ToxSci Advance Access originally published online on June 20, 2006
Toxicological Sciences 2006 93(1):196-204; doi:10.1093/toxsci/kfl040
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Methoxychlor Induces Atresia of Antral Follicles in ER
-Overexpressing Mice
* Program in Toxicology, Department of Epidemiology and Preventive Medicine, University of Maryland, Baltimore, Maryland 21201; Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, Washington, DC 20057; and Department of Physiology, University of Maryland, Baltimore, Maryland 21201
1 To whom correspondence should be addressed at Program in Toxicology, Department of Epidemiology and Preventive Medicine, University of Maryland, 660 West Redwood Street, Howard Hall 133B, Baltimore, MD 21201. Fax: (410) 706-1503. E-mail: jflaws{at}epi.umaryland.edu.
Received April 17, 2006; accepted June 12, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Methoxychlor (MXC) is a pesticide that is known to bind to estrogen receptor alpha (ER
) and to induce atresia of antral ovarian follicles. Although studies have shown that MXC is toxic to the ovary, we hypothesize that perturbation to the estrogen-signaling system (i.e., increase or decrease in estrogen sensitivity) might alter ovarian responsiveness to MXC. Thus, we examined whether ER overexpression alters the ability of MXC to increase follicle atresia. To do so, we employed a transgenic mouse model in which ER can be inducibly overexpressed in animal tissues (ER overexpressors). We dosed female controls and ER overexpressors with sesame oil (vehicle control) or MXC (32 and 64 mg/kg/day) for 20 days. After dosing, the ovaries were collected for histological evaluation of follicle numbers and follicle atresia, while blood was collected for measurements of hormones. Estrous cycles were determined in all animals to ensure that all were terminated during estrus. Although there were no significant effects of MXC on the numbers of primordial, primary, and preantral follicles in both controls and ER overexpressors, there was an effect on antral follicles. Specifically, our data indicate that 32 and 64 mg/kg MXC increased the percentage of atretic follicles compared to vehicle in both control and ER overexpressor groups. Moreover, there was a clear trend toward greater sensitivity to 64 mg/kg MXC in ER-overexpressing mice compared to control animals. Specifically, at the 64-mg/kg MXC dose, ER-overexpressing mice had a significantly higher percentage of atretic follicles compared to control animals (controls = 21.5 ± 3%, n = 5; ER overexpressors = 37 ± 23%, n = 9, p 
0.05 vs. controls). After 20 days of dosing, there were no differences in estradiol levels between controls and ER-overexpressing mice in all treatment groups. Follicle-stimulating hormone (FSH) levels were similar in sesame oil–treated control mice and control mice treated with 32 mg/kg MXC, while control mice treated with 64 mg/kg MXC had significantly lower levels of FSH compared to sesame oil–treated controls (sesame oil = 4.31 ± 0.7, MXC [64 mg/kg/day] = 1.89 ± 0.4, n = 3, p 0.02 vs. sesame oil). ER-overexpressing mice treated with sesame oil, 32 or 64 mg/kg MXC, had similar FSH levels. Thus, we observed an increased percentage of atretic antral follicles in ER-overexpressing mice treated with MXC compared to control mice treated with the same compound, suggesting that the ER-signaling pathway plays an important role in MXC-induced atresia. The trend toward greater sensitivity to MXC in ER-overexpressing mice compared to control animals cannot be explained by alterations in estradiol and/or FSH levels.
Key Words: methoxychlor; mouse model; follicle atresia; ER.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In the past few years, considerable attention has been given to the possibility that chemicals in the environment pose a hazard to human reproductive health (Danzo, 1998
). One such compound is 1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane (methoxychlor [MXC]). MXC is a pesticide widely used against insects that attack fruits, vegetables, and home gardens (U.S. EPA, 2001). One report indicates that MXC is found in 1.2% of composite food samples in the United States (Duggan et al., 1983) and in 20–30% of water samples in Poland (Badach et al., 2000). Current data suggest that low levels of MXC (ranging from 0.001 to 0.004 mg/kg) are present in dairy products, grains, fruits, and vegetables (ATSDR, 2002). Further, studies indicate that higher levels of MXC (ranging from 10 to 120 mg/kg) are present in fish (ATSDR, 2002).
Several investigators have reported that MXC adversely affects reproductive endpoints in a variety of mammalian and nonmammalian species (Borgeest et al., 2002, 2004; Gray et al., 1989; Miller et al., 2005; Swartz and Corkern, 1992; Swartz and Eroschenko, 1998). In one study, Swartz and Eroschenko (1998) reported that mice exposed to 14–714 mg/kg MXC neonatally had problems with mating, ovulation, and achieving pregnancy upon reaching sexual maturity. Furthermore, Gray et al. (1989) dosed rats with vehicle or 14–714 mg/kg/day of MXC and found that all doses of MXC significantly reduced the rate of pregnancy, the highest doses by 25%. In another study, Swartz and Corkern (1992) dosed pregnant CD-1 mice with 5.0 mg MXC to determine the effects of MXC on female offspring. The authors concluded that adult mice exposed in utero to MXC had a higher number of atretic follicles compared to controls (Swartz and Corkern, 1992).
Recent in vivo and in vitro studies suggest that MXC is directly toxic to antral follicles in the rodent ovary (Borgeest et al., 2002, 2004; Miller et al., 2005). Borgeest et al. (2002, 2004) showed that exposure to 32 and 64 mg/kg MXC in vivo results in antral follicle–specific toxicity, characterized by a decreased number of healthy antral follicles and an increased percentage of antral follicles undergoing atresia versus controls. Furthermore, Miller et al. (2005) showed that exposure of isolated antral follicles to 10–100 µg/ml MXC in vitro inhibits follicle growth and increases atresia in a dose-dependent manner.
