Am J Physiol Endocrinol Metab 290: E1145–E1154, 2006.
First published January 10, 2006; doi:10.1152/ajpendo.00142.2005.
Regulation of human male germ cell death by modulators of ATP production
Krista Erkkila,1,2 Sauli Kyttanen,1 Marten Wikstrom,3 Kimmo Taari,4
Amiya P. Sinha Hikim,2 Ronald S. Swerdloff,2 and Leo Dunkel1,5
1
Program for Developmental and Reproductive Biology, Biomedicum Helsinki, and Hospital for Children
and Adolescents, 3Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki;
4
Department of Urology, Helsinki University Hospital, Helsinki; 5Department of Pediatrics, Kuopio University
Hospital, University of Kuopio, Kuopio, Finland; and 2Division of Endocrinology, Department of Medicine,
Harbor-University of California Los Angeles Medical Center and Los Angeles Biomedical Research Institute,
David Geffen Medical School at the University of California Los Angeles, Torrance, California
Submitted 30 March 2005; accepted in final form 4 January 2006
testis; spermatogenesis; apoptosis; mitochondria; oxidative phosphorylation
THE ENERGY METABOLISM and catabolism in the testis involve a
unique network of reactions and include several testis-specific
enzymes, hormonal regulation, and essential cell-to-cell interactions (1, 8, 27, 29, 41, 43, 55, 56, 58). The proper functioning
of this network is critical for testicular physiology. Another
crucial regulator of normal testicular function is appropriate
germ cell death, and the disruption of this orderly process is
associated with several male reproductive disorders (15, 16,
Address for reprint requests and other correspondence: K. Erkkila, Division
of Endocrinology, Harbor-UCLA Medical Center and Los Angeles Biomedical
Institute, Box 446 (1000 W. Carson St.), Torrance, CA 90509 (e-mail:
[email protected]; [email protected]).
http://www.ajpendo.org
30, 38, 47, 54, 60). How these two entities, i.e., energy
metabolism/catabolism and male germ cell death, are linked is
presently unclear.
The cell types of the seminiferous epithelium differ from
each other in their sensitivity to death-inducing signals and
with their preferred substrates for energy metabolism. Spermatogonia, mature spermatozoa, and the somatic Sertoli cells
exhibit high glycolytic activity, whereas spermatocytes and
spermatids produce ATP mainly by mitochondrial oxidative
phosphorylation (OXPHOS; see Refs. 3, 28, 31, 37, 45, 53,
64). Interestingly, the cell types that use OXPHOS for energy
production (i.e., spermatids and spermatocytes) are sensitive to
death-inducing signals such as hormonal deprivation, Fas activation, and elevated temperature (19, 24, 34, 47, 48, 51, 60,
60). The OXPHOS of the inner mitochondrial membrane (IM)
is comprised of the electron transport chain (the respiratory
chain) and the ATP synthase (F0F1-ATPase) (4, 9, 52). The
electron transport chain, in turn, consists of the protein complexes I to IV through which the electrons shuttle, for example,
by cytochrome c (cyt c) and pass sequentially to molecular
oxygen. The respiratory chain generates a proton concentration
gradient (change in pH) and a transmembrane potential (⌬⌿m)
across the IM, which then provide energy for synthesizing ATP
by the ATP synthase (4, 9, 52).
The components of the OXPHOS are not only involved in
energy production per se but are also the targets and the
modulators of cell death events (5, 9, 17, 23, 25, 36, 39, 40, 42,
52, 57, 65). Many of the apoptotic pathways trigger an increase
in the permeability of the mitochondrial IM [permeability
transition (PT)] and of the outer membrane, leading to the
dissipation of the ⌬⌿m and the proton gradient and in the
release of factors, such as cyt c, to the cytosol where these
proteins activate later apoptotic cascades (5, 25, 36, 42). Loss
of the IM gradients together with the release of proteins is
thought to result in disruption of electron transport and OXPHOS and a consequent drop in ATP production during the
apoptotic process (5, 26, 42). Accordingly, apoptosis of human
male germ cells, induced by hormone- and serum-free tissue
culture conditions, is associated with PT and a decrease in ATP
levels (21, 22). Thus ATP production is a target of apoptotic
events of male germ cells.
The fact that potassium cyanide (KCN), an inhibitor of
mitochondrial respiration, effectively suppresses testicular
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Erkkila, Krista, Sauli Kyttanen, Marten Wikstrom, Kimmo Taari, Amiya P. Sinha Hikim, Ronald S. Swerdloff, and Leo Dunkel.
Regulation of human male germ cell death by modulators of ATP
production. Am J Physiol Endocrinol Metab 290: E1145–E1154, 2006.
