Methoxychlor Induces Atresia and Decreases Estradiol Levels in Antral Follicles Simultaneously.

Abstract

The endocrine disruptor methoxychlor (MXC) is a chlorinated organic pesticide which was first registered for use in 1948 and now is widely used in many countries against various species of insects that attack field crops, trees, vegetables, fruits, gardens, stored grain, livestock and domestic pets. MXC exposure is highest in people working in manufacturing industries, people living near farms, livestock, and in pet owners treating pests with MXC. MXC exposure causes adverse effects on reproductive functions in adult females, including altered estrous cyclicity, reduced fertility, and ovarian atrophy. MXC has been shown to target antral follicles. Specifically, it decreases the number of healthy antral follicles and increases the number of atretic antral follicles in adult female mice. Previous studies have shown that MXC induces atresia and decreases estradiol levels after 96 hours of follicle culture. The goal of the present study was to determine whether MXC induces atresia and decreases estradiol levels earlier than 96 hours of culture. Further, the present study was designed to determine whether MXC-induced changes in atresia precede MXC-induced changes in estradiol levels or vice versa. To complete these goals, antral follicles were mechanically isolated from ovaries of CD1 female mice aged 35-40 days. The isolated antral follicles (10-15 per treatment) were cultured in supplemented alpha-minimum essential media in the presence of dimethylsulfoxide (control; DMSO) or MXC (1, 10, 100 µg/ml) for 24 and 48 hours at 37°C and 5%CO2. After culture, the media was collected and follicles were fixed in Dietrick solution. Fixed follicles were embedded in plastic blocks, cut into 2 µm sections, stained with Lee's methylene blue-basic fuchsin, and graded for atresia on a scale of 1 to 4 based on the percentage of apoptotic bodies (1=0%, 2=<10%, 3=10-30%, and 4=>30% apoptotic bodies). The media was subjected to measurements of estradiol levels by enzyme-linked immunosorbent assays. The results show that MXC does not induce atresia at 24 hours, but it does induce atresia by 48 hours (24 hours: DMSO = 1.73 ± 0.11, MXC1 µg/ml = 1.59 ± 0.16, MXC10 µg/ml = 1.78 ± 0.20, MXC100 µg/ml = 1.90 ± 0.16; 48 hours: DMSO = 1.25 ± 0.14, MXC1 µg/ml = 1.79 ± 0.09, MXC10 µg/ml = 2.22 ± 0.15, MXC100 µg/ml = 4 ± 0; n=3 separate experiments; p ≤ 0.05 for DMSO versus MXC at 48 hours). The results also indicate that MXC does not alter estradiol levels at 24 hours, but it does decrease estradiol levels by 48 hours (24 hours: DMSO = 17.10 ± 4.98, MXC1 µg/ml = 18.73 ± 2.50, MXC10 µg/ml = 26.46 ± 7.95, MXC100 µg/ml = 18.63 ± 4.27 pg/ml; 48 hours: DMSO = 599.38 ± 68.97, MXC1 µg/ml = 225.65 ± 18.79, MXC10 µg/ml = 120.48 ± 35.31, MXC100 µg/ml = 32.85 ± 2.97 pg/ml; n=3 separate experiments; p ≤ 0.05 for DMSO versus MXC at 48 hours). Collectively, these data suggest that MXC decreases estradiol levels and induces atresia as early as 48 hours and that these effects of MXC on antral follicles occur simultaneously. Support: NIH R01 ES012893 and the Environmental Toxicology Program at UIUC. (poster)