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Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205
Address all correspondence and requests for reprints to: Dr. Jong-Min Kim, Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins University School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205.
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Introduction |
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Caspases play a critical role in the execution of apoptosis in a number of cell types (8). Most of the caspases are synthesized as inactive proenzymes that are processed to active (cleaved) forms in cells undergoing apoptosis. Caspase cleavage occurs by self-proteolysis and/or results from the actions other proteins (8). The cleaved forms consist of large (17–20 kDa) and small (10–12 kDa) subunits. Caspases can be classified as initiators (caspase-8, -9, and -10) or effectors (caspase-3, -6, and -7) (9). Under the appropriate stimulus, caspase-8 and/or -10 and caspase-9, which are themselves activated by Fas-associated death domain (FADD) and Apaf, respectively, activate effector caspases that, in turn, degrade or activate their cellular substrates [e.g. cytoplasmic structural proteins such as actin or nuclear proteins such as poly(ADP-ribose) polymerase] (9).
Among the caspases, caspase-3 (also known as CPP32) appears to be a key protease in the apoptotic pathway (10). Activated caspase-3 targets DNA fragmentation factor (DFF), which is integrally involved in degrading DNA (11). In vitro, inhibitors of caspase-3 have been shown to prevent caspase-3 activity, and thus apoptosis (12). We hypothesized herein that Leydig cell apoptosis induced by EDS might be mediated by caspase-3 activation. To test this, we examined the activation and localization of caspase-3 in relationship to the apoptotic death of the Leydig cells. We show that EDS-induced apoptosis of Leydig cells correlates with a decrease in procaspase-3 and an increase in the cleaved, active form of caspase-3, and that both in vivo and in vitro, inhibition of the cleavage of procaspase prevented apoptosis. Additionally, immunohistochemical analysis revealed that caspase-3, localized in the cytoplasm of control cells, translocates to a nuclear location at the time of caspase-3 activation. Taken together, these results suggest that Leydig cell apoptosis, induced by EDS, is mediated by caspase-3 activation and its accompanying nuclear translocation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]-dATP and enhanced
chemiluminescence (ECL) Western blotting detection kits were obtained
from Amersham Pharmacia Biotech (Arlington Heights, IL).
The caspase-3 inhibitor (Ac-DEVD-CHO) and substrate
[Ac-DEVD-aminomethylcoumarin (AMC)] were purchased from
BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).
Protein assay kits and horseradish peroxidase (HRP)-conjugated goat
antirabbit secondary antibody were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Biotinylated 16-dUTP,
HRP-conjugated streptavidin, and terminal deoxynucleotidyl transferase
(TdT) were purchased from Roche Molecular Biochemicals
(Indianapolis, IN). FCS, gentamicin, HBSS, medium 199, penicillin, and
streptomycin were obtained from Life Technologies, Inc./BRL (Grand Island, NY). Klenow enzyme was purchased from
New England Biolabs, Inc. (Beverly, MA). Rabbit antihuman
caspase-3 antibody was obtained from PharMingen (San
Diego, CA). Rabbit IgG and rabbit peroxidase kits were purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Sigma (St. Louis, MO) was the source of acrylamide,
agarose, aprotinin, BSA (fraction V),
3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate
(CHAPS), DMEM/Ham’s F-12, dimethylsulfoxide (DMSO),
dithiothreitol, hCG, leupeptin, 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT), normal goat serum, and soybean
trypsin inhibitor (STI). Avidin/Biotin Blocking and VECTOR VIP
Substrate kits were obtained from Vector Laboratories, Inc. (Burlingame, CA). Collagenase (type I) was purchased from
Worthington Biochemical Corp. (Lakewood, NJ).
Experimental protocol
Adult male Sprague Dawley rats (250–300 g) were purchased from
Harlan Sprague Dawley, Inc. (Indianapolis, IN). Rats were
housed in a climate-controlled (22 C) animal room with a constant 14-h
light, 10-h dark cycle and had free access to rat chow (Link Klein,
Baltimore, MD) and water. All procedures were in accordance with
protocols approved by The Johns Hopkins University animal care and use
committee. Rats (n = 6/group) were injected (ip) either with EDS
(85 mg/kg BW in DMSO-water, 1:3, vol/vol) or with an equivalent of
volume of vehicle (DMSO-water, 1:3, vol/vol). At 0, 3, 6, 12, or
24 h after EDS injection, rats were killed by cervical
dislocation, and testes were removed. The left testis was fixed in 4%
neutral buffered formaldehyde (pH 7.4) for histological processing, and
the right testis was immediately placed on ice in dissociation buffer
(medium 199 buffered with 8.5 mM sodium bicarbonate and 9.3
mM HEPES containing 0.1% BSA and 25 mg/liter STI,
pH 7.4) for subsequent isolation of Leydig cells.
