Biochemical & Proteomic Analysis of Cumulus Cell Secreted Zinc Inhibitory Factor

描述（由申请人提供）：生育能力受损通常是由排卵期过渡期间的问题引起的。减数分裂缺陷，排卵失败和卵子质量差都有助于降低生育力。卵母细胞-颗粒细胞通讯对受精和胚胎发育非常重要。然而，对卵泡中特定的旁分泌信号和分泌因子调控的机制还不完全清楚。LH峰引起卵丘-卵母细胞复合体（COC）的显著变化，包括卵丘细胞的扩张和卵母细胞的胞质和核成熟。最近的证据强烈支持锌介导的过程中的作用，卵母细胞成熟和受精的调节。卵丘细胞分泌锌抑制因子（ZIF），有效抑制排卵前卵母细胞中锌的积累。该产物是一种新的调节卵泡锌代谢的旁分泌信号。本提案的目的是对卵丘细胞产生的ZIF进行生物化学和蛋白质组学分析。在具体目标1中，我们将卵丘细胞分泌产物按表观分子量进行分级。接下来，将通过灵敏的纳米液相色谱质谱（nano LC MS）方法分析含有最高ZIF活性的级分中的所有分泌蛋白。在目的2中，将测试含有高ZIF活性的条件培养基的制备物对卵母细胞生育力的影响。这些目标的共同结果将开发出一种有价值的试剂，能够在体外成熟/受精过程中精确控制锌稳态。

DESCRIPTION (provided by applicant): Compromised fertility is often caused by problems during the periovulatory transition. Meiotic defects, ovulation failure and poor egg quality all contribute to decreased fertility. Oocyte-granulosa cell communication is very important for fertilization and embryo development. However, the specific paracrine signals and the mechanisms controlled by secreted factors in the follicle are not completely know or understood. The LH surge causes dramatic changes in the cumulus-oocyte complex (COC), including expansion of the cumulus cells and oocyte cytoplasmic and nuclear maturation. Recent evidence strongly supports a role for zinc-mediated processes in the regulation of oocyte maturation and fertilization. Cumulus cells secrete a zinc inhibitory factor (ZIF) that potently suppresses zinc accumulation in the oocyte before ovulation. This product is a novel paracrine signal regulating zinc metabolism in the follicle. The objective of this proposal is the biochemica and proteomic analysis of ZIF produced by cumulus cells. In specific aim 1, we will fractionate cumulus cell secreted products according to apparent molecular weight. Next, all secreted proteins in the fraction containing the highest ZIF activity will be analyzed by a sensitive nano-liquid chromatography mass spectrometry (nano LC MS) method. In aim 2, the effect on oocyte fertility of a preparation of conditioned medium containing high ZIF activity will be tested. Together results from these aims will develop a valuable reagent able to precisely control zinc homeostasis during in vitro maturation/fertilization procedures.