Abstract
Treatment with the anticancer drug cyclophosphamide (CPA) destroys ovarian follicles. The active metabolites of CPA are detoxified by conjugation with glutathione (GSH). We tested the hypotheses that CPA causes apoptosis in ovarian follicles and that suppression of ovarian GSH synthesis before CPA administration enhances CPA-induced apoptosis. Proestrous rats were given two injections, 2 h apart, with (1) saline, then saline; (2) saline, then 50 mg/kg CPA; (3) saline, then 300 mg/kg CPA; or (4) 5 mmol/kg buthionine sulfoximine (BSO) to inhibit glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and then 50 mg/kg CPA. Statistically significantly increased DNA fragmentation by agarose gel electrophoresis and granulosa cell apoptosis by TUNEL were observed in the CPA-treated ovaries 24 h after the second injection, but BSO did not enhance the effect of 50 mg/kg CPA. We next tested the hypothesis that CPA depresses ovarian GSH concentration and expression of the rate-limiting enzyme in GSH synthesis, GCL. Proestrous rats were injected with 300 or 50 mg/kg CPA or vehicle and were sacrificed 8 or 24 h later. After CPA treatment, ovarian and hepatic GSH levels decreased significantly, and ovarian GCL subunit mRNA levels increased significantly. There were no significant changes in GCL subunit protein levels. Finally, we tested the hypothesis that GSH depletion causes apoptosis in ovarian follicles. Proestrous or estrous rats were injected with 5 mmol/kg BSO or saline at 0700 and 1900 h. There was a significant increase in the percentage of histologically atretic follicles and a nonsignificant increase in the percentage of apoptotic, TUNEL-positive follicles 24 h after onset of BSO treatment. Our results demonstrate that CPA destroys ovarian follicles by inducing granulosa cell apoptosis and that CPA treatment causes a decline in ovarian GSH levels. More pronounced GSH suppression achieved after BSO treatment did not cause a statistically significant increase in follicular apoptosis. Thus, GSH depletion does not seem to be the mechanism by which CPA causes follicular apoptosis.
Highlights
Glutathione (GSH) is the most abundant nonprotein thiol in mammalian cells
We have previously demonstrated, using in situ hybridization, that Gclm mRNA is strongly expressed in the granulosa cells and oocytes of healthy, growing follicles, whereas atretic follicles, which have granulosa cells that stain for DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), lack Gclm expression [19]
TUNEL demonstrated that CPA treatment dose-dependently increased granulosa cell apoptosis in preantral follicles ( p < .001, effect of treatment) and antral follicles ( p < .001, effect of treatment) (Fig. 2), but not in primary or primordial follicles
Summary
Glutathione (GSH) is the most abundant nonprotein thiol in mammalian cells. It plays important roles in maintaining the intracellular environment by assisting with amino acid transport, regulating enzyme activity, maintaining membrane structural integrity, and protecting against exogenous and endogenous toxicants [1]. It is likely that ovarian GSH plays an important role in protecting ovarian follicles from damage by exogenous toxicants. Cellular levels of GSH are maintained by two tightly coupled enzymatic processes involving a rate-limiting step, catalyzed by glutamate cysteine ligase (GCL), to form g-glutamylcysteine, followed by addition of glycine to make glutathione [4,5]. The enzymatically active form of GCL is a heterodimer, composed of a catalytic (GCLc) and a modifier (GCLm) subunit [4,5]
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