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Toxicology |
Program in Toxicology,3 Department of Epidemiology and Preventive Medicine
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Department of Physiology,4 University of Maryland, Baltimore, Maryland 21201
Department of Physiology,5 University of Arizona, Tucson, Arizona 85724
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, environment, follicle, ovary, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Bcl-2 and Bax proteins are well characterized, and studies have shown that they are important regulators in the ovary [6–9]. Yang and Rajamahendran demonstrated that progesterone-induced antral follicle atresia was associated with an increased ratio of Bax levels to Bcl-2 levels in bovine follicles [6]. Another study showed that overexpression of Bcl-2 during embryonic life resulted in a greater number of primordial follicles at birth [7]. Similarly, a Bcl- 2 knockout mouse model was found to have fewer than the normal complement of primordial follicles at birth [8]. Mice deficient in Bax, a proapoptotic protein, were found to have greater numbers of ovarian primordial follicles that were maintained into advanced chronological age [9].
In addition to being important apoptotic regulators in the ovary, Bcl-2 and Bax have been shown to be involved in chemically induced apoptosis [4]. For example, dimethylbenz[a]anthracene, a polyaromatic hydrocarbon that has been shown to induce ovarian atresia in juvenile catfish [10], also has been shown to induce Bax expression and apoptosis in mouse oocytes [11].
Atresia can occur at any stage of follicular development, but antral follicles are most vulnerable to atresia. Antral follicles are major producers of estrogen in the ovary and are also responsive to gonadotropin stimulation. These follicles will begin to die without proper hormonal stimuli. Specifically, follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol are major survival factors for antral follicles [12]. Thus, it is possible that antral follicles may undergo an increased rate of atresia due to an alteration in hormones or because fewer antral follicles are available for estrogen production, which might in turn lead to altered levels of gonadotropins.
The purpose of this study was twofold, first to examine whether MXC-induced atresia occurred through alterations in Bcl-2 or Bax, and second, to examine the effect of MXC on selected hormone and/or receptor levels. Specifically, this work tested the hypothesis that Bcl-2 expression is downregulated and/or Bax expression is upregulated by MXC, and further that an increase in Bcl-2 protein expression would protect against MXC-induced atresia, and the elimination of Bax expression would protect against MXC- induced atresia. In addition, this work tested the hypothesis that MXC increased atresia by altering levels of estradiol, FSH, or LH, hormones vital to antral follicle survival [13, 14] or that MXC affects the levels of estrogen receptors and/or FSH receptors because the levels of these receptors are also important for follicular survival [14].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MXC (99% pure) was purchased from Chemservice (West Chester, PA) in a powdered form. Stock solutions were prepared by mixing the appropriate amount of MXC with a constant volume of sesame oil. For the 8 mg/kg dose, 50 mg of MXC was mixed with 10 ml sesame oil; for the 16 mg/kg dose, 100 mg MXC was mixed with 10 ml sesame oil; for the 32 mg/kg dose, 200 mg MXC was mixed with 10 ml sesame oil; for the 64 mg/kg dose, 400 mg MXC was mixed with 10 ml sesame oil. Thus, the amount of chemical was concentrated with each subsequent dose so that mice received comparable injections based on their weights. This eliminated the need to give a greater volume injection to mice receiving higher doses than those receiving lower doses.
Animals
Cycling female CD-1 mice between the ages of 32 and 36 days were obtained from Charles River Laboratories (Wilmington, MA) and housed five animals per cage at the University of Maryland School of Medicine Central Animal Facility. In addition, our laboratory currently maintains breeding colonies of Bcl-2 overexpressing mice and Bax-deficient, or Bax knockout (BaxKO), mice. The Bcl-2 overexpressing mice were developed by Flaws et al. and are in a FVB background [7], and the Bax deficient mice were generated by Knudson et al. and are in a C57BL/6 background [15]. Females from these colonies between the ages of 30 and 66 days were housed (3–6 animals per cage) at the University of Maryland Central Animal Facility. Transgenic mice were genotyped using polymerase chain reaction (PCR)-based assays that have been previously described [7, 15]. Only wild-type mice and mice with homozygous deletion or overexpression were used in experiments. All animals were provided food and water for ad libitum consumption. Temperature was maintained at 22 ± 1°C and animals were subjected to 12L:12D cycles.
