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Endocrinology Vol. 143, No. 3 1058-1065
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

17ß-Estradiol Affords Protection against 4-Vinylcyclohexene Diepoxide-Induced Ovarian Follicle Loss in Fischer-344 Rats

Kary E. Thompson, I. Glenn Sipes, Ben D. Greenstein and Patricia B. Hoyer

Departments of Physiology (K.E.T., P.B.H.) and Pharmacology and Toxicology (I.G.S.), Southwest Environmental Health Sciences Center (I.G.S., P.B.H.), and Arthritis Center (B.D.G.), University of Arizona, Tucson, Arizona 85724

Address all correspondence and requests for reprints to: Dr. Patricia B. Hoyer, Department of Physiology, University of Arizona, 1501 North Campbell Avenue, Tucson, Arizona 85724-5051. E-mail: . hoyer{at}u.arizona.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Repeated dosing with 4-vinylcyclohexene diepoxide (VCD) accelerates atresia via apoptosis in primordial and primary follicles in ovaries of rats. The mechanisms that control atresia and VCD-induced toxicity are unknown; however, they could involve 17ß-E2. Atresia slows as animals enter puberty, whereas circulating E2 levels increase with the the onset of cyclicity. This inverse relationship suggests that E2 may be involved in the control of atresia. Therefore, this study was designed to determine whether treatment of immature rats with E2 could protect follicles normally destroyed by VCD-induced apoptosis. Female F344 rats were treated daily with E2, ER analogs, and/or VCD for 15 d. VCD alone caused a 50% reduction in primordial and primary follicles. Coinjection of E2 (0.1 mg/kg) and VCD (80 mg/kg) selectively protected primary follicles from VCD-induced follicle loss. This protection was mimicked by an ER agonist, genistein (0.1 mg/kg), and prevented by an ER antagonist, 4-hydroxytamoxifen (2 mg/kg). VCD treatment increased caspase-3-like activity, whereas concurrent treatment with genistein and VCD restored caspase-3-like activity to control levels. VCD treatment had no effect on circulating E2 levels, uterine weight, or E2 binding to the ER, nor could it directly displace E2 from ERß. These observations support the idea that ER-mediated protection against VCD-induced follicle toxicity is obtained by reducing apoptosis in small preantral follicles, although VCD does not appear to directly interact with ER.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REPEATED DAILY DOSING with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) destroys primary and primordial (small preantral) follicles in the ovaries of rats and mice (1, 2). Mammals are born with a finite number of primordial follicles, and the depletion of this follicle pool results in ovarian failure (menopause in humans). Therefore, any toxicant that targets these small follicles extensively can cause premature ovarian failure.

Previous studies have demonstrated that VCD causes follicle loss by apoptosis and accelerates the natural process of atresia (3, 4). However, the exact mechanisms of VCD-induced follicle toxicity are unknown. Although it is not thought to act as a direct endocrine disrupter, VCD has previously been found to reduce uterine weights, interrupt normal estrous cycles in adult rats, and eventually cause premature ovarian failure in rats and mice (2, 5, 6). These effects are most likely the result of reduced follicular development as the pool of small follicles for recruitment is depleted. Although human exposure to VCD at concentrations found to cause toxicity in rodents is unlikely, VCD is used as a model compound. Because it is specific for small preantral follicles and alters a natural process in the ovary, VCD has been used to study follicle toxicants that target small preantral follicles and as a tool to dissect the atretic signaling pathway.

The specific factors that control the determination between follicular survival vs. death by atresia (apoptosis) are not well understood. Additionally, those factors that regulate follicular survival appear to differ between various stages of follicles. Many studies have examined the mechanism in large antral follicles. The primary site of induction of apoptosis in follicular atresia is thought to be within granulosa cells in larger follicles and is known to involve several factors (7). However, factors that regulate survival vs. atresia in small preantral follicles are not as well known.

In the past, small preantral follicles were not shown to express ER{alpha} (8). Thus, it was assumed for many years that the control of development in these follicles is steroid independent. However, ERß has recently been discovered and reported to be expressed in granulosa cells of small preovulatory ovarian follicles (9, 10). Thus, the possibility of a regulatory role in preantral follicle development seems more plausible. A role for 17ß-E2 in controlling apoptosis has been well established in many tissues throughout the body. It was found to suppress apoptosis in the rabbit corpus luteum via an impact on expression of the bcl-2 gene family (11). 17ß-E2 has also been found to exert antiapoptotic effects in endothelial cells (12), peripheral blood mononuclear cells (13), neurons (14), MCF-7 breast cancer cells (15), osteocytes (16), and cardiac myocytes (17).

