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.
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.
