The oocyte's progression through meiosis: Involvement of a heart disease-associated protein

卵母细胞减数分裂的进展：心脏病相关蛋白的参与

• 批准号: 10018056
• 负责人: JANICE P EVANS
• 金额: $ 32.94万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-13 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10018056
• 关键词: Actins; Affect; Binding; Biology; Cardiac Myocytes; Cardiomyopathies; Complement; Cytoplasm; Data; Defect; Dilated Cardiomyopathy; Disease; Educational workshop; Embryo; Embryonic Development; Event; F-Actin; Failure; Female; Female infertility; Fertility; Foundations; Functional disorder; G Actin; Genes; Germ Cells; Goals; Health; Heart; Heart Diseases; Human; Image; Immunoglobulin Domain; Impairment; In Vitro; Infertility; Knockout Mice; LIM Domain; Letters; Life; Light; LoxP-flanked allele; Mechanics; Mediating; Meiosis; Metaphase; Methods; Microfilaments; Mission; Mitosis; Modeling; Molecular; Movement; Mus; Muscle; Muscle function; Mutate; Mutation; National Institute of Child Health and Human Development; Oocytes; Ovary; Pathway interactions; Patients; Phenotype; Phosphotransferases; Positioning Attribute; Process; Proteins; RNA Interference; Regulatory Pathway; Reproduction; Reproductive Health; Research; Role; Severities; Sex Differences; Site; Skeletal Muscle; Structure; Testing; Thinking; Translating; Woman; Work; actin depolymerizing factor; base; cell type; cofilin; conditional knockout; depolymerization; egg; female fertility; in vivo; insight; knock-down; knockout gene; live cell imaging; novel; public health relevance; reproductive; subfertility

SUMMARY Successful embryonic development is dependent on the female gamete progressing correctly through meiosis. Assembly and positioning of the meiotic spindle is a crucial part of this process, with gene knockouts that impair these processes causing female infertility. Oocyte spindle organization and positioning is orchestrated by actin, involving actin-associated proteins in a cytoplasmic meshwork and in the oocyte cortex. Our research on actin-associated proteins in oocytes has identified nexilin as involved in these events, with data presented here showing that RNAi-mediated knockdown of nexilin results in meiotic arrest and aberrant organization of oocyte actin. We also have evidence that loss of nexilin affects the actin regulatory pathway involving the LIM-domain containing kinase (LIMK) and its substrate, the actin-depolymerization factor cofilin. The LIMK-cofilin pathway affects the depolymerization of F-actin filaments to monomeric G-actin, and thus this is a promising mechanism by which nexilin could impact actin-dependent processes. Nexilin is of broader relevance as well, due to its role in dilated and hypertropic cardiomyopathies (DCM and HCM, respectively). Thus, the impact of the research proposed here is wide-ranging, with relevance to reproduction, oocyte biology, muscle function, and cardiomyopathies. With onset of DCM typically being in one's 40s-60s, we hypothesize that a function-disrupting mutation in the NEXN gene could be a cause of female infertility during reproductive years, and then of heart disease later in life. Given that little is known about nexilin, our overall goal is to elucidate the functions of nexilin, its connection to the LIMK-cofilin pathway, and how nexilin dysfunction contributes to abnormalities in mammalian oocytes. We will achieve these goals with following Specific Aims. In Aim 1, we will build on our data from RNAi-mediated knockdown nexilin in oocytes, and develop an oocyte-specific nexilin conditional knockout (cKO) model, to analyze the effects of loss of nexilin activity in oocytes, in vivo and in vitro. Aim 2 will use state-of-the-art studies in cellular mechanics, live-cell imaging, and quantitative analyses to elucidate the mechanisms underlying the defects in spindle organization and translocation associated with nexilin deficiency. This aim will test the hypotheses that aberrant spindle positioning associated with deficiencies in nexilin or the LIMK-cofilin pathway are attributed to (a) aberrant tension for cortical anchoring for spindle pulling to the oocyte periphery, or (b) defects in actin-based movement of the spindle in the oocyte cytoplasm. Aim 3 will investigate how mutated forms of nexilin affect oocytes, eggs, and early embryos. This work will be an invaluable assessment of the severity of different disease-associated forms, and also provide answers to the question of if a woman has one of these NEXN mutations, what would the effects be on her oocytes? Overall, this project will shed light on a poorly understood but significant health-relevant protein by elucidating nexilin functions in oocytes and in general. In turn, this work will translate to understanding nexilin functions in cardiomyocytes and other cell types.

概括 成功的胚胎发育取决于雌配子通过减数分裂的正确进展。 减数分裂纺锤体的组装和定位是这一过程的关键部分，基因敲除 损害这些过程导致女性不孕。卵母细胞纺锤体的组织和定位是精心安排的 通过肌动蛋白，涉及细胞质网和卵母细胞皮质中的肌动蛋白相关蛋白。我们的 对卵母细胞肌动蛋白相关蛋白的研究已确定 nexilin 参与这些事件，并有数据 这里提出的结果表明 RNAi 介导的 nexilin 敲低会导致减数分裂停滞和异常 卵母细胞肌动蛋白的组织。我们还有证据表明 nexilin 的缺失会影响肌动蛋白调节途径 涉及含有 LIM 结构域的激酶 (LIMK) 及其底物肌动蛋白解聚因子 肌切蛋白。 LIMK-cofilin 途径影响 F-肌动蛋白丝解聚为单体 G-肌动蛋白，并且 因此，这是一种很有前途的机制，nexilin 可以通过该机制影响肌动蛋白依赖性过程。内西林是 由于其在扩张型心肌病和肥厚性心肌病（DCM 和 HCM、 分别）。因此，这里提出的研究的影响是广泛的，与繁殖相关， 卵母细胞生物学、肌肉功能和心肌病。 DCM 通常在 40 岁至 60 岁之间发病， 我们假设 NEXN 基因中的功能破坏性突变可能是女性不孕的原因 在育龄期，然后在晚年患心脏病。鉴于人们对 nexilin 知之甚少，我们 总体目标是阐明 nexilin 的功能、其与 LIMK-cofilin 通路的联系，以及 nexilin 如何发挥作用 功能障碍导致哺乳动物卵母细胞异常。我们将通过以下方式实现这些目标 具体目标。在目标 1 中，我们将基于卵母细胞中 RNAi 介导的 nexilin 敲低数据，以及 开发卵母细胞特异性 nexilin 条件敲除 (cKO) 模型，以分析 nexilin 丢失的影响 卵母细胞体内和体外的活性。目标 2 将利用细胞力学、活细胞等领域的最先进研究 成像和定量分析，以阐明纺锤体组织缺陷的潜在机制 和与 nexilin 缺乏相关的易位。该目标将检验异常主轴的假设 与 nexilin 或 LIMK-cofilin 通路缺陷相关的定位归因于 (a) 异常 用于将纺锤体拉至卵母细胞外围的皮质锚定张力，或 (b) 基于肌动蛋白的缺陷 纺锤体在卵母细胞胞质中的运动。目标 3 将研究 nexilin 的突变形式如何影响 卵母细胞、卵子和早期胚胎。这项工作将对不同问题的严重性进行宝贵的评估 疾病相关形式，并提供女性是否患有这些 NEXN 之一的问题的答案 突变，会对她的卵母细胞产生什么影响？总的来说，这个项目将揭示一个糟糕的情况 通过阐明卵母细胞和一般情况下的 Nexilin 功能，我们发现了一种理解但重要的健康相关蛋白质。在 反过来，这项工作将转化为了解心肌细胞和其他细胞类型中的 nexilin 功能。