Cellular senescence and epigenomic remodeling in ovarian aging

项目摘要/摘要 卵巢功能的下降与健康寿命和寿命的缩短密切相关。数不胜数 研究已将卵巢功能障碍与全身器官老化联系起来，尽管对此知之甚少 涉及启动和维持年龄相关性卵巢功能衰退的基本、内在机制。这些 知识差距是开发旨在减轻年龄相关性卵巢疾病的治疗方法的关键障碍 失败了。卵巢衰老的特征是静止的原始卵泡逐渐枯竭，这是 最终导致排卵的不规则模式和卵巢内分泌环境的破坏。这个项目将 系统地探索细胞衰老和表观遗传学/基因组中的细胞类型特异性变化 组织通过使用新的转基因NuTRAP模型来促进年龄相关的卵巢衰竭 允许从卵母细胞、颗粒细胞或卵泡膜细胞中分离核酸(DNA和RNA) 而不需要细胞分选。该项目的成功完成将决定：1)细胞 衰老和表观遗传修饰在原始卵泡衰竭中起作用，2)年龄相关的细胞 衰老和表观遗传修饰以细胞类型特定的方式沿着不同的时间线发生，3)如果 表观遗传变化在细胞衰老出现之前或之后，以及4)如果衰老的去除 细胞和/或通过敏感药物治疗抑制SASP可以延长生殖寿命。我们 假设包裹原始卵泡的颗粒细胞积累了有害的细胞和表观遗传学 变化，经历类似衰老的过渡，从而加速原始卵泡的生长和 成熟，从而导致精疲力竭。我们将通过以下目标来检验这一假设。具体目标1： 描述卵母细胞、颗粒细胞和膜细胞中细胞衰老标志物的细胞类型特异性变化 来自老化的卵巢。我们预测衰老细胞负担和炎症介质将随着 随着年龄的增长，颗粒细胞是这种表型的主要来源。具体目标2：确定细胞的特征 卵母细胞、颗粒和卵膜内表观遗传学改变和转录谱的类型特异性变化 来自老化卵巢的细胞。我们预测，大多数与年龄相关的变化将是特定于细胞的， 不同细胞类型的“表观遗传时钟”以不同的速度前进。我们还预计颗粒细胞将 显示表观遗传学和转录图谱的最大变化，这将对应于 卵巢原始卵泡储备。目标3：确定卵巢内衰老细胞的清除是否恢复 卵巢功能，延长生殖适合度。我们预测，感光处理将增加这一数字。 随着年龄的增长，卵母细胞积累的DNA损伤会更少。我们也 预计这将产生更多的胚胎，而DNA损伤更少，从而增加 年龄较大的雌性小鼠怀孕、产仔和产仔的数量。这项研究的最终目的是 制定临床干预措施，延长生殖寿命，使全身健康受益。

PROJECT SUMMARY/ABSTRACT Declines in ovarian function are closely associated with reductions in healthspan and longevity. Numerous studies have linked ovarian dysfunction to systemic organismal aging, although very little is known with regard to the basic, intrinsic mechanisms that initiate and perpetuate age-related ovarian functional declines. These knowledge gaps represent a critical barrier to developing treatments aimed at attenuating age-related ovarian failure. Ovarian aging is characterized by the progressive depletion of quiescent primordial follicles, which eventually leads to irregular patterns of ovulation and disruption of the ovarian endocrine milieu. This project will systemically explore how cell-type specific changes in cellular senescence and epigenetics/genomic organization contribute to age-related ovarian failure through the use of novel transgenic NuTRAP models that allow for the isolation of nucleic acids (DNA & RNA) specifically from oocytes, granulosa cells, or theca cells without the need for cell sorting. Successful completion of this project will determine: 1) what role cellular senescence and epigenetic modifications play in primordial follicle exhaustion, 2) if age-related cellular senescence and epigenetic modifications occur in a cell-type specific fashion along differing timelines, 3) if epigenetic changes precede or follow the emergence of cellular senescence, and 4) if the removal of senescent cells and/or the suppression of the SASP through senolytic drug treatment can extend reproductive lifespan. We hypothesize that granulosa cells encapsulating primordial follicles accumulate deleterious cellular and epigenetic alterations, undergo a senescence-like transition, and thereby accelerate primordial follicle growth and maturation, thereby leading to exhaustion. We will test this hypothesis through following aims. Specific Aim 1: Characterize cell type-specific changes in markers of cellular senescence in oocytes, granulosa, and theca cells from the aging ovary. We predict that senescence cell burden and inflammatory mediators will increase with advancing age and that granulosa cells are a major source of this phenotype. Specific Aim 2: Characterize cell type-specific changes in epigenetic alterations and transcriptional profiles within oocytes, granulosa, and theca cells from the aging ovary. We predict that the majority of age-related changes will be cell-specific and that `epigenetic clocks' of distinct cell types advance at different rates. We also anticipate that granulosa cells will display the greatest changes in epigenetic and transcriptional profiles and that this will correspond to declines in ovarian primordial follicle reserve. Aim 3: Determine if the clearance of senescent cells within the ovary restores ovarian function and prolongs reproductive fitness. We predict that senolytic treatment will increase the number of primordial follicles and that oocytes will accumulate less DNA damage with chronological aging. We also anticipate that this will result in a greater production of embryos with less DNA damage, thereby increasing the number of pregnancies, litters, and pups born to older female mice. The ultimate goal of this research is to develop clinical interventions that extend reproductive lifespan for systemic health benefits.