First published January 10, 2006; doi:10.1152/ajpendo.00142.2005.—
The understanding of testicular physiology, pathology, and male
fertility issues requires knowledge of male germ cell death and energy
production. Here, we induced human male germ cell apoptosis (detected by Southern blot analysis of DNA fragmentation, TUNEL,
activation of caspases-3 and -9, and electron microscopy) by incubating seminiferous tubule segments under hormone- and serum-free
conditions. Inhibitors of complexes I to IV of mitochondrial respiration, exposure to anoxia, and inhibition of F0F1-ATPase (with oligomycin) decreased the ATP levels (analyzed by HPLC) and suppressed
apoptosis at 4 h. Uncoupler 2,4-dinitrophenol (DNP) and oligomycin
combination also suppressed death at 4 h, as did the DNP alone.
Inhibition of glycolysis by 2-deoxyglucose neither suppressed nor
further induced apoptosis nor altered the antiapoptotic effects of the
mitochondrial inhibitors. Furthermore, Fas system activation did not
modify the effects of mitochondrial modulators. After 24 h, delayed
male germ cell apoptosis was observed despite the presence of the
mitochondrial inhibitors. We conclude that the mitochondrial ATP
production machinery plays an important role in regulating in vitroinduced primary pathways of human male germ apoptosis. The ATP
synthesized by the F0F1-ATPase seems to be the crucial death regulator, rather than any of the complexes (I-IV) alone, the functional
electron transport chain, or the membrane potential. We also conclude
that there seem to be secondary pathways of human testicular cell
apoptosis that do not require mitochondrial ATP production. The
present study emphasizes the role of the main catabolic pathways in
the complex network of regulating events of male germ cell life and
death.
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MATERIALS AND METHODS
Patients. Testicular tissue was obtained from 41 adult men aged
52– 89 yr undergoing orchidectomy as a treatment for prostate cancer.
The patients had received neither hormonal nor chemotherapeutic
medication nor had they received radiotherapy before the operation,
and none of them had suffered from cryptorchidism. The operations
were performed between November 1998 and July 2005 at the
Department of Urology, Helsinki University Central Hospital (Helsinki, Finland). The Ethics Committees of the Departments of Pediatrics and of Urology, University of Helsinki, approved the study
protocol (number 14/95).
Tissue preparation and induction of apoptosis. To maintain the
physiological cell-to-cell interactions of testicular cells, segments of
seminiferous tubules were cultured instead of isolated cells. The tissue
culture, including the apoptosis-inducing conditions and the time
points, was based on our previously described in vitro model (18 –20).
Briefly, immediately after the testis tissues from the operations were
obtained, segments of seminiferous tubules (1–5 mm in length) were
microdissected in petri dishes containing tissue culture medium (Nutrient mixture Ham’s F-10 containing 6.1 mmol/l of D-glucose and 1.0
mmol/l of sodium pyruvate; GIBCO Europe, Paisley, UK) supplemented with 0.1% of human serum albumin (Sigma Chemical, St.
Louis, MO) and 10 ␮g/ ml of gentamicin (GIBCO). For induction of
apoptosis, segments of seminiferous tubules were transferred to culture plates containing the hormone- and serum-free culture medium
described above and incubated for 4 or 24 h at 34°C in humidified
room air with CO2 adjusted to 5%.
Inhibition of the complexes of mitochondrial respiratory chain. To
evaluate the effects of the different complexes of the mitochondrial
respiratory chain on human testicular cell apoptosis, specific inhibitors
of the complexes were added to the culture medium. NADH-ubiquinone oxidoreductase (complex I) was inhibited with rotenone; succinate-ubiquinone oxidoreductase (complex II) with thenoyltrifluoroacetone (TTFA); ubiquinol-cyt c oxidoreductase (the bc1-complex or
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complex III) with antimycin A; and cyt c oxidase (cytochrome aa3 or
complex IV) with KCN (all derived from Sigma Chemical). The final
concentrations of rotenone were 10 and 100 ␮M, of TTFA 100 and
200 ␮M, of antimycin A 10 and 20 ␮M, and of KCN 5 and 10 mM.
In all experiments, the pH was neutralized before culture.
Exposure to anoxia. Exposure to anoxia was performed by culturing the samples in humidified tight glass chambers under continuous
gas flow (minimum 1 l/h) from gas bottles containing 5% of CO2 and
95% of nitrogen (maximum ⬍10 parts/million of 02; Aga, Espoo,
Finland).
Inhibition of the F0F1-ATP synthase and uncoupling of OXPHOS.
The mitochondrial F0F1-ATP synthase was inhibited by adding oligomycin ABC (Sigma) to the cultures at final concentrations of 200
and 400 ␮M. Uncoupling of OXPHOS was achieved with 2,4dinitrophenol (DNP; Sigma) at final concentrations of 200 and 400
␮M. To create a situation in which the respiratory chain is active, but
where ATP synthase is inhibited, a combination of uncoupler (DNP)
and the F0F1-ATP synthase inhibitor oligomycin was used. In all
experiments, the pH was neutralized before culture.