Ovarian atresia, which occurs via apoptosis, is a dynamic process. The rate of physiological atresia slows as a female enters puberty, whereas circulating E2 levels increase from undetectable (<10 pg/ml in rats) to adult levels (30–60 pg/ml in rats) with the onset of estrous cycles (18). This inverse relationship between the rate of atresia and circulating 17ß-E2 suggests a role for E2 in the normal control of atresia. Exposure to VCD is known to accelerate atresia. Therefore, the following study was designed to test the hypothesis that VCD-targeted small preantral follicles in immature animals can be protected from VCD-induced apoptosis by concurrent treatment with E2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Medium 199, TRIzol, and {phi}X 174 RF DNA/HaeIII fragments were purchased from Life Technologies, Inc. (Grand Island, NY). VCD (purity, >99%), collagenase (Clostridium histolytium type I), deoxyribonuclease (type I from bovine pancreas), BSA, tRNA (type X-SA from bakers yeast), genistein, 4-hydroxytamoxifen, 17ß-E2, sesame oil, and other nonspecified reagents were purchased from Sigma (St. Louis, MO). AMV-reverse transcriptase and buffer were purchased from Promega Corp. (Madison, WI). AmpliTaq Gold polymerase, PCR buffer, MgCl2, and nucleotides were purchased from Perkin-Elmer Corp. (Norwalk, CT). Ac-Asp-Glu-Val-Asp-Amino-4-methyl-coumarin and the caspase-3 inhibitor (aldehyde) were purchased from Alexis Co. (San Diego, CA). Radiolabeled [{alpha}-32P]dCTP (3000 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL), and [{gamma}-32P]ATP was purchased from DuPont (NEN Life Science Products, Boston, MA).

Animals
Immature female Fischer 344 rats (21 d old) were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN), housed in plastic cages, and maintained on 12-h light/dark cycles at a controlled temperature of 22 ± 2 C. Animals were allowed to acclimate to the animal facilities for 1 wk before initiation of treatment. Rats were provided food (Purina rat chow, Ralston Purina Co., St. Louis, MO) and water ad libitum. All experiments were approved by the University of Arizona institutional animal care and use committee and conformed to the Guide for the Care and Use of Experimental Animals.

Animal treatment
Immature rats (28 d old) were weighed and treated daily for 15 d with one or more of the following treatments: sesame oil (ip, vehicle control), VCD (80 mg/kg, ip), 17ß-E2 (0.1 mg/kg, sc), genistein (0.1 mg/kg, ip), or 4-hydroxytamoxifen (2 mg/kg, ip). The dose of VCD used has previously been determined to destroy 50% of small preantral follicles (3, 4). The dose of E2 used was determined by a dose-response study examining follicle counts (data not shown). The dose of genistein was chosen to be the same as the E2 dose, because genistein has been found to bind ERß as well as stimulate ER ({alpha} and ß)-mediated transcription at similar levels as the endogenous ligand E2 (19). The dose of the ER antagonist was selected because 4-hydroxytamoxifen has been shown to inhibit E2-induced uterine weight increases at 2.02 mg/kg (20). Four hours after the final dose, animals were killed by CO2 inhalation, and ovaries, uterus, and trunk blood were collected.

RIA
Serum 17ß-E2 was measured by RIA. Briefly, duplicate serum samples (250 µl) were extracted twice with diethyl ether. Samples were incubated with an E2 antibody (Sigma) and 3H-labeled E2 (NEN Life Science Products; 9 pg/tube) in assay buffer [0.05 M Tris-HCl (pH 8.0), 0.1 M NaCl, 0.1% gelatin, and 0.1% sodium azide] overnight at 4 C. Samples were then incubated with 200 µl charcoal solution (0.5% activated charcoal and 0.05% dextran T-70, in assay buffer) for 15 min and centrifuged at 6900 x g for 10 min at 4 C. Supernatant was poured into scintillation vials, and counts per min were determined in a scintillation counter (model 5801, Beckman Coulter, Inc., Fullerton, CA). The mean sensitivity of the assays was 8.5 pg/tube. The within-assay coefficient of variation was 4.8%, and the between-assay coefficient of variation was 5.8%.

