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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, Johns Hopkins University School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205. E-mail: jmkim{at}jhsph.edu
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Abstract |
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Introduction |
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The caspases, well conserved from nematodes to mammals, are processed to active (cleaved) forms in cells undergoing apoptosis and thus play a crucial role in the transduction of apoptotic signals in cells that are destined to die (13). Caspase activation occurs by self-proteolysis and/or results from the actions of other proteins (14). Ligation of Fas ligand to Fas in the cellular membrane and an increase in the Bax/Bcl-2 ratio in the mitochondrial membrane trigger activation of caspase-8 and caspase-9, respectively (15). Once activated, these caspases transduce a signal to effector caspases, including caspases-3, -6, and -7 (15). This causes degradation of the cellular substrates of these caspases, including cytoplasmic structural proteins such as actin and cytokeratins and/or nuclear proteins such as poly(ADP- ribose) polymerase (PARP) and lamins (16, 17, 18, 19). Among the effector caspases, activated caspase-3 appears to induce activation of caspase-activated deoxyribonuclease (CAD; also called DNA fragmentation factor-40 or caspase-activated nuclease), which is integrally involved in degrading DNA (20, 21, 22).
In the present study we examined the roles of caspase-3 and CAD activation in relation to the apoptotic death of germ cells. We show that testosterone withdrawal results in spermatocyte apoptosis, and that this is correlated to activation of caspase-3 as well as increased CAD protein expression. Additionally, immunohistochemical analysis revealed that both activated caspase-3 and CAD become localized to the nuclei of spermatocytes at the time of apoptosis. Taken together these results suggest that spermatocyte apoptosis, resulting from reduced intratesticular testosterone, is mediated by caspase-3 activation and CAD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]Deoxy-ATP,
gold-conjugated goat antirabbit IgG, Intense silver enhancement system,
and the enhanced chemiluminescence Western blotting detection kit were
obtained from Amersham Pharmacia Biotech (Arlington
Heights, IL). ApopTag peroxidase in situ apoptosis detection
kits were purchased from Intergen (Gaithersburg, MD).
Horseradish peroxidase-conjugated goat antirabbit and antimouse
secondary antibodies were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Rabbit antimouse CAD antibody was obtained
from Calbiochem (La Jolla, CA). Agarose, DMEM/Ham’s F-12,
and HBSS 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
and rabbit antihuman active form-specific antibody were obtained from
PharMingen (San Diego, CA). Rabbit antihuman PARP
antibody, rabbit IgG, and mouse IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Sigma (St.
Louis, MO) was the source of acrylamide, aprotinin, BSA (fraction V),
dithiothreitol (DTT), hyaluronidase, leupeptin, mouse antipan actin
antibody, normal goat serum, and soybean trypsin inhibitor (STI).
Collagenase (type I) was purchased from Worthington Biochemical Corp. (Lakewood, NJ).
Experimental protocol
Adult male Sprague Dawley rats (12 wk old) were purchased from
Harlan Sprague Dawley, Inc. (Indianapolis, IN). Rats were
housed in a climate-controlled (22 C) animal room at a constant 14-h
light, 10-h dark cycle, with free access to rat chow (Link Klein,
Baltimore, MD) and water. All procedures were performed in accordance
with protocols approved by The Johns Hopkins University animal care and
use committee. Rats (n = 6–9/group) received subdermal 2.5-cm
testosterone (T)- and 0.1-cm 17ß-estradiol (E)-filled
polydimethylsiloxane capsules, or empty polydimethylsiloxane capsules
(control), according to previously described methods (23).
On d 1, 3, 5, 7, 14, and 28 after implantation, rats were killed by
carbon dioxide asphyxiation, and testes were removed. The left testis
was used for collection of interstitial fluid (IF; see below). The
right testis was placed on ice in dissociation buffer (DMEM/Ham’s F-12
buffered with 14.3 mM sodium bicarbonate and 15
mM HEPES containing 0.33 mg/ml STI, pH 7.3) for subsequent
isolation of germ cells. For histological studies, rats were
anesthetized with sodium pentobarbital, and testes were fixed by
perfusing of Bouin’s fixative through the dorsal aorta.
Collection of IF and RIA
IF was collected according to a previously described method
(24). Briefly, the tunica albuginea was incised at one
pole, and testes were centrifuged at low speed (54 x
g, 10 min, 4 C) to drain IF. Immediately after collection,
IF was snap-frozen in liquid nitrogen and stored frozen at -80 C
before assay for testosterone. Serum and IF testosterone concentrations
were determined in duplicate samples by a previously described RIA
procedure (24). The sensitivity of the assay was 10
pg/tube, with intra- and interassay coefficients of variation of 11.2%
and 9.6%, respectively.