Leydig cell isolation
Leydig cells were isolated as described previously (13), with
slight modifications. Briefly, decapsulated testes were incubated with
dissociation buffer containing 0.25 mg/ml collagenase at 34 C in a
shaking water bath (90 cycles/min) for 15 min. After dissociation, the
seminiferous tubules were removed by filtration through 100-µm pore
size nylon mesh. The filtrate was centrifuged (250 x
g, 10 min), the pellet was resuspended in buffered HBSS, and
the suspension was mixed with isoosmotic Percoll in HBSS [11:1
(vol/vol) dilution of Percoll in 10x Ca2+-
and Mg2+-free HBSS]. After centrifugation
(20,000 x g, 60 min, 4 C), fractions of 1.068 g/ml and
heavier were collected and washed with dissociation buffer. The cells
subsequently were incubated in plastic culture dishes (5 min, 34 C) to
minimize potential contamination by testicular macrophages. Leydig cell
purity was assessed by cytochemical staining for 3ß-hydroxysteroid
dehydrogenase (3ßHSD), as described previously (14). The cell purity
was consistently about 90%.
Morphological assessment of apoptosis
Freshly isolated Leydig cells were vitally stained with
propidium iodide (50 µg/ml in PBS, 5 min) and subsequently fixed with
4% neutral buffered formaldehyde (pH 7.4). Nuclear morphology was
assessed under a fluorescence microscope.
Leydig cell culture
This was performed as reported previously (15) with minor
modifications. In brief, isolated Leydig cells were resuspended in
DMEM/Ham’s F-12 culture medium containing 15 mM HEPES,
0.1% FCS, 0.1 ng/ml hCG, 5 µg/ml gentamicin, 50 U/ml penicillin, and
50 µg/ml streptomycin. Cells were seeded on Falcon culture plates
(Becton Dickinson and Co., Lincoln Park, NJ) for DNA
fragmentation (1.2 x 106 cells/well) and
MTT (5 x 104 cells/well) assays. To assess
the effects of EDS in vitro, cells were plated for 12 h
at 34 C in a humidified 5% CO2-95% air
atmosphere. Two hours before EDS treatment, either cell-permeable
caspase-3 inhibitor (Ac-DEVD-CHO; final concentration, 30
µM) or an equal volume of vehicle (DMSO) was
added. Then, either EDS (500 µg/ml) or an equivalent volume of
vehicle (DMSO) was added to the cultures for 3 h. For all
treatments, the final DMSO concentration was no more than 0.5%.
DNA fragmentation analysis
Total DNA was extracted from freshly isolated or cultured Leydig
cells. In brief, cells were homogenized in sample buffer, and
homogenates were incubated successively in 0.6% SDS (65 C; 30 min) and
35 mM potassium acetate (0 C; 60 min) and centrifuged
(5,000 x g, 4 C; 10 min). The supernatants were
extracted in phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol/vol).
Nucleic acid in the aqueous phase was precipitated and collected by
centrifugation (14,000 x g, 4 C, 30 min). RNA in the
nucleic acid preparation was removed by ribonuclease A (10 µg/ml)
treatment (37 C, 60 min). DNA content was determined by absorbance at
260 nm.
To enhance the visualization of DNA laddering, DNA was radiolabeled
using Klenow enzyme (16). In brief, DNA (500 ng) was incubated with
Klenow enzyme (5 U in 10 mM Tris and 5 mM
MgCl2) and 0.5 µCi
[-32P]dATP (3000 Ci/mmol) for 15 min at room
temperature, and the reaction was terminated with the addition of EDTA
(pH 8.0). Labeled DNA was separated on 1.8% agarose gels and
visualized after drying and exposure to x-ray film at -70 C. After
autoradiography, low mol wt DNA (<4 kb) was quantified
densitometrically.
