HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hu, X. Articles by Hoyer, P. B. Search for Related Content PubMed PubMed Citation Articles by Hu, X. Articles by Hoyer, P. B. Agricola Articles by Hu, X. Articles by Hoyer, P. B.

Expression and Redistribution of Cellular Bad, Bax, and Bcl-x_L Protein Is Associated with VCD-Induced Ovotoxicity in Rats¹

^a Department of Physiology and ^b Pharmacology and Toxicology, ^c Southwest Environmental Health Sciences Center, The University of Arizona, Tucson, Arizona 85724

ABSTRACT

Previous studies have shown that 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in rats is likely caused by acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small preantral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of several Bcl-2 family member proteins induced by dosing of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1 day; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15 days; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25–100 µm) and large (100–250 µm) preantral follicles were isolated, and subcellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the proapoptotic protein, Bad, were greater in both cytosolic and mitochondrial fractions of small preantral follicles collected from 15-day VCD-treated rats (cytosol, 1.97 ± 0.16; mitochondria, 2.20 ± 0.24, VCD/control, P < 0.05). After 15 days of daily VCD dosing, total cellular antiapoptotic Bcl-x_L protein levels were unaffected in small preantral follicles, but its distribution in mitochondrial and cytosolic components was altered (mitochondria, 0.635 ± 0.08; cytosol, 1.39 ± 0.14, VCD/control, P < 0.05). Likewise, VCD did not affect protein levels of proapoptotic Bax in small follicles on Day 15. However, consistent with a Bax-mediated mechanism of apoptosis, the relative ratio of Bax/Bcl-x_L in the mitochondrial fraction of small preantral follicles was significantly increased by VCD dosing (1.62 ± 0.21, VCD/control, P < 0.05). Immunofluorescence staining intensity evaluated by confocal microscopy visualized cytochrome c protein in the cytosolic compartment in granulosa cells of preantral follicles in various stages of development. Relative to controls, within the population of small preantral follicles, staining intensity was less (P < 0.05) and presumably more diffuse, specifically in stage 1 primary follicles from VCD-treated animals (15 days). VCD caused none of these effects in large preantral follicles or liver (not targeted by VCD). These data provide evidence that the apoptosis induced by VCD in ovarian small preantral follicles of rats is associated with increased expression of Bad protein, redistribution of Bcl-x_L protein and cytochrome c from the mitochondria to the cytosolic compartment, and an increase in the Bax/Bcl-x_L ratio in the mitochondria. These observations are consistent with the involvement of Bcl-2 gene family members in VCD-induced acceleration of atresia.

apoptosis, follicle, ovary, signal transduction, toxicology

INTRODUCTION

Females are born with a finite number of primordial follicles that cannot be further generated after birth. The number of follicles that will ever be selected for development to ovulation is small compared with the total number of primordial follicles present at birth [1]. Instead, the vast majority undergo degeneration at various developmental stages by a process known as atresia [2]. Follicular atresia occurs continuously in the ovary from birth until the supply of follicles is depleted, and ovarian failure (menopause in women) occurs [3]. Menopause is known to be associated with a variety of health problems; therefore, ovotoxic chemicals that destroy primordial follicles can pose a long-term risk in women by causing early menopause [4].

One chemical that has been shown to destroy oocytes contained in primordial and primary (small preantral) follicles in rats and mice is 4-vinylcyclohexene diepoxide (VCD), although it is uncertain whether VCD acts on oocytes directly or indirectly via targeting of granulosa cells. VCD is a metabolite of 4-vinylcyclohexene (VCH), which is formed from the dimerization of 1,3-butadiene as a byproduct in the manufacture of pesticides, flame retardants, rubber, and plastics [5]. How exposure to VCH or VCD affects ovarian function in occupationally exposed women is not known, because epidemiological studies have not been reported. However, industrial workers exposed acutely to VCH complain of nasal irritation and headaches, and those exposed to VCD complain of minor to moderate skin irritation [6]. Therefore, human exposure to VCH or VCD may represent a potential general health hazard. The conditions of exposure of rats we employ are unlikely to be routes and doses of exposure in humans. However, ovotoxicity in animals caused by VCH or VCD has also been observed via inhalation, dermal, and oral exposures [7–9].

Following 30 days of daily dosing with VCD, the majority of small preantral follicles in immature and adult rats are destroyed [10]. Previous studies have determined that repeated dosing with VCD (80 mg/kg, i.p.) is required to cause ovotoxicity, and the earliest evidence of impending VCD-induced follicle damage is observed following 10 daily doses [11]. After 15 days of dosing, loss of about 50% of small preantral (primordial and primary) ovarian follicles has occurred [11–13].

Observations in these previous reports have led to the conclusion that VCD causes follicle loss by accelerating the overall rate of follicular atresia, a process known to occur via apoptosis [11–15]. Apoptosis, an active process of cellular self-destruction, involves a precisely controlled series of events that become activated in a cell poised to die [16]. One recent study also reported that the activity of the apoptosis-associated executor enzyme, caspase-3, is increased in small preantral follicles during VCD-induced ovotoxicity [15].

