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

Abstract

Methoxychlor (MXC), an organochlorine pesticide, inhibits growth and induces atresia of antral follicles in rodents. MXC metabolites, mono-OH MXC (mono-OH) and bis-OH MXC (HPTE), are thought to be more toxic than the parent compound. Although studies have examined the effects of MXC in rodents, few studies have evaluated the effects of MXC in primates. Therefore, the present study tested the hypothesis that MXC, mono-OH, and HPTE inhibit growth and induce atresia of baboon antral follicles. To test this hypothesis, antral follicles were isolated from adult baboon ovaries and cultured with vehicle (dimethylsulfoxide; DMSO), MXC (1–100 μg/ml), mono-OH (0.1–10 μg/ml), or HPTE (0.1–10 μg/ml) for 96 hr. Growth was monitored at 24 hr intervals. After culture, follicles were processed for histological evaluation of atresia. MXC, mono-OH, and HPTE significantly inhibited follicular growth and increased atresia compared to DMSO. Moreover, the adverse effects of MXC and its metabolites on growth and atresia in baboon antral follicles were observed at lower (100-fold) doses than those causing similar effects in rodents. These data suggest that MXC and its metabolites inhibit growth and induce atresia of baboon antral follicles, and that primate follicles are more sensitive to MXC than rodent follicles.

Introduction

Female mammals are born with finite number of follicles (Hirshfield, 1991). At the time of birth, human females have about 400,000 primordial follicles and by age 30, the number is reduced to 25,000 through ovulation and natural cell death (Moffet, 1993). These primordial follicles must grow and mature through different stages known as the primary, preantral, and antral stages to be capable of ovulation. The antral follicle is the penultimate stage required for normal ovulation and synthesis/secretion of the major sex steroid hormones, which are essential for reproduction (Hirshfield, 1991).

Any chemical that disturbs follicular growth or atresia (an apoptotic process) can alter reproductive and endocrine function. The degree and type of damage to the ovary depends on the type of follicle that is targeted by the chemical. Administration of a toxicant that destroys primordial follicles may result in permanent infertility because primordial follicles cannot be replenished. Administration of a toxicant that targets primary follicles may result in temporary or permanent infertility. Temporary infertility occurs if the toxicant is removed, the primordial follicles are unaffected, and a new cohort of primordial follicles is able to grow and replace the damaged primary follicles. Permanent infertility occurs if the chemical is not removed because there will be no primary follicles to grow to the larger stages.

Administration of a toxicant that destroys antral follicles may result in temporary infertility if the toxicant is removed and undamaged primordial, primary, and preantral follicles are able to grow to take the place of damaged antral follicles. Permanent infertility occurs if the chemical is not removed because there will be no antral follicles to release ova for fertilization. Further, administration of a toxicant that increases atresia of follicles at any stage could accelerate the rate at which follicles are depleted from the ovary and result in premature or early reproductive senescence.

Methoxychlor (MXC) is an organochlorine pesticide that is used in many countries against insects that attack fruits, vegetables, and home gardens. Thus, humans as well as wildlife species are exposed to this chemical. MXC is a known reproductive toxicant. Investigators have reported that MXC exposure reduces fertility in a variety of species (Martinez and Swartz, 1992; Swartz and Corkern, 1992; Borgeest et al., 2002a). Several studies indicate that MXC reduces fertility because it causes ovarian atrophy and decreases the ability of ovarian cells to synthesize and secrete hormones (Martinez and Swartz, 1992; Swartz and Corkern, 1992). In addition, MXC induces atresia of antral follicles in rodents (Borgeest et al., 2002a, b; Borgeest et al., 2004; Miller et al., 2005). Borgeest et al. (2002b, 2004) have shown that MXC specifically targets antral follicles by reducing their number and increasing the percentage of atretic antral follicles in mice. Miller et al. (2005) have shown that MXC inhibits growth and induces atresia of mouse antral follicles in culture.

MXC is metabolized in the body to mono-hydroxy MXC (mono-OH), bis-hydroxy MXC (HPTE), catechol MXC, and tris-hydroxy MXC by cytochrome P-450 enzymes (Figure 1). HPTE is known to act as an estrogen receptor (ER) α agonist and an ERβ antagonist in human hepatoma cells (Gaido et al., 2000). HPTE and mono-OH are proposed to be more toxic than the parent compound in other tissues, (Gaido et al., 2000) but one study has shown that mono-OH is more toxic to mouse antral follicles compared to MXC and HPTE (Miller et al., 2006).

While previous studies have examined the effects of MXC and/or its metabolites in rodents and cell lines, no studies have examined the effects of MXC on primate ovarian follicles. Such studies are important for better understanding species differences in response to environmental chemicals. By better understanding species differences in response to chemicals, it may be possible to determine which model is best for predicting toxicity of similar chemicals or for extrapolating data to other species. Thus, the goal of these studies was to establish a culture system using follicles from non-human primate model, the baboon, and then to use this system to determine the effects of MXC and its metabolites on primate follicles. Specifically, this study tested the hypothesis that MXC and its metabolites (HPTE and mono-OH) inhibit growth and induce atresia of baboon antral follicles.