Protein extraction and Western blot analysis
Freshly isolated Leydig cells were lysed with ice-cold PBS (pH
7.4) containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS,
and protease inhibitors (1 mM phenylmethylsulfonylfluoride,
10 mg/ml aprotinin, and 10 mg/ml leupeptin). Lysates were centrifuged
(13,000 x g, 4 C, 30 min). The protein content of the
supernatant was determined by protein assay (Bio-Rad Laboratories, Inc.). Equal amounts of protein (20 µg) were
resolved by 15% SDS-PAGE and transferred to nitrocellulose membranes.
After blocking, the membranes were incubated (1 h, room temperature)
with 0.2 µg/ml rabbit polyclonal antihuman caspase-3 antibody and
then (30 min, room temperature) with HRP-conjugated secondary antibody
(1:3000). Peroxidase activity was visualized with the Amersham Pharmacia Biotech ECL system according to the
manufacturer’s instructions. The caspase-3 protein content was
determined densitometrically.
Caspase-3 activity assay
Caspase-3 activity was measured as described previously (12)
with minor modifications. In brief, freshly isolated Leydig cells
(2 x 106 cells) were homogenized in lysis
buffer (10 mM HEPES/KOH, 2 mM EDTA, 0.1%
CHAPS, 5 mM dithiothreitol, 1 mM
phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, and 50 µg/ml
leupeptin) and centrifuged (10,000 x g, 10 min). The
supernatant was added to the reaction mixture [10
mM HEPES/KOH (pH 7.4), 0.1% CHAPS, 5
mM dithiothreitol, 10% sucrose, 10
µM DEVD- AMC]. After incubation (37 C, 60
min), the fluorescence resulting from free AMC, generated as a result
of cleavage of the aspartate-AMC bond, was measured with a microplate
reader (HTS7000+ Bio Assay Reader,
Perkin-Elmer Corp., Norwalk, CT) using 360-nm excitation
and 450-nm emission filters. To inhibit caspase-3 activity,
DEVD-aldehyde (CHO) was added (final inhibitor concentration, 1
µM).
MTT cell viability assay
MTT cell viability assays were performed as described previously
(17). One hour after EDS treatment of Leydig cells (5 x
104 cells in 96-well plates), 5 µl MTT agent in
PBS were added, and incubation was continued for 2 h. Then, 50
µl 0.05 N HCl in isopropanol were added, and cells were
solubilized. Absorbance was measured at 570–650 nm using an automated
96-well plate reader (Molecular Devices, Sunnyvale, CA).
All assays were repeated four times in triplicate.
TdT-mediated deoxy-UTP-biotin nick end labeling (TUNEL)
Apoptotic cells were detected by slight modification of the
in situ TUNEL method (18). In brief, deparaffinized testis
sections were incubated in TdT buffer (10 U TdT and 1 nmol biotinylated
16-dUTP) at 37 C (60 min) in a humidified chamber. The enzyme reaction
was stopped by dipping slides into 2 x SCC (5 min). The
biotinylated dUTP molecules incorporated into nuclear DNA was
visualized by incubating slides with HRP-conjugated streptavidin
(diluted 1:100, room temperature, 30 min) followed by diaminobenzidene
(DAB). The sections were counterstained with methyl green. In negative
control slides, TdT enzyme or biotinylated 16-dUTP was omitted.
Immunohistochemistry for caspase-3
For caspase-3 immunohistochemistry, deparaffinized and hydrated
testis sections were treated in 3%
H2O2 for 5 min and rinsed
with PBS for 15 min. The sections were blocked with 1.5% normal goat
serum in PBS and then incubated (45 min, room temperature) with rabbit
polyclonal antihuman caspase-3 (0.5 µg/ml) in 1.5% normal goat serum
in PBS. The sections then were incubated with biotin-conjugated goat
antirabbit IgG (1:200, 1 h, room temperature),
avidin-biotin-peroxidase complex (Santa Cruz Biotechnology, Inc., rabbit peroxidase kit; 1 h) and DAB solution.
Sections were counterstained with hematoxylin. For negative controls,
rabbit IgG (1 µg/ml) instead of the primary antibody was added to the
reaction.
For in situ double detection of apoptosis and caspase-3, the TUNEL and immunohistochemical techniques were conducted in sequence. After TUNEL staining, the slides were incubated in 3% H2O2 (5 min) and subsequently with avidin/biotin blocking solution according to the manufacturer’s instructions. After routine immunohistochemical staining (described above), the caspase-3 signal was differentially detected using the VIP Substrate Kit (Vector Laboratories, Inc.) instead of DAB. The tissue was counterstained with methyl green.