Dosing
CD-1 mice were dosed beginning at 39 days old with 8–64 mg kg–1 day–1 of MXC or sesame oil (vehicle control). Bcl-2 overexpressing mice were divided into two groups for dosing, one group receiving 64 mg/kg MXC and the other receiving sesame oil. Their wild-type littermates were also divided into two groups: one group receiving 64 mg/kg MXC and the other group receiving sesame oil. Similarly, BaxKOs and their wild- type littermates were divided into two groups, with one receiving 64 mg/ kg MXC and the other receiving sesame oil. We elected to use the 64 mg/ kg MXC dose in the transgenic animal experiments because our experiments in CD-1 mice showed that this dose was the most effective in inducing atresia without overt toxicity. Transgenics and their wild-type counterparts were aged between 30 and 66 days at the start of the dosing experiments.
All animals were dosed for 20 continuous days via intraperitoneal injection and were killed during estrus in the early part of the day (between 0800 and 1300 h). Vaginal cytology was used to determine the day of estrus. The University of Maryland School of Medicine Institutional Animal Use and Care Committee approved all procedures involving animal care, killing, and tissue collection.
Histological Evaluation of the Number of Follicles and Corpora Lutea
After dosing, ovaries were collected and fixed in Dietrick solution for 24 h. After fixation, the tissues were dehydrated, embedded in Paraplast (VWR Scientific, West Chester, PA), serially sectioned (8 µm), mounted on glass slides, and stained with Weigert hematoxylin-picric acid methylene blue. Ovarian sections were sampled and follicles were counted according to published methods [7, 16, 17]. Briefly, a stratified sample consisting of every 10th section was used to estimate the total numbers of antral follicles per ovary. The selected sections from one ovary were randomized and the number of antral follicles was counted in the entire section. Only follicles with a visible nucleolus were counted to avoid double counting. Sections were counted without knowledge of treatment.
Ovarian follicles were categorized as described by Flaws et al. [7]. Follicles were classified as antral if they contained five or more layers of granulosa cells and a clearly defined antral space. In some cases, antral follicles showed no antral space in cross section, but were scored as antral if they contained five granulosa cell layers. Antral follicles were classified atretic using standard morphological criteria [18]. Briefly, follicles were considered atretic if 10% of the granulosa cells were apoptotic (defined by the appearance of pyknotic bodies in the granulosa cell layer), the granulosa cell layer was disorganized, the oocyte was degenerating or its nucleus was fragmented.
To estimate the number of corpora lutea (CL) per ovary, sections were used to count the number of CL without knowledge of treatment group. To avoid double counting, each CL was followed through consecutive sections to ensure that it was only counted once.
Immunohistochemistry
After dosing, ovaries were fixed in 4% paraformaldehyde for 24 h. After fixation, the tissues were dehydrated, embedded in Paraplast (VWR Scientific, Baltimore, MD), serially sectioned (5 µm), and mounted on silane-treated (Sigma Aldrich, St. Louis, MO) glass slides. The sections were processed for immunolocalization of Bcl-2 and Bax using a commercially available polyclonal antibody against Bcl-2 (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and a polyclonal antibody against Bax (1:50 dilution; Santa Cruz Biotechnology) as the primary antibodies. The primary antibodies were found to be specific for their respective protein in previous studies conducted in our laboratory [17]. Furthermore, a titration of antibody concentration was conducted to ensure specificity in our tissues (dilutions 1:20–1:200). In some experiments, sections were processed for immunolocalization of ER
and ERß using a commercially available antibody against ER (1:80 dilution; Santa Cruz Biotechnology,) or ERß (1:360 dilution; Zymed Laboratories, San Franscisco, CA). The secondary antibody and visualization reagents were used according to the manufacturer's instructions from the HistoMouse-SP Kit (Zymed Laboratories). As a control for background staining, two methods were used: incubation with secondary antibody, but no primary antibody, or incubation with normal rabbit serum followed by secondary antibody for confirmation of results. Both types of negative controls produced the same result. Six to eight tissue sections were analyzed per slide.
