GSH: Protecting Ovarian Follicles from Oxidant Injury

描述（由申请人提供） 环境毒物对卵母细胞的破坏可导致永久性丧失 因为雌性哺乳动物拥有有限的卵母细胞。 一 毒物破坏卵泡和卵母细胞的关键机制是 通过氧化损伤。 谷胱甘肽（GSH）在保护 细胞免受内源性和外源性试剂诱导的氧化损伤。 因此，了解GSH在卵巢中的功能对于 了解谷胱甘肽在保护卵巢免受有毒物质侵害中的作用， 令人惊讶是，对这些功能知之甚少。 校长 这项提案的调查员，Luderer博士，是唯一有资格解决 这些问题，拥有医学博士学位，学位和博士学位生殖 内分泌学以及职业和环境医学培训。 的 本提案中概述的研究和职业发展活动将 使吕德勒博士能够实现她的目标， 计划解决卵巢毒物的作用机制，翻译 研究人员将这一发现应用于人类卵巢，并测试潜在的预防机制。 职业发展活动，包括正式课程工作、 分子生物学和毒理学，以及与导师的一对一互动， 共同赞助者，将与本研究计划同步进行， 探讨谷胱甘肽在保护卵泡免受氧化损伤中的作用。 该研究计划基于初步工作，表明mRNA 谷氨酸半胱氨酸连接酶（GLCL，也称为谷氨酸半胱氨酸连接酶）调节亚基的表达 称为γ-谷氨酰半胱氨酸合成酶），GSH中的限速酶 合成，仅限于颗粒细胞和卵母细胞的形态 健康的卵泡这些发现构成了主要假设的基础， 这一建议，指出谷胱甘肽在卵巢中的关键作用是保护 从应激源中发育卵泡，如产生的活性氧 在正常的卵泡发育或暴露于有毒物质，这可能 促进卵泡闭锁的凋亡过程。 几种方法将 本研究旨在验证以下假设：1）原位GLCL mRNA和蛋白 表达将与细胞凋亡的原位末端标记相关， 2）GSH在卵泡凋亡中的机制作用 将通过化学阻断GSH合成进行检查，3） 培养的卵泡中的氧化应激和凋亡将是 在正常或毒性耗尽的GSH水平存在下测定，4） GSH对内源性和毒物诱导的损伤的保护作用 卵泡将使用转基因模型进行测试，其中GLCL亚基基因 在颗粒细胞中过度表达或被敲除。 拟议的研究 将提供一种创新的方法来理解机制， GSH保护卵巢免受氧化损伤。 这些研究将 此外，还为实现更长期的理解目标奠定了基础。 卵巢毒物引起人类不育的机制， 国家环境健康科学研究所的一项研究 优先领域。 最后是研究计划和职业发展活动 本提案中概述的内容将使Luderer博士能够成功地 从受指导的研究人员到独立的科学家-临床医生的转变。

DESCRIPTION (provided by applicant) Destruction of oocytes by environmental toxicants can result in permanent loss of fertility because female mammals possess a finite complement of oocytes. A key mechanism by which toxicants may destroy ovarian follicles and oocytes is via oxidant injury. Glutathione (GSH) plays critical roles in protecting cells from oxidant injury induced by both endogenous and exogenous agents. Understanding the function(s) of GSH in the ovary is therefore essential to understanding the role of GSH in protection from ovarian toxicants, but surprisingly little is known about these function(s). The principal investigator of this proposal, Dr. Luderer, is uniquely qualified to address these questions, possessing both an M.D. degree and a Ph.D. in reproductive endocrinology, and training in occupational and environmental medicine. The research and career development activities outlined in this proposal will enable Dr. Luderer to achieve her goal of running a successful research program resolving the mechanisms of action of ovarian toxicants, translating the findings to the human ovary, and testing potential preventive mechanisms. The career development activities, including formal course work, training in molecular biology and toxicology, and one-on-one interactions with mentors and co-sponsors, will be carried out in tandem with this research plan designed to address the role of GSH in protecting ovarian follicles from oxidant injury. The research plan is based on preliminary work demonstrating that mRNA expression of the regulatory subunit of glutamate cysteine ligase (GLCL, also called gamma-glutamylcysteine synthetase), the rate-limiting enzyme in GSH synthesis, is limited to granulosa cells and oocytes of morphologically healthy follicles. These findings form the basis of the primary hypothesis of this proposal, stating that a key role of GSH in the ovary is to protect developing follicles from stressors, such as reactive oxygen species produced during normal follicular development or exposure to toxicants, that may promote the apoptotic process of follicular atresia. Several approaches will be utilized to test this hypothesis: 1) In situ GLCL mRNA and protein expression will be correlated with in situ end-labeling for apoptosis in cycling rat ovaries, 2) the mechanistic role of GSH in follicular apoptosis will be examined by chemically blocking GSH synthesis, 3) the extent of oxidative stress and apoptosis in cultured ovarian follicles will be determined in the presence of normal or toxicant-depleted GSH levels, 4) the protective role of GSH against endogenous and toxicant-induced injury to follicles will be tested using transgenic models in which GLCL subunit genes in granulosa cells are over-expressed or knocked out. The proposed studies will provide an innovative approach to understanding the mechanism by which GSH protects the ovary against oxidative damage. These studies will additionally set the stage for achieving the longer-term goal of understanding the mechanisms by which ovarian toxicants cause human infertility, addressing one of the National Institute of Environmental Health Sciences' research priority areas. Finally, the research plan and career development activities outlined in this proposal will enable Dr. Luderer to successfully make the transition from mentored researcher to independent scientist-clinician.