| Mammalian females are born with a finite stock of oocytes, which are associated with granulosa cells to form follicles. Radiation therapy has enhanced the survival of women afflicted with certain malignant cancers. Unfortunately, radiotherapy is toxic to follicles and can cause infertility. Generation of reactive oxygen species has been implicated in the toxicity of ionizing radiation in several cell types. We have shown that depletion of the antioxidant glutathione sensitizes follicles and granulosa cells to toxicant-induced apoptosis and that supplementation of glutathione is protective. The first and rate-limiting reaction in glutathione biosynthesis is catalyzed by glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a regulatory subunit (GCLM). Previous work in other systems has shown that overexpression of Gclc and/or Gclm increases total intracellular GSH levels. We adopted a two-pronged experimental paradigm to test the hypothesis that overexpression of Gclc and/or Gclm, to increase glutathione biosynthesis, would protect oocytes and granulosa cells against apoptosis induced by oxidative stress and ionizing radiation. We first generated transgenic mice that selectively overexpressed Gclc in their developing oocytes. We proposed to study the effects of ovarian toxicants and ionizing radiation on their oocytes to determine if there was protective effect of increased glutathione biosynthesis. Though we generated several mouse lines, we were unsuccessful in identifying a line that displayed significantly increased oocyte glutathione levels relative to wildtype mice. We then transfected an immortalized line of human granulosa cells with vectors designed for the constitutive expression of Gclc , Gclm, or both Gclc and Gclm. Total glutathione levels were significantly increased in the transfected cells. We then showed that exposure of untransfected granulosa cells to therapeutic doses of ionizing radiation rapidly increased intracellular ROS, followed by induction of apoptosis. Further, we showed that granulosa cells with increased glutathione, due to stable overexpression of GCL subunits, were protected against oxidant and radiation-induced apoptosis. These results provide support for the hypothesis that GSH protects granulosa cells against ionizing radiation and contribute to our understanding of the effects of radiation on normal ovarian follicles. |