Quantification of immunohistochemical results was carried out with a Zeiss microscope fitted with an MTI CCD72 camera (Dage-MTI, Michigan City, IN) connected to a Macintosh computer, and the NIH Image software program for image analysis. Optical densities were read by the NIH Image analysis software as pixels, which ranged in value from 0 (white) to 255 (black). A standard curve was generated in this grayscale range using a fourth-degree polynomial (R = 1.000) for a relative pixel density range of 0–0.60 units. A blue filter was used on the microscope to filter out the blue counterstain of the sections. Standardized levels remained calibrated for all sections analyzed. Representative stained ovarian sections were chosen and three measurements of pixel density were made of the granulosa and thecal layers of every antral follicle in that section. For instance, in a section containing seven antral follicles, a total of 21 measurements of the granulosa cell layer and 21 measurements of the thecal layer were made. Means and standard error of the means were determined for pixel density measurements of granulosa and thecal layers from three individual ovarian sections of sesame control and 64-mg kg–1 day–1 MXC-treated mice.
Estradiol Assays
Blood was collected during estrus from CD-1 mice after dosing and was subjected to enzyme-linked immunosorbent assay (ELISA) for measurement of estradiol levels. ELISA kits and reagents were obtained from Diagnostic Systems Laboratories, Inc. (Webster, TX). The assay was run using the manufacturer's instructions and published methods [19]. All samples were run in duplicate. The minimum detection limit, as stated in the instructions of the kit, was 7 pg/ml. The average intraassay coefficient of variation was 4.2% and the average interassay coefficient of variation was 8.2%.
Follicle Stimulating Hormone and Luteinizing Hormone Assays
Blood was collected from CD-1 mice during estrus and FSH and LH assays were carried out by radioimmunoassay (RIA) using reagents from the National Hormone and Pituitary Distribution Program. For FSH, we conducted measurements at all doses (8–64 mg/kg MXC); however, insufficient blood remained to measure LH at all doses, so we chose to measure the control and 32 mg/kg dose, a dose at which we observed an effect of MXC on the ovary [2]. Rat FSH and LH hormone antigen, rat FSH and LH antiserum, and mouse FSH and LH reference preparation were provided by the National Institute of Diabetes and Digestive and Kidney Diseases. Iodination reagents (IODO-BEADS 28665, 28666) were purchased from Pierce (Rockford, IL). For both FSH and LH, a standard curve was prepared and cold standards and samples (100 µl) were added to labeled tubes along with primary antibody (FSH at 1:1400 dilution and LH at 1:500) and iodinated FSH or LH. Samples were shaken and stored at 4°C overnight. On Day 2, secondary antibody was added (1:10 dilution) along with 2% normal rabbit serum (Sigma Aldrich, St. Louis, MO) and incubated at room temperature for 5 min. The tubes were centrifuged for 15 min at 3000 rpm, supernatant was decanted, and pellets were counted in a gamma counter for 1 min each. All samples were run in duplicate. Sensitivity for the FSH assay was 200 pg/ml with inter- and intraassay coefficients of variation of 2.7% and 6.7% respectively. Sensitivity for the LH assay was 86 pg/ml with inter- and intraassay coefficients of variation of 5.3% and 2.5% respectively.
