Methoxychlor and Its Metabolites Inhibit Growth and Induce Atresia of Baboon Antral Follicles

Chemicals

MXC powder (99%) was purchased from Chemservice (West Chester, PA). HPTE and mono-OH were purchased from Cedra Corporation (Austin, TX). Stock solutions of MXC, HPTE, and mono-OH were prepared using dimethylsulfoxide (DMSO) (Sigma, St. Louis, MO) as the solvent, and in various concentrations (133, 13.3, 1.33, and 0.133 mg/ml) that allowed an equal volume to be added to culture wells for each treatment group to control for solvent concentration. Final concentrations of MXC in culture were 1, 10, and 100 μg per ml. Final concentrations of HPTE and mono-OH in culture were 0.1, 1, and 10 μg per ml. The concentrations of MXC (1–100 μg/ml) were based on a previous study by Miller et al. (2005), which showed inhibition of growth and atresia of mouse antral follicles using similar concentrations. The 10-fold lower concentrations of HPTE and mono-OH were used because these metabolites of MXC are thought to be more toxic than the parent compound (Gaido et al., 2000) and one would expect the amount of the metabolites reaching the follicles after MXC being metabolized to be lower than the parent compound.

Animals

Normal cycling adult female baboons (Papio anubis) purchased from the Southwest Foundation for Biomedical Research (San Antonio, TX), and weighing 15–18 kg were housed individually in large aluminum primate cages and maintained in a controlled environment (12L:12D). Baboons received high-protein monkey chow (Teklad-Harlan, St. Louis, MO) supplemented daily with fresh fruit and vitamins. Water was provided ad libitum. Menstrual cycle history was determined from the daily pattern of perineal turgescence (Albrecht, 1980) and serum estradiol profiles. Animals were cared for and used strictly in accordance with U.S. Department of Agriculture regulations and the Guide for the Care and Use of Laboratory Animals prepared by the National Research Council (National Academy Press, revised 1996). The experimental protocol used in this study was approved by the Institutional Animal Care and Use Committee of the University of Maryland, School of Medicine.

Following ketamine (10mg/kg BW im) sedation, baboons were anesthetized with a mixture of halothane (1.0–1.5%): nitrous oxide (0.4 L/min): oxygen (2.0 L/min), and bilaterally ovariectomized under aseptic conditions via a 5- to 6-cm midline abdominal incision. Whole ovaries were carefully dissected and placed immediately into warm (37°C) sterile α-minimal essential media (α-MEM, Invitrogen, Carlsbad, CA) for transport to the laboratory and for the mechanical isolation of antral follicles.

Follicle Culture

Antral follicles (determined by appearance and relative size) were isolated mechanically from baboon ovaries and cleaned of interstitial tissue using fine watchmaker forceps. About 75–100 antral follicles (between 800–1500 μm) were obtained from each pair of ovaries, and a total of 4 separate pairs of baboon ovaries were used in this study. Upon isolation, follicles were randomly allocated to treatment groups and placed individually in wells of a 96-well culture plate with unsupplemented α-MEM prior to treatment. Each experiment contained a minimum of 10–16 follicles per treatment regimen. MXC (1–100 μg/ml), HPTE (0.1–10 μg/ml), mono-OH (0.1–10 μg/ml), and DMSO controls were individually prepared in supplemented α-MEM, with an equal volume of chemical added for each dose to control for the amount of vehicle in each preparation.

For experimental treatment, unsupplemented α-MEM was removed from each well and replaced with 150 μl supplemented α-MEM containing either MXC (1–100 μg/ml), HPTE (0.1–10 μg/ml), mono-OH (0.1–10 μg/ml), or DMSO vehicle. Supplemented α-MEM was prepared with: 1% ITS (10 ng/ml insulin, 5.5 ng/ml transferrin, 5.5 ng/ml selenium), 100 U/ml penicillin, 100 mg/ml streptomycin, 5 IU/ml human recombinant follicle-stimulating hormone (FSH; Dr. A. F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA), and 5% fetal calf serum (Atlanta Biologicals, Lawrenceville, GA). Follicles were incubated for 96 hrs at 37°C in 95% air and 5% CO₂. DMSO concentrations in all experiments were kept below 0.075%, which is below the level toxic to cultured follicles. Non-treated (NT) controls were used in each growth experiment to control for culture conditions.