Detection of apoptotic cells and immunolocalization of caspase-3
and CAD
Apoptotic cells were detected by terminal
deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling
(TUNEL), using an in situ detection kit (ApopTag,
Intergen) according to the manufacturer’s instruction.
For caspase-3 and CAD immunohistochemistry, deparaffinized and hydrated
testis sections were rinsed with PBS for 15 min. The sections were
blocked with normal goat serum (1.5% in PBS) and then incubated (45
min, room temperature) with caspase-3 antibody specific for the active
form (1.0 µg/ml) or with CAD antibody (0.5 µg/ml) in normal goat
serum (1.5% in PBS). The sections then were incubated with gold (1
nm)-conjugated goat antirabbit IgG (1:200; 30 min, room temperature)
and developed with silver enhancement solution (Amersham Pharmacia Biotech silver enhancement system, 5 min). Sections
were counterstained with methyl green. For negative controls, rabbit
IgG (1 µg/ml) instead of the primary antibody was added to the
reaction.
Germ cell isolation
Germ cells were isolated as described previously
(25) with slight modifications. In brief, decapsulated
testes were incubated with dissociation buffer containing collagenase
(1 mg/ml) and hyaluronidase (2 mg/ml) at 34 C in a shaking water bath
(105 cycles/min) for 25 min. After dissociation, the germ cells were
released from seminiferous tubules by gentle pipetting for 5 min. The
cell suspension was then filtered through 50-µm nylon mesh, the
filtrate was centrifuged (1,000 x g, 5 min), and the
pellet was resuspended in germ cell isolation medium [GCIM
(DMEM/Ham’s F-12 buffered with 14.3 mM sodium
bicarbonate and 15 mM HEPES containing 11
mM sodium lactate, 1 mM
sodium pyruvate, and 0.025 mg/ml STI, pH 7.3)]. The cells were
filtered through 30-µm pore size nylon mesh, centrifuged (1,000
x g, 5 min), resuspended in GCIM containing 2% BSA, and
incubated (34 C, 30 min) for sedimentation of Sertoli cells and clumped
cells. The supernatant was centrifuged (1,000 x g, 5
min), and the cells were resuspended in GCIM and filtered through
20-µm pore size nylon mesh. Finally, cells were mixed with isoosmotic
Percoll in HBSS (11:1, vol/vol, of Percoll in 10x
Ca2+,Mg2+-free HBSS). After
centrifugation (20,000 x g, 30 min, 4 C), fractions of
1.041–1.053 g/ml were collected and washed with GCIM. Cells were
counted on a hemocytometer and either immediately used for biochemical
analysis or stored at -80 C after snap-freezing. Germ cell purity was
assessed after Giemsa staining of smeared cells or hematoxylin-eosin
staining of sections of germ cell pellets. The germ cell purity was
consistently about 85–95%. Residual bodies represented the major
contaminant. No elongated spermatids or spermatozoa were seen.
DNA fragmentation analysis
Total DNA was extracted from testicular tissues or isolated germ
cells as previously described (26), and DNA concentration
was determined by absorbance at 260 nm. To enhance the visualization of
DNA laddering, DNA was radiolabeled using Klenow enzyme
(27). In brief, DNA (500 ng) was incubated with Klenow
enzyme (5 U in 10 mM Tris and 5 mM
MgCl2) and with 0.5 µCi
[-32P]deoxy-ATP (3000 Ci/mmol) for 15 min at
room temperature. The reaction was terminated by the addition of EDTA
(pH 8.0). Labeled DNA was separated on 1.8% agarose gels and
visualized by exposing dried gels to x-ray film at -70 C. After
autoradiography, low mol wt DNA (<4 kb) was quantified by
ß-scintillation counting of bands cut from the gels.
Isolation of germ cell nuclei
Germ cell nuclei were isolated as previously described
(28). Briefly, germ cells (3 x
106 cells) were homogenized in 2 vol prechilled
solution A [250 mM sucrose, 1 mM DTT, 80
mM KCl, 15 mM NaCl, 5 mM EDTA, 15
mM PIPES-NaOH, 0.5 mM spermidine, 0.2
mM spermine, 1 mM phenylmethylsulfonylfluoride
(PMSF), 10 mg/ml aprotinin, and 10 mg/ml leupeptin (pH 7.4)], using a
Dounce homogenizer (12–15 strokes; Kontes Co., Vineland, NJ). The
homogenates were filtered through four layers of cheesecloth, and 2 vol
solution B [2.3 M sucrose, 1 mM DTT, 80
mM KCl, 15 mM NaCl, 5 mM EDTA, 15
mM PIPES-NaOH, 0.5 mM spermidine, 0.2
mM spermine, 1 mM PMSF, 10 mg/ml aprotinin, and
10 mg/ml leupeptin (pH 7.4)] were added. Filtrates were centrifuged at
22,000 rpm (1.5 h, 4 C), and the nuclei were recovered from the bottom
of the tube. The isolated nuclei were immediately used for protein
extraction or stored at -80 C after snap-freezing in liquid
nitrogen.