Western Blot Analysis
After dosing, ovaries were collected from mice and immediately snap frozen in a dry ice and ethanol bath. Each frozen sample was homogenized in lysis buffer (40 mM Tris-HCl, pH 8.0, 1% NP40, 150 mM NaCl, 2.5 mM EDTA, 20 mM NaF, 20% glycerol) containing a protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany). After homogenization, the amount of protein in each sample was evaluated using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Protein lysates (40 µg/lane) were subjected to Western blot analysis using a 1:200 dilution of polyclonal antibody specific for ER (Santa Cruz Biotechnology) or a polyclonal antibody specific for ERß (Zymed Laboratories) as the primary antibody. A 1:3000 dilution of horseradish peroxidase (HRP)- conjugated anti-mouse polyclonal antibody (Santa Cruz Biotechnology) was used as the secondary antibody. Immune complexes were visualized using an enhanced chemiluminescence (ECL) detection kit (Cell Signaling Technologies, Beverly, MA). To ensure that proteins were loaded equally in each lane, the blots were stripped using ImmunoPure elution buffer (Pierce) and incubated with a ß-actin (1:3000 dilution, Santa Cruz Biotechnology) and HRP-conjugated anti-mouse polyclonal antibody. Scanning densitometry was used to estimate the pixel density of individual bands using Molecular Analyst Software (Bio-Rad Laboratories, Hercules, CA).
RNA Isolation and Real-Time Polymerase Chain Reaction Analysis
To compare FSH receptor (FSHR) gene expression between MXC- treated and sesame oil-treated ovaries, we used real time-PCR. Briefly, ovaries were collected from sesame oil- and MXC-treated animals, snap frozen, and stored at –70°C until further processing. Total RNA was extracted from whole ovaries using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's protocol. Real-time quantitative PCR was conducted using a MJ Research (OPTICON) Real-Time PCR machine (MJ Research, Waltham, MA) and accompanying software according to the manufacturer's instructions. The OPTICON quantifies the amount of PCR product generated by measuring the dye (SYBR green) that fluoresces when bound to double-stranded DNA. A standard curve was generated from five serial dilutions of one of the samples, thus allowing analysis of the amount of cDNA in the exponential phase. FSHR primer sequences were: (forward) 5'-AGCAAGTTTGGCTGTTATGAGG-3', (reverse) 5'-GTTCTGGATGATTTAGAGG-3' [20]. An initial incubation of 95°C for 10 min was followed by denaturing at 94°C for 10 sec, annealing at 55°C for 10 sec, and a final extension at 72°C for 10 sec for 50 cycles followed by final extension at 72°C for 10 min. A standard curve was generated by the software, and ß-actin was used for each sample as an internal standard. Final values for FSHR expression were calculated as the ratio FSHR:ß-actin [20]. Primers specific for the mouse ß-actin were used as an internal control as previously described [21]. All experiments were performed in triplicate.
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 comparisons between sesame oil-treated and MXC-treated mice and between genotypes, we used analysis of variance when comparing more than two groups and the Student t-test when comparing two groups. In the Western blot comparisons, we normalized doses to controls and conducted analysis of variance and linear regression analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MXC caused a dose-dependent increase in the percentage of atretic antral follicles compared with controls at the 32 mg/kg dose (72% atretic follicles with MXC treatment compared with 57% atretic follicles with sesame oil treatment) and 64 mg/kg dose (87% atretic follicles with MXC treatment compared with 57% atretic follicles with sesame oil treatment) after 20 days of dosing (Fig. 1; n = 3–9, P 0.001). In addition, mice treated with 64 mg/kg MXC had fewer antral follicles than those treated with sesame oil. The sesame oil-treated animals had a total of 1632 ± 192 antral follicles, whereas animals treated with 64 mg/kg MXC had a total number of 936 ± 136 antral follicles (n = 3–9, P 0.01).
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As an estimate of ovulation capacity, the number of CL in controls and MXC-treated ovaries were counted. The results indicate that the average number of CLs per ovary declined significantly with MXC treatment. There was an average of 9.5 ± 0.9 CL in the sesame oil-treatment group, 6.0 ± 0.7 in the 32 mg kg–1 day–1 MXC-treatment group and 4.8 ± 0.9 in the 64 mg kg–1 day–1 MXC treatment group (n = 3–7, P 0.01). There was no overt toxicity nor any change in the weights of the animals at these doses of MXC.