Blockade of glycolysis. Cytosolic ATP production was inhibited by
preventing glycolysis with 2-deoxyglucose (2-DG, Sigma) at concentrations of 5 or 10 mmol/l. To inhibit both cytosolic and mitochondrial
ATP production, combinations of 2-DG and inhibitors of mitochondrial respiration (rotenone, TTFA, antimycin, KCN, or oligomycin)
were used in the cultures.
Activation of the Fas receptors. To evaluate whether activation of
the Fas system would modulate the effects of mitochondrial inhibitors,
or vice versa, agonistic anti-Fas antibody (Roche Molecular Biochemicals, Mannheim, Germany) was added in the cultures. The final
concentrations were 2 and 5 ␮g/ml, and it was used in the absence or
the presence of KCN, DNP, or oligomycin. In the pilot experiments,
recombinant human Fas ligand (Fas-L; Calbiochem-Oncogene Research Products, San Diego, CA) was also tested, instead of anti-Fas
antibody being activated. The final concentrations of Fas-L were 0.5
and 1.0 ␮g/ml.
Detection of cell death by Southern blot analysis of DNA fragmentation. Segments of seminiferous tubules were snap-frozen in liquid
nitrogen and stored at ⫺80°C until isolation of DNA. DNA was
extracted using the Apoptotic DNA Ladder Kit (Roche Molecular
Biochemicals), as previously described (50). After isolation, the DNA
was quantified by spectrophotometry (absorbance at 260 nm). DNA
samples (1 ␮g) were 3⬘-end-labeled with digoxigenin-dideoxy-UTP
(Roche) using the terminal transferase (Roche) reaction, subjected to
electrophoresis on 2% agarose gels, and blotted on nylon membranes.
DNA was cross-linked to the membranes by ultraviolet irradiation.
The membranes were washed and blocked with 1% Blocking reagent
(Roche) in maleic buffer (100 mmol/l maleic acid and 150 mmol/l
NaCl, pH 7.5). The 3⬘-end-labeled DNA on the membranes was
localized with alkaline phosphatase-conjugated anti-digoxigenin antibody (Anti-Digoxigenin-AP; Roche) as described previously (19).
After being washed with washing buffer (0.1% Tween 20 in maleic
acid buffer), the membrane was equilibrated in detection buffer (0.1
M Tris 䡠 HCl, 0.1 M NaCl, 50 mM MgCl2, pH 9.5), and the bound
antibody was detected by the chemiluminescence reaction (Roche) at
room temperature for 5 min and enhanced at 37°C for 15 min. X-ray
films were exposed to the chemiluminescence and scanned, and the
digitized information (optical density) was analyzed with Scion Image
analysis software. Low-molecular-weight DNA fractions (⬍1.3 kb) of
the 0-h sample were set at 1.0 (100%), to which the other settings
were compared. Thus the results are expressed in relation to the
starting (0-h) time point.
In situ end-labeling (TUNEL) of DNA and propidium iodide staining. Short segments of seminiferous tubules (1–3 mm in length) were
gently squashed under cover slips to enable the cells to move out from
the tubules and produce a monolayer of cells on a microscope slide.
These squash preparations were fixed as previously described (46).
Briefly, the microscope slides were frozen in liquid nitrogen, the cover
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germ cell death (22) strongly suggests that the ATP machinery
is not only the target of the death events but also a regulator of
male germ apoptosis. However, whether it is the ATP (or its
concentration) as such or whether it is a certain site of the
machinery that controls death is not known. For nontesticular
cells, there are multiple proposals for the crucial death-regulating site, including the ATP itself or certain OXPHOS component(s), such as individual complexes, the ⌬⌿m, or the ATP
synthase (7, 10, 11, 17, 25, 32, 39, 40, 52, 57).
The purpose of the present study was to evaluate the relationship(s) of the following two fundamental entities of testicular functions: the male germ cell death and the ATP-producing machinery. The goal was to find out whether the ATPproducing machinery or a particular part of OXPHOS would
regulate human testicular cell death. More specifically, we
aimed at investigating the roles of individual mitochondrial
complexes, the F0F1-ATP synthase, by using specific inhibitors
and of uncoupling of OXPHOS in human male germ cell
apoptosis. In addition, the study was designed to determine
whether blocking of glycolysis or activating the Fas system
would modulate male germ cell death, which was induced by
incubating segments of human seminiferous tubules under
hormone- and serum-free conditions. Characterizing and understanding the basic mechanisms involved in male germ cell
apoptosis are essential steps for the development of novel
therapeutic regiments to control accelerated apoptosis during
abnormal spermatogenesis as well as for more targeted approaches to male contraception.
