Biochemical & Proteomic Analysis of Cumulus Cell Secreted Zinc Inhibitory Factor

生化

• 批准号: 8583461
• 负责人: FRANCISCO J DIAZ
• 金额: $ 7.48万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-21 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8583461
• 关键词: Acute; Area; Biochemical; Cell Communication; Cells; Complex; Conditioned Culture Media; Data; Defect; Development; Diagnosis; Diet; EGF gene; Embryo; Embryonic Development; Excision; Failure; Female; Fertility; Fertilization; Functional disorder; Health; Heating; Homeostasis; Hour; In Vitro; Infertility; Knowledge; Label; Mediating; Meiosis; Metabolism; Metaphase; Methods; Molecular Weight; Mus; Nuclear; Oocytes; Ovarian; Ovulation; Paracrine Communication; Pathway interactions; Pentetic Acid; Preparation; Procedures; Process; Proteins; Proteomics; Pump; Reagent; Regulation; Reproductive Health; Reproductive Medicine; Research Activity; Role; Sampling; Somatic Cell; Technology; Testing; Transition Elements; Trypsin; Zinc; Zinc deficiency; egg; granulosa cell; improved; in vivo; liquid chromatography mass spectrometry; mass spectrometer; metal chelator; nano; novel; oocyte maturation; poor egg quality; premature; prevent; public health relevance; reproductive function; success; treatment duration; zygote

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.

描述(由申请者提供)：生育力受损通常是由于排卵转换期的问题引起的。减数分裂缺陷、排卵失败和鸡蛋质量差都会导致生育力下降。卵母细胞-颗粒细胞通讯对受精和胚胎发育非常重要。然而，卵泡内分泌因子调控的特异性旁分泌信号及其作用机制尚不完全清楚。黄体生成素的激增导致卵丘-卵母细胞复合体(COC)的剧烈变化，包括卵丘细胞的扩张以及卵母细胞的胞质和核成熟。最近的证据有力地支持了锌介导的过程在卵母细胞成熟和受精调节中的作用。卵丘细胞分泌一种锌抑制因子(ZIF)，能在排卵前有效地抑制锌在卵母细胞中的积累。该产品是一种新的旁分泌信号，调节卵泡中的锌代谢。这项建议的目的是对积云细胞产生的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的产物按表观分子量进行分级。接下来，含有最高ZIF活性的组份中的所有分泌蛋白质将用灵敏的纳米液色谱质谱(NanLC MS)方法进行分析。在目标2中，将测试一种含有高ZIF活性的条件培养液对卵母细胞受精率的影响。结合这些目标的结果，将开发一种有价值的试剂，能够在体外成熟/受精过程中精确控制锌的动态平衡。