Methoxychlor May Cause Ovarian Follicular Atresia and Proliferation of the Ovarian Epithelium in the Mouse

C. Borgeest*, D. Symonds*, L. P. Mayer{dagger}, P. B. Hoyer{dagger} and J. A. Flaws*,{ddagger},1

* Program in Toxicology, University of Maryland, Baltimore, Maryland 21201; {dagger} Department of Physiology, University of Arizona, Tucson, Arizona; and {ddagger} Department of Epidemiology and Preventive Medicine, University of Maryland, 660 W. Redwood Street, Baltimore, Maryland 21201

Received December 26, 2001; accepted April 2, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methoxychlor (MXC) is currently used to protect agricultural products from insects. Previous studies show that MXC adversely affects the ovary, but the target cells were not revealed by those studies. Therefore, the purpose of this study was to test the hypothesis that MXC induces ovarian changes by adversely affecting the antral follicles and the ovarian surface epithelium in the mouse. To test this hypothesis, cycling female CD-1 mice (39 days) were dosed with MXC (8, 16, or 32 mg/kg/day), kepone (KPN, 8 mg/kg/day, positive control), or sesame oil (vehicle control) via intraperitoneal injection for 10 or 20 days. Estrous cyclicity was evaluated daily via vaginal lavage. After dosing, ovaries were collected for histological evaluation of follicle numbers, atresia, and surface epithelial height. The results indicate that at the 20-day time point, MXC (32 mg/kg) and KPN (8 mg/kg) increased the percentage of atretic antral follicles (n= 4–9,p<= 0.001). MXC (32 mg/kg) also increased the height of the ovarian surface epithelium compared with controls (n= 7–10,p<= 0.045), and KPN increased the percentage of days in estrus (n= 6–10,p<= 0.0001). These data suggest that MXC and KPN increase antral follicle atresia, MXC increases surface epithelial height, and KPN affects vaginal cytology.

Key Words: methoxychlor; kepone; ovary; follicle; atresia; ovarian epithelium; estrous cycle.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methoxychlor (MXC) is a pesticide widely used against insects that attack fruits, vegetables, and home gardens (U.S. EPA, 2001Go). Several studies have found that MXC affects the female reproductive system, including the ovary. For example, one study found that MXC (200 mg/kg/day) increased lipid accumulation in interstitial and thecal cells of the ovaries in mice (Martinez and Swartz, 1992Go). Another study revealed that MXC caused an increase in pyknotic granulosa cells in mice (Swartz and Eroschenko, 1998Go). Adult mice exposed to MXC in utero have a higher percentage of atretic follicles than controls (Swartz and Corkern, 1992Go). MXC also reduces ovarian weights, the number of corpora lutea, and the capacity for superovulation in the mouse (Eroschenko et al., 1997Go).

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, 1997Go). 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, 1997Go). 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, 1997Go). 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., 2001Go)

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 (8–32 mg/kg) than those used in many previous studies on the effects of MXC (25–700 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., 1998Go; Hodges et al., 2000Go; Johnson et al., 1995Go; Swartz and Mall, 1989Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
MXC and KPN were purchased from Chemservice (West Chester, PA) in a powdered form. For the 8 mg/kg doses, 50 mg of chemical was mixed with 10 ml sesame oil; for the 16 mg/kg dose, 100 mg MXC was mixed with 10 ml sesame oil, and for the 32 mg/kg dose, 200 mg MXC was mixed with 10 ml sesame oil.

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 Bouin’s 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 Weigert’s hematoxylin-picric acid methylene blue. Ovarian sections were sampled and follicles were counted according to published methods (Flaws et al., 2001Go; Smith et al., 1991Go; Tomic et al., 2002Go). 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 20–30 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 2–4 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 o’clock 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., 1993Go). 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., 1993Go).

