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Regular Article |
c Departments of Physiology and
d Pharmacology and Toxicology,
e Southwest Environmental Health Sciences Center, The University of Arizona, Tucson, Arizona 85724
ABSTRACT
Previous studies have shown that ovotoxicity induced in rats by dosing with 4-vinylcyclohexene diepoxide (VCD) is likely via acceleration of the normal rate of atresia (apoptosis). The present study was designed to investigate the apoptosis-related caspase cascades as a component of this phenomenon in isolated ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., on Day 1; a time when ovotoxicity has not been initiated), or dosed daily for 15 days (80 mg/kg, i.p., on Day 15; a time when significant ovotoxicity is underway). Ovaries were collected after the final dose. Small preantral follicles (25–100 µm in diameter) were isolated, cellular fractions were prepared, and cleavage activity or protein expression levels of caspases-3, -8, and -9 were measured. Cytosolic caspase-3 activity was increased in small follicles (P < 0.01) by VCD treatment (Day 1, 2.86 ± 0.23; Day 15, 3.25 ± 0.64, VCD/control, n = 3). This activation was not seen in large or antral follicles (not targeted by VCD). Procaspase-3 protein was increased(P < 0.05) by VCD treatment 212% over controls in small ovarian follicles in Day 15, but not Day 1-dosed rats. Immunofluorescence staining intensity was evaluated by confocal microscopy. Caspase-3 protein, located in the cytosolic compartment of oocytes and granulosa cells of preantral follicles in various stages of development, was selectively increased (P < 0.05) in primordial and small primary follicles from Day 15 VCD-dosed rats. Caspase-8 activity was increased in small follicles in Day 15, but not in Day 1-treated rats; whereas caspase-9 activity was increased by VCD on Day 1 in the mitochondrial fraction. Thus, these data provide evidence that accelerated atresia induced in small ovarian follicles in rats by VCD is associated with activation of a caspase-mediated cascade.
apoptosis, follicle, ovary, signal transduction
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 eventually 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. Ovotoxic chemicals that destroy primordial follicles can cause early menopause, and thus pose a long-term risk in women [4].
One chemical that has been shown to specifically destroy oocytes contained in primordial and primary follicles in rats and mice is 4-vinylcyclohexene diepoxide (VCD). 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]. Following 30 days of daily dosing, the majority of small preantral follicles in immature as well as in adult rats are destroyed [6]. Previous studies have shown that 15 daily doses of VCD (80 mg/kg, i.p.) destroy about 50% of oocytes contained in small preantral (primordial and primary) ovarian follicles in rats [7–9]. Recent studies have focused on cellular changes associated with the VCD-induced follicle destruction. For example, in isolated fractions of small preantral follicles collected from rats dosed daily for 10 days, VCD caused low-molecular-weight DNA degradation, morphological changes, and enhanced expression of mRNA encoding the cell death enhancer, bax [7, 10]. These observations have led to the conclusion that VCD-induced follicle destruction is via physiological cell death, apoptosis. Atresia, the normal mechanism by which the majority of follicles degenerate during development, is also known to occur by apoptosis [11, 12]. Therefore, it has been proposed that VCD causes follicle loss by accelerating the overall rate of atresia [10].
Apoptosis can be triggered by diverse stimuli ranging from intracellular stress to extracellular receptor signaling. Recent evidence suggests that the distal execution machinery of apoptosis may be evolutionarily conserved [13]. A central component of this apoptotic machinery is a family of cysteine proteases called caspases [14]. Caspases are expressed as inactive precursors (procaspases) that are activated upon proteolytic cleavage. According to the current model, two classes of caspases, initiators and executors, are involved in apoptosis [15]. Proapoptotic signaling activates initiator caspases, such as caspase-2, -8, and -9. Activation of the initiator caspases is autocatalytic and requires the binding of specific cofactors [16]. Once activated, the initiator caspases cleave and activate the executor caspases (caspase-3, -6, and -7), which cause cellular collapse by cleaving a specific set of protein substrates. Within the execution phase, caspase-3 appears to be upstream of caspase-6 and -7 and, therefore, its activation represents a critical point in transmission of the apoptotic signal. However, the caspase cascade is not yet completely understood, and some caspases may serve both upstream and downstream functions [17]. Furthermore, in apoptotic cells the cleavage of more than 70 proteins by caspases has been reported [18]. Because these proteins are located in different intracellular compartments, it is reasonable to assume that the selective localization of procaspases or active-caspases in different subcellular compartments may play an important role in the development of the apoptotic process [19, 20].