Protein extraction and Western blot analysis
Isolated germ cells (3 x 106 cells)
or germ cell nuclei (from 3 x 106 cells)
were lysed with same amount of ice-cold PBS (pH 7.4) containing 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease
inhibitors (1 mM PMSF, 10 mg/ml aprotinin, and 10 mg/ml
leupeptin), and lysates were centrifuged (13,000 x g,
4 C, 30 min). The same volumes of supernatants (each containing 
50
µg protein) were resolved by SDS-PAGE and transferred to a
nitrocellulose membrane. After blocking, the membranes were incubated
(1 h, room temperature) with a primary antibody [anti caspase-3 (0.5
µg/ml), antiactive caspase-3 (0.5 µg/ml), anti-CAD (0.2 µg/ml),
anti-PARP (0.2 µg/ml), or antiactin (1.0 µg/ml)] and then with the
appropriate horseradish peroxidase-conjugated secondary antibody
(1:3,000) for 30 min. Peroxidase activity was visualized with the
Amersham Pharmacia Biotech enhanced
chemiluminescence system according to the manufacturer’s
instructions. Caspase-3 and CAD protein contents were determined
densitometrically.
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 significantly different at P <
0.05.
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Results |
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). By 7 d, the
intratesticular testosterone concentration had decreased to below 10
ng/ml, and it remained at or below this concentration through d 28.
Testicular weight also was reduced after TE administration (Fig. 1B),
with gradual reductions seen through d 7 and significant reduction by d
14 post-TE and thereafter. Consistent with the reduced testicular
weight, increased DNA fragmentation, a hallmark of apoptotic cell
death, was apparent by d 7 after TE administration (Fig. 1C). DNA
fragmentation was significantly higher than the control value by d 14,
and remained high on d 21 (Fig. 1, C and D) and d 28 (not shown).
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, compare a and
b) apparently resulted from reduced germ cell numbers compared
with controls (Fig. 2, compare c and d), in particular spermatocytes
and spermatids. In situ TUNEL staining revealed that
spermatocytes represented the predominant apoptotic cell type from 1–4
wk postimplantation (Fig. 2, e and f); spermatids were rarely, if ever,
TUNEL positive.
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). In control testes, immunoreactivity
for caspase-3 was generally observed scattered in the cytoplasm or in
the perinuclear region of germ cells (Fig. 3a, short
arrows), with more intense staining seen in the residual bodies
(Fig. 3a, arrowheads). After TE administration, intense
immunoreactivity was seen primarily in the nuclei of spermatocytes
(Fig. 3b, long arrows).
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). For these studies germ cells from
control testes and from testes of rats that had been administered TE
were used. A caspase-3 antibody that recognized both the pro form (32
kDa) and cleaved forms (20 and 17 kDa) was employed. Pro-caspase-3
protein (32 kDa) was found to increase significantly in germ cells
after TE administration to rats compared with control cells (Fig. 4, A
and B), but there was no significant difference in the activated
(cleaved) form of caspase-3 (20 and 17 kDa) between the two groups.
This was puzzling because isolated germ cells showed clear differences
in DNA fragmentation patterns (Fig. 4C). In light of the observation
that the active form of caspase-3 was localized to the nuclei of germ
cells (Fig. 3B
), we conducted Western blot analysis of protein
extracted from the nuclei of germ cells rather than from whole cells.
With nuclei as the starting material, a different picture emerged. As
shown in Fig. 4, D and E, significantly more active caspase-3 (17 kDa)
was seen in the nuclei of germ cells from the TE- administered rats
than from the controls.