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 Dunnett’s 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of MXC and KPN on Follicle Numbers
At the 10-day time point, there were no significant differences in the number of smaller follicles between controls and pesticide-treated ovaries (n = 3–9, p >= 0.28). Controls had 21,209 ± 2272 primordial follicles, the 8 mg/kg MXC group had 19,131 ± 2596 primordial follicles, the 16 mg/kg MXC group had 24,560 ± 3241 primordial follicles, the 32 mg/kg MXC group had 30,133 ± 670 primordial follicles, and the 8 mg/kg KPN group had 25,413 ± 4714 primordial follicles. Similarly, controls had an estimated 7698 ± 1202 primary follicles, the 8 mg/kg MXC group had 7188 ± 1405 primary follicles, the 16 mg/kg MXC had 9227 ± 2547 primary follicles, the 32 mg/kg MXC group had 4213 ± 218 primary follicles, and the 8 mg/kg KPN group had 6400 ± 742 primary follicles. Finally, controls had an estimated 1253 ± 168 preantral follicles, the 8 mg/kg MXC group had 1668 ± 312 preantral follicles, the 16 mg/kg MXC had 853 ± 213 preantral follicles, the 32 mg/kg MXC had 1280 ± 445 preantral follicles, and the 8 mg/kg KPN group had 1733 ± 271 preantral follicles.

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. 1Go, n = 5–10, p >= 0.99). There also was no significant difference in the numbers of primary follicles between control and MXC- and KPN-treated ovaries (Fig. 1Go, n = 5–10, 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. 1Go, n = 5–10, p <= 0.04).



View larger version (38K):
[in this window]
[in a new window]
 
FIG. 1. Effect of MXC and KPN on primordial, primary, and preantral follicle numbers. Ovaries were collected after 20 days of dosing from control, 8, 16, or 32 mg/kg MXC-treated and 8 mg/kg KPN-treated mice. Complete serial sections were prepared and subjected to histological examination for follicle numbers. Bars represent means and ± SEM. The letters a and b denote significant difference between the 16 and 32 mg/kg/day MXC and the 16 mg/kg/day MXC and 8 mg/kg/day KPN treatment groups (p <= 0.04); n = 5–10.

 
Although there were no significant effects of MXC and KPN on the number of primordial, primary, or preantral follicles, there was an effect of these chemicals on antral follicles. Figure 2Go shows the morphology of a healthy follicle from a control mouse. This follicle contained an intact oocyte, organized granulosa cell layers with few or no pyknotic cells, and an intact thecal layer. Figure 3Go shows the morphology of an atretic follicle from a mouse treated with 32 mg/kg methoxychlor. This follicle contained an oocyte partially separated from the cumulus granulosa cell layer, a disorganized granulosa cell layer, and the appearance of pyknotic bodies.



View larger version (148K):
[in this window]
[in a new window]
 
FIG. 2. Photomicrograph of a healthy antral follicle from control mouse. Ovaries were collected after 20 days of dosing from control, 8, 16, or 32 mg/kg MXC-treated and 8 mg/kg KPN-treated mice. Complete serial sections were prepared and subjected to histological examination for morphological abnormalities. Normal cells stain blue-green. O, oocyte; GC, granulosa cells. Original magnification x40.

 


View larger version (142K):
[in this window]
[in a new window]
 
FIG. 3. Photomicrograph of an atretic antral follicle from a mouse treated with 32 mg/kg/day MXC. Ovaries were collected after 20 days of dosing from control, 8, 16, or 32 mg/kg MXC-treated and 8 mg/kg KPN-treated mice. Complete serial sections were prepared and subjected to histological examination for morphological abnormalities. O, oocyte; GC, granulosa cells; arrows point to pyknotic (apoptotic) bodies. Original magnification x40.

 
When the numbers of healthy and atretic follicles were quantitated, MXC- and KPN-treated mice had fewer healthy antral follicles than controls. Control mice had a mean of 738 ± 58 healthy antral follicles, whereas mice treated with 32 mg/kg MXC had a mean of 480 ± 68 healthy antral follicles, and mice treated with 8 mg/kg KPN had a mean of 496 ± 59 healthy antral follicles (n = 5–10, p <= 0.01). There also was a significant increase in the percentage of atretic antral follicles compared with controls at the 32 mg/kg MXC dose and the 8 mg/kg KPN dose after 20 days of dosing (Fig. 4Go, n = 4–9, p <= 0.001).



View larger version (52K):
[in this window]
[in a new window]
 
FIG. 4. Effect of MXC and KPN on the percentage of atretic antral follicles per ovary. Ovaries were collected after 20 days of dosing from control, 8, 16, or 32 mg/kg MXC-treated and 8 mg/kg KPN-treated mice. Complete serial sections were prepared and subjected to histological examination for atresia. Bars represent means ± SEM, p >= 0.21, and n = 3–9 for the 10-day time point. *Significant difference from control (p <= 0.001); n = 4–9 for the 20-day time point.