Statistical analysis
Data were expressed as the mean ± SEM of three
or four separate experiments. Group differences were analyzed by
one-way ANOVA. In cases in which P < 0.05, differences
between individual treatment groups were determined by Tukey’s test.
Means were considered to be different at P < 0.05.
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Results |
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shows Leydig cells isolated
from the testes of control rats (a and c) and 24 h after
rats received a single injection of EDS (b and d). Cells were stained
cytochemically for 3ßHSD (Fig. 1, a and b) or vitally with propidium
iodide (Fig. 1, c and d). Intense staining for 3ßHSD was seen in the
cytoplasm of virtually all Leydig cells from control rats. In contrast,
only a very small number of cells from testes of EDS-treated rats were
stained (compare Fig. 1, a and b). There were obvious morphological
differences in the cells from the two groups; whereas the cells from
controls had rounded nuclei and intact cytoplasm, the cells from
EDS-treated rats had relatively small nuclei and much less cytoplasm.
Fluorescence microscopy of Leydig cells stained with propidium iodide
provided further morphological evidence of apoptosis; the nuclei of the
EDS-treated Leydig cells appeared fragmented (Fig. 1d), whereas the
nuclei of vehicle-treated (Fig. 1c) or untreated control cells (not
shown) appeared intact.
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). Increases in low mol wt DNA were
apparent at 0 and 24 h after EDS administration, whereas the
intensity of high mol wt DNA was reduced at these times (Fig. 2A).
Quantification by densitometry of low mol wt DNA from three separate
experiments (Fig. 2B) revealed no detectable differences through 6
h post-EDS, and then significant increases in DNA fragmentation at 12
and 24 h.
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, A and B), and caspase-3
activity was measured using fluorescence (AMC)-conjugated
caspase-3-specific substrate (Fig. 3C). Western blot analysis revealed
a 32-kDa caspase-3 band in the lysates of control Leydig cells. Jurkat
and HeLa cell lysates, which served as positive controls, also showed
this 32-kDa band, characteristic of caspase-3. An increase in this band
was seen at 3–6 h post-EDS (Fig. 3, A and B). At 12 and 24 h
post-EDS, the times at which increased DNA fragmentation was seen (Fig. 2
), Western blots of Leydig cell lysates revealed cleaved (active)
forms of caspase-3 proteins, at 20 and 17 kDa, with a significant
decrease in the 32-kDa band at these times (Fig. 3, A and B). Using
data derived from four separate experiments, caspase-3 activity in
Leydig cell homogenates was quantified by incubating the homogenates in
DEVD-AMC and measuring the fluorescence resulting from the generation
of free AMC (Fig. 3C). As would be predicted from the Western blots,
caspase-3 activity assay increased at 12 and 24 h post-EDS
treatment. This activity was suppressed in the presence of the specific
caspase-3 inhibitor, Ac-DEVD-CHO (Fig. 3C).
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). Cell death in the presence of EDS
was reduced significantly by the caspase-3 inhibitor Ac-DEVD-CHO (Fig. 4A). Figure 4B shows a representative autoradiogram of DNA
fragmentation in isolated Leydig cells incubated with EDS in the
presence or absence of caspase-3 inhibitor. Low levels of DNA
fragmentation were seen in control and inhibitor-treated cells.
Incubation of the cells in EDS increased the level of DNA fragmentation
substantially, an increase that was prevented by incubating cells with
inhibitor along with EDS.
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). Leydig cells in the testes of control
rats were TUNEL negative (Fig. 5a). These cells showed evidence of
caspase-3 immunoreactivity in the cytoplasm (Fig. 5b). By 12 h
after EDS, TUNEL-positive Leydig cells were observed in the
interstitial compartment (Fig. 5c, arrows), with intense
immunoreactivity for caspase-3 localized primarily in the
perinuclear regions (Fig. 5d, short arrow). By 24 h
post-EDS, well over half of the Leydig cells were TUNEL positive, with
most of the stained (apoptotic) nuclei smaller than those observed
earlier (Fig. 5e, arrows). Leydig cells at this time were
intensely stained for caspase-3, with the most intense immunoreactivity
localized within the nuclei (Fig. 5f, short arrows).
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). All TUNEL-positive cells exhibited
intense immunoreactivity for caspase-3 that was localized to the
nuclear region (arrows and inset b). In contrast,
TUNEL-negative cells (solid box) exhibited only weak
immunoreactivity for caspase-3, localized to the cytoplasm and
perinuclear region (arrowheads and inset a).