Currently, there is disagreement in the scientific community about the exact mechanism by which MXC causes these toxic effects. MXC is metabolized into mono- and bisphenol-demethylated derivatives, including a bisphenol metabolite, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichlorethane (HPTE), in vivo. The HPTE metabolite is thought to be responsible for the estrogenic action of MXC in vivo (Bulger et al., 1978; Kupfer and Bulger, 1987). Gaido et al. (1999) have shown that HPTE binds directly to estrogen receptors (ERs) and androgen receptors (ARs) in vitro and that it acts as an agonist for ER and antagonist for ERß and AR. Thus, the physiological effect of MXC treatment in vivo may be due to actions mediated by HPTE binding to ER, ERß, or AR. This hypothesis is supported by in vitro studies which have shown that ICI 182,780, an ER blocker, diminishes 3µM MXC-induced ovarian surface epithelium (OSE) growth (Symonds et al., 2006) and 1µM HPTE-induced uterine smooth muscle proliferation (Hodges et al., 2000), suggesting an ER-mediated mechanism for MXC in the OSE and uterus. However, while Miller et al. (2005) found that MXC exposure in vitro inhibits follicle growth and induces atresia, additional studies suggest that HPTE (0.01–10 µg/ml) does not appear to be involved in this mechanism (Miller et al., 2006).
MXC is also reported to have pharmacological activities unrelated to ER binding. Specifically, Ghosh et al. (1999) demonstrated that MXC (3.75, 5.7, 7.5, 10.5, 15, and 30 mg/kg) induces lactoferrin and glucose-6-phosphate dehydrogenase expression in ER knockout mice and that this effect is not blocked by the antiestrogen ICI 182,780. Furthermore, other investigators have shown, using microarray analysis, that MXC regulates the expression of a wide variety of genes (Larkin et al., 2003; Terasaka et al., 2004; Waters et al., 2001). For example, Larkin et al. (2003) demonstrated that MXC exposure upregulates at least six genes (Vtgs 1 and 2, choriogenins 2 and 3, ER, and coagulation factor 11) and downregulates at least three genes (transferrin, ß-actin, and alpha 1-microglobulin/bikunin precursor protein) in sheepshead minnows. Taken together, these studies implicate other pathways of action as well as an ER-mediated one in MXC-induced toxicity.
The objective of this study was to test the hypothesis that perturbation of the estrogen-signaling system (i.e., increase in estrogen sensitivity) alters ovarian responsiveness to MXC. Specifically, we examined whether ER overexpression alters the ability of MXC to increase follicle atresia. To do so, we employed a line of transgenic mice in which conditional expression of ER can be targeted to specific tissues (tet-op-ER mice) (Hruska et al., 2002). The tet-op-ER mice have been previously characterized and utilized in several studies including those on the effect of ER overexpression on mammary tumor formation (Tilli et al., 2003) and the development of ductal carcinoma in situ (Frech et al., 2005). For this study, tet-op-ER mice were mated with tet-op-tTA/tet-op-luciferase mice (Shockett et al., 1995) to produce triple-transgenic mice tet-op-tTA/tet-op-luciferase/tet-op-ER. These triple-transgenic mice overexpress ER in a variety of tissues and are hereafter referred to as ER overexpressors. In addition, tet-op-tTA/tet-op-luciferase mice (Shockett et al., 1995) were used as control mice. This work further examined the effects of MXC on estrous cyclicity and on the levels of estradiol and follicle-stimulating hormone (FSH), hormones vital to antral follicle survival, in ER overexpressors and controls.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Generation of ER
-overexpressing and control mice.Transgenic mice carrying a transgene composed of a coding sequence for murine ER
placed under the regulatory control of a tet-op promoter (tet-op-ER mice) (Hruska et al., 2002) were mated to tet-op-tTA/tet-op-luciferase mice (Shockett et al., 1995) to produce triple-transgenic mice tet-op-tTA/tet-op-luciferase/tet-op-ER. Transcriptional activation of the ER transgene is only achieved in the presence of a tetracycline-responsive transactivator (tTA) protein (Fig. 1). In the triple-transgenic mice that were produced, tTA protein is produced through a positive feedback mechanism. Initial transcription of the tTA protein results from leaky transcription from the tet-op promoter producing only small amounts of tTA protein. However, these small amounts then positively feedback on the tet-op promoter and further increase tTA protein production (Shockett et al., 1995). The tTA protein produced also binds to the tet-op promoter (in the tet-op-ER transgene and in the tet-op-luciferase) driving the expression of transgenic ER and luciferase. Tet-op-tTA/tet-op-luciferase mice (Shockett et al., 1995) were used as control mice in this study.
|
Screening/genotyping mice.
Mice were genotyped using polymerase chain reaction (PCR)–based assays. Briefly, ear punch tissues from pups were lysed in proteinase K buffer (50mM Tris-HCl, 20mM NaCl, 1mM EDTA, and 1% SDS, pH 8.0) containing 1 µl of 20 mg/ml proteinase K (Qiagen Inc., Valencia, CA). Digestion was carried out at 100°C for 3 min. The lysate was then subjected to PCR using primers (1) 5'-CGAGCTCGGTACCCGGGTCG-3' and (2) 5'-GAACACAGTGGGCTTGCTGTTT-3' for tet-op-tTA and primers (1) 5'-CGAGCTCGGTACCCGGGTCG-3' and (2) 5'-GCAAAAGTGAGTATGGTGCC-3' for tet-op-ER
. The conditions for tet-op-tTA PCR were 94°C for 3 min of initial denaturation followed by 35 cycles at 94°C for 60 s, 61°C for 60 s, 72°C for 180 s, and final extension at 72°C for 10 min. The conditions for tet-op-ER PCR were 94°C for 3 min of initial denaturation followed by 35 cycles at 94°C for 60 s, 57°C for 90 s, and 72°C for 120 s. PCR products were then subjected to agarose gel electophoresis. The presence of a 372-bp fragment indicated that the mice were controls (tet-op-tTA/tet-op-luciferase) and the presence of both 372- and 369-bp bands indicated that the mice were ER overexpressors (tet-op-tTA/tet-op-luciferase/tet-op-ER).