Analysis of Follicle Growth

Antral follicles were cultured for 96 hrs and follicle growth was examined at 24 hr intervals using published methods (Cortvrindt et al., 2002; Miller et al., 2005; Gupta et al., 2006). In short, control follicles placed in culture attach themselves to the bottom of the culture plate, and granulosa cells proliferate to form a “dome-shaped” cover over the follicle, resulting in an increased in follicular size. Measurements of follicle size were then taken on two perpendicular axes with an inverted microscope equipped with a calibrated ocular micrometer. Follicle diameter measurements were averaged among treatment groups and plotted to compare the effects of chemical treatments on growth over time. Data were presented as percent change/time. As another indicator of follicle growth, the thickness of the granulosa cell layers was compared in control versus treated follicles. All slides were evaluated and scored without knowledge of treatment group.

Histological Evaluation of Atresia

Upon termination of follicular cultures, supplemented α-MEM was removed from each well and Dietrick’s solution was immediately added to fix the follicles. Follicles were fixed for at least 24 hrs in Dietrick’s solution and transferred in histology cassettes to 70% ethanol. The tissues were dehydrated, embedded in Paraplast (VWR Scientific, West Chester, PA), serially sectioned (5 μm), mounted on glass slides, and stained with Weigert’s hematoxylin and methyl blue:picric acid. Each follicle section was examined for level of atresia (follicle death) as evidenced by the presence of pyknotic bodies and reported at the highest level observed throughout the tissue. Follicles were rated on a scale of 1–4 for the presence of pyknotic bodies: 1 = healthy, 2 = less than 10% pyknotic bodies (early atresia), 3 = 10–30% pyknotic bodies (mid atresia), 4 = greater than 30% pyknotic bodies (late atresia), as previously described by Miller et al. (2005). Ratings were averaged and plotted to compare the effect of chemical treatments on atresia levels.

Statistical Analysis

All data were analyzed using SPSS statistical software (SPSS, Inc., Chicago, IL). For all comparisons, statistical significance was assigned at p ≤ 0.05. For multiple comparisons between controls and treatment groups, we used analysis of variance (ANOVA), followed by Tukey’s post hoc test, or we conducted multiple regression analysis. At least three separate experiments were conducted for each treatment regimen prior to data analysis. All data are presented as means ± standard error of the means (SEM).

Follicle Culture

Using a follicle culture assay, we determined that baboon antral follicles can be grown and maintained in culture in the absence of chemical treatment, without visible signs of cell death. Representative pictures of baboon antral follicles in culture are shown in Figure 2. Freshly isolated antral follicles were intact and consisted of a clearly visible oocyte, multiple layers of granulosa cells, and a theca cell layer. After 96 hrs in culture, non-treated and DMSO treated follicles grew in size, with theca cells attaching to the bottom of the culture well and granulosa cells proliferating to increase follicle diameter. After MXC, HPTE, or mono-OH treatment for 96 hrs, antral follicles did not grow compared to DMSO controls. While theca cells seemed to attach, the attachments were not as dense and firm as those in DMSO controls. Further, granulosa cells did not proliferate and the follicles became dark in appearance.

Effect of MXC, HPTE, and Mono-OH on Follicle Growth

Using the follicle culture assay, the effect of MXC on follicle growth was evaluated for 96 hrs. Follicles treated with vehicle control showed normal growth, however, significant inhibition of antral follicle growth was observed with MXC-treatment (1, 10 and 100 μg/ml) compared to DMSO control follicles at 72 and 96 hrs (Figure 3A). At 72 hrs, DMSO control follicles increased in size by 11.7 ± 0.7%, whereas MXC 1, 10 and 100 μg/ml treated follicles only increased in size by 10.3 ± 1.8, 5.2 ± 1.6, and 0.9 ± 0.4%, respectively. At 96 hrs, DMSO control follicles increased in size by 28.7 ± 0.2%, whereas MXC 1, 10 and 100 μg/ml treated follicles only increased in size by 17.0 ± 2.7, 4.8 ± 0.2, and 0.4 ± 0.3%, respectively (n = 23 follicles per treatment, p ≤ 0.03). Further, the granulosa cell layer thickness for DMSO-treated follicles was 72.56 ± 1.64 μm (n = 6 follicles), whereas the granulosa cell layer thickness for the MXC-treated groups was essentially 0 because the granulosa cells were no longer in layers and appeared dispersed.

