* Program in Toxicology, University of Maryland, Baltimore, Maryland 21201;
Department of Physiology, University of Arizona, Tucson, Arizona; and
Department of Epidemiology and Preventive Medicine, University of Maryland, 660 W. Redwood Street, Baltimore, Maryland 21201
Received December 26, 2001; accepted April 2, 2002
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ABSTRACT |
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Key Words: methoxychlor; kepone; ovary; follicle; atresia; ovarian epithelium; estrous cycle.
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INTRODUCTION |
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Although these experiments yield important toxicological information, they are limited in mechanistic information about the effects of MXC on the ovary. For example, ovarian weights were often used as a measure of ovarian function, but this endpoint is not sensitive enough to detect subtle changes in the percentage of atretic follicles or decreases in the number of smaller follicles. Furthermore, ovarian weights do not reveal which cells within the ovary (oocytes, granulosa, thecal, surface epithelial cells, or some combination of cell types) or which follicle types (primordial, primary, preantral, or antral) are targeted by MXC.
It is important to identify specific cells/follicles targeted by MXC because the target tissue can be used to predict the impact of the chemical on reproduction (Hirshfield, 1997). For example, a toxicant that destroys primordial follicles can lead to permanent infertility because of the eventual depletion of this large, nonrenewable pool of follicles. On the other hand, a toxicant that damages primary or preantral follicles may lead to temporary infertility, assuming the primordial follicles are unaffected and the toxic insult is removed in enough time to allow recruitment of primordial follicles to the antral stage (Hirshfield, 1997
). Furthermore, a toxicant that damages antral follicles may lead to changes in hormone levels and/or cyclicity because these follicles are responsible for synthesis of estrogens that help regulate cyclicity (Hirshfield, 1997
). Finally, a toxicant that affects the ovarian surface epithelium may alter the risk of ovarian cancer, as this is thought to be the primary site of ovarian cancer development (Auersperg et al., 2001
)
Therefore, the purpose of this study was to test the hypothesis that MXC adversely affects the ovarian follicles and surface epithelium in the mouse. We examined ovarian damage by testing whether MXC altered follicle numbers, the percentage of atretic follicles, or the thickness of the ovarian epithelial layer. In addition, we evaluated estrous cyclicity in the mice to determine whether MXC disrupted the estrous cycle. Finally, we used lower overall doses in these studies (832 mg/kg) than those used in many previous studies on the effects of MXC (25700 mg/kg/day). In our studies, the organochlorine pesticide kepone (KPN) was used as a positive control because it is a lipophilic, persistent chemical that has been shown to damage the ovary and disrupt estrous cyclicity in mice (Das et al., 1998; Hodges et al., 2000
; Johnson et al., 1995
; Swartz and Mall, 1989
). We chose to dose with 8 mg/kg/day KPN because this dose was sufficient to cause follicular atresia. For MXC, we started the dosing at 8 mg/kg/day to correlate with the dose of KPN selected for the study. We also chose to use two additional doses of MXC to obtain dose-response information.
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MATERIALS AND METHODS |
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Animals.
Cycling female CD-1 mice were housed five animals per cage at the University of Maryland School of Medicine Central Animal Facility and provided food and water for ad libitum consumption. Temperature was maintained at 22.2°C, and animals were subjected to 12-h light-dark cycles. Vaginal cytology was analyzed to determine the day of the cycle beginning at 35 days of age. Mice (beginning at 39 days of age) were dosed via intraperitoneal injection with 8, 16, or 32 mg/kg/day MXC, 8 mg/kg/day KPN, or sesame oil (vehicle) for 10 or 20 continuous days, and euthanized during estrus in the early part of the day (between 8 A.M. and 1 P.M.). To determine if 20 days of continuous dosing was causing undue stress, a second control group received no vehicle or pesticide treatment for 20 days. The University of Maryland School of Medicine Institutional Animal Use and Care Committee approved all procedures involving animal care, euthanasia, and tissue collection.
Histological evaluation of follicle numbers.
Ovaries were collected and fixed in Bouins solution for 24 h and transferred to 70% alcohol. After fixation, the tissues were dehydrated, embedded in Paraplast (VWR Scientific, West Chester, PA), serially sectioned (8 mm), mounted on glass slides, and stained with Weigerts hematoxylin-picric acid methylene blue. Ovarian sections were sampled and follicles were counted according to published methods (Flaws et al., 2001; Smith et al., 1991
; Tomic et al., 2002
). Briefly, a stratified sample consisting of every tenth section was used to estimate the total numbers of primordial, primary, preantral, and antral follicles per ovary. The selected sections from one ovary (approximately 2030 sections per ovary) were randomized and the number of primordial, primary, preantral, and 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. (2001). Briefly, follicles were classified as primordial if they contained an intact oocyte with a visible nucleolus surrounded by a single layer of fusiform-shaped granulosa cells. Follicles were classified as primary if they consisted of an oocyte with a visible nucleolus and a single layer of cuboidal granulosa cells. Follicles were classified as preantral if they contained an oocyte with a visible nucleolus and 24 layers of granulosa cells with no antral space. Follicles were classified as antral if they contained three 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 5 granulosa cell layers. Antral follicles were considered atretic if they contained at least 20 apoptotic granulosa cells (defined by the appearance of apoptotic bodies in the granulosa cell layer), disorganized granulosa cells, a degenerating oocyte, or fragmentation of the oocyte nucleus.