 
Effect of MXC and KPN on the Ovarian Surface Epithelium
The highest dose of MXC (32 mg/kg) significantly increased the height of the ovarian surface epithelium compared with control (Fig. 5AGo, n, = 7–10, p <= 0.045), but this effect was not seen at the two lower doses of MXC or with KPN (Fig. 5Go, n = 7–10, p > 0.12). After 20 days of dosing, the average thickness of the epithelial layer in control mice was 6.5 ± 0.54 mm, and in the KPN-treated mice it was 7.8 ± 0.58 mm, while the average thickness of the epithelial layer in mice treated with 32 mg/kg MXC was 9.7 ± 1.3 mm.



View larger version (25K):
[in this window]
[in a new window]
 
FIG. 5. Effect of MXC and KPN on ovarian epithelial height. Ovaries were collected after 20 days of dosing from control, 8, 16 or 32 mg/kg MXC- and 8 mg/kg KPN-treated mice. Complete serial sections were prepared and subjected to histological examination for epithelial height. Bars represent mean ± SEM. (A) Comparison between control and 8, 16 or 32 mg/kg MXC. *Significant difference from control (p <= 0.045); n = 7–10. (B) Comparison between control and 8 mg/kg KPN (p >= 0.12); n = 7–10.

 
Effect of MXC and KPN on Estrous Cyclicity
The percentage of days in estrus for each treatment group is shown in Figure 6Go. Mice dosed with 8 mg/kg KPN experienced a significant increase in the percentage of days in estrus compared with controls and MXC-treated mice (n = 6–10, p <= 0.0001). Specifically, after 8–10 days of dosing, KPN-treated animals went into persistent estrus, whereas control mice and those dosed with MXC continued to pass through diestrus, proestrus, and estrus. For the 20-day dosing period, KPN-treated animals spent approximately 57% of their days in estrus, whereas the control and MXC-treated animals spent 20–30% of their days in estrus (n = 6–10, p <= 0.0001).



View larger version (34K):
[in this window]
[in a new window]
 
FIG. 6. Effect of MXC and KPN on percentage days in estrus. Mice were either not dosed (control) or dosed with sesame oil (sesame control), 8, 16, or 32 mg/kg MXC or 8 mg/kg KPN. Vaginal cytology was monitored prior to dosing and during dosing as described in Materials and Methods section. Bars represent means ± SEM. *Significant difference from control (p <= 0.0001); n = 6–10.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To our knowledge, this is the first work to show that MXC does not affect numbers of primordial, primary, and preantral follicles, but it does increase the percentage of atretic antral follicles and the height of the ovarian surface epithelium. The reason that MXC appears to selectively target antral follicles and surface epithelium is unknown. It is possible that these tissues may be targeted because they express estrogen receptors, and MXC is thought to work via interaction with these receptors (Bulger et al., 1978Go; Kuiper et al., 1996Go). The major liver metabolite of MXC, known as 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, has been shown in vitro to be an estrogen receptor-{alpha} (ER{alpha}) agonist and an estrogen receptor-ß (ERß) antagonist (Gaido et al., 1999Go). Antral follicles express high levels of ERß, and the surface epithelium expresses high levels of ER{alpha} (Couse et al., 2000Go). The differential expression of the two estrogen receptor subtypes in these distinct tissues may explain why MXC increases follicular atresia, which is thought to be an apoptotic process (Hughes and Gorospe, 1991Go), whereas it increases ovarian surface epithelial height, which is thought to be either a proliferative or an antiapoptotic process (Bai et al., 2000Go; Choi et al., 2001Go). However, other factors may be involved: KPN also increased follicular atresia in our experiments, but it is not thought to be an ERß agonist, the major form of estrogen receptor in the follicles (Jefferson et al., 2000Go; Kuiper et al., 1998Go; Mowa and Iwanaga, 2000Go).

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., 2000Go). 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., 1993Go). MXC may also affect follicle-stimulating hormone (FSH) levels, as FSH is an important survival factor for early antral follicles (Chun et al., 1996Go). 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., 1998Go, 2002Go). 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., 1999Go).

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 50–200 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.


