| As highly specialized female germ cells,mammalian oocytes are the progenitor cell of ontogenesis.The quality of matured oocyte is directly related to embryonic development capacity after fertilization.Oocytes in vitro maturation are the basis of a series of reproductive technologies,including in vitro fertilization,in vitro production of embryo and embryo transfer.However,the maturation efficiency,blastocyst developmental rate and pregnancy rate during in vitro maturation are low compared to that of in vivo maturation of oocyte.The main reasons are dyssynchrony of nuclear and cytoplasmic maturation,inefficient cytoplasmic maturation and deficient of in vitro maturation system.Futhermore,the molecular mechanism of oocyte maturation processes remains elusive.Competence of maturation,fertilizaiton and embryonic development are acquired during the period of oocyte growth and maturation.Therefore,proteomics technology is applied to study the dynamic change rule of protein anabolism during in vitro maturation of oocyte,which will contribute to discover the molecular mechanism of oocyte maturation,and it is great helpful to improve the oocyte quality of in vitro maturation.In this study,local characteristic buffalo was selected as the object,iTRAQ quantitative proteomics technology was applied to compare the proteome of immature and mature oocytes,normal cytoplasm and abnormal cytoplasm M?oocytes during in vitro maturation,and the proteome expression profile of immature and mature buffalo oocytes were constructed.The function and biological pathways of the proteins were studied by bioinformatics analysis tools,and the key proteins related to buffalo oocyte maturation were confired,which would furthermore discover the molecular mechanism of buffalo oocyte during in vitro maturation.First,we applied one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with liquid chromatography tandem mass spectrometry to construct the proteome expression profile of immature and mature buffalo oocytes.We identified 647(FDR<1%)and 570(FDR<1%)proteins from immature and mature buffalo oocytes,respectively.414 proteins of which were common identified,representing 51.6%of the total identified proteins.Nine abundance proteins were identified in the two cell type,8 of which were the same between two groups,including major vault protein,glutathione S-transferase mu3,predicted:protein-arginine deiminase type-6,peroxiredoxin-2,nucleoside diphosphate kinase B,predicted:dynein light chain 1,aldose reductase,predicted:glyceraldehyde-3-phosphate dehydrogenase-like isoform2.Bioinformatics analysis revealed that the common identified proteins between immature and mature oocytes were involved in more than 23 metabolism pathways,such as pyruvate metabolism,glycolysis,citrate cycle,valine,leucine and isoleucine degradation,propanoate metabolism,fatty acid metabolism and pentose phosphate pathway.It revealed the critical role of the pathways relating to energy metabolism process during buffalo oocyte maturation,which provided energy source for oocytes maturation and completion of meiosis.The unique identified proteins of immature oocytes were related to oxidative phosphorylation,ribosome,and proteasome pathway(p<0.05).The unique identified proteins of mature oocytes were related to DNA replication,amino sugar and nucleotide sugar metabolism pathway(p<0.05).The results demonstrated that some proteins expressed through the whole period of oocyte maturation,which suggesting their important roles during maturation.In addition,some unique proteins expressed at different development stage,and maintained during oocytes,growth and development.Second,the proteomes of immature and mature oocytes,normal cytoplasm and abnormal cytoplasm M? oocytes were quantitatively analyzed by iTRAQ-based proteome technology.A total number of 3857(FDR<1%)proteins were identified and 3245 proteins of which were quantified.In total,3287 proteins had more than 2-peptide,representing 85%of the total identified proteins.A total of 173 proteins were differentially expressed between immature and normal cytoplasm M? oocytes,and 108 proteins were up-regulated and 65 proteins were down-regulated.Totally,146 proteins were differentially expressed between normal cytoplasm and abnormal cytoplasm M? oocytes,and 111 proteins were up-regulated and 35 proteins were down-regulated.Bioinformatics analysis of the differentially expressed proteins between immature oocytes and normal cytoplasm M? oocytes revealed that up-regulated proteins involved in biological processes,including microtubule-based process,protein transport,cellular protein catabolic process and cell cycle.Bioinformatics analysis of the differentially expressed proteins between normal cytoplasm and abnormal cytoplasm M? oocytes revealed that up-regulated proteins involved in biological processes,including oxidation reduction,translation,protein transport,oxidative phosphorylation,and small GTPase mediated signal transduction.Compared to GV with abnormal cytoplasm M?oocytes,key kinases involving in electron respiratory chain of oxidative phosphorylation signal pathway were significant up-regulated in normal cytoplasm M? oocytes.It was reported that the level of oxidative phosphorylation strongly influenced the oocyte quality,and was an important aspect of meiotic and developmental competence in buffalo oocyte.It also indicated that the energy requirement was needed during oocyte maturation,which also reflected that the metabolic was needed for the oocytes in preparation for following fertilization.During the above research processes,we found that low molecular weight(LMW),low abundance proteins which performed important phosiological function,their signal were interfered and inhibited by high abundance proteins using mass spectrometer analysis,resulting in these LMW and low abundance proteins would't be identified.So we developed a "Gel-filter" method to efficiently separate and enrich LMW,low abundance proteins and remove high abundance proteins.The result showed that,the density of LMW protein bands increased with the increase of serum sample amount,indicating the increase of protein amount.And the relative abundance of proteins was totally identical after the treatment of gel-filter method.Furthermore,comparison with other three common approaches used MS.A total of 559(FDR<1%)serum LMW proteins were identified from gel-filter method,which were the highest one among the four approaches.The number of LMW proteins and peptides identified with Gel-filter method was the highest one in each molecular weight range bin.We categorized common identified proteins based on their theoretical molecular weight and relative abundance represented by their spectral counts in mass spectrometry analysis.As expected,the Gel-filter method performed relative broad abundance distribution on all of the molecular weight range bins.In addition,the higher sequence coverage of LMW proteins was obtained by Gel-filter method.In order to evaluate the recovery rate of LMW proteins with different strategies treatment,the protein Glutathione-S-transferase(GST)which was expressed in the light stable isotope labeled E.coli,was spiked in serum sample and dealt with different approaches.Mass spectrometer analysis indicated that Gel-filter method performed higher recovery rate of GST protein,with 33.1 ± 0.01%against 18.7 ± 0.01%and 9.6 ± 0.03%for differential solubilization(DS)and ProteoMiner methods,respectively.Finally,Gel-filter method was proved to be as a simple,rapid,efficiently and good reproducibility method and a useful tool for further proteomics research. |
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