Within certain types of cells, translation of the signal for apoptosis occurs at an intracellular checkpoint involving the Bcl-2 family of proteins [17, 18]. The Bcl-2 oncogene [19] has been associated with resistance to apoptosis in a variety of mammalian systems [20]. Several members of the Bcl-2 protein family can homodimerize and heterodimerize through specific conserved domains to modulate cell death signals [21–23]. Bcl-2 and its homologue, Bcl-x_L, function to prevent cell death, whereas Bax and Bak accelerate the cell death signal [24–26]. A "rheostat" theory has been proposed in which the ratio of proapoptotic to antiapoptotic proteins determines the susceptibility of a given cell to apoptosis [17].

Much information is available about receptor-mediated cellular regulation in large, antral ovarian follicles. However, little has been studied or reported as to what factors mediate viability/growth, or death/atresia in small preantral follicles (primordial and primary). It has been reported, however, that tyrosine kinase receptors mediated by growth factors and cytokines figure prominently in survival of preantral follicles [27, 28]. Conversely, the onset of cell death (apoptosis) has been associated with tumor necrosis factor alpha and Fas ligand [29].

Although little is known about the intracellular mechanism or mechanisms involved in VCD-induced apoptosis, our previous findings with bax mRNA [12, 14] and preliminary screening experiments by microarray suggested the involvement of several members of the Bcl-2 family in VCD-induced acceleration of atresia. Therefore, the present study has been designed to test the hypothesis that VCD dosing alters the expression and distribution of Bad, Bcl-x_L, and Bax proteins in small ovarian follicles.

MATERIALS AND METHODS

Reagents

Polyclonal primary antibody for Bad and phospho-Bad (Ser 112/136) was purchased from Cell Signaling Technology (Beverly, MA). Monoclonal primary antibody for Bcl-x_L was purchased from Transduction Laboratories (Lexington, KY). Polyclonal primary antibodies for Bax (N-20) and cytochrome c (H-104) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-rabbit and anti-mouse secondary antibodies conjugated with horseradish peroxidase were purchased from New England BioLabs, Inc. (Beverly, MA). Medium 199 (M199) was purchased from Gibco, Inc. (Grand Island, NY). Dimeric cyanine nucleic acid dye (YOYO-1) was purchased from Molecular Probes, Inc. (Eugene, OR). Cy5-conjugated streptavidin was purchased from Jackson Immuno-Research Laboratories, Inc. (West Grove, PA). Bio-Rad DC protein assay kits were purchased from Bio-Rad Laboratories (Hercules, CA). VCD (purity >99%) and all other chemicals were reagent grade and purchased from Sigma Chemical Co. (St. Louis, MO).

Animals and Tissue Collection

Immature (21 days old) female Fisher 344 rats were obtained from Harlan Laboratories (Indianapolis, IN), housed in plastic cages, given food and water ad libitum, and maintained on a 12L:12D cycle. Animals were allowed to acclimate for 1 wk before experiments began. All experiments were approved by the university's Institutional Animal Care and Use Committee. Rats (28 days old) were given daily i.p. injections of either sesame oil (2.5 ml/kg, vehicle control) or VCD dissolved in sesame oil (80 mg/kg, 0.57 mmol/kg) for 1 day or 15 days as described previously [11, 30]. Four hours following the final dose, rats were killed by inhalation of CO₂. Tissues (liver and ovaries) were excised, and ovarian preantral follicles were isolated. The percentage of atretic follicles that were collected on Day 1 ranged from 17% to 25% and on Day 15 from 40% to 60% [12, 13].

Follicle Isolation

Small preantral follicles (fraction 1, 25–100 µm in diameter) and large preantral follicles (fraction 2, 100–250 µm in diameter) were prepared by gentle enzymatic dissociation of ovaries and sorting with micropipettes as previously described [30]. Pools of follicles were collected from both ovaries of six rats in each treatment (control or VCD) for each observation (n). Following isolation, follicles were washed twice with M199 and used for separation of subcellular fractions. The purity of the isolated follicles evaluated microscopically was >99%, and the viability of isolated follicles evaluated microscopically by trypan blue dye-exclusion was >99% [30, 31].