    ACKNOWLEDGMENTS
 
We thank Ms. Janice Babus for her help with histology and dosing and Drs. Dragana Tomic and Kathleen Hruska, Ms. Jamie Benedict, and Mr. Charles Greenfeld for their help with dosing. We also thank Ms. Lynn Van Ruiten for her computer support and Ms. Maura Whiteman for her help with statistics. Supported by National Institutes of Health HD 38955 to J.A.F., a Women’s Health Research grant to C.B., and National Institute of Environmental Health Sciences training grant T32-ES07263.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (410) 706-1503. E-mail: flaws{at}epi.umaryland.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., and Leung, P. C. (2001). Ovarian surface epithelium: Biology, endocrinology, and pathology. Endocr. Rev. 22, 255–288.[Abstract/Free Full Text]

Bai, W., Oliveros-Sauders, B., Wang, Q., Acevedo-Duncan, M. E., and Nicosia, S. V. (2000). Estrogen stimulation of ovarian surface epithelial cell proliferation.In Vitro Cell Dev. Biol. Anim. 36, 657–666.[ISI][Medline]

Billig, H., Furuta, I., and Hsueh, A. J. (1993). Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 133, 2204–2212.[Abstract]

Bulger, W. H., Muccitelli, R. M., and Kupfer, D. (1978). Interactions of methoxychlor, methoxychlor base-soluble contaminant, and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane with rat uterine estrogen receptor. J. Toxicol. Environ. Health 4, 881–893.[ISI][Medline]

Choi, K. C., Kang, S. K., Tai, C. J., Auersperg, N., and Leung, P. C. (2001). Estradiol up-regulates antiapoptotic Bcl-2 messenger ribonucleic acid and protein in tumorigenic ovarian surface epithelium cells. Endocrinology 142, 2351–2360.[Abstract/Free Full Text]

Chun, S-Y., Eisenhauer, K. M., Minami, S., Billig, H., Perlas, E., and Hsueh, A. J. (1996). Hormonal regulation of apoptosis in early antral follicles: Follicle-stimulating hormone as a major survival factor. Endocrinology 137, 1447–1456.[Abstract]

Cooper, R. L., Goldman, J. M., and Vandenbergh, J. G. (1993). Monitoring the estrous cycle in the laboratory rodent by vaginal lavage. In Methods in Toxicology (R.E. Chapin and J. Heindel, Eds.), pp. 45–56. Academic Press, Orlando, FL.

Couse, J. F., Hewitt, C., and Korach, K. S. (2000). Receptor null mice reveal contrasting roles for estrogen receptor-{alpha} and -ß in reproductive tissues. J. Steroid Biochem. Mol. Biol. 74, 287–296.[ISI][Medline]

Das, S. K., Tan, J., Johnson, D. C., and Dey, S. K. (1998). Differential spatiotemporal regulation of lactoferrin and progesterone receptor genes in the mouse uterus by primary estrogen, catechol estrogen, and xenoestrogen. Endocrinology 139, 2905–2915.[Abstract/Free Full Text]

Eroschenko, V. P., Swartz, W. J., and Ford, L. C. (1997). Decreased superovulation in adult mice following neonatal exposures to technical methoxychlor. Reprod. Toxicol. 11, 807–814.[ISI][Medline]

Flaws, J. A., Hirshfield, A. N., Hewitt, J. A., Babus, J. K., and Furth, P.A. (2001). Effect of bcl-2 on the primordial follicle endowment in the mouse ovary. Biol. Reprod. 64, 1153–1159.[Abstract/Free Full Text]

Gaido, K. W., Leonard, L. S., Maness, S. C., Hall, J. M., McDonnell, D. P., Saville, B., and Safe, S. (1999). Differential interaction of the methoxychlor metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1,-trichloroethane with estrogen receptors-{alpha} and -ß. Endocrinology 140, 5746–5753.[Abstract/Free Full Text]

Gray, L. E. Jr., Ostby, J., Cooper, R. L., and Kelce, W. R. (1999). The estrogenic and antiandrogenic pesticide methoxychlor alters the reproductive tract and behavior without affecting pituitary size or LH and prolactin secretion in male rats. Toxicol. Ind. Health 15, 37–47.[ISI][Medline]

Hirshfield, A. N. (1997). Overview of ovarian follicular development: considerations for the toxicologist. Environ. Mol. Mutagen. 29, 10–15.[ISI][Medline]

