成熟前后水牛卵母细胞蛋白质组的初步研究

Proteomics Analysis of Immature and Mature Buffalo Oocytes

应用聚丙烯酰胺凝胶电泳联合反相液相色谱-串联质谱(RP-LC-MS/MS)蛋白质组学技术,研究GV期和MII期水牛卵母细胞蛋白质组,以期为揭示水牛卵母细胞体外成熟分子机理、筛选与卵母细胞成熟质量相关蛋白质奠定理论基础。从GV期和MII期水牛卵母细胞中分别鉴定到647和570种蛋白质(FDR<1%)。其中,鉴定相同蛋白质414种;GV期和MII期卵母细胞特有鉴定蛋白质分别为233种和156种。通过谱图计数法获得鉴定蛋白质的半定量信息,从GV期和MII期水牛卵母细胞中筛选到9种高丰度蛋白质。其中8种为相同蛋白质,包括穹窿体蛋白、谷胱苷肽S-转移酶M3、蛋白精氨酸脱亚氨酶6、过氧化物氧化还原酶2、二磷核苷酸激酶B、动力蛋白轻链1、醛糖还原酶、类甘油醛-3-三磷酸脱氢酶同源物2等。热激蛋白90α和谷胱甘肽S-转移酶P分别为GV期和MII期水牛卵母细胞特有的高丰度蛋白质,这些蛋白质可能与水牛卵母细胞成熟过程密切相关。GO(Gene Ontology)和KEGG分析显示,鉴定相同蛋白质主要集中在丙酮酸盐代谢、三羧酸循环(TCA)、糖酵解/糖原生成、磷酸戊糖途径等能量代谢通路,提示水牛卵母细胞成熟过程中可能需要维持高的能量代谢水平,以保证卵母细胞减数分裂的完成。GV期细胞特有鉴定蛋白质主要集中在氧化磷酸化、核糖体以及蛋白酶体等KEGG通路,表明GV期卵母细胞可能具有高的氧化磷酸化和蛋白合成水平,蛋白酶体在推动水牛卵母细胞成熟进程中可能发挥重要作用。MII期细胞特有鉴定蛋白质主要集中在DNA复制和氨基糖、核苷糖代谢等KEGG通路,提示水牛卵母细胞可能在为后续的受精过程和早期卵裂进行遗传物质的准备。综上表明,成熟前后水牛卵母细胞蛋白质表达模式,为进一步深入了解水牛卵母细胞成熟分子机理奠定理论基础。

To reveal the molecular mechanism underlying in vitro maturation of the buffalo oocyte and select the proteins associated with the oocyte maturation quality,sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled to reverse-phase liquid chromatography tandem mass spectrometry（RP-LC-MS/MS）proteomics technology were applied to identify the buffalo oocyte proteome at germinal vesicle（GV）stage and metaphase II（MII）stages.In total,647 and 570pro-teins（FDR〈1%）were identified from GV and MII stage buffalo oocytes,respectively.Among them,414 proteins were common identified,233 and 156proteins were uniquely identified,respectively.The semi-quantitative information of the identified proteins was obtained using spectral counting method.Nine high abundance proteins were identified from GV and MII stage buffalo oocytes,eight of which were common proteins,including MVP,GATM3,PADI6,PRDX2,NE2,DYNLL1,AKR1B1 and LOC786101.HSP90αand GSTP1 were unique high abundance proteins identified in GV and MII stage buffalo oocytes,respectively.It indicated that they might be closely related to buffalo oocyte maturation.Results of GO（Gene Ontology）and KEGG analysis showed that these common identified proteins involved in metabolism pathways,such as pyruvate metabolism,glycolysis,tricarboxylic acid cycle（TCA）,and pentose phosphate pathway,etc,which suggested that high metabolism level was needed to complete meiotic maturation in buffalo oocytes.Proteins involved in oxidative phosphorylation,ribosome,and proteasome pathway were uniquely identified in GV stage oocytes,indicating that high level of oxidative phosphorylation and protein synthesis might exist in GV stage oocyte,and the proteasome pathway might be essential for the maturation progression of buffalo oocytes.Proteins involved in DNA replication,amino sugar and nucleotide sugar metabolism pathway were uniquely identified in MII stage oocytes,demonstrating that the oocytes might need to prepare genetic material for the fertilization and early cleavage divisions.Overall,above results revealed the protein expression profile of buffalo oocytes before and after maturation,they are useful to understand the molecular mechanisms underlying buffalo oocyte maturation.