Histology and oocyte counting
Ovaries were trimmed of fat, fixed in Bouin’s fixative, embedded, sectioned (5 µm), mounted, and stained with hematoxylin and eosin. In every 40th section, preantral follicles (containing an oocyte nucleus) were classified as primordial, primary, or secondary and counted as previously described (1, 21). For descriptive purposes, primordial and primary follicles are collectively referred to as small preantral follicles. On the average, 10.4 ± 0.3 sections/ovary were evaluated.

Follicle isolation
Ovaries were trimmed of fat, cut into small pieces, and dissociated with BSA, deoxyribonuclease, and collagenase as previously described (2). Ovarian digests were filtered through a 250-µm pore size screen to exclude large antral follicles. Preantral follicles were then hand-sorted by size into fraction 1 (25–100 µm; small preantral follicles containing primordial, primary, and some small secondary follicles) and fraction 2 (100–250 µm; large preantral follicles consisting of larger secondary follicles), snap-frozen, and stored at -70 C. For each treatment, 12 ovaries from 6 rats were pooled and used as a single observation (n). Each experiment used at least three separate groups of animals for each treatment.

RNA preparation
Total RNA was extracted from fraction 1 and 2 follicles by the one-step TRIzol method (22). RNA was resuspended in diethylpyrocarbonate-treated water and quantified using a spectrophotometer at 260 and 280 nm (DU-64, Beckman Coulter, Inc., Fullerton, CA).

RT-PCR
RNA (0.75 µg) was reverse transcribed with AMV-RT as previously described (4). Five percent of an RT reaction was amplified in the presence of 32P-labeled dCTP with primers specific for either rat ER{alpha} (forward, 5'-AATTCTGACAATCGACGCCAG-3'; reverse, 5'-GTGCTTCAACATTCTCCCTCCTC-3'; 344-bp product; 95 C for 60 sec, 65 C for 60 sec, and 72 C for 60 sec for 30 cycles) or ERß (forward, 5'-AAAGCCAAGAGAAACGGTGGGCAT-3'; reverse, 5'-GCCAATCATGTGCACCAGTTCCT-3'; 204-bp product; 95 C for 60 sec, 65 C for 60 sec, and 72 C for 60 sec for 30 cycles) (23). PCR products were separated by 6% PAGE and compared with {phi}X 174 RF DNA/HaeIII DNA fragments radio-end-labeled with [{gamma}-32P]ATP. Products were verified by oligonucleotide sequencing.

Immunofluorescence and confocal microscopy
Ovaries were fixed in 4% buffered formalin, paraffin-embedded, sectioned (5 µm), and deparaffinized. Microwave antigen retrieval in citrate buffer was completed, and tissue sections were blocked with 5% BSA/PBS for 5 min. Either anti-ER{alpha} (rabbit polyclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution, 1:10) or anti-ERß (rabbit polyclonal, Upstate Biotechnology, Inc., Lake Placid, NY; dilution, 1:20) was applied for 1 h, followed by a biotinylated goat antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) at a 1:100 dilution (1 h). Cy5-streptavidin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was applied for 1 h at a 1:50 dilution. Sections were treated with ribonuclease A (100 µg/ml; Sigma) for 1 h, followed by YOYO-1 (Molecular Probes, Inc., Eugene, OR) staining (5 nM) for 10 min. Slides were repeatedly washed with PBS, coverslipped with aqueous mounting medium, and stored in the dark at 4 C until viewed on a Leica Corp. (Heidelburg, Germany) confocal microscope at 488 and 647 nm with a xenon light source. All incubations were completed at room temperature. In each experiment an immunonegative section was performed for each ovary stained.

ER assay
Rats (n = 6) were treated daily (ip) for 15 d with either vehicle control (sesame oil) or VCD (80 mg/kg). For each observation, both ovaries from one animal were homogenized in buffer (0.01 M NaPO4, 0.25 M sucrose, and 0.2% sodium azide) supplemented with 0.1 M MgCl2 and 0.1 M ß-mercaptoethanol. Homogenates were centrifuged at 100,000 x g for 1 h at 4 C, and the supernatant was saved as the cytosolic fraction. Sixty microliters of the cytosolic fraction were assayed for number of ER as previously described (24). Scatchard analysis was used to determine Kd and ER number.