Effect of MXC on Bcl-2 and Bax Levels
Using immunohistochemical analysis, Bcl-2 staining was found in the oocyte, granulosa, and thecal cells of the follicles in sesame oil- and MXC-treated ovaries. MXC (64 mg kg–1 day–1 for 20 days) did not affect the expression of Bcl-2 in the antral follicles of the ovary. There was no difference with regard to follicle staining whether the follicles were healthy or atretic. Small (primordial and primary) follicles were unstained, although staining was present in some larger preantral follicles (Fig. 2, A–D). Image analysis confirmed there was no difference in staining in either the granulosa or thecal cell layers between the sesame oil- and MXC-treated ovaries (n = 3, P = 0.5).
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Immunohistochemical staining for Bax was very light in the granulosa and thecal cells of follicles from the sesame oil-treated mice; however, the staining in the granulosa and especially in the thecal cells was very intense in the follicles and interstitium of the MXC-treated (64 mg kg–1 day–1 for 20 days) mice in both healthy and atretic follicles. As with the Bcl-2 staining, the primordial and primary follicles were largely unstained, although the larger preantral follicles did show Bax staining (Fig. 3, A–D). Image analysis confirmed that there was greater staining in both the granulosa and thecal cell layers. Mean pixel density in the granulosa cell layer was 0.31 ± 0.01 and 0.36 ±0.01 in the sesame oil and the MXC-treated animals, respectively (n = 3; P 0.001). Mean pixel density in the thecal cell layer was 0.31 ± 0.01 and 0.38 ± 0.01 in the sesame oil- and MXC-treated animals, respectively (n = 3; P 0.001).
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Effect of MXC on the Ovaries of Mice That Overexpress Bcl-2 Protein
In wild-type mice, MXC (64 mg kg–1 day–1 for 20 days) significantly increased the percentage of atretic follicles compared with controls (Fig. 4). Wild-type mice treated with MXC had 59% atretic follicles, whereas wild-type mice treated with sesame oil had 43% atretic follicles (Fig. 4A; n = 8; P 0.03). In Bcl-2 overexpressing mice, MXC was unable to significantly increase the percentage of atretic follicles compared with the sesame oil-treated Bcl-2 overexpressing mice. MXC-treated Bcl-2 overexpressing mice had 48% atretic follicles and sesame oil-treated Bcl- 2 overexpressing mice had 50% atretic follicles (Fig. 4A; n = 7; P = 0.7). In addition, levels of atresia in MXC- treated Bcl-2 overexpressing mice were no different than the levels in sesame oil-treated wild-type mice (48% vs. 43%; Fig. 4A; n = 7–8; P = 0.4).
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MXC-treated Bcl-2 overexpressing mice had significantly more antral follicles (1726 ± 155) than MXC-treated wild-type mice (1330 ± 99; n = 7–9; P 0.05), and of these, the number of healthy antral follicles in the Bcl-2 overexpressing mice was greater than those of the MXC- treated wild-type mice (903 ± 123 vs. 540 ± 72, respectively; Fig. 4B; n = 7–9; P 0.03).
Effect of MXC on the Ovaries of Mice Deficient in Bax Protein
In wild-type mice treated with MXC (64 mg kg–1 day–1 for 20 days), there was a significant increase in the percentage of atretic follicles compared with sesame oil-treated counterparts (73% atretic follicles in the MXC-treated wild- type mice vs. 49% in the sesame oil-treated wild-type mice; Fig. 5; n = 3–5; P 0.0001). In BaxKO mice, however, MXC was unable to increase the percentage of atretic follicles (31% atretic follicles in the MXC-treated BaxKO vs. 27% in the sesame oil-treated BaxKO mice; Fig. 5; n = 3– 5; P = 0.5).
