Age and molecular mechanisms contributing to aneuploidy in oocytes

着丝粒遗传悖论是，尽管着丝粒通过指导 染色体分离，着丝粒本身不是由特定的DNA序列编码。相反地， 着丝粒在表观遗传学上由含有组蛋白H3变体的核小体的存在来定义 CENP-A。蛋白质是标记，这为卵母细胞中保持着丝粒同一性提出了问题 因为卵母细胞在恢复细胞之前停滞在前期I数年或数十年（在人类中） 完成减数分裂。在循环体细胞中，CENP-A在DNA扩增过程中在姐妹染色单体之间分配， 在G1期早期复制和重新加载，从而在细胞周期之间维持CENP-A水平。这些 这些机制不能解释卵母细胞中着丝粒的特性是如何维持的，因为没有已知的 根据已建立的用于在G1中加载的范例，组装新的CENP-A核小体的机制。 卵母细胞的独特之处在于它们停滞在前期I，然后必须保持着丝粒直到细胞分裂。 在荷尔蒙的刺激下，周期会重新开始。原则上，两个因素可能有助于维持 着丝粒身份：（1）CENP-A核小体的内在稳定性和（2）可能的前期I装载 这是一种替代CENP-A的机制，随着年龄的增长而消失。我们的初步数据显示， 捐款.目的1将定义CENP-A在动物生殖寿命内的稳定性，确定 的加载机制，并确定这种加载机制的功能意义。 我们之前的生物物理和结构研究表明，CENP-A核小体的刚性要高10倍 比他们的H3对应物要多目标2将检验CENP-A的结构刚性是 其在卵母细胞中的长期稳定性和着丝粒遗传。我们将识别点突变， 折衷结构刚性和稳定性，但正常定位并在循环中支持着丝粒功能 体细胞我们还将产生选定突变体的敲入动物，以测试功能后果 降低了维持卵母细胞中着丝粒身份和功能的稳定性。总的来说，我们的实验将 首次深入了解哺乳动物雌性生殖系中着丝粒遗传的机制， 并将原子水平的结构洞察力与着丝粒身份的长期维持联系起来， 生殖适应性我们的方法是高度跨学科的，结合结构生物学和生物物理学， 在体内生殖生物学，以了解卵母细胞如何维持结构组装在进入 减数分裂前期I必须在几年或几十年后的卵母细胞成熟过程中发挥作用。等 鉴于生育率下降与孕产妇死亡率上升有关， 年龄和妇女推迟生育时间。

The centromere inheritance paradox is that although centromeres control genetic inheritance by directing chromosome segregation, centromeres themselves are not encoded by a particular DNA sequence. Instead, centromeres are defined epigenetically by the presence of nucleosomes containing the histone H3 variant CENP-A. That a protein is the mark poses a problem for maintaining centromere identity during oocyte development because oocytes arrest in prophase I for years or decades (in humans) before resuming the cell cycle to complete meiosis. In cycling somatic cells, CENP-A partitions between sister chromatids during DNA replication and reloads in early G1 phase, which maintains CENP-A levels between cell cycles. These mechanisms do not explain how centromere identity is maintained in the oocyte because there is no known mechanism to assemble new CENP-A nucleosomes according to the established paradigm for loading in G1. Oocytes are unique in that they arrest in prophase I, and then centromeres must be maintained until the cell cycle resumes in response to hormonal stimuli. In principle, two factors could contribute to maintain centromere identity: (1) the intrinsic stability of CENP-A nucleosomes and (2) a possible prophase I loading mechanism to replace CENP-A that is lost with age. Our preliminary data show that both make significant contributions. Aim 1 will define CENP-A stability over the reproductive lifespan of the animal, determine the mechanism of loading during prophase I, and determine the functional significance of this loading mechanism. Our previous biophysical and structural studies showed that CENP-A nucleosomes are ~10-fold more rigid than their canonical H3 counterparts. Aim 2 will test the hypothesis that structural rigidity of CENP-A underlies its long-term stability and centromere inheritance in the oocyte. We will identify point mutations that compromise structural rigidity and stability but localize normally and support centromere function in cycling somatic cells. We will also generate knock-in animals of selected mutants to test the functional consequences of reduced stability for maintaining centromere identity and function in oocytes. Overall, our experiments will provide the first insight into mechanisms underlying centromere inheritance in the mammalian female germline, and connect atomic level structural insights to long-term maintenance of centromere identity and ultimately reproductive fitness. Our approach is highly interdisciplinary, combining structural biology and biophysics with in vivo reproductive biology, to gain insight into how oocytes maintain structures assembled during entry into meiotic prophase I that must then function years or decades later during oocyte maturation. Such understanding has clear health implications given the decline in fertility associated with increasing maternal age and women delaying the time of childbirth.