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). Cleavage of PARP to a band of 85 kDa
occurred in germ cells from TE-administered rats (Fig. 5A). CAD
protein, which would be expected to be dissociated from its inhibitor
protein, ICAD, by activated caspase-3 during the terminal stages of
apoptosis, was significantly up-regulated (195 ± 18%; n =
4) in germ cells after TE (Fig. 5B). This up-regulation was coincident
with caspase-3 activation, cleavage of caspase-3 substrates, and
increased DNA fragmentation of germ cells. Silver-enhanced
immunohistochemical methods were used to localize CAD protein (Fig. 6). The nuclei of apoptotic
(TUNEL-stained) spermatocytes (Fig. 6b) in testes from TE-treated rats
showed intense immunoreactivity for CAD (Fig. 6d). In contrast, only
the cytoplasm or perinuclear regions of nonapoptotic spermatocytes
(Fig. 6a) from controls stained for CAD (Fig. 6c).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, 14 caspases have been implicated in the apoptotic pathway cascade (34). Among these, caspase-3 is considered to be a major execution protease (35, 36). The cleavage of caspase-3 from its pro form to its active form has been shown to be critical for its role in apoptosis (37). Caspase-3 cleavage can be triggered by active caspase-8, processed from its pro form after Fas ligation (38), or by caspase-9, processed from its pro form after increased ratios of heterodimerized Bax/Bcl-2 (39). It has been shown in a number of cell types that, once activated, caspase-3 cleaves numerous cellular proteins associated with the cytoskeleton [actin (16) and cytokeratin (17)], cell cycle regulation [Mdm2 (40) and Rb (41)], DNA repair [PARP (18)], and DNA degradation [ICAD/CAD complex (42)]. Among these substrates, it has been suggested that active caspase-3 causes CAD to be dissociated from its inhibitor protein, ICAD (also called DNA fragmentation factor-45) (42, 43). The liberated CAD then is thought to translocate into the nucleus to cause DNA fragmentation (20).
We show herein, by high resolution silver-enhanced immunogold histochemistry and an antibody that recognized the active form of caspase-3, that caspase-3 protein is localized to germ cells, and that after reduction of intratesticular testosterone, intense caspase-3 immunoreactivity translocates from the cytoplasm to become concentrated in the nuclei of spermatocytes. This observation suggests that, as in Leydig cells (26), nuclear translocation of activated caspase-3 may be required for caspase-3 to function in germ cell apoptosis. Consistent with the immunohistochemical findings, Western blot analysis of germ cell lysates revealed a significant increase in pro-caspase-3 protein expression after TE administration, which suggests that, as in Leydig cells (26), expression of pro-caspase-3 may be required before its activation in vivo after the cells are exposed to death signal(s). We were surprised to find no difference in activated (cleaved) caspase-3 between control and TE-treated groups when whole germ cells were examined. However, when proteins extracted from germ cell nuclei were examined by Western blot, a significant increase in the active form of caspase-3 was detected with TE administration. This further suggested that nuclear translocation of the active form of caspase-3 protein may trigger the terminal stages of apoptosis.
Having demonstrated activated caspase-3 protein in germ cell nuclei, we determined whether target substrates of caspase-3 were degraded as a function of reduced intratesticular testosterone. PARP, one such substrate, was indeed found to be degraded (cleaved) in germ cells of the TE-treated rats, further suggesting that caspase-3 activity present in germ cells was related to their apoptosis.
Activated caspase-3 has been reported to induce activation of CAD, an event that is integrally involved in degrading DNA (20, 44). We show that CAD protein content was significantly increased in germ cells after rats were administered TE and that, as with caspase-3, CAD immunoreactivity was localized to the nuclei of apoptotic spermatocytes. From these findings we suggest that an increase in CAD protein is an important indicator of its level of dissociation from ICAD, and that the translocation of CAD into the nucleus is a critical step in the induction of DNA fragmentation.
Taken together, these results suggest that germ cell apoptosis resulting from reduced intratesticular testosterone concentration is caspase-3 activation dependent, and further, that the translocation of active caspase-3 and CAD to the nucleus may be a prerequisite for DNA degradation and subsequent germ cell apoptosis.
In other systems, caspases-8 and -9 are activated by ligation of Fas ligand to Fas and by an increase in the Bax/Bcl-2 ratio, respectively. As yet, there is uncertainty about whether germ cell apoptosis in the rat begins with ligation of Fas ligand to Fas, changes in the Bcl-2 family, or both. To address this important issue, we currently are investigating the possible activation of caspase-8, caspase-9, or both, and the mechanisms by which activation occurs.
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Acknowledgments |
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Footnotes |
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Abbreviation: CAD, Caspase-activated deoxyribonuclease; DTT, dithiothreitol; ICAD, inhibitor of CAD; IF, interstitial fluid; PARP, poly(ADP-ribose) polymerase; PMSF, phenylmethylsulfonylfluoride; STI, soybean trypsin inhibitor; TE, testosterone and estradiol; TUNEL, terminal deoxynucleotidyltransferase- mediated deoxy-UTP nick end labeling.
Received November 30, 2000.
Accepted for publication May 15, 2001.
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but not CPP32: multiple interleukin
1ß-converting enzyme-related proteases with distinct substrate
recognition properties are active in apoptosis. Proc Natl Acad Sci USA 93:8395–8400This article has been cited by other articles:
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