HUMAN MALE GERM CELL DEATH AND ATP PRODUCTION
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min. The compounds were identified and quantified by comparison
with the retention times and peak areas of known standards, calibrated
by spectrophotometry. The AN concentrations were expressed in
relation to testis tissue wet weight (␮mol/g of testis).
Statistical analysis. The experiments were repeated on at least three
independent occasions. For statistical comparisons, data obtained
from the replicate experiments (means ⫾ SE) were analyzed by
one-way ANOVA followed by the post hoc test with Bonferroni
correction. A P value ⬍0.05 was considered significant.
RESULTS
Inhibitors of mitochondrial respiratory complexes prevent
human testicular apoptosis and suppress the ATP levels at 4 h.
In line with our previous results (18 –20), incubation of human
seminiferous tubule segments under serum- and hormone-free
culture conditions induced apoptosis in male germ cells (Figs.
1 and 2). Apoptosis was detected by Southern blot analysis of
DNA fragmentation (Fig. 1, A and B), by activation of the
caspase-3 (as previously described and Refs. 61 and 62) and
caspase-9 (Fig. 1F), and by in situ end labeling of the apoptotic
DNA (TUNEL; Fig. 2, A–D). The apoptotic nature of cell
death and the identity of dying cells were further confirmed by
EM. Consistent with our previous studies (17–19), the dying
cells were identified as mainly spermatocytes and spermatids.
The testicular cell death was effectively prevented by all the
specific inhibitors of individual mitochondrial complexes (IIV) at 4 h (Figs. 1 and 2). Low-molecular-weight DNA
fragmentation was suppressed by 82% (P ⬍ 0.001), 82% (P ⬍
0.001), 70% (P ⬍ 0.001), and 78% (P ⬍ 0.001) after 4 h of
exposure to the inhibitor of OXPHOS complex I (100 ␮M
rotenone), complex II (200 ␮M TTFA), complex III (20 ␮M
antimycin A), and complex IV (10 mM KCN), respectively,
relative to the 4-h control (Fig. 1, A and B). The lower
concentrations of the inhibitors (10 ␮M rotenone, 100 ␮M
TTFA, 10 ␮M antimycin, and 5 mM KCN) also had antiapoptotic effects that did not significantly differ from those obtained
with the higher concentrations of the inhibitors (data not
shown). In addition to the apoptotic laddering in the Southern
blot analysis of DNA fragmentation, some unspecific smearing
of DNA, indicating necrotic death, was seen in occasional
experiments (data not shown). Consistent with the Southern
blot analysis and TUNEL, most of the testicular cells showed
normal morphology under the EM (Fig. 2, E and H, top) after
treatment with the mitochondrial inhibitors (rotenone, TTFA,
antimycin A, KCN, or oligomycin) for 4 h. Occasional spermatocytes and spermatids showed signs of typical apoptosis
(Fig. 2F). Germ cells showing atypical ultrastructural morphology or necrosis were rarely seen (Fig. 2, G and H). This was
further confirmed by propidium iodide staining for cell viability (data not shown). In addition, the suppression of the caspase
activation by the OXPHOS inhibitors (Fig. 1F) demonstrates
that the effects of these compounds on the apoptotic pathways
occur both at the level of caspase activation and DNA fragmentation.
Each inhibitor significantly suppressed the ATP level compared with the control samples but did not totally deplete ATP
(Fig. 1C). The ADP levels were also diminished significantly
compared with the 4-h control samples (Fig. 1D), whereas the
AMP levels showed more variability, and the decline did not
reach statistical significance (Fig. 1E).
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slips were removed, and the frozen slides were dipped in ice-cold
ethanol and then fixed for 10 min in formalin and washed in PBS. The
slides were then kept in ethanol-acetic acid (2:1) for 5 min at ⫺20°C,
then washed in PBS, dehydrated, and stored at ⫺20°C until they were
stained. DNA in situ 3⬘-end-labeling was performed as described
earlier (50) with modifications. Briefly, after rehydration and permeabilization in a microwave oven for 5 min in 10 mmol/l citric acid (pH
6.0), the samples were preincubated with terminal transferase reaction
buffer (1 mol/l potassium cacodylate, 125 mmol/l Tris 䡠 HCl, and 1.25
mg/ml BSA, pH 6.6). The DNA in the samples was 3⬘-end-labeled
with Dig-dd-UTP (Roche) by the terminal transferase (Roche) reaction for 1 h at 37°C. For the negative controls, the terminal transferase
enzyme was replaced with the same volume of distilled water. The
samples were then treated with blocking solution [2% Blocking
reagent (Roche) in 150 mmol/l NaCl and 100 mmol/l Tris 䡠 HCl, pH
7.5]. Antidigoxigenin antibody conjugated with horseradish peroxidase (Anti-Digoxigenin-POD; Roche) was used to detect the Dig-ddUTP-labeled DNA. The bound antibody was then localized, using
diaminobenzidine (Sigma), after which the slides were weakly counterstained with hematoxylin and dehydrated. Alternatively to TUNEL,
the samples were stained with propidium iodide according to the
manufacturer’s protocol (Vector Laboratories, Burlingame, CA) to
test the viability of the cells.
