GSH: Protecting Ovarian Follicles from Oxidant Injury

描述(由申请人提供) 环境毒物破坏卵母细胞可导致永久性丧失 因为雌性哺乳动物拥有有限数量的卵母细胞。一个 毒物破坏卵泡和卵母细胞的关键机制是 通过氧化剂损伤。谷胱甘肽(GSH)在保护 细胞免受内源性和外源性因素的氧化损伤。 因此，了解谷胱甘肽在卵巢中的功能(S)对于 了解GSH在保护卵巢毒物中的作用，但 令人惊讶的是，人们对这些功能知之甚少(S)。校长 这项提案的调查者路德勒博士是唯一有资格解决 这些问题，既有医学博士学位，又有生殖医学博士学位 内分泌学，以及职业和环境医学培训。这个 本提案中概述的研究和职业发展活动将 使路德勒博士能够实现她的目标，即成功地进行研究 解决卵巢毒物作用机制的程序，翻译 对人类卵巢的发现，并测试潜在的预防机制。 职业发展活动，包括正规课程工作、培训 分子生物学和毒理学，以及与导师和 共同赞助者，将与这项研究计划同步进行，旨在 阐述GSH在保护卵泡免受氧化剂损伤中的作用。 该研究计划是基于前期工作证明了mRNA 谷氨酸半胱氨酸连接酶调节亚单位(GLCL，又称GLCL)的表达 谷氨酰半胱氨酸合成酶)，谷胱甘肽的限速酶 合成，在形态上仅限于颗粒细胞和卵母细胞 健康的卵泡。这些发现构成了以下基本假设的基础 这项建议指出，GSH在卵巢中的一个关键作用是保护 应激源发育卵泡，如产生的活性氧 在正常的卵泡发育或暴露于毒物期间，这可能 促进卵泡闭锁的细胞凋亡过程。有几种方法将 用来检验这一假说：1)原位GLCL mRNA和蛋白 表达与原位末端标记的细胞凋亡相关 2)GSH在卵泡细胞凋亡中的作用机制 将通过化学阻断GSH合成来检查，3)程度 体外培养的卵泡的氧化应激和细胞凋亡 在正常或毒物耗竭的GSH水平下测定，4) 谷胱甘肽对内源性和毒物性损伤的保护作用 将使用转基因模型对卵泡进行测试，在转基因模型中，GLCL亚单位基因 颗粒细胞过度表达或被敲除。建议进行的研究 将提供一种创新的方法来理解 GSH保护卵巢免受氧化损伤。这些研究将 此外，还为实现理解的长期目标奠定了基础 卵巢毒物导致人类不孕不育的机制 国家环境健康科学研究所的研究之一 优先领域。最后，研究计划和职业发展活动 这份提案中概述的内容将使Luderer博士能够成功地 从导师研究员到独立科学家-临床医生的过渡。

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.