Detection of ER levels and luciferase activity in ovaries and uterus.
To confirm that the transgenic mice overexpressed ER in the tissues of interest, ovaries (n = 4) and uteri (n = 4) were collected from ER overexpressors and control mice and subjected to quantitative real-time PCR for ER. Real-time PCR analysis was performed as previously described (Tomic et al., 2004). Total RNA was isolated from tissue using the RNeasy Mini Kit (Qiagen Inc.) according to the manufacturer's protocol. Reverse transcriptase generation of cDNA was performed with 0.5–1 µg of total RNA using Omniscript RT Kit (Qiagen Inc.) with random primers according to the manufacturer's protocols. Real-time PCR was conducted using an MJ Research (Bio-Rad Laboratories, Hercules, CA) (OPTICON) Real-Time PCR machine 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 purified PCR product. Primer sequences for ER were (forward) 5'-AATTCTGACAATCGACGCCAG-3' and (reverse) 5'-GTGCTTCAACATTCTCCCTCCTC-3' (Tomic et al., 2004; Weihua et al., 2000). Primers specific for mouse ß-actin were used as an internal control as previously described (Tomic et al., 2004; Weihua et al., 2000). For each primer, a melting curve was obtained. An initial incubation of 95°C for 10 min was followed by 40–50 cycles of 94°C for 10 s, 55–60°C for 10–20 s, and 72°C for 10–30 s, with final extension at 72°C for 10 min. Arbitrary numbers were assigned for each standard. Values were calculated for the experimental samples from the standard curve. ß-Actin mRNA was measured in each sample and used to normalize ratios between samples.
To measure relative luciferase activity (an indirect indicator of ER overexpression) in ovaries and uteri from ER overexpressors, tissues (n = 3) were snap frozen and then subsequently thawed and homogenized in Reporter Lysis Buffer in an adaptation of previously described procedures (Furth et al., 1994). Homogenized tissues were centrifuged, Luciferase Assay Reagent was added to the supernatant, and luciferase activity was measured using the Luciferase Assay System with Reporter Lysis Buffer (Promega, Madison, WI). Relative luciferase units (RLU) were determined, normalized to protein concentration, and expressed as RLU per milligram of protein. Protein concentration was quantified by BCA protein assay (Pierce, Rockford, IL).
Dosing regimen.
Mice were dosed via ip injection with 32 mg/kg MXC, 64 mg/kg MXC, or sesame oil (vehicle) for 20 continuous days and euthanized during estrus. The selected doses of MXC were based on several studies indicating that these doses are environmentally relevant and/or pose a threat to the female reproductive system (Borgeest et al., 2002, 2004). Environmental levels of MXC range from 40–160 ppm in waters downstream of MXC-sprayed areas (Wallner et al., 1969) to 0.1–4.0 ppb/day in human dietary exposures (ATSDR, 2002). MXC was purchased from ChemService (West Chester, PA) in powdered form. For the 32-mg/kg dose, 200 mg MXC was mixed with 10 ml sesame oil, and for the 64-mg/kg dose, 400 mg MXC was mixed with 10 ml sesame oil. All animals were housed in the University of Maryland's core animal facility and provided food and water ad libitum. Mice were monitored daily, and feeding behavior was indirectly documented by monitoring body weight. Temperature was maintained at 22.2°C, and animals were subjected to 12 h light-dark cycles. The University of Maryland School of Medicine Institutional Animal Use and Care Committee approved all procedures involving animal care, euthanasia, and tissue collection.
Cyclicity measurements.
During dosing, estrous cycles were monitored by analysis of vaginal cytology according to procedures described 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 solution. Vaginal cells were analyzed by light microscopy according to standard morphological criteria (Cooper et al., 1993). 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 (Borgeest et al., 2004; Cooper et al., 1993).
Uterine and vaginal weight measurements.
Mice were weighed to the nearest 0.1 g every day during the experiment. Upon termination, uteri and vaginas were removed and weighed to the nearest 0.1 mg.
Histological evaluation of follicle numbers.
After dosing, ovaries were collected and fixed in Kahle's solution (4% formalin, 28% ethanol, and 0.34N glacial acetic acid), and one ovary from each animal was serially sectioned (8 µm), mounted on glass slides, and stained with Weigert's hematoxylin–picric acid methylene blue. A stratified sample consisting of every 10th section was used to estimate the total number of primordial, primary, preantral, and antral follicles as previously described (Borgeest et al., 2002; Flaws et al., 2001; Smith et al., 1991; Tomic et al., 2002). Only follicles with a visible nucleus were counted to avoid double counting. The number of follicles in the marked sections was multiplied by 10 (because every 10th section was used) and subsequently by 8 (accounting for section thickness) to obtain an estimate of the total number of follicles in each ovary.
Ovarian follicles were categorized as described by Borgeest et al. (2002). Follicles were classified as primordial if they contained an intact oocyte with a visible nucleus surrounded by a single ring of fusiform granulosa cells. Follicles were counted as primary if they consisted of an oocyte with a visible nucleus and a single layer of cuboidal granulosa cells. Follicles were counted as preantral if they contained oocytes with a visible nucleus and more than one layer of granulosa cells. Finally, follicles were counted as antral if they contained five or more layers of granulosa cells and a clearly defined antral space. All sections were evaluated without knowledge of the genotype of the animals or treatment group.
Measurement of follicular atresia.