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Although there was an increase in the percentage of atretic follicles in MXC-treated wild-type mice vs. sesame oil-treated wild-type mice, there was no significant change in the total number of antral follicles between MXC-treated wild-type mice and sesame oil-treated wild-type mice (n = 3–5). In contrast, there was a striking increase in the total number of atretic antral follicles between MXC-treated wild-type mice (1387 ±141) when compared with the MXC-treated BaxKO mice (480 ± 33; n = 3–5, P 0.001).
Effect of MXC on Estradiol, FSH, and LH Levels
After 20 days of dosing, there were no differences in estradiol levels between sesame oil-treated mice and mice dosed with 8, 16, 32, or 64 mg kg–1 day–1 MXC (Fig. 6A; n = 6–9, P = 0.74). FSH levels also did not differ between sesame oil-treated mice and mice treated with 8, 16, 32, or 64 mg kg–1 day–1 MXC (Fig. 6B; n = 6–9; P = 0.39). Finally, LH levels were unchanged between sesame oil- treated mice and those treated with 32 mg kg–1 day–1 MXC (Fig. 6C; n = 3; P = 0.2).
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Effect of MXC on ER, ERß, and FSH Receptor Levels
After 20 days of dosing, there were no significant differences in ER or ERß protein levels between mice treated with sesame oil or mice treated with 8, 16, 32, or 64 mg kg–1 day–1 MXC (Fig. 7, A and B). In order to confirm these results, immunohistochemical analysis of ER and ERß staining intensity was performed on ovarian sections from mice treated with sesame oil and 64 mg kg–1 day–1 MXC. Similarly, there were no differences in ER or ERß levels between the sesame oil- and MXC-treated animals (data not shown). When ERß staining was analyzed in the follicles, there were no differences in staining intensity among the different follicle types. In the ER-stained ovaries, there was no major staining in the follicles. Finally, there were no significant differences in the levels of FSHR mRNA between mice treated with sesame oil and 64 mg kg–1 day–1 MXC. The mean pixel density of the ß-actin corrected values for FSHR in the sesame oil-treated ovaries was 1.1 ± 0.2, and for MXC-treated ovaries, it was 1.42 ± 0.2 (n = 3; P = 0.3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our study, the levels of Bcl-2 protein were not affected by MXC treatment of CD-1 mice; nevertheless, Bcl-2 overexpression did confer some resistance to MXC because the Bcl-2 overexpressing mice had significantly more total antral follicles as well as healthy antral follicles than the MXC-treated wild-type mice. In addition, Bcl-2 overexpressers treated with MXC did not have a greater percentage of atretic follicles than Bcl-2 overexpressers treated with sesame oil. We speculate that, after sustained MXC exposure, signaling pathways may be activated that result in an upregulation of Bax, thus leading to a change in the ratio of proapoptotic Bax to antiapoptotic Bcl-2, and that this may result in an increased likelihood of cell death.
There has been little work in vivo investigating the effects of pesticides on Bcl-2 family members and none, to our knowledge, investigating the effects of MXC on Bcl-2 family members in the ovary. Still, some studies have looked at other chemicals, and previous studies conducted in Bax-deficient mouse models found some protection from toxicant-induced apoptosis in primordial follicles and oocytes. For example, Takai et al. found that Bax-deficient mice were partially protected by the effects of the industrial chemical and primordial follicle toxicant, 4-vinylcyclohexene diepoxide [23]. Bax-deficient mice were also protected from destruction of oocytes by the chemotherapeutic agent doxorubicin [9]. Previous studies also have utilized transgenic mouse models that overexpress Bcl-2 to test whether Bcl-2 overexpression protects against toxicant-induced apoptosis, although none have looked at MXC. For instance, the EW36 cell line, a human cell line that overexpresses Bcl-2, was found to be more resistant to apoptosis induced by a variety of toxicants [24]. Morita et al. found that oocytes obtained from transgenic mice expressing higher amounts of Bcl-2 (with overexpression being driven by the zona pellucida 3 gene promoter) were more resistant to chemotherapy-induced apoptosis [25].