Evaluation of caspase activation. In addition to our previously
reported detection of the activation of caspase-3 during the cultures
(61, 62), we evaluated the activation of caspase-9 by Western blot
analysis, which was performed by the NuPage Bis-Tris gel system
(Invitrogen, Frederick, MD) according to the manufacturer’s instructions. In brief, total tissue proteins (50 ␮g) were separated on 4 –12%
NuPage Bis-Tris gradient gels, and electrophoresis was performed at
180 volts. The proteins from the gels were transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA),
and the transfer was checked by staining with 0.2% Ponceau S in 3%
TCA. After being blocked, caspase-9 was detected with caspase-9
rabbit polyclonal antibody (H-170; Santa Cruz Biotechnology, Santa
Cruz, CA), which recognizes both the precursor and the cleaved forms
of this caspase. After washes, the membranes were incubated with
peroxidase-conjugated anti-rabbit IgGs (Jackson ImmunoResearch
Laboratories, West Grove, PA). The bound secondary antibodies were
located with an electron chemiluminescence (ECL plus) detection kit
(Amersham Pharmacia Biotech, Uppsala, Sweden). The loading was
controlled by reprobing with ␣-tubulin antibody (Sigma).
Electron microscopy. For electron microscopy (EM), segments of
seminiferous tubules were fixed in 2.5% glutaraldehyde in 0.1 mol/l
phosphate buffer, pH 7.2, postfixed with 1% osmium tetroxide in 0.1
mol/l phosphate buffer, dehydrated, and embedded in epoxy resin.
Tissue blocks were then sectioned at 50 nm with a Reichert E
ultramicrotome (Reichert Jung, Vienna, Austria). The samples were
stained with uranyl acetate and lead citrate with a Leica EMstain
apparatus (Leica, Vienna, Austria). Observations were made with a
JEOL JEM 1200 EX transmission electron microscope (JEOL, Tokyo,
Japan).
Determination of adenine nucleotides by HPLC. Samples of testicular tissue were snap-frozen in liquid nitrogen. To extract the
adenine (ANs; ATP, ADP, and AMP), the tissues were homogenized
in 0.42 N ice-cold perchloric acid. The homogenates were then
neutralized with 4.42 N KOH and centrifuged. During these procedures, the samples were kept on ice. The AN concentrations in the
supernatants were determined by HPLC using a Shimadzu LC 10AD
vp liquid chromatograph with a reversed-phase column (Ultra Techsphere 5 ODS; Labtronic Oy, Vantaa, Finland) and an ultraviolet
detector set at 254 nm. The published method (12) was modified as
follows: buffer A (0.1 M KH2PO4 and 8.0 mM tetrabutylammonium
hydrogen sulfate, pH 6.0) was run at 1.5 ml/min for 2.5 min, followed
by a linear increase during 10 min to 100% buffer B (buffer A with
30% methanol), which was continued for 2.5 min and followed by a
linear increase during 1 min to 100% buffer A, which was run for 4
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Fig. 1. Inhibitors of mitochondrial complexes I to
IV and gas anoxia inhibit apoptosis in the human
testis at 4 h. Segments of human seminiferous tubules were incubated under hormone- and serumfree culture conditions. Mitochondrial complex I
was inhibited with rotenone (rote, 100 ␮M), complex II with thenoyltrifluoroacetone (TTFA, 100
␮M), complex III with antimycin A (antim A, 20
␮M), and complex IV with potassium cyanide
(KCN, 10 mM). Exposure to anoxia was performed
by culturing the samples in 0% O2 in humidified
tight glass chambers under continuous flow of gas
containing 5% of CO2 and 95% of nitrogen. A:
Southern blot analysis of DNA fragmentation. After
4 h of culture, all of the inhibitors of mitochondrial
complexes I to IV and exposure to gas anoxia
effectively suppressed testicular apoptosis, which
was induced by the culture conditions (4-h control).
