Mechanisms of Spindle Assembly in Oocytes

卵母细胞中纺锤体的组装机制

• 批准号: 9374195
• 负责人: MARIA M. VIVEIROS
• 金额: $ 7.5万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-07 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9374195
• 关键词: Address; Age; Aging; Aneuploidy; Binding Proteins; Centrioles; Centrosome; Chromosome Segregation; Chromosomes; Chromosomes, Human, Pair 6; Complex; Congenital Abnormality; Congenital Disorders; Data; Defect; Down Syndrome; Embryo; Exhibits; Female; Future; Genetic; Genetic Models; Guanosine Triphosphate Phosphohydrolases; Human; Knowledge; Maternal Age; Mediating; Meiosis; Metaphase; Microtubule Polymerization; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic spindle; Modeling; Mus; Oocytes; Patients; Ploidies; Pregnancy loss; Process; Proteins; RNA Interference; Reporting; Resolution; Role; Scaffolding Protein; Small Interfering RNA; Structure; Study models; Testing; Transgenic Mice; Transgenic Organisms; Woman; advanced maternal age; blastocyst; cell type; chromosome number abnormality; experimental study; fetal; gamma Tubulin; genetic regulatory protein; insight; knock-down; live cell imaging; mouse model; mutant; pericentrin; polymerization; protein complex; protein expression

PROJECT SUMMARY An abnormal chromosome number (aneuploidy) in developing embryos is the leading cause of birth defects and pregnancy loss in women. The majority of aneuploidies are attributed to error-prone meiotic division in oocytes and increase significantly with advanced maternal age. Accurate chromosome segregation is critically dependent on assembly of the microtubule (MT) spindle apparatus and the establishment of correct chromosome-MT interactions. Our studies in mice demonstrate that disruption of meiotic spindle stability can promote chromosome segregation errors, which are not fully resolved -despite spindle checkpoint (SAC) activation. Notably, meiotic spindle formation differs from mitosis as mammalian oocytes (mouse and human) lack typical centrosomes, with centriole loss occurring during fetal stages. Alternatively, spindle MT formation can occur from (i) unique acentriolar microtubule-organizing centers (aMTOCs) and (ii) aMTOC-independent mechanisms in mouse oocytes. In previous studies we identified pericentin (Pcnt) as an essential aMTOC scaffolding protein. Thus, to test aMTOC function we developed a unique oocyte-conditional Pcnt knockdown mouse model using a transgenic RNAi approach. Our recent analysis of oocytes from Tg mice demonstrates that meiotic division is highly error-prone in the absence of maternal Pcnt and disrupted aMTOCs, leading to female subfertility. Importantly, a new study reports that human oocytes (obtained from IVF patients), lack pericentrin and show strikingly similar meiotic errors as our Tg mice. The experiments outlined in this proposal will use this unique genetic model to address the underlying mechanism(s) of spindle formation in oocytes. In Aim 1 we will test the function of key aMTOC-independent mechanisms that promote meiotic spindle formation. We will (i) establish whether Ran activity regulates spindle formation in Pcnt-deficient mouse oocytes and (ii) undertake the first studies to test if the Augmin complex-mediated MT amplification also functions to promote spindle stability in mammalian oocytes. Why human oocytes reportedly lack pericentrin and why aMTOC-independent spindle formation is unstable in oocytes is not known. Thus, experiments in Aim 2 will (i) test the hypothesis that essential aMTOC-associated proteins (pericentrin and γ-tubulin) are disrupted with increasing maternal age. Additionally, we will (ii) determine whether MT dynamics differ in Pcnt-deficient oocytes, leading to spindle instability. Knowledge gained from these studies will provide critical new insight into (i) the key functional mechanisms that promote meiotic spindle formation and (ii) whether disruption of spindle assembly/stability contributes to age-associated aneuploidy in oocytes.

项目摘要 发育胚胎中的异常染色体数（非整倍性）是出生的主要原因 女性的缺陷和妊娠损失。大多数非整倍性归因于容易出错的减数分裂性 卵母细胞的分裂并随着高级母期年龄的显着增加。准确的染色体 隔离至关取决于微管（MT）纺锤体的组装和 建立正确的染色体-MT相互作用。我们在小鼠中的研究表明 减数分裂的纺锤体稳定性可以促进染色体隔离错误，这些错误尚未完全解决-DDITE 主轴检查点（SAC）激活。值得注意的是，有丝分裂的减数分裂纺锤体形成差异为哺乳动物 卵母细胞（小鼠和人）缺乏典型的中心体，胎儿阶段发生中心损失。 或者，纺锤体MT形成可能是从（i）独特的acentriolar微管组织中心发生的 （AMTOC）和（ii）小鼠卵母细胞中非AMTOC无关的机制。在先前的研究中，我们确定了 中心蛋白（PCNT）是必不可少的AMTOC支架蛋白。为了测试AMTOC函数，我们开发了 使用转基因RNAi方法的独特卵母细胞条件敲低小鼠模型。我们的 对TG小鼠的卵母细胞的最新分析表明，减数分裂分裂在高度错误中 缺乏材料PCNT和破坏的AMTOC，从而导致女性属性。重要的是，一项新研究 报告说，人类卵母细胞（从IVF患者获得），缺乏包中心素，并且表现出非常相似的 减数分裂错误作为我们的TG小鼠。该提案中概述的实验将使用此独特的通用 解决卵母细胞纺锤体形成的潜在机制的模型。在目标1中，我们将 测试促进减数分裂纺锤体形成的关键AMTOC独立机制的功能。我们将 （i）确定RAN活动是否调节PCNT缺陷小鼠卵母细胞中的主轴形成和（ii） 进行第一批研究，以测试Augmin复合物介导的MT扩增是否也起作用 促进哺乳动物卵母细胞的纺锤稳定性。据报道，为什么人类卵母细胞缺乏丁香蛋白以及为什么 在卵母细胞中，非AMTOC独立的纺锤体形成是不稳定的。那是AIM 2中的实验 （i）检验基本与AMTOC相关蛋白（塞霉素和γ-微管蛋白）的假设是 随着产妇年龄的增长而破坏。此外，我们将（ii）确定MT动力学是否不同 PCNT缺乏卵母细胞，导致纺锤不稳定性。从这些研究中获得的知识将提供 对（i）促进减数分裂纺锤体形成和（ii）的关键功能机制的关键新见解 纺锤体组件/稳定性的破坏是否有助于卵母细胞中与年龄相关的肾上腺素。