Preparation of Mitochondria and Cytosolic Fractions

The protocol was based on the method of Bossy-Wetzel [32] with minor modifications. Briefly, fresh liver or follicles were resuspended in ice-cold separation buffer (200 mM mannitol, 50 mM sucrose, 10 mM KCl, 1 mM EDTA, 10 mM Hepes-KOH pH 7.4, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin), and homogenized with a Dounce homogenizer. Samples were centrifuged in Eppendorf tubes (at 900 x g for 5 min at 4°C) to remove nuclei, followed by centrifugation at 10 000 x g (25 min at 4°C) to obtain a membrane pellet enriched in mitochondria. The supernatant was collected and used as the cytosolic fraction. The mitochondrial pellet was resuspended in 20 µl PBS, 0.2% Triton X-100. In experiments in which the phosphorylation status of Bad protein in isolated follicles was analyzed, 50 mM sodium fluoride and 1 mM sodium vanadate were added to the lysis buffer to inhibit, respectively, the protein kinase and phosphatase activity. One millimolar sodium vanadate was also added to Western blot transferring buffer to inhibit phosphatase activity. The resuspended mitochondrial fraction and the cytosolic fraction were either used immediately or stored at -20°C for assay within 1 mo. Mitochondrial content of the fractions was estimated by assay for cytochrome oxidase activity, known to be specific for the mitochondrial inner membrane [33]. The oxidation of cytochrome c was used to assess cytochrome oxidase activity (U = the optical density of the completely oxidized sample was subtracted from that at any given time and the logarithm of this difference was plotted against time). The average enzyme activity per milligram of protein was 481.6 U in the mitochondrial fraction, 4.7 U in the cytosolic fraction, and 68.6 U in whole cell lysate. The efficiency of mitochondria preparation was about 88%. Protein levels in the samples were determined with DC Bio-Rad protein assay kits.

Western Blot Analysis

Protein was resolved over a 15% polyacrylamide gel and transferred to a nitrocellulose membrane. The blot was preincubated in blocking buffer (5% nonfat dry milk, 1% Tween 20, in 20 mM TBS pH 8.0) for 1 h at room temperature, then incubated with appropriate primary antibodies in blocking buffer from 1 h at room temperature to overnight at 4°C followed by incubation with anti-rabbit or anti-mouse secondary antibodies conjugated with horseradish peroxidase and detected by chemiluminescence and autoradiography using x-ray film. Antibody against cytochrome c showed one (molecular weight 14 kDa) or two (dimer of cytochrome c, molecular weight 28 kDa) bands. The antibodies against Bcl-x_L, Bax, and Bad demonstrated several bands with the most intense staining at the predicted molecular weight in each case (26 kDa, 23 kDa, and 28 kDa, respectively). In each sample, quantification was made only on the band displaying the proper molecular weight.

Quantification for Western Blot Assay

Prior to each Western blot assay of a specific protein, a linear curve was generated to determine the optimal amount of protein for loading for that antibody. This assay was also used to determine specificity of the antibody and confirm that a protein was recognized at the predicted molecular weight. The loading protein ranged from 10 to 40 µg for all antibodies. In each experiment, equal amounts of protein were loaded for all samples, and all groups in one experiment were loaded on the same gel. For quantification, a screening was performed on blots with x-ray film using different times of exposure to optimize the signal so as not to quantify the overexposed bands. Densitometric measurements were performed using the Eagle Eye II system with Eaglesight software version 3.2 (Stratagene, La Jolla, CA).

Immunofluorescence by Confocal Microscopy

Both ovaries were fixed for 8–12 h in 4% buffered formalin, dehydrated, and embedded in paraffin. Five-micrometer sections were prepared and deparaffinized. All blocking, antibody detection, RNA digestion, and nucleic acid staining steps were performed at room temperature. Tissue sections were blocked with 5% BSA/PBS for 10 min, followed by goat serum (1:10 dilution) for 10 min. The primary antibody, polyclonal rabbit cytochrome c antibody (1:50–100 dilution), was applied for 1 h, followed by a biotinylated anti-rabbit secondary antibody (1 h), and then incubated with Cy5-Streptavidin at 1:100 dilution (1 h). Optimal primary antibody dilution was determined in a preliminary staining. For visualization of genomic DNA, tissue was reacted with ribonuclease A (DNase-free) at a concentration of 100 µg/ml for 1 h, followed by YOYO-1 staining (5 nM) for 10 min. After extensive PBS washes, aqueous mounting medium and coverslips were applied. Stained sections were stored at 4°C in the dark to maintain fluorescence until viewed (within 1 wk). Ovarian sections were viewed on a Leica confocal microscope using a xenon light source, and the intensity was determined using an argon-krypton laser projected through the tissue into a photo-multiplier tube at wavelengths of 488 (YOYO-1) and 647 nm (Cy5). Fluorescence staining of antigens was measured as the average color intensity of follicles using Scion Image Software (National Institutes of Health, Bethesda, MD). Staining intensity in each field was normalized to a nonstaining area of tissue. No autofluorescence was seen in unstained, coverslipped ovarian sections observed at 647 nm. Classification of preantral follicles was as follows: primordial (oocyte surrounded by a small number of squamous granulosa cells), stage 1 primary (oocyte surrounded by one layer of fewer than 20 granulosa cells), stage 2 primary (oocyte surrounded by one layer of more than 20 granulosa cells), and growing (oocyte surrounded by more than one layer of granulosa cells with no visible antrum).