Atresia was compared in controls and ER overexpressors by morphometric assessment of the percentage of atretic follicles as previously described by Borgeest et al. (2002). Briefly, ovaries were harvested from controls and ER-overexpressing mice after 20 days of dosing. The ovaries were fixed in Kahle's solution and processed for histological evaluation as described above. Healthy and atretic follicles were classified using strict morphological criteria and without knowledge of genotype or treatment (Borgeest et al., 2002; Mossman and Duke, 1973). Follicles were classified as healthy if they contained an intact oocyte, organized granulosa cell layers, and few (less than 10%) pyknotic bodies. 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. The percentage of atretic follicles was estimated by dividing the total number of atretic antral follicles by the total number of antral follicles (both healthy and atretic) and multiplying by 100.
Hormone assays.
Blood samples were obtained from controls and ER-overexpressing animals treated with 32 mg/kg MXC, 64 mg/kg MXC, or sesame oil and subjected to measurements of 17ß-estradiol (estradiol) and FSH levels. Blood was collected on the same day of the estrous cycle (estrus) at 9:00 A.M. to minimize natural fluctuation in hormone levels. Estradiol assays were performed using an enzyme-linked immunosorbant assay kit obtained from Diagnostic System Laboratories, Inc. (Webster, TX) as described previously (Borgeest et al., 2004; Tomic et al., 2004). The supplied protocol was followed without modifications and all samples were run in duplicate. The minimum detection limit, as stated in the instructions of the kit, was 7 pg/ml. The average intra-assay coefficient of variation was 4.2%, and the average interassay coefficient of variation was 8.2%.
Plasma FSH was measured by radioimmunoassay using reagents from the National Hormone and Pituitary Distribution Program (Tomic et al., 2004). Iodination reagents (IODO-BEADS 28665, 28666) were purchased from Pierce. A standard curve was prepared, and cold standards and samples (100 µm) were added to labeled tubes along with primary antibody (1:1400 dilution) and iodinated FSH. Samples were 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 interassay and intra-assay coefficients of variation of 2.7 and 6.7%, respectively.
Statistical analysis.
All data were analyzed using SPSS Statistical Software (SPSS, Inc., Chicago, IL). The mean numbers of primordial, primary, preantral, and antral follicles per ovary, as well as the mean percentage of atretic follicles per ovary, were calculated using ovaries from at least three different animals. Similarly, means ± SEMs for hormone levels and percentage days in estrus were calculated using at least three mice per group. Differences between means were evaluated by one-way ANOVA if more than two groups were compared and t-tests were used for single comparisons, with statistical significance assigned at p 0.05. When a significant p value was obtained by ANOVA, the Scheffe test was used in the post hoc analysis.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
ER
Overexpression in Ovaries and UteriSince the original paper by Shockett et al. (1995)
did not determine if the tet-op-tTA positive feedback system could induce conditional gene expression in ovaries and uteri, relative luciferase activity in ovaries and uteri was measured in the triple-transgenic ER-overexpressing mice and compared after normalization to 1 mg of protein as presented by Shockett et al. (1995). Relative luciferase activity in the ovary (398,950 ± 530,370) was much higher than that in the uterus (25,000 ± 15,850). Relative to other tissues, these results make the ovaries one of the highest expressing tissues in this system.
To confirm that the transgenic mice overexpressed ER, ovaries and uteri were collected from ER overexpressors and control mice and subjected to quantitative real-time PCR for ER (Fig. 2). Ovaries collected from ER overexpressors had a 2.3-fold increase in ER mRNA compared to ovaries isolated from control animals (Fig. 2A, controls = 0.18 ± 0.011 genomic units [gu], n = 4; ER overexpressors = 0.42 ± 0.07 gu, n = 4, p 0.05 vs. controls). In addition, uteri collected from ER overexpressors had a 44-fold increase in ER mRNA compared to uteri isolated from control animals (Fig. 2B, controls = 0.63 ± 0.3 gu, n = 4; ER overexpressors = 30.8 ± 3.9 gu, n = 4, p 0.05 vs. controls).
|
Effect of MXC Treatments on Estrous Cyclicity
The percentage of days in estrus for mice treated with sesame oil and 64 mg/kg MXC is shown in Fig. 3. MXC treatment significantly increased time spent in estrus in both controls and ER
-overexpressing mice. During the 20-day dosing period, control mice treated with sesame oil spent 26.7 ± 1.8% of those days in estrus (n = 3), while 64 mg/kg MXC–treated animals spent 51 ± 3% of those days in estrus (n = 3, p 0.05 vs. sesame oil). In the ER overexpressor group, mice treated with sesame oil spent 32 ± 1.87% of those days in estrus (n = 5), while mice treated with 64 mg/kg MXC spent 45 ± 1.98% of those days in estrus (n = 7, p 0.05 vs. sesame oil).
|
Effect of MXC Treatments on Body, Uterine, and Vaginal Weights
No significant differences were observed in animal weights at the beginning or at the end of the treatment period or in feeding behaviors between treatment groups during the experiment. There were no significant differences between estrous uterine and vaginal weights in mice treated with sesame oil (controls = 0.14 ± 0.01 g, n = 8; ER
overexpressors = 0.14 ± 0.01 g, n = 11), 32 mg/kg MXC (controls = 0.16 ± 0.01 g, n = 6; ER overexpressors = 0.16 ± 0.01 g, n = 10), or 64 mg/kg MXC (controls = 0.18 ± 0.01 g, n = 5; ER overexpressors = 0.17 ± 0.01 g, n = 9) in either the controls or the ER-overexpressing animals.