The present study was designed to examine the effect of VCD dosing on caspase-3 expression and activation in small preantral follicles in order to identify a possible mechanism associated with VCD-induced apoptosis. In addition, the possible involvement of caspase-8 and caspase-9 was also investigated.
MATERIALS AND METHODS
Reagents
Primary antibodies for caspase-3 (H-277), caspase-8 (T-16), and caspase-9 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-rabbit and anti-goat secondary antibodies conjugated with horseradish peroxidase were purchased from New England BioLabs, Inc. (Beverly, MA). Caspase-3 substrate (Ac-DEVD-AMC) and caspase-9 substrate (Ac-LEHD-AMC) were purchased from Alexis Company (San Diego, CA). Caspase-8 substrate (Ac-IETD-AFC) was purchased from BD PharMingen (San Diego, CA). 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 ImmunoResearch 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 Company (St. Louis, MO).
Animals and Tissue Collection
Immature (21-day-old) female Fisher 344 rats were obtained from Harlan (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 animal experiments were approved by the university's Institutional Animal Care and Use Committee, and were conducted in accord with the highest standards of humane animal care as outlined in Guidelines for Care and Use of Experimental Animals. Rats (28-day-old) were given daily i.p. doses (2.5 ml/kg) of either sesame oil (vehicle control) or VCD dissolved in sesame oil (80 mg/kg; 0.57 mmol/kg) as described previously [7, 8]. Four hours following the final dose on Day 1 or Day 15, rats were killed by CO2 inhalation and tissues (liver and ovaries) were excised. Various fractions of follicles were isolated from the ovaries.
Follicle Isolation
Small preantral follicles (fraction 1, 25–100 µm in diameter), large preantral follicles (fraction 2, 100–250 µm in diameter), and antral follicles (fraction 3, >250 µm in diameter) were prepared by gentle enzymatic dissociation of ovaries and sorting with micropipettes as previously described [21]. 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 medium and used for separation of subcellular fractions.
Preparation of Mitochondria and Cytosolic Fractions
The protocol was based on the method of Bossy-Wetzel and Green [22] 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 hand-held homogenizer. Samples were transferred to Eppendorf centrifuge tubes, centrifuged at 900 x g for 5 min to remove nuclei, followed by centrifugation at 10 000 x g for 25 min at 4°C to obtain the heavy 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. 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 [23]. The mitochondrial fraction contained substantial cytochrome oxidase activity, whereas negligible cytochrome oxidase activity was detected in the cytosolic fraction. Protein concentration was determined by DC Bio-Rad protein assay kits using the manufacturer's protocol in both the mitochondrial and cytosolic fractions.
Western Blot Analysis
Protein (20
40 µg) 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 Tris-buffered saline pH 8.0) for 1 h at room temperature, then incubated with appropriate polyclonal primary antibodies in blocking buffer from 1 h to overnight at 4°C followed by incubation with anti-rabbit or anti-goat secondary antibody conjugated with horseradish peroxidase and detected by chemiluminescence and autoradiography using x-ray film. Densitometric measurements of the bands in Western blot analysis were performed using the Eagle Eye II system with Eaglesight software version 3.2 (Stratagene, La Jolla, CA).
Immunofluorescence and Confocal Microscopy
Both ovaries were fixed for 8 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 caspase-3 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 TCS 4D confocal microscope (Leica Inc., Heidelberg, Germany) using a xenon light source, and the intensity was determined using an argon-krypton laser projected through the tissue into a photomultiplier 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.
Caspase-3-, -8-, and -9-Like Protease Activity Measurement
The cleavage activity of each caspase-like protease was measured as described by Garcia-Calvo et al. [24] and Stennicke and Salvesen [25] with slight modifications. Briefly, the enzymatic reaction was carried out at 37°C in protease assay buffer (20 mM PIPES, 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% [w/v] 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate, and 10% sucrose pH 7.2). Cellular protein (60–180 µg) was incubated with 50 µM of caspase substrate (Ac-DEVD-AMC for caspase-3, Ac-IETD-AFC for caspase-8, or Ac-LEHD-AMC for caspase-9) at 37°C for 45 min. Substrate cleavage was detected by measurement of the fluorescence of free 7-amino-4-methylcoumarin (AMC) or 7-amino-4-trifluoromethylcoumarin (AFC) with an F-2000 fluorescence spectrophotometer (Hitachi, Ltd., Tokyo, Japan). AMC was detected at 460 nm emission upon excitation at 380 nm, and AFC was detected at 505 nm emission upon excitation at 400 nm.