B: quantification of low-molecular-weight DNA
(⬍1.3 kb) fragmentation. C–E: HPLC analysis. As
previously described (22), apoptotic death was associated with declines in the ATP, ADP, and AMP
levels (0 vs. 4-h control). The ATP and ADP levels
measured from the seminiferous tubules were further and significantly diminished by the inhibitors of
the mitochondrial complexes compared with the
control cultures (4-h control), whereas the AMP
decline was not significant. The ATP decline mediated by exposure to gas anoxia was not significant
either. F: Western blot analysis, which is representative of 3 independent experiments, shows activation of caspase-9 (casp 9) during apoptosis and its
inhibition by mitochondrial suppressors such as
KCN. In B–E, each value represents a mean of
replicate experiments ⫾ SE. *P ⬍ 0.05, **P ⬍
0.01, and ***P ⬍ 0.001. NS, not significant. Nos. in
parentheses indicate the no. of replicate experiments
in each treatment.
Exposure to gas anoxia suppresses germ cell death after 4 h
of culture. To further assess the role of mitochondrial respiration, the samples were exposed to anoxic conditions. Culture of
the testicular samples for 4 h in these conditions significantly
suppressed germ cell death and resulted in a 69% (P ⬍ 0.01)
decrease of DNA laddering relative to the 4-h control sample
(Fig. 1, A and B). Exposure to anoxia resulted in a decline of
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the ATP levels (Fig. 1C) and of ADP and AMP (data not
shown), but these effects were not significant when compared
with control samples. There are several possible explanations
for this relatively slight AN decline. For example, although the
glass chamber in which the samples were cultured was very
tight and the anoxic gas flow was continuous, the possibility of
some minor oxygen leakage cannot be excluded totally. More-
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HUMAN MALE GERM CELL DEATH AND ATP PRODUCTION
over, the samples were not totally anoxic during the microdissection before the incubation period, which differs from the
case with mitochondrial inhibitors, which were present already
during microdissection. Nevertheless, anoxic conditions during
the 4-h incubation effectively suppressed germ cell apoptosis.
Inhibition of the F0F1-ATP synthase and/or uncoupling of
mitochondrial respiration suppress apoptotic DNA fragmentation at 4 h. Similar to the effect of respiratory chain inhibitors
and anoxia, oligomycin, an inhibitor of the ATP-forming
mitochondrial ATP synthase (F0F1-ATPase), suppressed apoptotic DNA fragmentation at 4 h (Fig. 3, A and B). A combination of oligomycin and DNP, which allows activity of the
respiratory chain while ATP synthase is blocked, also prevented male germ cell death at 4 h, as did DNP by itself (Fig.
3, A and B). Oligomycin and DNP separately, and their combination, again significantly suppressed the ATP (and ADP and
AMP) levels, as measured by HPLC and compared with the
4-h control samples (Fig. 3C). These effects of oligomycin and
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DNP were similar with the lower concentration of inhibitor
(see MATERIALS AND METHODS).
Inhibition of glycolysis decreases ATP levels but does not
suppress germ cell apoptosis. To study the role of glycolysis in
testicular cell death, 2-DG, an antimetabolite of glucose, was
added to the culture. Exposure to 2-DG (5 and 10 mM)
decreased the ATP levels measured by HPLC compared with
the samples cultured without 2-DG (4 h control vs. 4 h 2-DG;
Fig. 4C). However, the decline in ATP concentration was
statistically significantly less than that observed with the mitochondrial inhibitors (Fig. 4C). Also, 2-DG did not notably
affect apoptotic DNA laddering (Fig. 4, A and B). Apparently,
a blockade of glycolysis did neither inhibit nor further stimulate apoptosis in the present model.
A combination of 2-DG with an inhibitor of mitochondrial
respiration (rotenone, TTFA, antimycin, KCN, or oligomycin)
was then used to create a situation where no ATP production
would take place via glycolysis or OXPHOS. Such combinations depleted the ATP levels (Fig. 4C) and suppressed the
apoptotic DNA laddering at 4 h (Fig. 4, A and B), much like the
cases with the inhibitors of mitochondrial OXPHOS alone. In
the Southern blot analysis, some mild unspecific smearing of
DNA, indicating necrotic death, was seen sporadically (data
not shown).
Exposure to the inhibitors of mitochondrial ATP production
(⫾2-DG) results in delayed testicular apoptosis at 24 h. After
24 h of incubation, the inhibitors of mitochondrial ATP production (rotenone, TTFA, antimycin A, and oligomycin), with
or without 2-DG, still suppressed germ cell death (Fig. 4D),
although this effect was no longer significant, and clear DNA
laddering indicating delayed testicular apoptosis was observed
(Fig. 4, D and E). The levels of ATP (Fig. 4F), ADP, and AMP
(data not shown) were further depleted compared with the 4-h
samples. In Southern blot analysis, in addition to laddering,
some unspecific smearing of DNA, indicating necrotic death,
was seen in occasional experiments (data not shown). EM
results were consistent with the Southern blot analysis and
showed typical apoptotic death in numerous germ cells (data
not shown) at 24 h. In addition, sporadic cells appeared
necrotic or intoxicated, with dark and dense nuclei with irregular clumping of chromatin, swollen unrecognizable cytoplasmic organelles, and nondetectable plasma membranes (data not
shown), which suggests that some individual testicular cells
may be more sensitive to the toxic/necrotic effects than others
(data not shown).
