Methoxychlor Alters Pro-Apoptotic and Anti-Apoptotic Factors in the Antral Follicles of the Mouse Ovary.

The organochlorine pesticide methoxychlor (MXC) is an endocrine disrupting chemical that has been shown to affect reproductive functions in adult females by causing persistent estrous cyclicity, reduced fertility, and ovarian atrophy. MXC is widely used in many countries to prevent and kill various species of insects that attack field crops, trees, vegetables, fruits, gardens, stored grain, livestock, and domestic pets. MXC reduces fertility by increasing atresia of antral follicles, and by decreasing numbers of healthy antral follicles in adult female mice. However, the mechanism by which MXC causes follicular atresia is unknown. Previous studies have shown that MXC causes atresia, in part by increasing the pro-apoptotic factor Bax and decreasing the anti-apoptotic factor Bcl2 in antral follicles. Further, Bcl2 overexpression and Bax deletion help protect antral follicles from MXC-induced atresia. Presently, it is not known whether MXC alters other pro-apoptotic and anti-apoptotic factors in the antral follicles. Thus, this work was designed to test the hypothesis that MXC alters other pro-apoptotic (Bok, Bad, and Casp3) and anti-apoptotic factors (Bcl2l1 and Mcl1) in addition to Bcl2 and Bax. To test this hypothesis, 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 α-minimum essential media in the presence of dimethylsulfoxide (control; DMSO) or MXC (1, 10, 100 µg/ml) for 24, 48, and 96 hours at 37°C and 5% CO₂. At the end of the culture, follicles were collected and snap frozen. RNA was extracted from frozen follicles, cDNA was synthesized and then subjected to quantitative real-time PCR for the measurement of mRNA levels of Bok, Bad, Casp3, Bax, Bcl2, Mcl1, and Bcl2. The results indicate that at 24 h, MXC decreased Bok and Bcl2l1 mRNA levels (P ≤ 0.05, n=3) and increased Bax mRNA levels (P ≤ 0.05, n = 3). MXC did not affect Bad, Casp3, Mcl1, and Bcl2 mRNA levels at this time point. At 48 h, MXC decreased Casp3 mRNA levels (P ≤ 0.05, n = 3), but did not affect expression of the other pro-apoptotic and anti-apoptotic genes. At 96 h, MXC significantly increased expression of Bcl2, Bcl2l1, and Bax mRNA levels (P ≤ 0.05, n = 3), but did not affect Bok, Bad, Casp3, and Mcl1 mRNA levels. Collectively, these results show that MXC alters both pro-apoptotic factors and anti-apoptotic factors in cultured antral follicles. These data suggest that MXC may induce atresia by altering the balance between several pro-apoptotic and anti-apoptotic factors in antral follicles. Support: NIH R01 ES 019178 and the Environmental Toxicology Program, UIUC.

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