Statistical Analysis

All experiments involving follicle isolations were repeated with separate groups of rats (six rats/group) for independent observations. Quantitative analysis from all experiments was calculated by the following method. The data were analyzed statistically using individual optical density (OD) values (control and VCD) that were obtained with the Eagle Eye II system. The mean OD values were then expressed as a ratio (VCD/control) to emphasize the relative changes in protein level as a function of VCD treatment of rats. Mean differences were analyzed using analysis of variance and, where appropriate, the Fisher PLSD posthoc test. For all statistical analysis, significance was assigned P < 0.05.

RESULTS

Effect of VCD Dosing on Follicular Content of Bax, Bcl-x_L, and Bad Proteins

Total cellular protein from small ovarian follicles (targeted by VCD) or large ovarian follicles and liver (not targeted by VCD), were analyzed by Western blot analyses using anti-Bax, anti-Bcl-x_L, and anti-Bad primary antibodies. A single dose of VCD caused no effect on Bad, Bcl-x_L, or Bax protein in any tissue examined. Following daily dosing with VCD for 15 days, Bad protein levels in small preantral follicles were increased (P < 0.05), but not Bcl-x_L or Bax (Fig. 1). There were no effects of VCD in large preantral follicles at any time. In liver, Bax protein was decreased (P < 0.05) by VCD dosing on Day 15, whereas there was no effect on Bcl-x_L or Bad (Fig. 1). Bcl-x_L antibody detected two protein bands in ovarian follicles but only one in liver.

View larger version (64K): [in this window] [in a new window]   FIG. 1. Effect of VCD dosing in rats on Bcl-2 related proteins. Autoradiogram showing total cellular proteins prepared from small (25–100 µm) or large (100–250 µm) preantral ovarian follicles, or liver collected from F344 rats 4 h following repeated (15-day) daily dosing with VCD (80 mg/kg, i.p.) or vehicle control. Western blot analysis was performed using primary anti-Bax, anti-Bcl-xL, antiphosphorylated Bad, or anti-Bad antibodies as described in Materials and Methods. The gel is representative of three independent experiments

The phosphorylation status of Bad protein is known to affect its apoptotic activity; therefore, this was evaluated using an anti-phospho-Bad antibody. After 15 days of dosing with VCD, there was more phosphorylated Bad protein in small preantral follicles compared with control (Fig. 1, Table 1). However, the ratio of phosphorylated:total Bad protein was not different. Thus, there was also an increase in nonphosphorylated Bad (Table 1). The level of phosphorylated Bad was not altered by VCD in large preantral follicles or liver.

Subcellular Distribution of Bax, Bcl-x_L, and Bad in Small Ovarian Follicles Following VCD Dosing

To determine the effect of VCD dosing on the subcellular distribution of Bax, Bcl-x_L, and Bad, fractions of mitochondria and cytosol were prepared from isolated target or nontarget tissues, and the levels of Bcl-2-related proteins were determined by Western blot analyses. After a single dose of VCD (1 day), Bax, Bcl-x_L, and Bad proteins in mitochondrial fractions were not different from control. After daily dosing with VCD for 15 days, Bcl-x_L protein levels were decreased (P < 0.05) in the mitochondria and increased (P < 0.05) in the cytosolic fraction when compared with control (Fig. 2). No change in subcellular distribution of Bax protein was seen with VCD treatment. Relative to control, Bad protein was increased (P < 0.05) by VCD dosing in both fractions (Fig. 2).

View larger version (25K): [in this window] [in a new window]   FIG. 2. VCD-induced alteration in subcellular localization of Bcl-2 family member proteins. Cytosolic and mitochondrial fractions were prepared from isolated small or large ovarian follicles or liver tissue collected from F344 rats following 15 days of daily dosing with VCD (80 mg/kg, i.p.) or vehicle control. A representative immunoblot is shown in A) mitochondrial or B) cytosolic fractions that were analyzed by Western blot analysis using primary antibodies against Bcl-xL or Bad as described in Materials and Methods. C) Levels of Bcl-xL and Bad protein in cytosol and mitochondria. Data are expressed as effects of VCD versus control. *P < 0.01 compared with control, n = 3

VCD-Induced Alteration of Bax/Bcl-x_L in Mitochondria

A calculation of the Bax/Bcl-x_L ratio in mitochondria versus cytosolic compartments was made (Table 2). Repeated daily dosing with VCD (15 days) did not alter the ratio of Bax/Bcl-x_L in total cellular protein, but it was increased (P < 0.05) in the mitochondrial fraction, and decreased (P < 0.05) in the cytosol in small preantral follicles. There was no effect of VCD in large preantral follicles, but the cytosolic Bax/Bcl-x_L ratio was decreased by VCD in liver. Unlike small preantral follicles, this was not accompanied in liver by an increase in the mitochondrial fraction.