In addition, the effect of HPTE on follicle growth was evaluated for 96 hrs. Follicles treated with vehicle control showed normal growth, however, significant inhibition of antral follicle growth was observed with HPTE-treatment (0.1, 1 and 10 μg/ml) compared to DMSO control follicles at 72 and 96 hrs (Figure 3B). At 72 hrs, DMSO control follicles increased in size by 17.2 ± 1.1%, whereas HPTE 0.1, 1 and 10 μg/ml treated follicles only increased in size by 5.3 ± 0.2, 3.2 ± 0.3, and 1.9 ± 0.3% respectively. At 96 hrs, DMSO control follicles increased in size by 30.1 ± 0.8%, whereas HPTE 0.1, 1 and 10 μg/ml treated follicles only increased in size by 14.0 ± 0.6, 4.1 ± 0.2, and 2.2 ± 0.3%, respectively (n = 23 follicles per treatment, p ≤ 0.03). Further, the granulosa cell layer thickness for DMSO-treated follicles was 72.56 ± 1.64 μm (n = 6 follicles). Granulosa cell layer thickness for the HPTE-treated groups, however, was negligible as the granulosa cells were dispersed or non-existent and did not retain normal morphological appearance compared to the DMSO-treated group.

Further, the effect of mono-OH on follicle growth was evaluated for 96 hrs. Follicles treated with vehicle control showed normal growth, however, significant inhibition of antral follicle growth was observed with mono-OH-treatment (0.1, 1 and 10 μg/ml) compared to DMSO control follicles at 72 and 96 hrs (Figure 3C). At 72 hrs, DMSO control follicles increased in size by 17.2 ± 1.1%, whereas mono-OH 0.1, 1 and 10 μg/ml treated follicles only increased in size by 9.0 ± 0.5, 4.9 ± 0.3, and 2.3 ± 0.2% respectively. At 96 hrs, DMSO control follicles increased in size by 30.1 ± 0.8%, whereas mono-OH 0.1, 1 and 10 μg/ml treated follicles only increased in size by 16.3 ± 0.8, 5.9 ± 0.8, and 2.2 ± 0.6%, respectively (n = 23 follicles per treatment, p ≤ 0.03). As with the MXC-treated and HPTE-treated follicles, it was not possible to measure the thickness of the granulosa cell layers in mono-OH-treated follicles as the layers broke down and the granulosa cells became either dispersed or non-existent.

Effect of MXC, HPTE, and Mono-OH Treatment on Follicle Atresia

After culture, follicles were collected to determine the degree of atresia in treated and control follicles. Representative photographs of histological sections of control antral follicles are shown in Figure 4. There was little or no atresia (as evidenced by the lack of apoptotic bodies and disorganized cell layers) observed in DMSO-treated follicles (Figure 4).

Representative photographs of histological sections from MXC-treated antral follicles are shown in Figure 5A. MXC (1–100 μg/ml) increased follicular atresia compared to DMSO controls in a concentration dependent manner (Figure 5B). The atresia ratings were 1.7 ± 0.2 for DMSO, 3.2 ± 0.2 for MXC 1 μg/ml, 3.7 ± 0.1 for MXC 10 μg/ml, and 3.9 ± 0.1 for MXC 100 μg/ml-treated follicles (Figure 5B) (n = 6 follicles per treatment, p ≤ 0.001).

Representative photographs of histological sections of HPTE-treated antral follicles are shown in Figure 6A. HPTE (0.1–10 μg/ml) increased follicular atresia compared to DMSO controls in a concentration-dependent manner (Figure 6B). The atresia ratings were 1.4 ± 0.2 for DMSO, 2.7 ± 0.2 for HPTE 0.1 μg/ml, 3.3 ± 0.1 for HPTE 1 μg/ml, and 4.0 ± 0.0 for HPTE 10 μg/ml-treated follicles (Figure 6B) (n = 6 follicles per treatment, p ≤ 0.001).

Representative photographs of histological sections of mono-OH-treated antral follicles are shown in Figure 7A. Mono-OH (1 and 10 μg/ml) increased follicular atresia compared to DMSO controls in a concentration-dependent manner (Figure 7B). The atresia ratings were 1.0 ± 0.0 for DMSO, 1.4 ± 0.2 for mono-OH 0.1 μg/ml, 2.5 ± 0.2 for mono-OH 1 μg/ml, and 3.6 ± 0.2 for mono-OH 10 μg/ml-treated follicles (Figure 7B) (n = 6 follicles per treatment, p ≤ 0.001).