Vitale Alejandra M, Calvert Meredith E Kennedy, Mallavarapu Mallika, Yurttas Piraye, Perlin Julie, Herr John, Coonrod Scott
Weill Medical College of Cornell University, New York, New York 10021, USA.
Mol Reprod Dev. 2007 May;74(5):608-16. doi: 10.1002/mrd.20648.
In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin alpha2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin alpha2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage.
为了更好地理解卵母细胞的功能，我们利用二维（2D）电泳和质谱技术来鉴定在小鼠卵母细胞成熟过程中差异表达的蛋白质。从500个生发泡（GV）期和中期II（MII）期停滞的卵母细胞中提取蛋白质，在2D电泳凝胶上进行分离，并用银染色。凝胶分析表明，有12种蛋白质在GV期和MII期之间似乎存在差异表达。然后从2D凝胶上切下这些蛋白质条带，通过质谱鉴定为：转化酸性卷曲螺旋蛋白3（TACC3）、热休克蛋白105（HSP105）、程序性细胞死亡6相互作用蛋白（PDCD6IP）、应激诱导磷蛋白（STI1）、输入蛋白α2、腺苷酸琥珀酸合酶（ADDS）、Nudix、纺锤体相关蛋白、脂质运载蛋白、溶菌酶、翻译控制肿瘤蛋白（TCTP）和核质蛋白2（NPM2）。有趣的是，与GV期卵母细胞凝胶相比，PDCD6IP、输入蛋白α2、纺锤体相关蛋白和NPM2在MII期卵母细胞凝胶上的分子量似乎略大且酸性更强，这表明它们可能在卵母细胞成熟过程中发生了翻译后修饰。鉴于NPM2是一种卵母细胞特异性蛋白，我们选择进一步研究其在卵母细胞成熟和植入前发育过程中的特性。实时RT-PCR显示，受精时NPM2 mRNA水平迅速下降。间接免疫荧光分析表明，除了在MII期停滞的卵母细胞中定位于皮质外，NPM2在GV期卵母细胞和植入前胚胎的所有阶段均定位于细胞核。然后我们对小鼠卵母细胞和植入前胚胎进行了一维（1D）蛋白质印迹分析，发现正如2D凝胶比较所暗示的那样，NPM2在从GV期到MII期的转变过程中经历了磷酸酶敏感的电泳迁移率变化。迁移较慢的NPM2形式也存在于原核胚胎中，但到二细胞期，大多数NPM2以迁移较快的形式存在，并持续到囊胚期。
