Control of Meiotic Spindle Assembly in Mammalian Oocytes

描述(由申请人提供)：胚胎发育中的染色体数目异常(非整倍体)是导致女性先天性出生缺陷和妊娠丢失的主要遗传原因。大多数非整倍体是由于卵母细胞减数分裂过程中发生的染色体分离错误所致，并随着母体年龄的增加而显著增加。染色体分离严重依赖于微管纺锤体的组装以及稳定的染色体-微管相互作用的建立。老年女性卵母细胞中的纺锤体被破坏已经被发现有一段时间了，但潜在的原因仍然不清楚。值得注意的是，我们最近在小鼠身上的研究表明，纺锤体稳定性的破坏可以促进染色体分离错误，尽管卵母细胞中的纺锤体组装检查点(SAC)被激活，但这些错误并未完全解决。这些发现表明，纺锤体稳定性的缺陷不会促进减数分裂的完全停止，可能会对非整倍体的形成构成潜在的重大风险。然而，尽管它很重要，但对哺乳动物卵母细胞中纺锤体组装/组织的调控却知之甚少。在目前的研究中，我们确定了周中心素在稳定的减数分裂纺锤体形成中的关键作用。这项建议中概述的实验解决了卵母细胞减数分裂纺锤体形成的潜在机制，并将测试这些机制是否会随着母亲年龄的增加而被破坏。在目标1中，我们建议使用已建立的转基因RNAi方法，通过产生一个卵母细胞条件基因敲除小鼠来测试周围素的功能。这一独特的遗传模型将被用来测试两个核心问题。(1)周围素在调节卵母细胞MTOC的组织和功能中起关键作用吗？(2)MTOC介导的微管成核对卵母细胞稳定的减数分裂纺锤体形成是必不可少的吗？在目标2中，我们建议确定来自老年雌性小鼠的卵母细胞是否在减数分裂纺锤体组织和/或稳定性方面表现出缺陷。重要的是，我们将测试这些缺陷是否可归因于MTOC功能或纺锤体微管稳定性的改变。从这些研究中获得的知识将为人类非整倍体的基础提供重要的洞察力，并为分析卵母细胞质量提供改进的参数。 公共卫生相关性：胚胎发育中的染色体数目异常(非整倍体)是导致女性先天性出生缺陷和妊娠丢失的主要遗传原因。大多数非整倍体是由于卵母细胞减数分裂过程中发生的染色体分离错误所致，并随着母体年龄的增加而显著增加。准确的染色体分离取决于微管纺锤体的组装和稳定的染色体-微管相互作用的建立。在这项研究中，我们建议(1)评估减数分裂纺锤体形成的潜在机制和(2)检验减数分裂纺锤体稳定性缺陷导致与年龄相关的卵母细胞非整倍体的假设。

DESCRIPTION (provided by applicant): An abnormal chromosome number (aneuploidy) in developing embryos is the leading genetic cause of congenital birth defects and pregnancy loss in women. The majority of aneuploidies are attributed to errors in chromosome segregation that occur during meiotic division in oocytes, and increase significantly with maternal age. Chromosome segregation is critically dependent on assembly of the microtubule spindle apparatus as well as the establishment of stable chromosome-microtubule interactions. Disrupted spindles in oocytes from older women have been recognized for some time, yet the underlying causes remain unknown. Notably, our recent studies in mice demonstrate that disruption of spindle stability can promote chromosome segregation errors, which are not fully resolved -despite activation of the spindle assembly checkpoint (SAC) in oocytes. These findings indicate that defects in spindle stability, which do not promote complete meiotic arrest, likely pose a potentially significant risk in contributing to aneuploidy. Yet, despite its fundamental importance, regulation of spindle assembly/organization in mammalian oocytes is poorly understood. In current studies, we identified a critical role for pericentrin in stable meiotic spindle formation. The experiments outlined in this proposal address the underlying mechanisms of meiotic spindle formation in oocytes, and will test whether these mechanisms are disrupted with increasing maternal age. In Aim 1, we propose to test the function of pericentrin by generating an oocyte-conditional knockdown mouse using an established transgenic RNAi approach. This unique genetic model will be used to test two central questions. (1) Does pericentrin plays a key role in regulating oocyte MTOC organization and function? (2) Is MTOC-mediated microtubule nucleation essential for stable meiotic spindle formation in oocytes? In Aim 2, we propose to determine whether oocytes from older female mice exhibit defects in meiotic spindle organization and/or stability. Importantly we will test if these defects are attributable to alterations in MTOC function or spindle microtubule stability. Knowledge gained from these studies will provide critical insight into the basis of human aneuploidy and provide improved parameters for the analysis of oocyte quality. PUBLIC HEALTH RELEVANCE: An abnormal chromosome number (aneuploidy) in developing embryos is the leading genetic cause of congenital birth defects and pregnancy loss in women. The majority of aneuploidies are attributed to chromosome segregation errors that occur during meiotic division in oocytes, and increase significantly with maternal age. Accurate chromosome segregation is critically dependent on assembly of the microtubule spindle apparatus and the establishment of stable chromosome-microtubule interactions. In this study we propose to (1) assess the underlying mechanisms of meiotic spindle formation and (2) test the hypothesis that defects in meiotic spindle stability contribute to age-associated aneuploidy in oocytes.