Mono-hydroxy methoxychlor alters levels of key sex steroids and steroidogenic enzymes in cultured mouse antral follicles

Abstract

Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by decreasing antral follicle numbers and increasing follicular death. MXC is metabolized in the body to mono-hydroxy MXC (mono-OH). Little is known about the effects of mono-OH on the ovary. Thus, this work tested the hypothesis that mono-OH exposure decreases production of 17β-estradiol (E₂) by cultured mouse antral follicles. Antral follicles were isolated from CD-1 mice (age 35-39 days) and exposed to dimethylsulfoxide (DMSO), or mono-OH (0.1-10 μg/mL) for 96 h. Media and follicles were collected for analysis of sex steroid levels and mRNA expression, respectively. Mono-OH treatment (10 μg/mL) decreased E(2) (DMSO: 3009.72±744.99 ng/mL; mono-OH 0.1 μg/mL: 1679.66±461.99 ng/mL; 1 μg/mL: 1752.72±532.41 ng/mL; 10 μg/mL: 45.89±33.83 ng/mL), testosterone (DMSO: 15.43±2.86 ng/mL; mono-OH 0.1μg/mL: 17.17±4.71 ng/mL; 1 μg/mL: 13.64±3.53 ng/mL; 10 μg/mL: 1.29±0.23 ng/mL), androstenedione (DMSO: 1.92±0.34 ng/mL; mono-OH 0.1 μg/mL: 1.49±0.43ng/mL; 1 μg/mL: 0.64±0.31 ng/mL; 10 μg/mL: 0.12±0.06 ng/mL) and progesterone (DMSO: 24.11±4.21 ng/mL; mono-OH 0.1μg/mL: 26.77±4.41 ng/mL; 1 μg/mL: 20.90±3.75 ng/mL; 10 μg/mL: 9.44±2.97 ng/mL) levels. Mono-OH did not alter expression of Star, Hsd3b1, Hsd17b1 and Cyp1b1, but it did reduce levels of Cyp11a1, Cyp17a1 and Cyp19a1 mRNA. Collectively, these data suggest that mono-OH significantly decreases levels of key sex steroid hormones and the expression of enzymes required for steroidogenesis.

Figure 1. Mono-OH treatment decreases levels of 17β-estradiol in media from cultured mouse antral follicles

Isolated mouse antral follicles were cultured for 96 h in the presence of 0.1, 1 and 10 μg/mL mono-OH or DMSO as a vehicle control. Following culture, media samples from individual follicles were subjected to measurement of 17β-estradiol by ELISA as described in Materials and Methods. Data are expressed as mean ± SEM calculated from follicles in 3 separate culture experiments. Asterisk (*) indicates P<0.05.

Figure 2. Mono-OH treatment decreases levels of precursors of 17β-estradiol in media from cultured mouse antral follicles

Following culture, media samples from individual follicles were subjected to measurement of (A) progesterone, (B) androstenedione and (C) testosterone by ELISA as described in Materials and Methods. Data are expressed as mean ± SEM calculated from follicles in 3–6 separate culture experiments. Asterisk (*) indicates P<0.05.

Figure 3. Mono-OH treatment decreases expression of key steroidogenic enzymes in cultured mouse antral follicles

Following 96 h, antral follicles were removed from culture and processed for real-time qPCR as described in Materials and Methods. Messenger RNA levels for (A) steroidogenic acute regulatory protein, Star; (B) cholesterol side chain cleavage enzyme, Cyp11a1; (C) 3β-hydroxysteroid dehydrogenase, Hsd3b1; (D) 17α-hydroxylase/17,20-lyase, Cyp17a1; (E) 17β-hydroxysteroid dehydrogenase, Hsd17b1; and (F) aromatase, Cyp19a1 were determined and normalized to β-actin (Actb). Data are expressed as mean relative expression ratios ± SEM calculated from 3–5 separate culture experiments. Asterisk (*) indicates P<0.05.

Figure 4. Mono-OH treatment does not alter levels of an estrogen-metabolizing enzyme in cultured mouse antral follicles

Messenger RNA levels for cytochrome P450, family 1, subfamily b, polypeptide 1 (Cyp1b1) were determined and normalized to β-actin (Actb). Data are expressed as mean relative expression ratios ± SEM calculated from 5 separate culture experiments.