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女配子通过减数分裂正确进展。 减数分裂主轴的组装和定位是此过程的关键部分，基因敲除 损害这些导致女性不育症的过程。卵母细胞主轴组织和定位是精心策划的 通过肌动蛋白，涉及肌动蛋白相关的蛋白质中的细胞质网状蛋白和卵母细胞皮质中。我们的 卵母细胞中与肌动蛋白相关蛋白的研究已确定Nexilin涉及这些事件，并附有数据 这里介绍了RNAi介导的Nexilin的敲低导致减数分裂和异常 卵母细胞肌动蛋白的组织。我们还有证据表明尿素的丧失会影响肌动蛋白调节途径 涉及包含激酶（LIMK）及其底物的lim域肌动蛋白 - 分解因子 Cofilin。 limk-cofilin途径会影响F-肌动蛋白丝对单体G-肌动蛋白的解聚，而 因此，这是一种有前途的机制，通过它可以影响依赖肌动蛋白的过程。 Nexilin是 由于其在扩张和肥大的心肌病中的作用，也更广泛的相关性（DCM和HCM， 分别）。因此，这里提出的研究的影响是广泛的，与繁殖有关， 卵母细胞生物学，肌肉功能和心肌病。随着DCM的发作通常在40年代 - 60年代， 我们假设Nexn基因中的功能中断突变可能是女性不育症的原因 在生殖年份，然后是心脏病的后期。鉴于对Nexilin知之甚少 总体目标是阐明Nexilin的功能，其与limk-cofilin途径的联系以及Nexilin如何 功能障碍会导致哺乳动物卵母细胞异常。我们将通过以下 具体目标。在AIM 1中，我们将基于卵母细胞中RNAi介导的敲低Nexilin的数据，并建立 开发卵母细胞特异性的Nexilin条件基因敲除（CKO）模型，以分析Nexilin损失的影响 卵母细胞的活性，体内和体外。 AIM 2将在细胞力学中使用最先进的研究，活细胞 成像和定量分析以阐明主轴组织缺陷的基础机制 和与Nexilin缺乏症相关的易位。这个目标将检验异常纺锤体的假设 与Nexilin或Limk-Cofilin途径中缺陷相关的定位归因于（a）异常 皮层锚固的张力，用于纺锤体的螺旋细胞外围，或（b）基于肌动蛋白的缺陷 纺锤体细胞质中纺锤体的运动。 AIM 3将调查变异形式的Nexilin如何影响 卵母细胞，鸡蛋和早期胚胎。这项工作将是对不同严重性的宝贵评估 与疾病相关的形式，还为女性是否有这些NEXN之一的问题提供了答案 突变，对她的卵母细胞的影响会产生什么影响？总体而言，该项目将揭示一个糟糕的 通过阐明卵母细胞和一般而言的Nexilin功能，可以理解但与健康相关的蛋白质。在 转身，这项工作将转化为了解心肌细胞和其他细胞类型中的尿素蛋白功能。