Effect of VCD Dosing on Subcellular Distribution of Cytochrome c

Cytochrome c localization in ovarian follicles was visualized by confocal microscopy with immunohistochemistry using an anti-cytochrome c polyclonal antibody. Western blot analysis was used to verify the results. Cytochrome c was observed in the cytosolic compartment of granulosa cells in follicles at a variety of developmental stages. Compared with control, follicles from rat ovaries collected after a single dose (1 day) of VCD showed no difference in cytochrome c staining. However, following 15 days of daily dosing, the intensity of cytochrome c staining in granulosa cells in stage 1 primary follicles was selectively reduced, relative to control (Fig. 3). This observation is consistent with a diffusion of cytochrome c from mitochondria into the cytosol. This effect was not observed in other follicle types (Table 3). By Western blot analysis, there was a shift (P = 0.058) of cytochrome c from the mitochondria to the cytosol in small preantral follicles collected from VCD-dosed rats (15 days). VCD had no effect in liver or large preantral follicles (Fig. 4).

View larger version (55K): [in this window] [in a new window]   FIG. 3. Effect of VCD on distribution of cytochrome c in stage 1 primary ovarian follicles of rats. Four hours following 15-day daily doses, ovaries were collected from A) vehicle control or B) VCD-treated rats. Ovaries were processed and visualized by confocal microscopy as described in Materials and Methods. Genomic DNA in all cells was stained with YOYO-1 (green) to visualize individual cell nuclei. Cytochrome c was stained with anti-cytochrome c antibody, and detected with Cy5 (red)-conjugated streptavidin. Staining for cytochrome c is observed in the cytoplasm of granulosa cells of follicles in various developmental stages with punctated pattern. Immunonegative control (C) shows YOYO and Cy5 staining in the absence of anti-cytochrome c antibody

View larger version (24K): [in this window] [in a new window]   FIG. 4. Western blot analysis of cytochrome c distribution in isolated rat ovarian follicles. Subcellular fractions (cytosol and mitochondria) were prepared from isolated small (25–100 µm) or large (100–250 µm) preantral ovarian follicles or liver tissue collected from F344 rats following 15 days of daily dosing with VCD (80 mg/kg, i.p.) or vehicle control. Cytosolic or mitochondrial fractions were prepared and analyzed using primary antibody against cytochrome c as described in Materials and Methods. Data are expressed as effect of VCD treatment versus vehicle control, n = 3. * Indicates P = 0.058 compared with control

DISCUSSION

The Bcl-2 family of proteins is important in the cell death pathway of a variety of species and cell types [34]. Complex interactions among the various Bcl-2 family members determine the outcome in terms of cell progression toward either apoptotic cell death or survival [35]. Recent studies of follicular atresia have reported a number of cellular pathways that are involved in normal apoptosis in granulosa cells collected from large antral follicles. Similar to the findings presented here, these studies have also determined that members of the Bcl-2 family of protooncogenes are involved [36, 37]. However, evidence regarding the function and interactions of these proteins is mostly derived from studies using homogeneous cell lines. Less is known about their expression and regulation within the physiological context. The present study is the first to examine the expression and redistribution of Bcl-2 related proteins in rat small preantral ovarian follicles during ovotoxicity induced by VCD-dosing.

Up-Regulation of Bad Protein Expression Is a Critical Event in VCD-Induced Ovotoxicity

Western blot analysis evaluated the subcellular distribution of Bcl-x_L, Bax, and Bad proteins in small preantral follicles collected from VCD-treated rats (1 day and 15 days). No significant effects in any of these proteins were seen in small follicles collected on Day 1. This is consistent with the fact that VCD-induced ovotoxicity is not seen at that time [14]. On Day 15, however, VCD caused a significant increase in expression of Bad protein in mitochondrial and cytosolic subcellular fractions. This suggests that expression of Bad may be a key regulatory point in VCD-induced apoptosis.

It was recently demonstrated that Bad can act as a molecular switch between signal transduction pathways and the cell death process, and that the proapoptotic activity of Bad is modulated by its degree of phosphorylation [38]. By this model, in the nonphosphorylated state, Bad preferentially dimerizes with Bcl-2 or Bcl-x_L and prevents their antiapoptotic activity. Therefore, an increase in nonphosphorylated Bad is consistent with increased apoptosis. The data presented here show that VCD dosing (15 days) has no effect on the relative ratio of phosphorylated Bad to nonphosphorylated Bad in small preantral follicles. However, because expression of total Bad protein was increased, more nonphosphorylated protein has become available. This suggests that an increase in Bad-sequestered Bcl-x_L would result.

