Investigation into the Dynamic Zn Proteome during Mammalian Oocyte Maturation

 描述（由申请人提供）：锌的大量重组与多种人体生理活动的调节有关。最近，人们发现，细胞内和细胞外的锌流是至关重要的哺乳动物卵母细胞的发育和受精前和受精后发生。然而，人们对分子生物学的了解很少。 锌依赖性转换事件背后的机制决定卵母细胞成熟。没有这些知识，生物医学界就没有能力评估什么是“好鸡蛋”，也没有能力开发潜在的生殖疗法。因此，本研究的目的是通过跟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。我推测，在成熟过程中细胞内锌含量的变化驱动细胞周期的进展，通过满足金属的要求，在成熟过程中的关键锌蛋白。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，其中它们的金属辅因子的存在或不存在作为调节开关。这一假设将通过采用放射性锌示踪剂来跟踪和量化Zn2+的积累和再分布来解决。此外，这些研究还将阐明受精时被称为“锌火花”的流出事件的生理意义。“最后，锌金属蛋白质组的变化将使用放射性锌示踪剂结合天然电泳来表征，以揭示哪些锌蛋白在卵母细胞成熟过程中被激活。从这项研究中获得的数据将作为一个框架，建立一个新的锌依赖性机制，决定细胞周期的进展，在卵母细胞，从而提供新的手段评估卵母细胞的健康。从该研究项目中获得的见解也将适用于其他锌依赖性生理学。该奖项将支持广泛的多学科培训活动和经验，旨在为研究型大学的终身职位提供强有力的准备。培训活动将提高科学交流、指导、实验室管理等领域的关键能力，并扩大技术技能。

 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.