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.
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