Effect of MXC Treatments on Follicle Numbers
There were no significant differences between the numbers of primordial, primary, and preantral follicles per ovary in mice treated with MXC (32 or 64 mg/kg) for 20 days, compared to sesame oil, in both control and ER-overexpressing groups (Figs. 4A, 4B, and 4C, n = 5–11 mice per treatment group).
|
Although there were no significant effects of MXC treatment on the number of primordial, primary, and preantral follicles in controls (n = 8 for sesame oil group, n = 6 for 32-mg/kg MXC group, and n = 5 for 64-mg/kg MXC group) and ER
overexpressors (n = 11 for sesame oil group, n = 10 for 32-mg/kg MXC group, and n = 9 for 64-mg/kg MXC group), there was an effect of this chemical on antral follicles (Fig. 5). In the control group, mice treated with MXC (32 or 64 mg/kg) had similar numbers of healthy antral follicles compared to sesame oil–treated controls (Fig. 5A). In the ER-overexpressing group, ER overexpressors treated with 32 mg/kg MXC had a similar number of healthy antral follicles compared to sesame oil–treated ER overexpressors, but those treated with 64 mg/kg MXC had significantly lower numbers of healthy antral follicles compared to sesame oil–treated ER overexpressors (Fig. 5A, sesame oil = 1520 ± 168, n = 11; 64 mg/kg MXC = 1000 ± 128, n = 9, p 0.05 vs. sesame oil).
|
When the numbers of atretic antral follicles per ovary were quantified, control mice treated with 32 mg/kg MXC had similar numbers of atretic antral follicles per ovary compared to sesame oil–treated controls, while control mice treated with 64 mg/kg MXC had significantly higher numbers of atretic follicles per ovary compared to sesame oil–treated controls (Fig. 5B, sesame oil = 160 ± 16, n = 8; 64 mg/kg MXC = 352 ± 48, n = 5, p
0.001 vs. sesame oil). ER-overexpressing mice treated with 32 or 64 mg/kg MXC had significantly higher numbers of atretic follicles per ovary compared to sesame oil–treated ER overexpressors (Fig. 5B, sesame oil = 144.8 ± 14.4, n = 11; 32 mg/kg MXC = 331.2 ± 40, n = 10, p 0.05 vs. sesame oil; 64 mg/kg MXC = 608 ± 83.2, n = 9, p 0.0001 vs. sesame oil). Further, 64 mg/kg MXC–treated ER overexpressors had significantly higher numbers of atretic antral follicles compared to 64 mg/kg MXC–treated controls (controls = 352 ± 48, n = 5; ER overexpressors = 608 ± 83.2, n = 9, p 0.05 vs. controls).
When the percentage of atretic follicles per ovary was calculated, MXC-treated mice had a significantly higher percentage of atretic follicles compared to sesame oil–treated animals in both control (sesame oil = 9.17 ± 1.14, n = 8; 32 mg/kg MXC = 19.53 ± 4.47, n = 6, p 0.05 vs. sesame oil; 64 mg/kg MXC = 21.58 ± 3, n = 5, p 0.05 vs. sesame oil) and ER overexpressor groups (sesame oil = 9.56 ± 1.22, n = 11; 32 mg/kg MXC = 19.5 ± 1.39, n = 10, p 0.05 vs. sesame oil; 64 mg/kg MXC = 37.23 ± 4.8, n = 9, p 0.0001 vs. sesame oil). Moreover, there was a clear trend toward greater sensitivity to MXC in ER-overexpressing mice compared to control animals (Fig. 5C). Specifically, at the 64-mg/kg MXC dose, ER-overexpressing mice had a significantly higher percentage of atretic follicles compared to control animals treated with 64 mg/kg MXC (Fig. 5C, controls = 21.5 ± 3%, n = 5; ER overexpressors = 37 ± 23%, n = 9, p 0.05 vs. controls).
Serum Estradiol and FSH Concentrations in Controls and ER-Overexpressing Mice
Follicular atresia is regulated by hormones such as estradiol and FSH (Billig et al., 1993; Chun et al., 1996; Hsueh et al., 1994). Thus, the levels of these hormones were compared in controls and ER-overexpressing mice (Fig. 6). After 20 days of dosing, there were no differences in estradiol levels between sesame oil–treated mice and mice treated with MXC in both control (Fig. 6A, sesame oil = 69.8 ± 10.9 pg/ml, n = 8; 32 mg/kg MXC = 64.8 ± 11.6 pg/ml, n = 6; and 64 mg/kg MXC = 88.93 ± 16.3 pg/ml, n = 3) and ER-overexpressing mice (Fig. 6A, sesame oil = 86.7 ± 17.9 pg/ml, n = 7; 32 mg/kg MXC = 71.1 ± 11.5 pg/ml, n = 6; and 64 mg/kg MXC = 83.5 ± 9.1 pg/ml, n = 5).
|
FSH levels were similar in sesame oil–treated control mice and control mice treated with 32 mg/kg MXC (Fig. 6B, sesame oil = 4.3 ± 0.77 ng/ml, n = 6; 32 mg/kg MXC = 3.48 ± 0.36 ng/ml, n = 6). In contrast, control mice treated with 64 mg/kg MXC had significantly lower levels of FSH compared to sesame oil–treated controls (Fig. 6B, sesame oil = 4.31 ± 0.7 ng/ml, n = 6; 64 mg/kg MXC = 1.89 ± 0.4 ng/ml, n = 3, p < 0.02 vs. sesame oil). ER
-overexpressing mice treated with sesame oil (n = 5), 32 mg/kg MXC (n = 5), or 64 mg/kg MXC (n = 5) had similar FSH levels. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Female fertility depends on the maintenance of a steady stream of growing ovarian follicles from the primordial to the antral stage. Antral follicle are the only follicle type capable of releasing an egg for fertilization and synthesizing sufficient estrogen to trigger ovulation. The estrogen produced by antral follicles is essential for normal menstrual and estrous cyclicity, maintenance of the female reproductive tract, and maintenance of nonreproductive tissues such as bones, vascular tissue, and the brain (Britt et al., 2002
; Couse et al., 1998; Findlay et al., 2001). Thus, any chemical that damages antral follicles will profoundly alter reproductive function.