Statistical Analysis
All experiments involving follicle isolations were repeated with separate groups of rats (six rats/group) for independent observations. Comparison between treated and corresponding control groups employed the unpaired Student t-test. Statistical analyses involving more than two groups utilized one-way ANOVA. Mean values were analyzed. However, to emphasize the effect of VCD dosing, values are presented as VCD/control. Error estimates are given as standard error of the mean (SEM). Statistical significance was assigned P < 0.05.
RESULTS
Caspase-3-Like Cleavage Activity
A determination was made of the effect of VCD dosing on relative caspase-3-like cleavage activity in small follicles. Compared with the vehicle control group, cytosolic caspase-3-like cleavage activity in isolated fractions of small follicles was increased (P < 0.01) following a single dose (Day 1) or 15 daily doses (Day 15) of VCD (Fig. 1). Furthermore, repeated daily dosing with VCD increased cytosolic caspase-3-like cleavage activity more than did a single dose (P < 0.05, Fig. 1). Following repeated (15-day) dosing with VCD, when caspase-3-like activity was increased in fraction 1 follicles, caspase-3-like cleavage activity was not different from controls in fraction 2 or fraction 3 follicles (Fig. 2). Therefore, VCD-induced caspase-3-like cleavage activity was specific to the small preantral follicles (fraction 1, 25–100 µm). In contrast to the ovaries, there was no effect of VCD on cytosolic caspase-3-like cleavage activity in livers on Day 1 or Day 15 (Fig. 1).
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Caspase-3 Protein Expression
The effect of VCD dosing on the expression of caspase-3 protein was determined by immunoblotting analysis. Compared with controls, on Day 1 of dosing the relative level of 32-kDa procaspase-3 protein in cytosolic fractions of small ovarian follicles was slightly decreased (0.88 ± 0.028, VCD/control, P < 0.05; Fig. 3, A and B). However, after 15 daily doses of VCD, expression was significantly increased (1.59 ± 0.064, VCD/control, P < 0.01; Fig. 3, A and B). Relative protein levels of the 17-kDa active caspase-3 subfragment in the cytosolic fractions of small ovarian follicles were significantly increased after both 1-day and 15-day doses of VCD (1.49 ± 0.061, 1.7 ± 0.086, VCD/control, respectively, P < 0.01; Fig. 3, A and B). Procaspase-3 was identified in both the cytosolic and mitochondrial fractions; however, active caspase-3 was detected only in the cytosolic fractions (data not shown).
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Distribution of Procaspase-3 in Small Follicles
An antibody that recognizes total caspase-3 (pro- and active-caspase-3) was used to visualize expression and distribution of the protein by confocal microscopy in ovaries from VCD-treated and control rats. Immunofluorescence staining of caspase-3 was observed in the cytosolic fraction of oocytes and granulosa cells of follicles in various stages of development (data not shown). Figure 4 shows this staining in small primary follicles. Relative immunofluorescent staining intensity of caspase-3 was measured using a confocal micrograph image. Staining was significantly increased in granulosa cells and oocytes, specifically in primordial and small primary follicles, but not in large primary follicles following treatment with VCD for 15 days compared with vehicle controls (Table 1). VCD also had no effect on secondary preantral or antral follicles (data not shown). There was no effect of VCD on follicles of any size in 1-day-dosed animals (data not shown). To further define the cellular location of caspase-3 and its redistribution induced by VCD treatment, Western blot analysis was performed on subfractionated cellular protein collected in the 15-day-dosed rats. The increased expression of caspase-3 protein induced by VCD treatment was specifically located in cytosol, not in mitochondria (data not shown).
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Protein Expression and Activity of Caspase-8 and Caspase-9
To investigate the possible involvement of upstream activators of caspase-3 in VCD-induced apoptosis, activities and protein levels of caspase-8 and -9 were measured in isolated follicles from VCD-treated rats.