VCD Dosing Induces the Translocation of Bcl-x_L from Mitochondria to Cytosol

A growing body of evidence supports the concept that translocation of Bax to the mitochondria in response to apoptotic stimuli induces the release of cytochrome c and the induction of apoptotic cell death [39]. Bcl-x_L protein has the potential to block Bax-induced events. This can occur by heterodimerization with mitochondria-associated Bax, which reduces its mitochondrial membrane targeting, and prevents cytochrome c release. The data presented here show that VCD dosing induces the translocation of Bcl-x_L from mitochondria to cytosol in small preantral follicles. Although the exact mechanism of this translocation is not known, this results in an increase in the ratio of Bax/Bcl-x_L in the mitochondria. The Bax/Bcl-x_L ratio was, interestingly, decreased in liver cytosol from VCD-treated rats, but unchanged in the mitochondrial compartment. This is reflective of a decrease in total cellular Bax protein, which is consistent with an earlier observation of a VCD-induced reduction in bax mRNA in liver. In that study, repeated dosing with VCD also caused an increase in bax mRNA expression in isolated small ovarian follicles after 10 days [14]. However, in the present study, there was not an elevation of total cellular Bax protein levels in small ovarian follicles at Day 15. The reason for these differences is not clear, but may reflect the loss of bax-expressing follicles that has occurred after 15 days of repeated dosing as compared with the 10-day time point, which displays the earliest signs of impending follicular destruction, although follicle loss has not yet occurred. Alternatively, small follicles at both time points have been collected 4 h following the final dose. Therefore, in response to the final dose, bax mRNA may be altered within 4 h, while changes in protein expression have not yet occurred.

VCD-Induced Cytochrome c Release Is Specifically Located in Small Primary Follicles

In vitro studies have demonstrated that the release of cytochrome c from mitochondria is central to mitochondria-dependent apoptotic processes. From what is known of this signaling pathway, cytochrome c in the cytoplasm can cause activation of the caspase-9/Apaf-1 "apoptosome," with subsequent activation of caspase-3 and induction of apoptosis [23]. The subcellular localization of cytochrome c was observed in the present study by immunohistochemistry. Relative to control, staining intensity was reduced (P < 0.05) in the cytoplasm of granulosa cells only in stage 1 primary follicles in VCD-dosed rats. This observation is consistent with leakage of cytochrome c from the mitochondria (relatively concentrated) to the cytosol (relatively diffuse). This possibility was investigated by Western blotting analysis in which total cytochrome c protein in isolated small ovarian follicles was not changed by VCD dosing; however, there was a shift of cytochrome c from mitochondria to the cytosol. Because the isolated follicle fraction used for Western blot analysis also contains primordial and some small growing follicles, the magnitude of response measured by Western blot analysis is somewhat masked by other nonresponsive cells. Taken together, these data support a role for VCD-induced leakage of cytochrome c from mitochondria into the cytosolic compartment.

In conclusion, the current study provides evidence that VCD-induced acceleration of follicular atresia in rat ovarian small preantral follicles is associated with increased expression of Bad protein, translocation of Bcl-x_L from the mitochondria to the cytoplasm, increased Bax:Bcl-x_L ratio in the mitochondria, and increased release of cytochrome c from mitochondria to cytosol. These effects were specifically localized to small preantral follicles (known targets of VCD-induced apoptosis). Furthermore, these events were seen in rats treated for 15 days, but not after a single dose of VCD (when VCD-induced ovotoxicity is not under way). Thus, the evidence to support the involvement of the Bcl-2 family of protooncogenes in VCD-induced acceleration of atresia is quite convincing. Further studies will investigate potential mechanisms by which the Bcl-2 pathway is activated.

FOOTNOTES

First decision: 23 May 2001.

¹ Supported by National Institutes of Health grant RO1-ESO9246 and Center Grant ESO6694 (X.H., P.C., and P.B.H.).

² Correspondence: Patricia B. Hoyer, Department of Physiology, The University of Arizona, P.O. Box 245051, Tucson, AZ 85724. FAX: 520 626 2382; hoyer{at}u.arizona.edu

Accepted: June 28, 2001.

Received: May 3, 2001.