Regarding the effect of MXC on hormonal parameters, MXC-induced atresia did not result in changes in estradiol, FSH, or LH, hormones known to be important antiapoptotic factors in follicles [12, 13, 14]. In spite of the decline in antral follicles that we observed in mice treated with MXC, we did not see a difference in estradiol levels between the sesame oil- and MXC-treated mice. This was initially surprising because antral follicles are a major producer of estradiol, but there may be several explanations for this finding. First, the reduction in antral follicles that we observed with MXC treatment may not be dramatic enough to alter endogenous estradiol levels. Second, it is possible that the remaining follicles compensate for the loss of follicles by increasing their estradiol production, (i.e., the few remaining antral follicles are producing high levels of estradiol to compensate for follicular loss). Finally, it is possible that MXC increases estrogen production by nonovarian tissues, such as fat.
Regarding the gonadotropins, previous studies examining the effect of MXC in male rats found that serum levels of LH were unaffected by 200–400 mg kg–1 day–1 MXC treatment for 44 wk [26]. On the other hand, Okazaki et al. observed a decline in LH and FSH levels in male rats treated for 28–31 days with 100 or 500 mg/kg MXC, respectively [27]. Also, the investigators observed a decline in LH levels in female rats treated with 100 or 500 mg/kg MXC for 28– 31 days [27]. The doses and duration of dosing in these previous studies were greater than those used in this study. Furthermore, the authors of the previous studies suggested that the effects of MXC on gonadotropins might be a secondary effect due to ovarian atrophy [27]. In other studies of female CD-1 mice, the effects of MXC on female reproduction were speculated to be due to estrogen mimicry and/ or damage to the hypothalamic-pituitary-ovarian axis [28, 29]. These studies, however, did not include measurements of estradiol, FSH, or LH. It appears from the results of our study that the increase in atresia and the decline in healthy antral follicles observed after MXC treatment are not due to a decrease in estradiol, FSH, or LH.
Our study shows that MXC treatment does not significantly affect the levels of ER, ERß, or FSHR in the ovary. This is the first in vivo study that we are aware of that has examined the effect of MXC on ER, ERß, or FSHR. As stated previously, estrogen is necessary for FSHR induction [14], and our results suggest that MXC does not mimic estrogen to induce, nor does it block estrogen to reduce FSHR content. Furthermore, our study suggests that MXC does not act as an estrogen to induce levels of estrogen receptor.
In conclusion, our data suggest that MXC-induced atresia involves the Bax and Bcl-2 protein signaling pathways. Our data also suggest that MXC does not induce atresia by altering levels of estradiol or the gonadotropins, nor does it alter levels of ER, ERß, or FSHR. There are many other members of the Bcl-2 family, and future studies should look at the expression of other Bcl-2 family members in treated follicles, such as Mcl-1 and Bok, as each is thought to be a major player in ovarian apoptosis. Such studies will lead to an increased understanding of how toxicants induce atresia, and possibly lead to treatments for toxicant-induced ovarian failure.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jodi A. Flaws, Department of Epidemiology and Preventive Medicine, 660 West Redwood Street, Baltimore, MD 21210-1596. FAX: 410 706 1503; jflaws{at}epi.umaryland.edu
Received: 2 September 2003.
First decision: 23 September 2003.