Caspase-8 Compared with controls, cytosolic caspase-8-like cleavage activity was increased in small follicles isolated from rats after 15 days of dosing with VCD, but not after a single dose (Fig. 5). Expression of procaspase-8 protein, as detected by Western blot, was not affected by treatment of rats with VCD for 1 day or 15 days (Fig. 6). As with caspase-3, there was no effect in liver.
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Caspase-9
Following a single dose of VCD, caspase-9-like cleavage activity was increased in the mitochondrial fraction and unaffected in the cytosolic fraction of isolated small follicles (P < 0.01; Fig. 7). Conversely, caspase-9 activity was unaffected in the mitochondrial fraction and was decreased 20% in the cytosolic fraction after 15 daily doses of VCD (P < 0.01; Fig. 7). No significant effect of VCD in procaspase-9 protein expression was seen at any time by Western blotting (data not shown).
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DISCUSSION
Activation of Caspase-3 Is Involved in VCD-Induced Ovotoxicity
Activation of caspase-3 is a central event in the apoptotic process upon which numerous signaling pathways converge and through which multiple downstream substrates are cleaved [26]. The present study is the first to examine caspase-3 enzyme activity in rat small preantral ovarian follicles, and to evaluate the effect of VCD dosing on caspase-3 distribution and activity during ovotoxicity. Following repeated dosing with VCD, increased caspase-3-like activity was specific to the VCD-targeted small preantral follicles (fraction 1). There was no change in caspase-3-like cleavage activity in the large non-VCD-targeted follicles (fractions 2 and 3). VCD also caused no change in caspase-3-like activity in the liver. Evaluation of caspase-3 protein distribution by confocal microscopy demonstrated that the VCD-induced caspase effect was specific for primordial and small primary follicles. Repeated exposures to VCD specifically destroys primordial and primary follicles (contained in fraction 1) by programmed cell death and does not damage large follicles or the liver [7–10, 21]. Therefore, the pattern of VCD-induced activation of caspase-3 activity is consistent with the known specificity of VCD-induced follicle loss.
Caspase-3-like activity in isolated small ovarian follicles was significantly increased by both a single dose and 15 daily doses of VCD, whereas the expression of procaspase-3 protein was enhanced only after 15 daily doses. These results show that the initial response to VCD dosing is activation of caspase-3 activity. However, following repeated dosing, expression of procaspase-3 protein is also induced. Our previous VCD results showed that a single dose of VCD initiates a protective response against the normal rate of atresia [10]. Thus, the present results suggest that caspase-3 that is activated by a single dose of VCD might not be directly associated with cell death, and may be overridden by protective components in vivo that are involved in setting the normal rate of atresia. Hsp70 has been reported to protect against apoptosis in the face of activated caspase-3 activity in an overexpressing WHEI-S cell line [27]. Alternatively, additional components required for caspase-3-directed apoptosis may not be available after a single dose of VCD and may be expressed after repeated dosing. At any rate, it appears that VCD-enhanced caspase-3 activity is not sufficient to initiate apoptosis. Possible physiological roles for the caspases in cells not committed to death have been discussed by Zeuner et al. [28]. Specifically, it was suggested that the "point of no return" in the apoptotic pathway may be downstream of caspase activation. Thus, apoptosis produced by VCD may require repeated activation of caspase-3 within the follicles. Furthermore, increased expression of procaspase-3 protein following repeated dosing with VCD may also play a critical role in VCD-induced ovotoxicity.
Studies from other laboratories have demonstrated that procaspase-3 is located in both the cytosolic and mitochondrial subcellular fractions in a variety of tissues in rats and mice. Thus, the distribution or localization of caspases within the cells may be associated with their activation [19–20, 29]. The Western blotting data from the current study provides evidence that procaspase-3 is located in both the cytosolic and mitochondrial fractions of rat small ovarian follicles, and that repeated dosing with VCD enhances the expression of procaspase-3 protein in the cytosolic but not the mitochondrial fraction. The immunocytochemistry data demonstrated that repeated dosing with VCD enhances the expression of caspase-3 protein in granulosa cells and oocytes, specifically in primordial and small primary follicles. Because this mimics the selectivity of follicle damage caused by VCD, this provides more convincing evidence that caspase-3 is involved in VCD-induced ovotoxicity.