The results of our study suggest that 32 and 64 mg/kg MXC treatments increase the percentage of atretic antral follicles compared to vehicle in both control and ER-overexpressing mice. This is in agreement with previous in vivo and in vitro studies indicating that MXC is directly toxic to antral follicles in the ovary of CD-1 mice (Borgeest et al., 2002; Miller et al., 2005). To our knowledge, however, the current studies are the first to show that the mechanism through which MXC induces antral follicle atresia in the mouse ovary may involve the ER-signaling pathway. Specifically, our data suggest that ER plays an important role in MXC-induced atresia. This conclusion is based on the observation that there was an increased percentage of atretic antral follicles in ER-overexpressing mice treated with MXC compared to control mice treated with the same chemical.
To further investigate the mechanism underlying greater sensitivity to MXC in ER-overexpressing mice compared to control animals, we measured and compared estradiol and FSH levels in MXC-treated control and ER-overexpressing animals. FSH and estradiol are major survival factors for antral follicles (Chun et al., 1996; Couse et al., 1998; Findlay et al., 2001). In our study, control mice treated with 32 and 64 mg/kg MXC had similar numbers of healthy antral follicles and similar levels of estradiol compared to sesame oil–treated controls. In ER overexpressors, the decline in healthy antral follicles in 64 mg/kg MXC–treated ER-overexpressing animals did not result in changes in estradiol levels. This was initially surprising because antral follicles are the major site of estrogen synthesis, but there may be several explanations for this finding. First, the reduction in healthy antral follicles that we observed in MXC-treated ER overexpressors may not be sufficient to significantly lower circulating estradiol levels. Second, it is possible that remaining antral follicles are synthesizing more estradiol than normal. Finally, MXC treatment may increase estrogen production by nonovarian tissues, such as fat, in ER-overexpressing animals. These data are consistent with earlier results from our laboratory which showed that estradiol levels were unchanged in CD-1 mice treated with 8–64 mg/kg MXC (Borgeest et al., 2004). Furthermore, we observed no difference between estradiol levels in MXC-treated ER-overexpressing mice compared to MXC-treated control mice. Thus, the trend toward greater sensitivity to MXC in ER-overexpressing mice compared to control animals cannot be explained by alterations in estradiol levels.
Regarding the effect of MXC exposure on FSH levels, a previous study by Okazaki et al. (2001) observed a decline in FSH levels in male rats treated with MXC. In another study, FSH serum levels in female CD-1 mice were unaffected by 8- to 64-mg/kg MXC treatment (Borgeest et al., 2004). In our study, serum FSH levels in both control and ER-overexpressing animals were unaffected by 32-mg/kg MXC treatment. However, it appears from the results of our study that 64 mg/kg MXC treatment significantly decreases FSH levels in control animals, but it does not significantly affect FSH levels in ER-overexpressing animals. Thus, it is possible that lower levels of FSH observed in 64 mg/kg MXC–treated control mice may increase the rate of follicle atresia in the ovary, as FSH is an important survival factor for early antral follicles (Chun et al., 1996). The reasons for differences in effect of MXC on FSH levels among studies are unknown, but may be related to strain differences in mice. Interestingly, we observed no difference between FSH levels in MXC-treated ER-overexpressing mice compared to MXC-treated control mice. Thus, the trend toward greater sensitivity to MXC in ER-overexpressing mice compared to control animals cannot be explained by alterations in FSH levels.
In addition to affecting hormone levels, MXC may mimic estrogen action in the ovary. Numerous studies have shown that MXC mimics estradiol action in the female rodent reproductive tract, causing uterotropic responses in ovariectomized rats and adverse developmental and reproductive effects in gestational or chronically exposed rodents (Alm et al., 1996; Chapin et al., 1997; Cummings and Gray, 1989; Gray et al., 1989; Hall et al., 1997). Furthermore, molecular studies in ovariectomized rats have shown that MXC and estradiol regulate the activity of many of the same uterine proteins, including epithermal growth factor receptor (Metcalf et al., 1996), uterine peroxidase (Cummings and Metcalf, 1994), and ER (Eroschenko et al., 1996). As estradiol has been shown to inhibit proliferation of granulosa cells and thecal cells (Kushner et al., 1990; Ranson et al., 1997; Sadrkhanloo et al., 1987), MXC-induced apoptosis of granulosa cells may be due to its estrogenic action in the ovary. Thus, since ER-overexpressing mice have more ER receptors compared to controls, they may be more sensitive to MXC than control mice.
In conclusion, our data suggest that MXC-induced atresia involves the ER-signaling pathway. Our data also suggest that MXC does not induce atresia by altering levels of estradiol. It is possible that lower levels of FSH observed in 64 mg/kg MXC–treated control mice may increase the rate of follicle atresia in the ovary. However, we observed no difference between estradiol and/or FSH levels in MXC-treated ER-overexpressing mice versus MXC-treated control mice. Thus, the trend toward greater sensitivity to MXC in ER-overexpressing mice compared to control animals cannot be explained by alterations in estradiol and/or FSH levels. Our future studies will address the molecular mechanism underlying MXC-induced atresia in ER-overexpressing animals.
|
ACKNOWLEDGMENTS |
|---|
We thank Dr Patricia Hoyer and Sam Marion for their assistance with the FSH assays and Dr Rosemary Schuh for her help with dose administration. We also acknowledge Lynn Lewis for her help with formatting this document and gratefully acknowledge the support of NIH R21 ES 013061 (J.A.F.), RO1 ES 012893 (J.A.F.), DOD W81XWH-05-1-0302 (MSF), and NIH RO1 CA89041 (P.A.F.).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Agency for Toxic Substances and Disease Registry (ATSDR) (2002). Toxicological Profile for Methoxychlor. pp. 161. U.S. Department of Health and Human Services Public Health Service, ATSDR, Atlanta, GA.
