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

摘要 成功的胚胎发育依赖于雌配子在减数分裂过程中的正确发育。 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，涉及细胞质网络和卵母细胞皮质中的肌动蛋白相关蛋白。我们的 对卵母细胞中肌动蛋白相关蛋白的研究已经证实奈西林与这些事件有关，数据显示 结果表明，RNAi介导的nexlin基因敲除导致减数分裂停滞和异常 卵母细胞肌动蛋白的组织。我们也有证据表明，nexlin的缺失会影响肌动蛋白的调控途径。 涉及含有LIM结构域的激酶(LIMK)及其底物--肌动蛋白解聚因子 科菲林。LIMK-cofilin途径影响F-肌动蛋白细丝解聚为单体G-肌动蛋白，并 因此，这是一个很有希望的机制，通过它可以影响肌动蛋白依赖的过程。奈西林是 由于其在扩张型和肥厚型心肌病(DCM和HCM， )。因此，这里提出的研究的影响是广泛的，与生殖有关， 卵母细胞生物学、肌肉功能和心肌病。DCM的发病年龄通常在40多岁到60多岁， 我们推测NEXN基因的功能中断突变可能是导致女性不孕的原因之一 在生殖年龄段，然后是晚年的心脏病。鉴于人们对奈西林知之甚少，我们的 总体目标是阐明Nexlin的功能，它与LIMK-cofilin通路的联系，以及Nexlin是如何 功能障碍会导致哺乳动物卵母细胞的异常。我们将通过以下方式实现这些目标 明确的目标。在目标1中，我们将建立在我们的数据基础上，从RNAi介导的卵母细胞中击倒nexin，以及 建立卵母细胞特异性奈西林条件性基因敲除(CKO)模型，分析奈西林缺失的影响 在体内和体外卵母细胞中的活性。目标2将使用最先进的细胞力学研究，活细胞 成像和定量分析以阐明纺锤体组织缺陷的潜在机制 与奈西林缺乏症相关的易位。这一目标将检验异常纺锤体的假设 与nexlin或LIMK-cofilin途径缺陷相关的定位被归因于(A)异常 皮质锚定以将纺锤体拉至卵母细胞外围的张力，或(B)基于肌动蛋白的缺陷 卵母细胞胞质中纺锤体的运动。目标3将研究奈西林的突变形式如何影响 卵母细胞、卵子和早期胚胎。这项工作将是对不同疾病严重性的宝贵评估 与疾病相关的形式，也为女性是否患有这些NEXN之一的问题提供了答案 突变，对她的卵母细胞会有什么影响？总体而言，这个项目将揭示一个糟糕的 通过阐明Nexlin在卵母细胞和一般情况下的功能，了解了与健康相关的重要蛋白质。在……里面 反过来，这项工作将转化为了解心肌细胞和其他类型细胞中的奈西林功能。

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.