Roles of Caspase-8 and -9 in VCD-Induced Ovotoxicity
Recent in vitro studies have shown that active caspase-8 may induce apoptosis following direct activation of procaspase-3 [30]. Alternatively, caspase-8 may activate procaspase-3 through indirect cleavage of other cytosolic factors, such as Bid [31]. The present results demonstrate that although a single dose of VCD does not activate caspase-8 in small follicles, repeated VCD dosing does. This is in contrast to caspase-3 activity, which was increased after 1-day and 15-day doses of VCD. This suggests that caspase-8 may be most directly involved in the pathways associated with VCD-induced apoptosis. In addition, increased expression of caspase-3 was measured on Day 15, but there was no increase in caspase-8 protein at that time, although activity was increased. This suggests that, as seen withcaspase-3, enzyme activation (Day 1) can precede increased enzyme expression (Day 15). It may be that upstream events associated with activation of caspase-8 are put in place only following repeated dosing. This provides further evidence for an apoptosis-associated involvement ofcaspase-8 in VCD-induced ovotoxicity.
Previous in vitro studies have also proposed an involvement of mitochondria and cytochrome c release in caspase-mediated apoptosis. Once released, as a cofactor with Apaf-1, cytochrome c associates with the complex causing activation of caspase-9 [31, 32]. As a result, activated caspase-9 can then directly cleave and activate procaspase-3. Unlike caspase-8, the present results indicated that caspase-9 activity was increased in small follicles collected after a single dose of VCD, but decreased after 15 daily doses. Furthermore, activation of caspase-9 induced by a single dose of VCD was localized in mitochondria but not cytosol, and reduction of caspase-9 activity induced by 15-day VCD dosing was located in cytosol but not mitochondria.
These findings provide some curious and contradictory results. The earlier response involving caspase-9 suggests that it may be involved in the initiation of a VCD-induced caspase cascade. Because Day 1 is the time at which VCD dosing protects against atresia, caspase-9 may participate in or be bypassed by this event. Alternatively, the reduction in caspase-9 activity seen in 15-day follicles may result from a selective loss of cells in the follicles that have been lost to ovotoxicity prior to Day 15. Moreover, caspase-8 activation may occur later and may result from caspase-3 activation, or may be more involved than caspase-9 in the upstream activation of caspase-3 during the time at which active follicle loss is occurring (15-day dosing). It must be considered that regulation of the caspase cascades in vivo may be more complicated than that which has been demonstrated in in vitro studies using established cell lines, which are not subject to physiological feedback regulation. That is, during the course of repeated dosing, the caspase-3 activation cascade may initially be mediated by caspase-9, but ultimately by caspase-8. The exact placement of these putative regulators of caspase-3 in the signaling pathway to apoptosis must be more closely studied to determine their roles in this process.
A potential factor that might influence in vivo effects on follicular development is hypothalamic/pituitary regulation of ovarian steroid production. However, a previous study provided evidence that VCD does not directly affect the number of estrous cycles during 30 days of dosing in rats the age of those studied here [33]. In addition, two studies have evaluated the effect of 30 days of dosing on subsequent reproductive function for up to a year in mice [34] and rats (unpublished results). Circulating levels of FSH (as a monitor of altered ovarian function) were not different between treated and control animals prior to Day 240 (mice) or Day 120 (rats), although substantial loss of primordial and primary follicles had occurred by Day 30. Thus, it is more likely that during the time of dosing (15 days) in the study reported here, differences in VCD-induced follicular responses are direct, rather than the result of a disruption of the hypothalamic/pituitary/ovarian regulatory axis.
In summary, the studies reported here demonstrate that activation of a caspase-3 associated cascade is induced in rat ovarian follicles by VCD dosing. Induction of caspase-3 activity and the distribution of protein expression are consistent with that observed in VCD-induced ovotoxicity. Furthermore, initiation of this cascade may be differentially regulated by caspase-8 and caspase-9. These findings provide convincing support that ovotoxicity induced by VCD is via caspase-mediated apoptosis.
FOOTNOTES
First decision: 17 January 2001.
1 Supported by National Institutes of Health grant RO1-ES09246 and center grant ESO6694. 
2 Correspondence: Patricia B. Hoyer, Department of Physiology, The University of Arizona, 1501 North Campbell Ave., P.O. Box 245051, Tucson, AZ 85724. FAX: 520 626 2382; hoyer{at}u.arizona.edu
Accepted: February 2, 2001.
Received: December 14, 2000.
REFERENCES
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|
|
|
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|
|
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|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
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|
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|
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|
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