocyte meiosis process and related signal pathways in lower animals(such as Xenopus and Drosophila),the function and mechanism in mammalian oocyte meiosis is less developed.Previous reports are mainly concentrated on mouse and bovine oocytes.However,there are many unknown about the function and mechanism of Aurora-A in porcine oocytes meiosis process.In vitro culture,western blot and immunofluorescent staining was applyed to detect the dynamic expression and subcellular localization of Aurora-A during porcine oocyte maturation.Base on this,Aurora-A specific inhibitors MLN8054 was used in the inhibition test to analyse the influence of Aurora-A on porcine oocyte maturation and its downstream phosphorylation-TACC3 expression,looking forward to put further insight in the molecular signal pathways of Aurora-A for the regulation of porcine oocytes meiosis,and in order to laying the theoretical foundation for more deeply understanding the molecular regulation mechanism during mammalian oocytes maturation.The main results are as follows:Experiment 1.Detection of Aurora-A protein expression during pig oocyte meiosisWe collected each period of meiosis of oocytes and assayed the expression of Aurora-A by western blot.It was shown that Aurora-A kinase was present in each periods of porcine oocyte maturation,and the quantity of this protein in GV(germinal vesicle)phases was higher than that in other meiotic maturation stages.The quantity of this protein decreased after GV,and increased at 44 h.Experiment 2.Aurora-A colocalization with a-tubulin in porcine oocytes maturationAs shown by immunofluorescence,Aurora-A mainly concentrated around the chromosomes and distributed uniformly at the germinal vesicle in GV oocytes.After GVBD(germinal vesicle breakdown),Aurora-A mainly accumulated at the microtubules of meiotic spindles,migrated along with spindles migration and most at spindle pole.Aurora-A concentrated at pbI and spindles of porcine oocytes until MII.Experiment 3.MLN8054 affects porcine oocyte maturation and meiosis processThe expansion was progressively weaker and the rate of oocyte maturation was obviously inhibited after treatment with Aurora-A specific inhibitors MLN8054.The Pro-MI rate of treatment groups was significantly increased(P<0.05),and the MI and AI-TI rate of MLN8054 treatment was significantly lower as compared with controls(P<0.05).Indeed,there were no oocytes at AI-TI stage,when MLN8054 concentration reached to 5?M.Experiment 4.MLN8054 affects spindle assembly and chromosome alignment in porcine oocytesWe observed spindle morphology and chromosomes alignment and counted the abnormal rates.For controls,chromosomes are arranged on the equatorial plate,and spindle morphology presented typical spindle-shaped of porcine oocyte,However,spindle morphology appears abnormal and were hardly seen in certain oocytes after treatment with MLN8054,concomitantly,the chromosomes irregular arranged.Experiment 5.Disruption of Aurora-A impacts the subsequent embryonic development of pig oocytesThe results showed that there was no significant difference of the rates of embryos cleavage between control group and treatment with MLN8054.However,the rates of embryos cleavage were gradiently reduced compared with control.In addition,the rates of embryos blastocyst were signifitanly reduced treatment with 3 or 5?M MLN8054,and gradiently reduced along with the increase of MLN8054 concentration.These results showed that its embryo development potential was inhibited after Aurora-A inhibition.Experiment 6,Effects of MLN8054 on p-Aurora-A expressionCompared with control group,p-Aurora-A expression in porcine oocytes was significantly reduced after MLN8054 treatment.The relative intensity of p-Aurora-A protein expression(p-Aurora-A/GAPDH)comfirmed the significantly decresed in p-Aurora-A protein expression(0.36/1;P<0.05).Experiment 7.Effects of MLN8054 on TACC3 localization and expressionp-TACC3 is present in each periods of porcine oocyte maturation.Its expression was significantly reduced after MLN8054 treatmentat any stage,and was most significantly decreased at 20h.Fluorescence staining results showed that p-TACC3 primarily accumulated at the spindle poles in control MI oocytes,while inhibiting Aurora-A activity resulted in the failure of p-TACC3 specific localization,as only a few signals were detected on spindle microtubules.