Alm, H., Tiemann, U., and Torner, H. (1996). Influence of organochlorine pesticides on development of mouse embryos in vitro. Reprod. Toxicol. 10, 321–326.[CrossRef][Web of Science][Medline]
Badach, H., Nazimek, T., Kaminski, R., and Turski, W. A. (2000). Organochlorine pesticides concentration in the drinking water from regions of extensive agriculture in Poland. Ann. Agric. Environ. Med. 7, 25–28.[Web of Science][Medline]
Billig, H., Furuta, I., and Hsueh, A. J. (1993). Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 133, 2204–2212.
Borgeest, C., Miller, K. P., Gupta, R., Greenfeld, C., Hruska, K. S., Hoyer, P., and Flaws, J. A. (2004). Methoxychlor-induced atresia in the mouse involves Bcl-2 family members, but not gonadotropins or estradiol. Biol. Reprod. 70, 1828–1835.
Borgeest, C., Symonds, D., Mayer, L. P., Hoyer, P. B., and Flaws, J. A. (2002). Methoxychlor may cause ovarian follicular atresia and proliferation of the ovarian epithelium in the mouse. Toxicol. Sci. 68, 473–478.
Britt, K. L., and Findlay, J. K. (2002). Estrogen actions in the ovary revisited. J. Endocrinol. 175, 269–276.[Abstract]
Bulger, W. H., Muccitelli, R. M., and Kupfer, D. (1978). Studies on the in vivo and in vitro estrogenic activities of methoxychlor and its metabolites. Role of hepatic mono-oxygenase in methoxychlor activation. Biochem. Pharmacol. 27, 2417–2423.[CrossRef][Web of Science][Medline]
Chapin, R. E., Harris, M. W., Davis, B. J., Ward, S. M., Wilson, R. E., Mauney, M. A., Lockhart, A. C., Smialowicz, R. J., Moser, V. C., Burka, L. T., et al. (1997). The effects of perinatal/juvenile methoxychlor exposure on adult rat nervous, immune, and reproductive system function. Fundam. Appl. Toxicol. 40, 138–157.[CrossRef][Web of Science][Medline]
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., and Vandenbergh, J. G. (1993). Monitoring of estrus cyclicity in the laboratory rodent by vaginal lavage. In Methods in Toxicology (R. E. Chapin and J. Heindel, Eds.), Vol. 3 B, pp. 45–56. Academic Press, Orlando.
Couse, J. F., and Korach, K. S. (1998). Exploring the role of sex steroids through studies of receptor deficient mice. J. Mol. Med. 76, 497–511.[CrossRef][Web of Science][Medline]
Cummings, A. M., and Gray, L. E., Jr. (1989). Antifertility effect of methoxychlor in female rats: Dose- and time-dependent blockade of pregnancy. Toxicol. Appl. Pharmacol. 97, 454–462.[CrossRef][Web of Science][Medline]
Cummings, A. M., and Metcalf, J. L. (1994). Mechanisms of the stimulation of rat uterine peroxidase activity by methoxyclor. Reprod. Toxicol. 8, 477–486.[CrossRef][Web of Science][Medline]
Danzo, B. J. (1998). The effects of environmental hormones on reproduction. Cell. Mol. Life Sci. 54, 1249–1264.[CrossRef][Web of Science][Medline]
Eroschenko, V. P., Rourke, A. W., and Sims, W. F. (1996). Estradiol or methoxychlor stimulates estrogen receptor (ER) expression in uteri. Reprod. Toxicol. 10, 265–271.[CrossRef][Web of Science][Medline]
Findlay, J. K., Britt, K., Kerr, J. B., O'Donnell, L., Jones, M. E., Drummond, A. E., and Simpson, E. R. (2001). The road to ovulation: The role of oestrogens. Reprod. Fertil. Dev. 13, 543–547.[CrossRef][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.
Duggan, R. E., Corneliussen, P. E., Duggan, M. B., McMahon, B. M., and Martin, R. J. (1983). Pesticide Residue Levels in Foods in the USA, July 1, 1969–June 30, 1976. Food and Drug Administration and Association of Official Analytical Chemists, Washington, D.C.
Frech, M. S., Halama, E. D., Tilli, M. T., Singh, B., Gunther, E. J., Chodosh, L. A., Flaws, J. A., and Furth, P. A. (2005). Deregulated estrogen receptor expression in mammary epithelial cells of transgenic mice results in the development of ductal carcinoma in situ. Cancer Res. 65, 1–5.
Furth, P. A., St Onge, L., Boger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H., and Hennighausen, L. (1994). Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc. Natl. Acad. Sci. 91, 9302–9306.
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 and ß. Endocrinology 140, 5746–5753.
Ghosh, D., Taylor, J. A., Green, J. A., and Lubahn, D. B. (1999). Methoxychlor stimulates estrogen-responsive messenger ribonucleic acids in mouse uterus through a non-estrogen receptor (non-ER) and non-ERß mechanism. Endocrinology 140, 3526–3533.
Gray, L. E., Jr, Ostby, J., Ferrell, J., Rehnberg, G., Linder, R., Cooper, R., Goldman, J., Slott, V., and Laskey, J. (1989). A dose-response analysis of methoxychlor-induced alterations of reproductive development and function in the rat. Fundam. Appl. Toxicol. 12, 92–108.[CrossRef][Web of Science][Medline]
Hall, D. L., Payne, L. A., Putnam, J. M., and Huet-Hudson, Y. M. (1997). Effect of methoxychlor on implantation and embryo development in the mouse. Reprod. Toxicol. 11, 703–708.[CrossRef][Web of Science][Medline]
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.
