METHOXYCHLOR EXPOSURE REDUCES THE LEVELS OF CELL CYCLE REGULATORS IN MOUSE OVARIAN ANTRAL FOLLICLES

Abstract

Mammalian females are born with a finite number of primordial follicles, of which a small fraction survive and reach the penultimate antral stage, while most die via apoptosis. Antral follicles are functional units for maturation and release of oocytes and for synthesis of sex steroid hormones such as estrogen. The organochlorine pesticide methoxychlor (MXC) is a toxicant that targets the mammalian ovary. Previous in vivo and in vitro studies with MXC showed that antral follicles were the primary targets of MXC with no apparent effect on other follicle types. Specifically, MXC exposure decreases antral follicle numbers and increases the percentage of atretic antral follicles by inducing oxidative stress. Furthermore, studies show that MXC inhibits antral follicle growth in vitro. The mechanism by which MXC inhibits growth of antral follicles is unknown. Granulosa cell proliferation is required for normal follicle growth and is controlled, in part, by cell cycle regulators such as proliferating cell nuclear antigen (PCNA), cyclins, and cyclin dependant kinases. Thus, this study tested the hypothesis that MXC inhibits follicle growth in vivo by reducing the levels of cell cycle regulators. Adult cycling mice were dosed with MXC (16, 32, or 64 mg/kg/day) or sesame oil (vehicle) for 20 days. Ovaries were collected and subjected to immunohistochemistry for PCNA staining. In addition, ovaries from vehicle- and MXC-treated mice were used to isolate antral follicles and subjected to real time polymerase chain reaction for measurement of mRNA levels of cyclin D2 (Ccnd2) and cyclin dependant kinase 4 (Cdk4). The results indicate that antral follicles from MXC-treated ovaries had significantly less staining for PCNA compared to controls (control = 71.2 ± 1.7% stained area/follicle; MXC 16 mg/kg/day = 51.9 ± 3.2% stained area/per follicle; MXC 32 mg/kg/day = 46.0 ± 2.7% stained area/per follicle; and MXC 64 mg/kg/day = 26.2 ± 3.2% stained area/per follicle; n = 3; P ≤ 0.05). Furthermore, MXC significantly reduced mRNA expression of Ccnd2 compared to controls (control = 0.85 ± 0.06 genomic equivalents (ge); MXC 16 mg/kg/day = 0.44 ± 0.08 ge; MXC 32 mg/kg/day = 0.46 ± 0.03 ge; MXC 64 mg/kg/day = 0.38 ± 0.09 ge; n = 3; P ≤ 0.05). Similarly, MXC significantly reduced mRNA expression of Cdk4 compared to controls (control = 0.89 ± 0.02 ge; MXC 16 mg/kg/day = 0.48 ± 0.02 ge; MXC 32 mg/kg/day = 0.56 ± 0.07 ge; MXC 64 mg/kg/day = 0.45 ± 0.03 ge; n = 3; P ≤ 0.05). Collectively, these data indicate that MXC exposure in vivo reduces the expression of PCNA, Ccnd2, and Cdk4 in antral follicles. Therefore, we postulate that MXC may inhibit the growth of antral follicles by decreasing the levels of cell cycle regulators in antral follicles. Supported by NIH R21 ES13061, R01 ES012893, and NIH HD46861. (platform)