REFERENCES

1. Richards J. Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol Rev 1980; 60:51-89[Free Full Text]
2. Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol 1991; 124:43-101[Medline]
3. Gosden R. Follicular status at the menopause. Hum Reprod 1987; 2:617-621[Abstract/Free Full Text]
4. Mattison DR, Evans MI, Schwimmer WB, White BJ, Jensen B, Schulman JD. Familial premature ovarian failure. Am J Hum Genet 1984; 36:1341-1348[Medline]
5. Rappaport SM, Fraser DA. Air sampling and analysis in rubber vulcanization area. Am Ind Hyg Assoc J 1977; 38:205-209[Medline]
6. Chabra RS, Elwell MR, Peter A. Toxicity of 4-vinylcyclohexene diepoxide after 13 weeks of dermal or oral exposure in rats and mice. Fundam Appl Toxicol 1990; 14:645-751
7. Bevan C, Stadler JC, Elliot GS, Frame SR, Baldwin JK, Leung H-W, Moran E, Panepinto AS. Subchronic toxicity of 4-vinylcyclohexene in rats and mice by inhalation exposure. Fundam Appl Toxicol 1996; 32:1-10[CrossRef][Medline]
8. National Toxicology Program (NTP). Reproductive Toxicology Testing by Continuous Breeding Test Protocol in Swiss (CD-1) Mice. NTIS Accession Number PB8915 2425/AS; 1989
9. Grizzle TB, George JD, Fail PA, Seely JC, Heindel JJ. Reproductive effects of 4-vinylcyclohexene in Swiss mice assessed by a continuous breeding protocol. Fundam Appl Toxicol 1994; 22:122-129[CrossRef][Medline]
10. Flaws JA, Doerr JK, Sipes IG, Hoyer PB. Destruction of preantral follicles in adult rats by 4-vinyl-1-cyclohexene diepoxide. Reprod Toxicol 1994; 8:509-514[CrossRef][Medline]
11. Springer LN, McAsey ME, Flaws JA, Tilly JL, Sipes IG, Hoyer PB. Involvement of apoptosis in 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats. Toxicol Appl Pharmacol 1996; 139:394-401[CrossRef][Medline]
12. Borman SM, VanDePol BJ, Kao SW, Thompson KE, Sipes IG, Hoyer PB. A single dose of the ovotoxicant 4-vinylcyclohexene diepoxide is protective in rat primary ovarian follicles. Toxicol Appl Pharmacol 1999; 158:244-252[CrossRef][Medline]
13. Kao SW, Sipes IG, Hoyer PB. Early effects of ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats and mice. Reprod Toxicol 1999; 13::67-75[CrossRef][Medline]
14. Springer LN, Tilly JL, Sipes IG, Hoyer PB. Enhanced expression of bax in small preantral follicles during 4-vinylcyclohexene diepoxide-induced ovotoxicity in the rat. Toxicol Appl Pharmacol 1996; 139:402-410[CrossRef][Medline]
15. Hu X, Christian PJ, Thompson KE, Sipes IG, Hoyer PB. Ovotoxicity induced in rats by 4-vinylcyclohexene diepoxides is associated with activation of the caspase cascade. Biol Reprod 2001; 65:87-93[Abstract/Free Full Text]
16. Steller H. Mechanisms and genes of cellular suicide. Science 1995; 267::1445-1449[Abstract/Free Full Text]
17. Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 1996; 88:386-401[Free Full Text]
18. Reed J. Bcl-2 family proteins: regulators of apoptosis and chemoresistance in hematologic malignancies. Semin Hematol 1997; 34:9-19[Medline]
19. Bakhshi A, Jensen JP, Goldman P, Wright JJ, McBride OW, Epstein AL, Korsmeyer SJ. Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 1985; 41:899-906[CrossRef][Medline]
20. White E. Life, death, and the pursuit of apoptosis. Genes Dev 1996; 10::1-15[Free Full Text]
21. Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994; 369:321-323[CrossRef][Medline]
22. Sato T, Hanada M, Bodrug S, Irie S, Iwama N, Boise LH, Thompson CB, Golemis E, Fong L, Wang HG. Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system. Proc Natl Acad Sci U S A 1994; 91:9238-9242[Abstract/Free Full Text]
23. Sedlak TW, Oltvai ZN, Yang E, Wang K, Boise LH, Thompson CB, Korsmeyer SJ. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc Natl Acad Sci U S A 1995; 92:7834-7838[Abstract/Free Full Text]
24. Oltvai Z, Milliman C, Korsmeyer S. Bcl-2 Heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993; 74:609-619[CrossRef][Medline]
25. Chittenden T, Harrington EA, O'Connor R, Flemington C, Lutz RJ, Evan GI, Guild BC. Induction of apoptosis by the Bcl-2 homologue Bak. Nature 1995; 374:733-736[CrossRef][Medline]
26. Chittenden T, Flemington C, Houghton AB, Ebb RG, Gallo GJ, Elangovan B, Chinnadurai G, Lutz RJ. A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J 1994; 14:5589-5596[Medline]
27. Roy SK, Greenwald GS. Follicular development through preantral stages: signalling via growth factors. J Reprod Fertil Suppl 1996; 50:83-94[Medline]
28. Packer AI, Hsu YC, Besmer P, Bachvarova RF. The ligand of the c-kit receptor promotes oocyte growth. Dev Biol 1994; 161:194-205[CrossRef][Medline]
29. Kondo H, Maruo T, Peng X, Mochizuki M. Immunological evidence for the expression of the Fas antigen in the infant and adult human ovary during follicular regression and atresia. J Clin Endocrinol Metab 1996; 81:2702-2710[Abstract]
30. Springer LN, Flaws JA, Sipes IG, Hoyer PB. Follicular mechanisms associated with 4-vinylcyclohexene diepoxide-induced ovotoxicity in rats. Reprod Toxicol 1996; 10:137-143[CrossRef][Medline]
31. Flaws JA, Salyers KL, Sipes IG, Hoyer PB. Reduced ability of rat preantral ovarian follicles to metabolize 4-vinyl-1-cyclohexene diepoxide in vitro. Toxicol Appl Pharmacol 1994; 126:286-294[CrossRef][Medline]
32. Bossy-Wetzel E, Green DR. Caspases induce cytochrome c release from mitochondria by activating cytosolic factors. J Biol Chem 1999; 274::17484-17490[Abstract/Free Full Text]
33. Cooperstein J, Lazarow A. A microspectrophotometric method for the determination of cytochrome oxidase. J Biol Chem 1951; 189:665-670[Free Full Text]
34. Golstein P. Controlling cell death. Science 1997; 275:1081-1082[Free Full Text]
35. Marone M, Ferrandina G, Macchia G, de Pasqua A, Benedetti-Panici P, Mancuso S, Scambia G. Bcl-2, Bax, Bcl-xL and Bcl-xS expression in neoplastic and normal endometrium. Oncology 2000; 58:161-168[CrossRef][Medline]
36. Tilly JL. Apoptosis and ovarian function. Rev Reprod 1996; 1:162-172[Abstract]
37. Hsu SY, Hsueh AJW. Tissue-specific Bcl-2 protein partners in apoptosis: an ovarian paradigm. Physiol Rev 2000; 80:593-614[Abstract/Free Full Text]
38. Zha J, Harada H, Yang E, Jockel J, Korsmeyer S. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 Not BCL-XL. Cell 1996; 87:619-628[CrossRef][Medline]
39. Pawlowski J, Kraft AS. Bax-induced apoptotic cell death. Proc Natl Acad Sci U S A 2000; 97:529-531[Free Full Text]

