冷冻猪GV期卵母细胞体外成熟后蛋白质组差异及调控策略研究

Porcine oocytes vitrified at the GV stage have been demonstrated to can maintain their normal ability to undergo in vitro maturation and fertilization; however, the embryo development of these cells is still low. The maternal proteins stored in oocytes have critical roles in embryonic development. The lower developmental competence of vitrified oocytes is probably due to abnormal maternal proteins. At present, few studies on this area have been carried out and focus only on one or more proteins. It is difficult to systematically elucidate the changes of constitute and function of proteins in vitrified oocytes. Therefore, protein expression changes of MII oocytes and cumulus cells derived from vitrified porcine GV oocytes are analyzed in this study using iTRAQ combined with the 2D LC-MS/MS analysis method, and the important proteins that determine the quality of vitrified oocytes after maturation are found. On the other hand, the vitrified porcine GV oocytes are treated with proteasome inhibitor MG132 during the late of maturation. In addition to the assessment of proteome, the maturation and developmental competence of oocytes are measured for confirming the beneficial effects of MG132 treatment. These findings explain the poor development of vitrified porcine GV oocytes based on proteome level and provide an available solution, and it possess theoretical and practical significance for further study and development of the oocytes vitrification technique.

猪GV期卵母细胞冷冻后虽然保有正常的减数分裂和体外受精能力，但胚胎发育却不尽如人意。卵母细胞储备的母源蛋白质对早期胚胎发育至关重要，冷冻可能导致这些蛋白异常表达，成为冷冻卵母细胞发育潜力下降的主要原因。目前这方面研究较少，并且集中于单个或少数蛋白质产物，难以系统透彻地阐释冷冻卵母细胞的蛋白质构成和功能变化。为此，本研究利用iTRAQ标记结合2D LC-MS/MS技术，系统分析冷冻猪GV期卵母细胞及包裹的卵丘颗粒细胞在体外成熟后的蛋白质表达差异，探寻影响其成熟质量的关键蛋白并进行功能验证；同时采用蛋白酶体抑制剂MG132作用于体外成熟后期，在差异蛋白质组分析基础上，进一步检测卵母细胞的成熟水平和发育潜力，明确MG132对冷冻卵母细胞质量的改善作用。研究结果将从蛋白质组水平阐明猪GV期卵母细胞冷冻后发育潜力下降原因并提供可行的解决方案，具有重要的理论和实践意义。

目前，有关冷冻卵母细胞的蛋白质组学特征尚无研究报道。本项目以玻璃化冷冻猪GV期卵母细胞为实验对象，采用TMT高通量蛋白质组测序技术，系统分析其体外成熟至MII期卵母细胞及相应颗粒细胞的蛋白质差异并进行生物信息学分析。研究结果显示，冷冻卵母细胞有153个差异蛋白，其中上调94个，下调59个。功能分类和富集分析发现，这些蛋白主要参与代谢、信号传导、刺激反应、补体级联等。冷冻颗粒细胞中有46个上调和42个下调蛋白表达差异显著。一些蛋白与细胞周期、卵子发生调控、类固醇生成、TGF-β信号通路有关联。此外，本项目也对GV期与MII期卵母细胞进行了蛋白组差异表达分析。共获得763个差异蛋白，其中上调和下调蛋白分别为450个和313个。生物信息学分析揭示了卵母细胞核、质成熟的多种调控机制，如纺锤体和染色体构型、细胞骨架重塑、表观遗传修饰、能量代谢等。我们采用PRM靶向蛋白定量技术也验证了这些TMT数据的准确性。最后，明确了蛋白酶体抑制剂MG132能够促进冷冻猪GV期卵母细胞的早期胚胎发育。本项目为研究卵母细胞的冷冻损伤及体外成熟机制提供蛋白质组学方面参考依据。