Effects of Glutathione Deficiency on Reproductive Aging and Fertility

谷胱甘肽缺乏对生殖衰老和生育能力的影响

• 批准号: 8912054
• 负责人: Rachel Perlas Cinco
• 金额: $ 3.67万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8912054
• 关键词: Age; Aging; Antioxidants; Atretic Follicles; Binding; Birth; Boxing; Breeding; Catalytic Domain; Cell Death; Cells; Cessation of life; Clinical; Data; Development; Embryo; Environment; Enzymes; Equilibrium; Etiology; Exhibits; Female; Fertility; Fluorescence; Future; Genes; Genetic; Genotype; Glutamate-Cysteine Ligase; Glutathione; Harvest; Homologous Gene; Image; Immunohistochemistry; In Vitro; Infertility; Knock-out; Knockout Mice; Laboratories; Life; Maintenance; Mating Types; Measures; Medical; Metabolic; Metabolism; Microscopy; Modeling; Mullerian-inhibiting substance receptor; Mus; NADH; Neonatal; Oocytes; Ovarian; Ovarian Follicle; Ovary; Oxidation-Reduction; Oxidative Stress; Partner in relationship; Phenotype; Phosphatidylinositols; Phosphotransferases; Play; Premature Ovarian Failure; Preventive; Primordial Follicle; Psychological Impact; Quality of life; Recruitment Activity; Regulation; Research; Role; Secondary to; Signal Transduction; Spatial Distribution; Staging; System; Testing; Therapeutic; Time; Tissues; Transgenic Organisms; Weaning; Woman; Work; Zona Pellucida; age related; cell type; child bearing; design; granulosa cell; in vivo; insight; male; mouse model; novel; postnatal; premature; public health relevance; reproductive; response; socioeconomics; trend; two-photon

 DESCRIPTION (provided by applicant): One percent of all women living today has or will develop premature ovarian failure (POF), defined as the precocious depletion of ovarian follicles before the age of 40. However, the mechanisms underlying POF remain poorly defined. Our previous work shows that normal ovarian aging is associated with increased ovarian oxidative stress and decreased antioxidant capacity. We have observed that Gclm -/- mice, genetically deficient in the modifier subunit of the rate-limiting enzyme in synthesis of the antioxidant glutathione (GSH), exhibit increased ovarian oxidative stress and accelerated ovarian aging. Gclm -/- mice show similar numbers of oocytes and ovarian follicles at birth and weaning compared to Gclm +/+ counterparts, but thereafter exhibit more rapid age-related declines in primordial and total follicle numbers. This makes the Gclm null mouse an ideal model to study premature ovarian failure. The aims in this proposal will identify the contribution of GSH to the proper maintenance of the primordial follicle pool and clarify effects of oxidative stress on the ovarian primordial follicle reserve, quiescent primordial to activated primary metabolism, and female fecundity. We will test our hypothesis that genetic deficiency in the antioxidant GSH causes prolonged oxidative stress that alters the redox equilibrium of the ovary, resulting in downstream effects that promote premature primordial follicle depletion by increasing primordial follicle activation and altering follicular metabolism and energetics in favor of accelerated ovarian aging. To identify GSH contribution to maintenance of the primordial follicle pool and fecundity, we propose the following specific aims: 1) Quantify primordial follicle activation and atresia in cultured neonatal ovaries of Gclm -/- and Gclm -/+ mice compared to Gclm+/+ mice, and assess phosphoinositide-3 kinase (PI3K) cellular signaling across genotypes. Neonatal ovary culture offers an effective model to investigate the response of primordial follicles to increased oxidative stress during normal culture conditions. [2) Perform Phasor FLIM at 740nm 2-photon excitation to characterize the levels of free-to-bound NADH during initial to early stages of follicular development and compare with immunolocalization of Silent mating type information regulation 2 homolog (Sirt1) and Forkhead Box O3a (Foxo3a) in stage matched follicles in both wildtype and Gclm +/+ vs Gclm -/- glutathione deficient mice.] 3) Establish if oocyte-specific and granulosa cell specific GSH depletion leads to premature ovarian failure and/or reduced fecundity, as observed in global Gclm null GSH deficient mice. In vivo requirements for GSH in maintenance of the primordial follicle pool will be identified utilizing tissue specific GSH depleted mouse models that have deletion in the catalytic subunit of the rate-limiting enzyme in GSH synthesis, Gclc. Mechanisms uncovered by the research performed in this proposal will provide new insights into POF etiology and contribute to future therapies and preventive measures.

 描述（由申请人提供）：今天生活的所有女性中有1%患有或将发展为卵巢早衰（POF），定义为40岁之前卵巢卵泡的过早消耗。然而，POF的机制仍然不清楚。我们以前的工作表明，正常的卵巢衰老与卵巢氧化应激增加和抗氧化能力下降有关。我们已经观察到，Gclm -/-小鼠，在合成抗氧化剂谷胱甘肽（GSH）的限速酶的修饰亚基遗传缺陷，表现出卵巢氧化应激增加和卵巢老化加速。与Gclm +/+小鼠相比，Gclm -/-小鼠在出生和断奶时显示出相似数量的卵母细胞和卵泡，但此后表现出更快的原始卵泡和总卵泡数量的年龄相关性下降。这使得Gclm null小鼠成为研究卵巢早衰的理想模型。本提案的目的是确定GSH对原始卵泡池适当维持的贡献，并阐明氧化应激对卵巢原始卵泡储备、静止原始至活化初级代谢和女性生育力的影响。我们将测试我们的假设，即抗氧化剂GSH的遗传缺陷会导致长期的氧化应激，改变卵巢的氧化还原平衡，导致下游效应，通过增加原始卵泡激活和改变卵泡代谢和能量学，促进过早的原始卵泡耗竭，有利于加速卵巢衰老。为了确定GSH对维持原始卵泡池和生育力的贡献，我们提出了以下具体目标：1）与Gclm+/+小鼠相比，量化Gclm -/-和Gclm -/+小鼠培养的新生儿卵巢中的原始卵泡激活和闭锁，并评估跨基因型的磷酸肌醇-3激酶（PI 3 K）细胞信号传导。新生儿卵巢培养提供了一个有效的模型来研究原始卵泡在正常培养条件下对增加的氧化应激的反应。[2)在740 nm双光子激发下进行Phasor FLIM，以表征卵泡发育的初始至早期阶段期间游离-结合NADH的水平，并与野生型和Gclm +/+ vs Gclm -/-谷胱甘肽缺陷小鼠中阶段匹配卵泡中沉默交配型信息调节2同源物（Sirt 1）和叉头盒O3 a（Foxo 3a）的免疫定位进行比较。] 3)确定卵母细胞特异性和颗粒细胞特异性GSH耗竭是否导致卵巢早衰和/或生育力降低，如在全局Gclm null GSH缺陷小鼠中观察到的。将利用组织特异性GSH耗竭小鼠模型鉴定维持原始卵泡池中GSH的体内需求，所述小鼠模型在GSH合成中的限速酶Gclc的催化亚基中具有缺失。本研究所揭示的机制将为POF病因学提供新的见解，并有助于未来的治疗和预防措施。