ERß competitor assay
The ability of VCD to displace an estrogen analog from ERß was measured as previously described (25) using the PanVera protocol. Briefly, a fluorescent estrogen (Fluormone ES2) was bound to ERß, various concentrations of VCD were added, and the shift in polarization as the fluorescent estrogen was displaced from the receptor was measured with the Beacon 2000 Fluorescence Polarization Instrument (PanVera Corp., Madison, WI).

Cellular fraction isolation
Cytosolic homogenates were isolated from fraction 1 follicles by homogenization in ice-cold lysis buffer [1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 0.1% SDS, 2 mM EDTA, and 50 mM NaF supplemented with the protease inhibitors phenylmethylsulfonylfluoride, aprotinin, and leupeptin]. Homogenized samples were incubated for 30–40 min on ice, and centrifuged at 14,000 rpm for 10 min. Supernatant was collected and stored at -20 C. Samples of cytosolic fractions were used for protein concentration determination using the enhanced protocol of the bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). Values were measured at 562 nm absorbance with a microplate reader using SOFTmax computer software (Molecular Devices, Sunnyvale, CA).

Western blotting
Fifty micrograms of fraction 1 follicle cellular homogenate from d 42 control rats were separated by 10% SDS-PAGE and transferred to nitrocellulose. Blots were blocked for 1 h in 5% dry milk in 0.5 M NaCl, 20 mM Tris, and 0.15% Tween 20 (TTBS) and then incubated in primary antibody (1:200 dilution) in 3% dry milk in TTBS for 1 h at 25 C. Blots were washed three times for 10 min each time in TTBS, and then horseradish peroxidase-conjugated secondary antibody (antirabbit IgG, 1:1000 dilution, Santa Cruz Biotechnology, Inc.) was added for 1 h at 25 C. Blots were washed extensively in TTBS and then detected.

Caspase-3-like activity
The cleavage activity of caspase-3 protease was measured as previously described with slight modifications (26). Protein (30 µg) was incubated at 37 C for 1 h in assay buffer [20 mM PIPES, 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% (wt/vol) 3-(3-cholamidopropylodimethylammanio-1-propane-sulfonate), and 10% sucrose, pH 7.2] supplemented with 50 M of the caspase-3 substrate Ac-DEVD–amino-4-methylcoumarin. Substrate cleavage was detected by measurement of the fluorescence of free 7-amino-4-methylcoumarin with an F-2000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) using excitation at 380 nm and detection emission at 460 nm. This assay was verified by use of a caspase-3 inhibitor (aldehyde).

Statistical analysis
For each experiment, the data (n >= 3) were averaged for each treatment. For the caspase-3-like activity and ER antagonist studies, treatment/control ratios were calculated and analyzed. Data are presented as the mean (or mean ratio) ± SE. Comparisons were made using one-way ANOVA and Fisher’s protected least significant difference) analysis. Although differences between all groups were determined by ANOVA, only significant differences from control are presented to emphasize treatment effects. Significance was assigned at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
After 15 d of repeated dosing, circulating levels of E2 were significantly higher (P < 0.0001; mean E2, 3.50 ± 0.24 ng/ml) in rats treated with E2 than in those not treated with E2 (<23 pg/ml). Treatment with E2 also increased uterine weight (P < 0.0001) compared with that in non-E2-treated animals (Table 1Go). Treatment with the ER analogs, genistein or 4-hydroxytamoxifen, or the follicle toxicant VCD had no effect on circulating E2 levels (data not shown). However, treatment with the ER antagonist, 4-hydroxytamoxifen, prevented E2-induced uterine weight increases, resulting in uterine weights equivalent to those in vehicle-treated control rats (Table 1Go).


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  		Table 1. Average uterine weight as a percentage of body weight after dosing between d 28 and 42 of age

 
After 15 d of daily dosing, VCD induced a significant loss (P < 0.05) of primordial and primary follicles compared with controls as previously described (2). When rats were coinjected with VCD and E2, there was a significant loss (P < 0.05) of primordial follicles; however, primary follicle numbers were equivalent to controls (Fig. 1Go). Because only primary follicles demonstrated this E2-induced protective effect, the following results will only be reported for this follicle population.