Accepted: 9 February 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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]anthracene on apoptosis and HSP70 expression in juvenile channel catfish (Ictalurus punctatus) ovary.. Aquat Toxicol 2001 54:39-50[CrossRef][Medline]This article has been cited by other articles:
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 R. K. Gupta, G. Aberdeen, J. K. Babus, E. D. Albrecht, and Jodi. A. Flaws Methoxychlor and Its Metabolites Inhibit Growth and Induce Atresia of Baboon Antral Follicles Toxicol Pathol, August 1, 2007; 35(5): 649 - 656. [Abstract] [Full Text] [PDF]
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 C. R Greenfeld, J. K Babus, P. A Furth, S. Marion, P. B Hoyer, and J. A Flaws BAX is involved in regulating follicular growth, but is dispensable for follicle atresia in adult mouse ovaries Reproduction, January 1, 2007; 133(1): 107 - 116. [Abstract] [Full Text] [PDF]
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 M. Uzumcu, P. E Kuhn, J. E Marano, A. E Armenti, and L. Passantino Early postnatal methoxychlor exposure inhibits folliculogenesis and stimulates anti-Mullerian hormone production in the rat ovary J. Endocrinol., December 1, 2006; 191(3): 549 - 558. [Abstract] [Full Text] [PDF]
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 R. K. Gupta, K. P. Miller, J. K. Babus, and J. A. Flaws Methoxychlor Inhibits Growth and Induces Atresia of Antral Follicles through an Oxidative Stress Pathway Toxicol. Sci., October 1, 2006; 93(2): 382 - 389. [Abstract] [Full Text] [PDF]
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K. P. Miller, R. K. Gupta, and J. A. Flaws Methoxychlor Metabolites May Cause Ovarian Toxicity Through Estrogen-Regulated Pathways Toxicol. Sci., September 1, 2006; 93(1): 180 - 188. [Abstract] [Full Text] [PDF]
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D. Tomic, M. S. Frech, J. K. Babus, R. K. Gupta, P. A. Furth, R. D. Koos, and J. A. Flaws Methoxychlor Induces Atresia of Antral Follicles in ER{alpha}-Overexpressing Mice Toxicol. Sci., September 1, 2006; 93(1): 196 - 204. [Abstract] [Full Text] [PDF]
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 G. S. Daftary and H. S. Taylor Endocrine Regulation of HOX Genes Endocr. Rev., June 1, 2006; 27(4): 331 - 355. [Abstract] [Full Text] [PDF]
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Y. Wu, W. G Foster, and E. V Younglai Rapid effects of pesticides on human granulosa-lutein cells Reproduction, February 1, 2006; 131(2): 299 - 310. [Abstract] [Full Text] [PDF]
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D. A. Symonds, K. P. Miller, D. Tomic, and J. A. Flaws Effect of Methoxychlor and Estradiol on Cytochrome P450 Enzymes in the Mouse Ovarian Surface Epithelium Toxicol. Sci., February 1, 2006; 89(2): 510 - 514. [Abstract] [Full Text] [PDF]
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R. A. Schuh, T. Kristian, R. K. Gupta, J. A. Flaws, and G. Fiskum Methoxychlor Inhibits Brain Mitochondrial Respiration and Increases Hydrogen Peroxide Production and CREB Phosphorylation Toxicol. Sci., December 1, 2005; 88(2): 495 - 504. [Abstract] [Full Text] [PDF]
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K. P. Miller, R. K. Gupta, C. R. Greenfeld, J. K. Babus, and J. A. Flaws Methoxychlor Directly Affects Ovarian Antral Follicle Growth and Atresia through Bcl-2- and Bax-Mediated Pathways Toxicol. Sci., November 1, 2005; 88(1): 213 - 221. [Abstract] [Full Text] [PDF]
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D. A. Symonds, D. Tomic, K. P. Miller, and J. A. Flaws Methoxychlor Induces Proliferation of the Mouse Ovarian Surface Epithelium Toxicol. Sci., February 1, 2005; 83(2): 355 - 362. [Abstract] [Full Text] [PDF]
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 D. Tomic, K. P. Miller, H. A. Kenny, T. K. Woodruff, P. Hoyer, and J. A. Flaws Ovarian Follicle Development Requires Smad3 Mol. Endocrinol., September 1, 2004; 18(9): 2224 - 2240. [Abstract] [Full Text] [PDF]
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