Materials and Methods

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).

Results

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).

Discussion

To our knowledge, this is the first study showing the effects of methoxychlor (MXC) and its metabolites, bis-hydroxy MXC (HPTE) and mono-hydroxy MXC (mono-OH), on baboon antral follicles. The results indicate that baboon antral follicles can be grown in culture successfully and that MXC and its metabolites (HPTE and mono-OH) inhibit growth and induce atresia of baboon antral follicles in culture.

MXC, an organochlorine pesticide, is a known reproductive toxicant. Most previous studies showing effects of MXC on reproductive organs such as the ovary have been done in rodents (Borgeest et al., 2002a, 2002b, 2004; Miller et al., 2005). These studies indicate that MXC increases the percentage of atretic antral follicles in mice when treated with MXC in vivo (Borgeest et al., 2002b, 2004), and it inhibits growth and induces atresia of cultured mouse antral follicles (Miller et al., 2005). In this study, we have shown that MXC inhibits growth and induces atresia of cultured baboon antral follicles. Interestingly, the adverse effects of MXC on growth and atresia in baboon antral follicles were observed at 100-fold lower doses than those causing similar effects in rodents (Miller et al., 2005). The reasons that baboon follicles are more sensitive to MXC than mouse follicles are unknown. It is possible, however, that baboon and mouse follicles have different cytochrome P450 enzymes in their follicles. Perhaps, the cytochrome P450 enzymes in the mouse ovary are better able to detoxify MXC than those in the baboon ovary.

While no other studies have reported the effects of MXC on baboon follicles, Golub et al. (2003) have shown that MXC treatment leads to premature emergence of a secondary sexual characteristic (retarded growth of the nipple), increased incidence of ovarian cysts/masses, and shorter follicular stages in rhesus monkeys (Golub et al., 2003). Further, studies have examined effects of other pesticides on non-human primates. Bourque et al. (1995) have shown that treatment with the pesticide hexachlorobenzene causes lesions in ovarian follicles in monkeys, which included lipid peroxidation, condensed mitochondria, degeneration of follicular cells, and appearance of abnormal spaces between the follicular cells (Bourque et al., 1995).

Our study is the first to show the effects of MXC metabolites (HPTE and mono-OH) on baboon antral follicles. HPTE and mono-OH inhibit growth and induce atresia of baboon antral follicles at a 10-fold lower concentration than the parent compound MXC. We hypothesized that the metabolites (HPTE and mono-OH) would be more toxic than the parent compound MXC based on previous binding studies (Bulger et al., 1978; Ousterhout et al., 1981; Bulger et al., 1985; Laws et al., 2000; Gaido et al., 2000). MXC, HPTE, and mono-OH have all been shown to compete with estradiol for binding to estrogen receptor (ER) to varying degrees; MXC has very low affinity for ER, while mono-OH and HPTE have higher affinity for ER (Bulger et al., 1978, 1985; Ousterhout et al., 1981; Laws et al., 2000). HPTE has been shown to be an ERα agonist and ERβ antagonist in HEP-G2 cells (Gaido et al., 2000). Specifically, HPTE has an EC50 value of 5 × 10⁻⁸ for human ERα and an EC value of 10⁻⁸ M for rat ERα (Gaido et al., 1999). Further, HPTE completely abolishes 17-β estradiol-induced ERβ activity (Gaido et al., 1999). It is possible that mono-OH might be acting through similar mechanism, although this has not been tested directly.

The mechanism by which MXC, HPTE, and mono-OH cause inhibition of growth and induce atresia of baboon antral follicles is unknown. Borgeest et al. (2004) have shown that the Bcl-2 pathway may be involved in MXC-induced atresia of antral follicles in mice. Recently, Li et al. (2006) showed that HPTE activates non-genomic MAP kinase and phosphoinositol-I-3 pathways in human MCF-7 cells (Li et al., 2006). Further, Gupta et al. (2006) have shown that oxidative stress pathways may regulate MXC-induced atresia in mice. Thus, it may be possible that MXC, HPTE, and mono-OH act through any or all of these pathways to inhibit growth and induce atresia of baboon antral follicles. Future studies are required to address the issue of mechanism of action of MXC and its metabolites on baboon follicles.

In addition, it is not known whether granulosa cells or thecal cells in follicles respond equally to MXC and its metabolites. In this study, it visually appears that MXC and its metabolites first affect the granulosa cells followed by the theca cells. We then see loss of structure of oocytes in our treated groups. Future studies need to be conducted to fully test whether granulosa cells or thecal cells are first targeted by the chemicals.

In conclusion, baboon antral follicles can be grown in culture and MXC and its metabolites, HPTE and mono-OH, inhibit growth and induce atresia of baboon antral follicles in culture. Further, this study indicates that primate follicles may be more sensitive to MXC and its metabolites than rodents.