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  		Figure 1. Effect of treatment with VCD and 17ß-E2 on rat small preantral ovarian follicles. F344 rats (d 28) were treated daily for 15 d with vehicle control (sesame oil), VCD (80 mg/kg, ip) or VCD and E2 (0.1 mg/kg, sc). Primordial and primary follicles were counted in every 40th section as described in Materials and Methods. Values are the mean total number of follicles counted in each ovary ± SE. *, Significant difference between control and treatment (P < 0.05). n >= 10.

 
After 15 d of treatment with E2 alone, there was a nonsignificant trend for primary follicles to be increased compared with controls (Fig. 2Go). Treatment with the ER agonist genistein demonstrated a pattern of results similar to that seen with the endogenous ER ligand, E2 (Fig. 2Go). Compared with controls, concurrent treatment with genistein and VCD resulted in no difference in primary follicle numbers (Fig. 2Go). After concurrent treatment with VCD, E2, and the ER antagonist 4-hydroxytamoxifen, there was a significant loss of primary follicles compared with controls (Fig. 3Go).



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  		Figure 2. Effect of treatment with VCD, 17ß-E2, and an ER agonist, genistein (Gen), on rat primary follicles. F344 rats (d 28) were treated daily for 15 d with one or more of the following treatments: vehicle control (sesame oil), VCD (80 mg/kg, ip), E2 (0.1 mg/kg, sc), or genistein (0.1 mg/kg, ip). Primary follicles were counted in every 40th section as described in Materials and Methods. Values are the mean total number of follicles counted in each ovary ± SE. *, Significant difference between control and treatment (P < 0.05). n >= 4.

 


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  		Figure 3. Effect of treatment with VCD, 17ß-E2, and an ER antagonist, 4-hydroxytamoxifen (Tam), on rat primary follicles. F344 rats (d 28) were treated daily for 15 d with vehicle control (sesame oil), VCD (80 mg/kg, ip) with or without E2 (0.1 mg/kg, sc), or Tam (2 mg/kg, ip), VCD, and E2. Primary follicles were counted in every 40th section as described in Materials and Methods. Values are the mean total number of follicles counted in each ovary as a percentage of the control ± SE. *, Significant difference between control and treatment (P < 0.05). n >= 4.

 
To examine the effect of the ER analog genistein on apoptosis, caspase-3-like activity was measured in isolated small preantral follicles. Repeated daily dosing with VCD caused an increase (P < 0.05) in caspase-3 activity compared with controls (Fig. 4Go). However, after treatment with either genistein or genistein and VCD, caspase-3-like activity levels were similar to control values (Fig. 4Go).



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  		Figure 4. Effect of treatment with VCD and an ER agonist, genistein (Gen), on rat small preantral ovarian follicle numbers and caspase-3-like activity. F344 rats (d 28) were treated daily for 15 d with vehicle control (sesame oil), VCD (80 mg/kg, ip), genistein (0.1 mg/kg, ip), or VCD and genistein. Primary follicles were counted in every 40th section of ovaries as described in Materials and Methods. Caspase-3-like activity was measured in isolated fraction 1 follicles (25–100 µm) as described in Materials and Methods. {square}, Mean follicles counted in each ovary ± SE (n >= 4); , mean fluorescence as a measure of caspase-3-like activity ± SE (n = 3). *, Significantly different from control (P < 0.05).

 
Because the results of treatment suggested an ER-mediated effect, the expression and localization of ER isoforms {alpha} and ß (ER{alpha} and ERß) were examined. Using RT-PCR, the presence of mRNA encoding ER{alpha} and ERß was detected in isolated fractions of small preantral follicles (25–100 µm; Fig. 5Go).



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  		Figure 5. RT-PCR of ER{alpha} and ERß in fraction 1 ovarian follicles (25–100 µm). Ovaries were collected from three groups of d 42 control rats and dissociated for handsorting of follicles. Each lane represents pooled follicles from six rats. ER{alpha} and ERß were amplified by RT-PCR and visualized with autoradiography as described in Materials and Methods.

 
To verify protein expression for ER{alpha} and ERß in VCD-targeted follicles, immunohistochemical staining and confocal microscopy were used. Consistent with the RT-PCR observations, ERß protein was more widely distributed in rat ovaries compared with ER{alpha} (Figs. 6Go, A and B). When images were merged, colocalization of the red stain for either ER{alpha} or ERß and the green nuclear stain YOYO-1 appeared yellow, indicating that the protein of interest is located in the nucleus. ER{alpha} protein was localized to the oocyte nuclei of all sizes of follicles (Fig. 6AGo), whereas ERß protein staining was seen in the cytoplasm and nucleus of both oocytes and granulosa cells in primordial and small primary follicles (Fig. 6BGo). However, in larger follicles, ERß staining was compartmentalized within the nucleus of oocytes and granulosa cells. VCD had no effect on the distribution or localization of either ER{alpha} or ERß. To verify the antibodies used in this analysis, Western blotting demonstrated a single band for both ER{alpha} and ERß at the expected size (Fig. 7Go).



