Age and molecular mechanisms contributing to aneuploidy in oocytes

着丝粒遗传悖论是，尽管着丝粒通过引导基因遗传来控制基因遗传 染色体分离，着丝粒本身不是由特定的DNA序列编码的。相反， 着丝粒是由含有组蛋白H3变异体的核小体的存在在表观遗传学上定义的 CENP-A蛋白质是标记，这给在卵母细胞中保持着丝粒的一致性带来了问题。 发育，因为卵母细胞在I期前期停滞数年或数十年(在人类中)，然后恢复细胞 完成减数分裂的周期。在体细胞周期中，CENP-A在DNA过程中在姐妹染色单体之间的分配 在G1期早期复制和重新加载，在细胞周期之间维持CENP-A水平。这些 机制不能解释着丝粒特性是如何在卵母细胞中保持的，因为没有已知的 根据已建立的范例组装新的CENP-A核小体以在G1中装载的机制。 卵母细胞的独特之处在于，它们在前期i停止，然后着丝粒必须保持到细胞 在荷尔蒙刺激下，周期重新开始。原则上，有两个因素可能有助于维持 着丝粒同一性：(1)CENP-A核小体的内在稳定性和(2)可能的前期I装载 取代随着年龄增长而消失的CENP-A的机制。我们的初步数据显示，两者都有重要意义 贡献。目标1将定义CENP-A在动物生殖寿命内的稳定性，确定 第一阶段的加载机制，并确定该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核小体的刚性是原来的10倍 比它们典型的H3同类病毒更多。目标2将检验CENP-A的结构刚性是基础的假设 它在卵母细胞中的长期稳定性和着丝粒遗传。我们将确定点突变 折衷结构的刚性和稳定性，但正常局部化，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 在卵母细胞中维持着丝粒特性和功能的稳定性降低。总的来说，我们的实验将 首次洞察了哺乳动物雌性生殖系着丝粒遗传的潜在机制， 并将原子级结构洞察与着丝粒身份的长期维护联系起来，最终 生殖适合度。我们的方法是高度跨学科的，结合了结构生物学和生物物理学 体内生殖生物学，以深入了解卵母细胞如何维持在进入 减数分裂前期I，随后必须在数年或数十年后的卵母细胞成熟期间发挥作用。是这样的 考虑到生育率的下降与孕产妇死亡率的增加相关，理解这一点具有明显的健康影响 年龄和妇女推迟分娩时间。

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.