Hughes, F. M., Jr., and Gorospe, W. C. (1991). Biochemical identification of apoptosis (programmed cell death) in granulosa cells: Evidence for a potential mechanism underlying follicular atresia. Endocrinology 129, 2415–2422.[Abstract]

Hodges, L. C., Bergerson, J. S., Hunter, D. S., and Walker, C. L. (2000). Estrogenic effects of organochlorine pesticides on uterine leiomyoma cells in vitro. Toxicol. Sci. 54, 355–364.[Abstract/Free Full Text]

Jefferson, W. N., Couse, J. F., Banks, E. P., Korach, K. S., and Newbold, R. R. (2000). Expression of estrogen receptor-ß is developmentally regulated in reproductive tissues of male and female mice. Biol. Reprod. 62, 310–317.[Abstract/Free Full Text]

Johnson, D. C., Banerjee, S., and Chatterjee, S. (1995). Estradiol and chlordecone (kepone) decrease adenosine 3'5'-cyclic monophosphate concentrations in the ovariectomized immature rat uterus. Proc. Soc. Exp. Biol. Med. 210, 33–38.[Abstract]

Kuiper, G. G. J. M., Carlsson, B., Grandien, K., Enmark, E., Häggblad, J., Nilsson, S., and Gustafsson, J. Å. (1996). Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors-{alpha} and -ß. Endocrinology 138, 863–870.[Abstract/Free Full Text]

Kuiper, G. G. J. M., Lemmen, J. G., Carlsson, B., Corton, J. C., Safe, S. H., van der Saag, P. T., van der Burg, B., and Gustafsson, J. Å. (1998). Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor-ß. Endocrinology 139, 4252–4263.[Abstract/Free Full Text]

Martinez, E. M., and Swartz, W. J. (1992). Effects of methoxychlor on the reproductive system of the adult female mouse: 2. Ultrastructural observations. Reprod. Toxicol. 6, 93–98.[ISI][Medline]

Mowa, C. N., and Iwanaga, T. (2000). Differential distribution of oestrogen receptor-{alpha} and -ß mRNAs in the female reproductive organ of rats as revealed by in situ hybridization. J. Endocrinol. 165, 59–66.[Abstract/Free Full Text]

Rodriguez, G. C., Nagarsheth, N. P., Lee, K. L., Bentley, R. C., Walmer, D. K., Cline, M., Whitaker, R. S., Isner, P., Berchuck, A., Dodge, R. K., and Hughes, C. L. (2002). Progestin-induced apoptosis in the macaque ovarian epithelium: Differential regulation of transforming growth factor-ß. J. Natl. Cancer Inst. 94, 50–60.[Abstract/Free Full Text]

Rodriguez, G. C., Walmer, D. K., Cline, M., Krigman, H., Lessey, B. A., Whitaker, R. S., Dodge, R., and Hughes, C. L. (1998). Effect of progestin on the ovarian epithelium of macaques: Cancer prevention through apoptosis? J. Soc. Gynecol. Investig. 5, 271–276.[ISI][Medline]

Smith, B. J., Plowchalk, D. R., Sipes, I. G., and Mattison, D. R. (1991). Comparison of random and serial sections in assessment of ovarian toxicity. Reprod. Toxicol. 5, 379–383.[ISI][Medline]

Swartz, W. J., and Corkern, M. (1992). Effects of methoxychlor treatment of pregnant mice on female offspring of the treated and subsequent pregnancies. Reprod. Toxicol. 6, 431–437.[ISI][Medline]

Swartz, W. J., and Eroschenko, V. P. (1998). Neonatal exposure to technical methoxychlor alters pregnancy outcome in female mice. Reprod. Toxicol. 12, 565–573.[ISI][Medline]

Swartz, W. J., and Mall, G. M. (1989). Chlordecone-induced follicular toxicity in mouse ovaries. Reprod. Toxicol. 3, 203–206.[ISI][Medline]

Tomic, D., Brodie, S. G., Deng, C., Hickey, R. J., Babus, J. K., Malkas, L. H., and Flaws, J. A. (2002). Smad3 may regulate follicular growth in the mouse ovary. Biol. Reprod. 66, 917–923.[Abstract/Free Full Text]

U.S. EPA (2001) Consumer factsheet on methoxychlor. U.S. Environmental Protection Agency. EPA Office of Water, 4-12-2001. Available at: http://www.epa.gov/safewater/dwh/c-soc/methoxyc.html. Accessed August 22, 2001.