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  		Figure 6. Distribution of ER{alpha} and ERß protein by confocal microscopy. Ovarian sections from d 42 control rats were incubated with either an anti-ER{alpha} or ERß antibody. Slides were analyzed on a Leica Corp. confocal microscope at x40 as described in Materials and Methods. The green stain (YOYO-1) displays DNA in all cell nuclei; the red stain (Cy-5) represents ER{alpha} (box A) or ERß (box B) protein. Colocalization of the red and green stains appears yellow. Boxes C and D represent the immunonegative slide, incubated with no primary antibody.

 


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  		Figure 7. Representative Western blot of ER{alpha} and ERß in fraction 1 follicles. Fifty micrograms of control fraction 1 follicle cellular homogenate were separated by 10% SDS-PAGE and then transferred to nitrocellulose. ER{alpha} and ERß protein were detected as described in Materials and Methods. Each antibody recognized a single band at the expected size (ER{alpha}, 66 kDa; ERß, 65 kDa).

 
As determined by a receptor assay, VCD treatment did not alter whole ovarian ER ({alpha}+ß) receptor binding of E2 (mean control Kd, 2.94 x 10-10 ± 0.32 x 10-10; mean VCD Kd, 3.76 x 10-10 ± 0.34 x 10-10) or total ovarian ER number (mean control, 5.11 x 10-12 ± 1.3 x 10-12 mol ER/mg protein; mean VCD, 4.78 x 10-12 ± 0.67 x 10-12 mol ER/mg protein). In a competitive binding assay, VCD was not able to displace E2 from ERß at concentrations less than 0.1 M (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
17ß-E2 has been found to be protective against apoptosis in several tissues and cell types. The mechanism of protection is unclear and appears to involve several pathways. E2 prevents apoptosis via modulation of gene expression (27). Antiapoptotic effects of E2 have been demonstrated using several techniques, including terminal dUTP nick-end labeling, dye exclusion, reduced caspase-3 activity, and reduced activity of two nuclear factor-{kappa}B transcription factors (p65/RelA and p50) (16). E2 can also work as a direct antioxidant because it has a phenolic group and can scavenge reactive oxygen species (28). This is thought to contribute to the protective effects of E2 in the brain and cardiovascular system. Previous studies in our laboratory indicated that VCD does not reduce glutathione levels or increase lipid peroxidation, as measured by the incidence of thiobarbituric acid-reactive substances in whole ovaries (29). However, Springer et al. (30) demonstrated that repeated treatment with VCD (10 d) induced expression of mRNA encoding the oxidative stress response gene manganese superoxide dismutase in isolated small follicles (targeted by VCD). Up-regulation of mRNA encoding manganese superoxide dismutase gene has been shown to be a general defense against multiple cellular stresses (31, 32). Therefore, VCD may damage follicles by glutathione-independent pathways of oxidative stress leading to apoptosis.

Based on follicle counting in the present study, E2 prevented or reversed VCD-induced follicle toxicity/apoptosis specifically in primary follicles. The effect was mimicked by an ER agonist, genistein. Using the ER antagonist, 4-hydroxytamoxifen, E2-induced protection against follicle toxicity was prevented. This finding provides support that E2 probably prevents follicle toxicity via a receptor-mediated pathway.

Previous research has found that VCD destroys primordial and primary follicles and enhances caspase-3-like activity specifically in isolated small preantral follicles (26). In the study reported here, treatment with genistein or genistein plus VCD had no effect on follicle number or capsase-3-like activity. The caspase-3-like activities demonstrate that there is a significantly greater amount of atretic follicles in the VCD-treated group compared with controls or genistein-treated rats. Therefore, genistein has no effect on follicle number or apoptosis by itself; however, it is able to prevent VCD-induced changes in follicle number and caspase-3-like activity. These observations suggest that the ER-mediated protection from VCD-induced follicle toxicity is via reduced apoptosis.

