Mechanisms of Spindle Assembly in Oocytes

项目总结 发育中的胚胎染色体数目异常(非整倍体)是导致出生的主要原因。 女性的缺陷和妊娠丢失。大多数非整倍体都归因于容易出错的减数分裂。 卵母细胞分裂，并随着孕龄的增加而显著增加。精确染色体 分离严重依赖于微管(MT)纺锤体的组装和 建立正确的染色体-MT相互作用。我们在小鼠身上的研究表明，对 减数分裂纺锤体的稳定性可以促进染色体分离错误，这些错误并没有完全解决--尽管 主轴检查点(SAC)激活。值得注意的是，减数分裂纺锤体的形成不同于哺乳动物的有丝分裂。 卵母细胞(小鼠和人)缺乏典型的中心体，中心粒丢失发生在胎儿阶段。 另外，纺锤形MT的形成可以来自(I)独特的无着丝点微管组织中心 (Ii)小鼠卵母细胞的aMTOC非依赖机制。在之前的研究中，我们发现 包膜蛋白(Pericentin，Pcnt)是一种重要的aMTOC支架蛋白。因此，为了测试我们开发的aMTOC函数 一种使用转基因RNAi方法的独特的卵母细胞条件Pcnt基因敲除小鼠模型。我们的 最近对转基因小鼠卵母细胞的分析表明，卵母细胞的减数分裂非常容易出错 母体Pcnt的缺失和aMTOCs的中断，导致女性亚生育。重要的是，一项新的研究 有报道称，人类卵母细胞(取自体外受精患者)缺乏胞外集落蛋白，并表现出惊人的相似性。 减数分裂错误就像我们的转基因小鼠。这项提案中概述的实验将使用这种独特的基因 模型来解决卵母细胞纺锤体形成的潜在机制(S)。在目标1中，我们将 测试促进减数分裂纺锤体形成的不依赖于aMTOC的关键机制的功能。我们会 (I)确定Ran活性是否调节Pcnt缺陷小鼠卵母细胞纺锤体的形成和(Ii) 进行第一批研究，以测试Augmin复合体介导的MT扩增是否也起作用 促进哺乳动物卵母细胞纺锤体的稳定性。为什么据报道人类卵母细胞缺乏周中心素？为什么 不依赖aMTOC的纺锤体形成在卵母细胞中是不稳定的，目前尚不清楚。因此，目标2中的实验 将(I)检验基本的aMTOC相关蛋白(围集素和γ-微管蛋白)是 随着产妇年龄的增加而中断。此外，我们将(Ii)确定MT动态是否在 Pcnt缺陷的卵母细胞，导致纺锤体不稳定。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的关键作用机制和(Ii)的关键新见解 纺锤体组装/稳定性的破坏是否导致与年龄相关的卵母细胞非整倍体。

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.