Activation of the Fas system does not induce apoptosis in the
presence of mitochondrial inhibitors. The antiapoptotic effects
of the mitochondrial inhibitors KCN, DNP (Fig. 5), or oligomycin were not modified by activation of the Fas system at 4 h.
Even though the activating anti-Fas antibody maintains its
activity in nontesticular systems, the use of antibodies may
have tissue-specific (and other, such as target specificity)
concerns. Of note, in terms of concerns, we do not, in this
particular tissue model, mean inabilities of the antibodies to
pass through the blood-testis barrier. This is because, first, we
are using segments of tubules in vitro, and the antibodies are
introduced to testicular cells not only from outside the tubules
but also from the inside, i.e., from lumen, and second because
we have previously used other antibodies, such as antagonist
antibodies to the Fas system (48), that do affect germ cell
apoptosis in the present culture model. Nevertheless, in the
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Fig. 2. In situ 3⬘-end labeling (ISEL, TUNEL) of DNA and morphological
changes under the electron microscope. A–D: ISEL (TUNEL). Segments of
human seminiferous tubules were cultured, squashed, and fixed, and the
apoptotic cells (brown) were detected by ISEL (TUNEL) A: Squash preparation. ■, Seminiferous tubule; F, cells producing a monolayer on an objective
class. B: sample taken immediately after orchidectomy (0 h) showing only
sporadic apoptotic cells (brown cells). C: consistent with the results of
Southern blot analysis, incubation of testicular tissue for 4 h under hormoneand serum-free conditions (4 h control) induced germ cell death (brown cells,
arrows), which was suppressed by the inhibitors of mitochondrial oxidative
phosphorylation (OXPHOS), such as by rotenone (D). E–H: electron micrographs taken after exposure to inhibitors of OXPHOS. E: normal pachytene
spermatocyte after 4 h culture. F: typical apoptotic changes, such as condensation of chromatin, in a spermatocyte at 4 h. The synaptonemal complex,
characteristic of spermatocytes, is marked with an arrow. G and H: sporadic
spermatocytes showed atypical cytosolic and nuclear alterations. Aggregates of
lysosomes and autophagosomes as well as swollen cellular organelles are seen
in cytosol. In the nuclei, in addition to granular condensation of chromatin,
abnormal membranous structures are observed (arrows in G and H). The
neighboring spermatocyte has maintained its normal morphology (H, cell on
top).
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present study, we used a recombinant human Fas-L protein,
instead of the antibody, in the preliminary experiments. The
results obtained with the use of either agonistic anti-Fas antibody (Fig. 5) or Fas-L (data not shown) (⫾KCN) did not differ
from each other, thus supporting the use of the activating
anti-Fas antibody in further studies.
DISCUSSION
Fig. 3. Inhibition of the F0F1-ATP synthase and/or uncoupling of the mitochondrial respiration suppress testicular apoptosis at 4 h. Segments of human
seminiferous tubules were incubated under hormone- and serum-free culture
conditions. Mitochondrial F0F1-ATP synthase was inhibited with oligomycin
(200 ␮M), and uncoupling was chemically performed with 2,4-dinitrophenol
(DNP, 200 ␮M). A: Southern blot analysis of DNA fragmentation. After 4 h of
culture, oligomycin effectively suppressed testicular apoptosis. A combination
of oligomycin and DNP, which allows activity of the respiratory chain while
ATP synthase is blocked, also inhibited male germ cell death at 4 h, as did the
DNP by itself. B: quantification of low-molecular-weight (mw) DNA (⬍1.3
kb) fragmentation. C: HPLC analysis. Compared with the 4-h controls,
testicular ATP levels were further decreased by oligomycin and/or DNP. Each
value represents a mean of replicate experiments ⫾ SE. **P ⬍ 0.01 and
***P ⬍ 0.001. Nos. in parentheses indicate the no. of replicate experiments in
each treatment.
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In the present investigation, we studied the roles of modulators of ATP production in human male germ cell death.