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Discussion |
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To date, 14 caspases have been implicated in the apoptotic pathway cascade (19, 20). Among these, caspase-3 is considered to be of particular importance (10, 21). The cleavage of caspase-3 from its pro form (procaspase-3) to its active form has been shown to be critical for its role in apoptosis (22). The activation of caspase-3 can be inhibited by inhibitors of apoptosis proteins such as Xiap (X-chromosome-linked inhibitor of apoptosis protein) (23, 24). Caspase-3 cleavage is triggered by active caspase-8 (FADD-related interleukin-converting enzyme), which is processed from its pro form after Fas ligation (25); the activation of caspase-8 is inhibited by p35 (26).
In the present study, we demonstrate for the first time that caspase-3 protein and activity are present in rat Leydig cells. In response to EDS, the cleavage of caspase-3 to its active form was seen during the period in which Leydig cells underwent apoptosis. Six hours post-EDS, before any evidence of apoptosis, the protein content of procaspase-3 (32 kDa) was significantly increased compared with that of control cells. It is possible that increased procaspase-3 may overcome the inhibitory effects of endogenous cell survival factors such as the Bcl-2 family and inhibitors of apoptosis proteins, and thus be a prerequisite for the subsequent procaspase-3 activation. Consistent with this, a recent study reported increased Bcl-xl protein content in Leydig cells by 6 h post-EDS (6), suggesting that caspase-3 activation could be suppressed when Bcl-xl predominates. We show that by 12 and 24 h post-EDS, the cleaved forms of caspase-3 proteins were present. The appearance of these proteins was accompanied by the disappearance of the 32-kDa procaspase-3. Moreover, the specific caspase-3 inhibitor, Ac-DEVD-CHO, suppressed caspase-3 activity in the lysates of Leydig cells isolated from EDS-treated rats and also suppressed apoptosis. Taken together, these results suggest that the cleaved bands detected by Western blot at 12 and 24 h post-EDS were the active forms of caspase-3, and that they originated from the proenzyme. The fact that the induction of Leydig cell apoptosis by EDS in vitro was suppressed when the cell-permeable caspase-3 inhibitor, Ac-DEVD-CHO, was present, strongly supports the contention that EDS kills Leydig cells via a caspase-3-dependent mechanism.
After exposure to EDS, intense caspase-3 immunoreactivity first was observed in the cytoplasm and later the nuclei of Leydig cells. Colocalization studies indicated that apoptotic cells, recognized by TUNEL staining, all showed intense nuclear staining for caspase-3. Taken together, these results suggest that the immunoreactivity seen in the nuclei of Leydig cells 12 and 24 h post-EDS reflected active caspase-3. Indeed, TUNEL and caspase-3 staining consistently were seen together in the same nuclei, whereas in cells that were TUNEL negative, caspase-3 was localized in the cytoplasm. This suggests that there is translocation of active caspase-3 to the nuclei of at least some, if not all, of the cells undergoing apoptosis. This interpretation is consistent with the results of previous studies showing the presence of active species of caspases in the nuclei of apoptotic HL-60 cells (27) as well as nuclear translocation and activation of procaspase-1 (28). However, active caspases have been detected in the cytosolic fraction of cells (27). As yet, therefore, it is not clear what role caspase translocation plays in apoptosis. One possibility is that translocated nuclear caspase-3 may play a role in the activation of nuclear proteins that accelerate the terminal nuclear event of apoptotic process, DNA fragmentation. Indeed, one target of activated caspase-3 is DFF (11, 29), activation of which by caspase-3 has been shown to be a key event that ultimately leads to cleavage of DNA in a number of cells.
Leydig cells rarely divide or die under normal physiological
conditions. In some cells (e.g. T cells and macrophages),
Fas ligand (FasL), a well known death factor of the tumor necrosis
factor- family (30, 31), may induce apoptosis in cells in which Fas
receptor is present. Recent studies have reported that there is
considerable Fas protein in the Leydig cells of untreated adult rats
(7). However, the presence of survival proteins such as Bcl-xl in
Leydig cells (6) may suppress the apoptotic signal cascade, including
the activation of caspases, that otherwise would be initiated by
activation of the Fas/FasL system. This could be one of the
explanations for the observation that Leydig cell apoptosis is
uncommon.
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Acknowledgments |
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Footnotes |
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Received November 9, 1999.
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