Measurement of epithelial height.
The height of the ovarian surface epithelium was measured with an ocular micrometer and a 40x objective. Measurements were made at 12, 3, 6, and 9 oclock positions on serial sections. A total of 60 consecutive observations were recorded for each sample. The average was multiplied by a calibration factor from a micrometer scale to arrive at the average height in microns.
Estrous cycles.
Estrous cycles were monitored by analysis of vaginal cytology according to procedure described previously by Cooper et al. (1993). Briefly, a plastic pipette was inserted into the vagina, with care not to stimulate the cervix, and the area was gently flushed with a phosphate-buffered saline (PBS) solution. Vaginal cells were analyzed by light microscopy according to standard morphological criteria (Cooper et al., 1993). Briefly, estrus was characterized by masses of cornified cells, early diestrus by a mixture of leukocytes and epithelial cells, diestrus by leukocytes, and proestrus by round, nucleated epithelial cells (Cooper et al., 1993
).
Statistical analysis.
All data were analyzed using SPSS statistical software (SPSS, Inc., Chicago, IL) using ANOVA with statistical significance assigned at p 0.05. When a significant p value was obtained with ANOVA, the Scheffé test was used in the post hoc analysis. When appropriate, we also ran post hoc analysis using the Dunnetts t (two-sided) test. In some experiments, nontreated and sesame-treated controls were combined because there were no significant differences between groups. All data are presented as mean ± SEM.
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RESULTS |
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At the 20-day time point, there also were no significant differences in numbers of primordial and preantral follicles between controls and the pesticide-treated groups (Fig. 1, n = 510, p
0.99). There also was no significant difference in the numbers of primary follicles between control and MXC- and KPN-treated ovaries (Fig. 1
, n = 510, p
0.081). The 32 mg/kg MXC and 8 mg/kg KPN-treated mice had significantly fewer primary follicles than the 16 mg/kg MXC-treated mice (Fig. 1
, n = 510, p
0.04).
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DISCUSSION |
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MXC may increase antral follicle atresia and ovarian epithelial proliferation either by directly targeting these tissues or indirectly via alteration of endogenous hormones. A previous study using an in vitro rabbit model found that estrogen causes proliferation of the ovarian surface epithelium (Bai et al., 2000). Our experiments have shown that MXC also increases the ovarian surface epithelium height. Therefore, it may be mimicking an estrogen in this regard. On the other hand, MXC may act as an androgen or antiestrogen in the ovarian follicles, as it is thought that androgens promote and estrogens inhibit granulosa cell apoptosis (Billig et al., 1993
). MXC may also affect follicle-stimulating hormone (FSH) levels, as FSH is an important survival factor for early antral follicles (Chun et al., 1996
). It is also possible that MXC may be interfering with other hormonal pathways, for instance, progesterone. Previous studies looking at the effects of progesterone in the macaque ovarian epithelium found that progesterone promotes apoptosis in those cells (Rodriguez et al., 1998
, 2002
). Alternatively, it is possible that MXC is acting independent of hormonal pathways. In a previous experiment involving male rats, it was found that MXC affected the central nervous system, epididymal sperm numbers, accessory sex glands, and mating without changing the secretion of luteinizing hormone, prolactin or testosterone; that is, MXC did not change systemic endocrine function (Gray et al., 1999
).
MXC and KPN appear to differ somewhat in their action. For instance, both pesticides increased atresia in antral follicles, but only MXC increased the height of the ovarian surface epithelium. It is possible that KPN may cause an increase in epithelial height at a higher dose, and that higher doses of MXC may cause an increase in the number of days in estrus, as Martinez and Swartz (1992) have reported that MXC increased vaginal cornification at 50200 mg/kg/day. However, our study is important because it shows that low doses of MXC and KPN can affect the ovary, and that low doses of KPN can affect the vaginal epithelium. In general, KPN appears to be the more potent of the two pesticides we tested, as the effects on follicular atresia at the 8 mg/kg dose of KPN were comparable to those at the 32 mg/kg dose of MXC.
In conclusion, the results from these experiments show that MXC affects antral follicles and the ovarian surface epithelium, and that KPN affects antral follicles and estrous cyclicity. The effect of MXC on the ovary may be dualistic, because it causes an apparent increase in atresia (i.e., apoptosis) in antral follicles and an increase in proliferation in the ovarian surface epithelium. Our future studies will address the mechanism for this dualistic action as well as the reasons for the different effects of MXC and KPN on the ovary and estrous cycle.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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