Investigation into the Dynamic Zn Proteome during Mammalian Oocyte Maturation

 描述(由申请人提供)：锌的大规模重组与多种人类生理活动的调节有关。最近，人们发现细胞内和细胞外的锌离子对哺乳动物卵母细胞的成功发育至关重要，并且发生在受精前和受精后。然而，人们对这种分子的了解很少。 决定卵母细胞成熟的锌依赖转换事件背后的机制。如果没有这方面的知识，生物医学界就缺乏评估什么是“好鸡蛋”和开发潜在生殖疗法的能力。因此，这项建议的目的是通过追踪卵母细胞成熟过程中锌的积累和定位来阐明细胞对锌的需求。我假设，成熟过程中细胞内锌含量的变化通过满足成熟过程中关键锌蛋白的金属需求来驱动细胞周期进展。这些蛋白质包括催化某些细胞过程所需的重要含锌酶，以及锌受体蛋白，在锌受体蛋白中，金属辅因子的存在或不存在起着调节开关的作用。这一假说将通过使用放射性锌示踪剂来跟踪和量化锌离子的积累和再分配来解决。此外，这些研究还将阐明受精过程中被称为“锌火花”的外排事件的生理学意义。最后，锌金属蛋白质组的变化将用放射性锌示踪剂结合自然电泳法来表征，以揭示哪些锌蛋白在卵母细胞成熟过程中被激活。这项研究的数据将作为建立一种新的锌依赖机制的框架，该机制决定了卵母细胞的细胞周期进程，从而为评估卵母细胞的健康状况提供了新的手段。从这项研究项目中获得的见解也将适用于其他依赖锌的生理学。这一奖项将支持广泛的多学科培训活动和经验，目标是为一所研究型大学的终身教职职位提供强有力的准备。培训活动除了扩大技术技能外，还将提高科学交流、指导、实验室管理等领域的关键能力。

 DESCRIPTION (provided by applicant): Massive reorganizations of zinc have been implicated in the regulation of a variety of human physiologic activities. Recently, it was discovered that intracellular and extracellular zinc fluxes are vital to successful mammalian oocyte development and occur both pre- and post-fertilization. However, there is little understanding of the molecular mechanisms behind the zinc-dependent switching events that dictate oocyte maturation. Without this knowledge, the biomedical community lacks the ability to assess what makes a "good egg" and develop potential reproductive therapies. Therefore, the goal of this proposal is to elucidate the cellular needs for zinc by tracking its accumulation and localization during oocyte maturation. I hypothesize that variation in intracellular zinc content during maturation drives cell cycle progression by fulfilling the metal requirement of key Zn-proteins in the maturation process. These proteins include vital Zn-containing enzymes needed to catalyze certain cellular processes as well as zinc-receptor proteins where the presence or absence of their metal cofactor serves as a regulatory switch. This hypothesis will be addressed by employing radioactive zinc tracers to track and quantify Zn2+ accumulation and redistribution. In addition, these studies will elucidate the physiological significance of the efflux event at fertilization known as the "zinc spark." Lastly, changes in zinc metalloproteome will be characterized using radioactive zinc tracers in conjunction with native electrophoresis to reveal which Zn-proteins are activated during oocyte maturation. The data obtained from this study will serve as a framework for establishing a novel Zn-dependent mechanism that dictates cell cycle progression in the oocyte, thus providing new means for assessing oocyte health. Insights gained from this research project will be applicable to other zinc-dependent physiologies as well. This award will support a broad array of multi-disciplinary training activities and experienc with the goal of providing robust preparation for a tenure-track position in a Research I university. Training activities will increase key competencies in the areas of scientific communication, mentoring, lab management, in addition to broadening technical skills.