The interactions of E2 and VCD on primary follicles are most likely a direct ovarian effect, rather than an indirect effect involving hepatic metabolism. Previous studies have not found VCD-induced liver damage. VCD treatment does not alter liver weights or enzyme activity of either aspartate aminotransferase or alanine aminotransferase (Mayer, L. P., and P. B. Hoyer, unpublished data). Additionally, VCD was not found to change circulating E2 levels; therefore, it seems unlikely that treatment with VCD alters E2 metabolism.

Because the protective effect of E2 against follicle toxicity may be via a receptor-mediated mechanism, demonstrating that expression of ER protein in small preantral follicles is important. Expression of both ER{alpha} and ERß was observed in VCD-targeted small preantral follicles, as demonstrated at the mRNA and protein levels. ERß appears to be more highly expressed than ER{alpha} in primordial and primary follicles. Additionally, relative to larger, more highly developed follicles, ERß displayed a unique pattern of distribution in these small follicles as visualized by confocal microscopy. The ER is generally considered to be a nuclear protein. However, ERß was found in the cytoplasm of both the oocyte and granulosa cells in primordial and primary follicles, whereas it was only localized within the nuclear compartment in secondary and antral follicles. As ERß staining was seen in the cytoplasm, this indicates that ERß could be exerting nongenomic effects. The specialized localization of this receptor suggests that there may be a different role for ERß in early stage preantral follicles compared with larger preantral and antral follicles.

17ß-E2 prevented VCD-induced follicle loss specifically in primary follicles, but not primordial follicles, which are also targeted by VCD. This is an interesting finding because both follicle types express ER{alpha} and ERß. Thus, VCD may be activating the recruitment of primordial follicles into the growing stages rather than killing them directly. Alternatively, although primordial follicles express the ER, the receptor may be a nonfunctional protein at this stage of development.

Based on structure alone, VCD would not be predicted to bind ER. However, a variety of chemicals with nonpredicted structures have been found to interact with the ER (33). Therefore, the ability of VCD to directly interact with ERß was investigated. VCD was not able to displace E2 from ERß at concentrations below 0.1 M. This provides strong evidence that VCD does not compete directly for binding to the ER.

The protective effect against VCD-induced follicle destruction required pharmacological doses of E2 (367 nmol/kg BW), whereas lower doses were ineffective (100-fold; data not shown). There are other reports in which E2 has been shown to only exert protective effects at nonphysiological concentrations. Pharmacological doses (>=0.2 mg/kg) of E2 were required to reduce plasma cholesterol levels and increase expression of low density lipoprotein receptor mRNA in rats (34). In another study, pretreatment with low doses of E2 (2.28 µg/d) did not prevent the death of rats due to ventricular fibrillation that was seen with high levels of ethinyl E2 (30–60 µg/d) (35). Furthermore, suppression of symptoms associated with autoimmune disorders requires pharmacological doses of E2, and it is thought that this occurs by an antiinflammatory mechanism (36, 37, 38, 39, 40).

In whole ovary homogenates, cytosolic ER binding of 17ß-E2 was unchanged after VCD treatment. Likewise, using confocal microscopy there was no change in staining intensity or distribution of ER{alpha} or ERß protein after VCD treatment compared with controls. Taken together, these findings support the idea that VCD is not directly affecting ER expression and binding.

Collectively, the results presented here support the idea that 17ß-E2 can protect against VCD-induced follicle loss. Additionally, this does not appear to result from a direct interaction between VCD and ER. This effect will be further investigated to elucidate the specific mechanism(s) of this protection. Understanding general mechanisms of toxicant-induced follicle loss as well as cell signaling in normal ovarian atresia can provide insight into factors that regulate the reproductive life span in women.


   Acknowledgments
 
The authors thank Karen Turney for her help with the ERß competition assay, Patricia Christian for her help with the confocal microscopy, Sam Marion for his help with the RIAs, Loretta Mayer for the liver enzyme data, and Debra Pierce and Ellen Cannady for their technical assistance.


   Footnotes
 
This work was supported by NIH Grant R01-ES-0924, Center Grant ES-06694, and NIH Training Grant T32-GM-08400-10.

Abbreviations: TTBS, 0.5 M NaCl, 20 mM Tris, and 0.15% Tween 20; VCD, 4-vinylcyclohexene diepoxide.

Received August 8, 2001.

Accepted for publication October 29, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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