Oxidative stress drives ovarian aging: modification by genetics and environment

描述（由申请人提供）：女性的生育能力随着年龄的增长而下降，很大程度上是由于卵巢卵泡数量的减少。卵巢功能的下降和最终停止，称为更年期，与其他不良后果有关，如心血管疾病和骨质疏松症的风险增加。因此，了解导致卵巢正常和加速衰老的因素是很重要的，这样我们就可以预防过早绝经及其不良后果。本研究旨在验证卵巢衰老的特征是卵巢抗氧化能力下降，导致活性氧增加，从而引发卵巢卵泡凋亡破坏的假设。此外，据推测，遗传降低抗氧化能力和暴露于环境中的卵巢毒物加速了这一过程。具体目的1：验证小鼠卵巢衰老与卵巢氧化应激和卵泡凋亡增加以及抗氧化能力下降有关的预测。将在年轻成年小鼠和衰老野生型小鼠的卵巢中测量脂质、蛋白质和DNA的氧化损伤以及抗氧化活性。细胞凋亡的原位测量和卵巢卵泡数量的组织形态学评估将用于评估卵巢衰老。活性氧种类、抗氧化水平和对促性腺激素的反应性将在培养的卵巢卵泡和颗粒细胞中进行测量，这些细胞来自年轻成年小鼠和老年小鼠。特异性目的2：验证抗氧化能力基因缺陷小鼠卵巢氧化损伤和细胞凋亡增加，卵巢衰老加速的预测。我们将使用Aim 1中概述的方法，测试谷氨酸半胱氨酸连接酶修饰子亚基（Gclm）的缺失对生育能力、卵泡数量、卵泡凋亡、活性氧水平、谷胱甘肽氧化还原状态和氧化损伤等年龄相关变化的影响。Gclm会大大减少抗氧化剂谷胱甘肽的合成。抗氧化剂补充将被测试拯救Gclm-/-卵泡。具体目的3：通过确定Gclm-/-小鼠是否对环境污染物苯并[a]芘诱导的卵巢早衰更敏感，来测试基因-环境相互作用在加速卵巢衰老中的作用。在用苯并[a]芘或对照物处理的小鼠中，将测量氧化损伤、细胞凋亡和卵巢卵泡数量的终点。还将比较Gclm-/-型和野生型小鼠培养的卵泡对苯并[a]芘体外处理的敏感性。通过补充抗氧化剂来拯救培养的卵泡免受苯并[a]芘的毒性，将进行试验。公共卫生相关性：尽管所有妇女都经历更年期，但决定更年期何时发生的因素尚不清楚。这些研究将阐明卵巢衰老是否与卵巢抗氧化能力下降和卵巢氧化应激增加有关，并测试一种重要抗氧化剂基因缺乏的小鼠是否会加速卵巢自发衰老和增加对化学诱导的卵巢早衰的易感性。这将有助于确定预防早期绝经及其伴随的不良后果的新目标这一更大的目标。

DESCRIPTION (provided by applicant): Fertility declines with age in women, due in large part to decreased ovarian follicle numbers. The decline and eventual cessation of ovarian function, termed menopause, is associated with other adverse consequences, such as increased risks of cardiovascular disease and osteoporosis. It is therefore important to understand the factors that contribute to normal and accelerated ovarian aging so that we can prevent early menopause and its adverse consequences. This proposal aims to test the hypothesis that ovarian aging is characterized by decreased ovarian antioxidant capacity, leading to increased reactive oxygen species, which initiate apoptotic destruction of ovarian follicles. Further, it is hypothesized that genetically decreased antioxidant capacity and exposure to ovarian toxicants in the environment accelerate this process. Specific Aim 1: To test the prediction that ovarian aging in mice is associated with increased ovarian oxidative stress and follicular apoptosis and decreased antioxidant capacity. Oxidative damage to lipids, proteins, and DNA, and antioxidant activities will be measured in ovaries of young adult and aging wild type mice. In situ measures of apoptosis and histomorphometric assessment of ovarian follicle numbers will be utilized to assess ovarian aging. Reactive oxygen species, antioxidant levels, and responsiveness to gonadotropins will be measured in cultured ovarian follicles and granulosa cells from young adult and aging mice. Specific Aim 2: To test the prediction that mice genetically deficient in antioxidant capacity have increased ovarian oxidative damage and apoptosis and accelerated ovarian aging. We will test the effects of deletion of glutamate cysteine ligase modifier subunit (Gclm), which greatly decreases synthesis of the antioxidant glutathione, on the age-associated changes in fertility, ovarian follicle numbers, follicular apoptosis, reactive oxygen species levels, glutathione redox state, and oxidative damage using the methods outlined for Aim 1. Antioxidant supplementation will be tested to rescue Gclm-/- follicles. Specific Aim 3: To test the role of gene-environment interaction in accelerated ovarian aging by determining whether Gclm-/- mice are more sensitive to premature ovarian failure induced by the environmental pollutant benzo[a]pyrene. Endpoints of oxidative damage, apoptosis, and ovarian follicle numbers will be measured as for Aim 1 in mice treated with benzo[a]pyrene or vehicle. The sensitivity of cultured follicles from Gclm-/- and wild type mice to in vitro treatment with benzo[a]pyrene will also be compared. Rescue of cultured follicles from benzo[a]pyrene toxicity by supplementatio with antioxidants will be tested. PUBLIC HEALTH RELEVANCE: Although all women undergo menopause, the factors that determine when it occurs are not well understood. These studies will elucidate whether ovarian aging is associated with decreased ovarian antioxidant capacity and increased ovarian oxidative stress and test whether mice genetically deficient in an important antioxidant have accelerated spontaneous ovarian senescence and increased susceptibility to chemically-induced premature ovarian failure. This will contribute to the larger goals of identifying new targets for prevention of early menopause and its attendant adverse consequences.