Hruska, K. S., Tilli, M. T., Ren, S., Cotarla, I., Kwong, T., Li, M., Fondell, J. D., Hewitt, J. A., Koos, R. D., Furth, P. A., et al. (2002). Conditional over-expression of estrogen receptor alpha in a transgenic mouse model. Transgenic Res. 11, 361–372.[CrossRef][Web of Science][Medline]
Hsueh, A. J., Billig, H., and Tsafriri, A. (1994). Ovarian follicle atresia: A hormonally controlled apoptotic process. Endocr. Rev. 15, 707–724.
Kupfer, D., and Bulger, W. H. (1987). Metabolic activation of pesticides with proestrogenic activity. Fed. Proc. 46, 1864–1869.[Web of Science][Medline]
Kushner, P. J., Hort, E., Shine, J., Baxter, J. D., and Greene, G. L. (1990). Construction of cell lines that express high levels of the human estrogen receptor and are killed by estrogens. Mol. Endocrinol. 4, 1465–1473.
Larkin, P., Folmar, L. C., Hemmer, M. J., Poston, A. J., and Denslow, N. D. (2003). Expression profiling of estrogenic compounds using a sheepshead minnow cDNA macroarray. EHP Toxicogenomics 111, 29–36.[Medline]
Metcalf, J. L., Laws, S. C., and Cummings, A. M. (1996). Methoxychlor mimics the action of 17 beta-estradiol on induction of uterine epidermal growth factor receptors in immature female rats. Reprod. Toxicol. 10, 393–399.[CrossRef][Web of Science][Medline]
Miller, K. P., Gupta, R. K., Greenfeld, C. R., Babus, J. K., and Flaws, J. A. (2005). Methoxychlor directly affects ovarian antral follicle growth and atresia through Bcl-2- and Bax-mediated pathways. Toxicol. Sci. 88, 213–221.
Miller, K. P., Gupta, R. K., and Flaws, J. A. (2006). Methoxychlor metabolites may cause ovarian toxicity through estrogen regulated pathways. Toxicol. Sci. June 7, 2006; EPub ahead of print.
Mossman, H. W., and Duke, K. L. (1973). Comparative Morphology of the mammalian Ovary. The University of Wisconsin Press, Madison, WI.
Okazaki, K., Okazaki, S., Nishimura, S., Nakamura, H., Kitamura, Y., Hatayama, K., Nakamura, A., Tsuda, T., Katsumata, T., Nishikawa, A., et al. (2001). A repeated 28-day oral dose toxicity study of methoxychlor in rats, based on the ‘enhanced OECD test guideline 407’ for screening endocrine-disrupting chemicals. Arch. Toxicol. 75, 513–521.[CrossRef][Web of Science][Medline]
Ranson, E. J., Picton, H. M., and Hunter, M. G. (1997). Effects of testosterone and oestradiol on [3H]-thymidine incorporation by porcine granulosa and theca cells. Anim. Reprod. Sci. 47, 229–236.[CrossRef][Web of Science][Medline]
Sadrkhanloo, R., Hofeditz, C., and Erickson, G. F. (1987). Evidence for widespread atresia in the hypophysectomized estrogen-treated rat. Endocrinology 120, 146–155.
Shockett, P., Difilippantonio, M., Hellman, N., and Schatz, D. G. (1995). A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice. Proc. Natl. Acad. Sci. 92, 6522–6526.
Smith, B. J., Plowchak, 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.[CrossRef][Web of Science][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.[CrossRef][Web of Science][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.[CrossRef][Web of Science][Medline]
Symonds, D. A., Miller, K. P., Tomic, D., and Flaws, J. A. (2006). Effect of methoxychlor and estradiol on cytochrome p450 enzymes in the mouse ovarian surface epithelium. Toxicol. Sci. 89, 510–514.
Terasaka, S., Aita, Y., Inoue, A., Hayashi, S., Nishigaki, M., Aoyagi, K., Sasaki, H., Wada-Kiyama, Y., Sakuma, Y., Akaba, S., et al. (2004). Using a customized DNA microarray for expression profiling of the estrogen-responsive genes to evaluate estrogen activity among natural estrogens and industrial chemicals. Environ. Health Perspect. 112, 773–781.[Web of Science][Medline]
Tilli, M. T., Frech, M. S., Steed, M. E., Hruska, K. S., Johnson, M. D., Flaws, J. A., and Furth, P. A. (2003). Introduction to estrogen receptor- into the tTA/TAg conditional mouse model precipitates the development of estrogen-responsive mammary adenocarcinoma. Am. J. Pathol. 163, 1713–1719.
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.
Tomic, D., Miller, K. P., Kenny, H. A., Woodruff, T. K., Hoyer, P., and Flaws, J. A. (2004). Ovarian follicle development requires Smad3. Mol. Endocrinol. 18, 2224–2240.
U.S. Environmental Protection Agency (U.S. EPA) (2001). Consumer fact sheet on methoxychlor. EPA Office of Water, 4-12-2001. Available at: http://www.epa.gov/safewater/dwh/c-soc/methoxyc.html. Accessed August 22, 2001.
Wallner, W. E., Leeling, N. C., and Zabik, M. J. (1969). The fate of methoxychlor applied by helicopter for smaller European elm bark beetle control. J. Econ. Entomol. 62, 1039–1042.
Waters, K. M., Safe, S., and Gaido, K. W. (2001). Differential gene expression in response to methoxychlor and estradiol through ER, ERß, and AR in reproductive tissues of female mice. Toxicol. Sci. 63, 47–56.
Weihua, Z., Saji, S., Makinen, S., Cheng, G., Jensen, E. V., Warner, M., and Gustafsson, J. A. (2000). Estrogen receptor (ER) beta, a modulator of ERalpha in the uterus. Proc. Natl. Acad. Sci. 97, 5936–5941.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||