This article has been cited by other articles:

				A. F. Keating, K. S. Rajapaksa, I. G. Sipes, and P. B. Hoyer Effect of CYP2E1 Gene Deletion in Mice on Expression of Microsomal Epoxide Hydrolase in Response to VCD Exposure Toxicol. Sci., October 1, 2008; 105(2): 351 - 359. [Abstract] [Full Text] [PDF]

				S. M. Fernandez, A. F. Keating, P. J. Christian, N. Sen, J. B. Hoying, H. L. Brooks, and P. B. Hoyer Involvement of the KIT/KITL Signaling Pathway in 4-Vinylcyclohexene Diepoxide-Induced Ovarian Follicle Loss in Rats Biol Reprod, August 1, 2008; 79(2): 318 - 327. [Abstract] [Full Text] [PDF]

				K. S. Rajapaksa, I. G. Sipes, and P. B. Hoyer Involvement of Microsomal Epoxide Hydrolase Enzyme in Ovotoxicity Caused by 7,12-Dimethylbenz[a]anthracene Toxicol. Sci., April 1, 2007; 96(2): 327 - 334. [Abstract] [Full Text] [PDF]

				C. R Greenfeld, J. K Babus, P. A Furth, S. Marion, P. B Hoyer, and J. A Flaws BAX is involved in regulating follicular growth, but is dispensable for follicle atresia in adult mouse ovaries Reproduction, January 1, 2007; 133(1): 107 - 116. [Abstract] [Full Text] [PDF]

				P. Desmeules and P. J. Devine Characterizing the Ovotoxicity of Cyclophosphamide Metabolites on Cultured Mouse Ovaries Toxicol. Sci., April 1, 2006; 90(2): 500 - 509. [Abstract] [Full Text] [PDF]

				K. E Valdez, S P. Cuneo, and A. M Turzillo Regulation of apoptosis in the atresia of dominant bovine follicles of the first follicular wave following ovulation Reproduction, July 1, 2005; 130(1): 71 - 81. [Abstract] [Full Text] [PDF]

				L. P. Mayer, P. J. Devine, C. A. Dyer, and P. B. Hoyer The Follicle-Deplete Mouse Ovary Produces Androgen Biol Reprod, July 1, 2004; 71(1): 130 - 138. [Abstract] [Full Text] [PDF]

				C. J. Guigon, S. Mazaud, M. G. Forest, S. Brailly-Tabard, N. Coudouel, and S. Magre Unaltered Development of the Initial Follicular Waves and Normal Pubertal Onset in Female Rats after Neonatal Deletion of the Follicular Reserve Endocrinology, August 1, 2003; 144(8): 3651 - 3662. [Abstract] [Full Text] [PDF]

				Y. Takai, J. Canning, G. I. Perez, J. K. Pru, J. J. Schlezinger, D. H. Sherr, R. N. Kolesnick, J. Yuan, R. A. Flavell, S. J. Korsmeyer, et al. Bax, Caspase-2, and Caspase-3 Are Required for Ovarian Follicle Loss Caused by 4-Vinylcyclohexene Diepoxide Exposure of Female Mice in Vivo Endocrinology, January 1, 2003; 144(1): 69 - 74. [Abstract] [Full Text] [PDF]

				X. Hu, J. A. Flaws, I. G. Sipes, and P. B. Hoyer Activation of Mitogen-Activated Protein Kinases and AP-1 Transcription Factor in Ovotoxicity Induced by 4-Vinylcyclohexene Diepoxide in Rats Biol Reprod, September 1, 2002; 67(3): 718 - 724. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hu, X. Articles by Hoyer, P. B. Search for Related Content PubMed PubMed Citation Articles by Hu, X. Articles by Hoyer, P. B. Agricola Articles by Hu, X. Articles by Hoyer, P. B.