Because cell-to-cell interactions play an important role in the
regulation of male germ cell energy metabolism, catabolism,
and death, as well as in the overall physiology and pathology
of the testis (6, 14, 27, 51, 60), incubation of isolated cells was
considered inappropriate. Therefore, we used a tissue culture
model in which segments of human seminiferous tubules are
cultured. The tissue culture model, like all in vitro models, has
limitations. However, it has the advantage of maintaining the
physiological contacts between the Sertoli cells and the germ
cells, which allows studies on apoptotic mechanisms involving
different cell types. Furthermore, physiological concentrations
of testicular compounds, testosterone, estradiol, and lactate,
prevent germ cell apoptosis in this model (18, 19, 50). To our
knowledge, there are currently no other methods that would be
more sophisticated for manipulating human male germ cell
death in an environment maintaining the physiological interactions. Therefore, we think that, at present, the in vitro tissue
culture model gives the best available way to investigate
apoptotic mechanisms within the human seminiferous tubules.
In the present study, each of the inhibitors of mitochondrial
complexes I to IV at 4 h (i.e., rotenone, TTFA, antimycin A,
and KCN, respectively; Fig. 1) effectively prevented the apoptotic death of spermatocytes and spermatids induced by hormone- and serum-free culture conditions. Hence, it is very
unlikely that any one of the complexes alone is crucial for the
primary apoptotic pathways triggered by the culture conditions. In other words, if one of the inhibitors alone, such as the
previously reported KCN (22), had shown the antiapoptotic
effect while others did not, it would have indicated the importance of the particular complex (e.g., IV), but this was not the
case. Thus our findings strongly suggest that the antiapoptotic
effect of the respiratory chain inhibitors must be explained by
the effect of each of them in blocking collective mitochondrial
ATP machinery activity. This conclusion is further supported
by the similar effect of anoxia, which also blocks the flux of
reducing equivalents in the respiratory chain.
ATP generation by the F0F1-ATP synthase is crucially
dependent on an active respiratory chain. Specific inhibition of
ATP synthase by oligomycin resulted in an antiapoptotic effect
of similar magnitude as that resulting from respiratory chain
HUMAN MALE GERM CELL DEATH AND ATP PRODUCTION
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Fig. 4. Inhibition of glycolysis neither suppresses germ cell apoptosis nor alters the testicular apoptosis, which is delayed by inhibitors of mitochondrial
OXPHOS. Segments of human seminiferous tubules were cultured for 4 or 24 h. Glycolysis was prevented by adding 2-DG (10 mM), an antimetabolite of
glucose, to the cultures. Complex I of the respiratory chain was inhibited with rotenone (100 ␮M) and F0F1-ATP synthase with oligomycin (oligo, 200 ␮M).
A: Southern blot analysis of DNA fragmentation. After 4 h of culture, blocking of glycolysis by 2-DG did not suppress (or further induce) testicular apoptosis
nor alter the antiapoptotic effects of the mitochondrial modulators. B: quantification of low-molecular-weight DNA (⬍1.3 kb) fragmentation. C: HPLC analysis.
Compared with the 4 h controls, testicular ATP levels were further decreased by 2-DG and/or by the mitochondrial inhibitors. D: after 24 h of incubation, the
inhibitors of mitochondrial ATP production (rote and oligo), with or without 2-DG, still suppressed germ cell death, but this antiapoptotic effect was no longer
significant, and clear DNA laddering indicating delayed testicular apoptosis was observed. E: quantification of low-molecular-weight DNA (⬍1.3 kb)
fragmentation. F: at 24 h, the ATP levels measured by HPLC were declined in the control cultures (24 h control) and further depleted by 2-DG, rotenone, and
oligomycin. Each value represents a mean of replicate experiments ⫾ SE. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001. Nos. in parentheses indicate the no. of
replicate experiments in each treatment.
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blockade, and the same was found with the uncoupler DNP.
These observations allow us to conclude that the observed
antiapoptotic effect is unlikely to be the result of, specifically,
a decrease in the membrane potential or pH gradient across the
IM, because, although the electrochemical proton gradient is
dissipated by blocking the respiratory chain, or by uncoupling,
it remains unaffected by oligomycin. The fact that the uncoupler DNP failed to abolish the antiapoptotic effect of oligomycin supports the conclusion that electron transfer in the respiratory chain, as such, is not a regulatory factor of testicular
apoptosis.
It seems unreasonable to assume that compounds with such
different primary action as, e.g., antimycin, oligomycin, and
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Fig. 5. Antiapoptotic effect of the mitochondrial inhibitor was not modified by
activating the Fas system. Segments of seminiferous tubules were cultured
under serum- and hormone-free culture conditions in the absence or presence
of mitochondrial uncoupler DNP (200 ␮M) and/or agonistic anti-Fas antibody
(5 mg/ml). A: Southern blot analysis of DNA fragmentation. Activation of the
Fas receptor by agonistic anti-Fas antibody did not induce apoptosis when
mitochondrial ATP production was impaired, nor did the mitochondrial uncoupler presensitize the testicular cells to Fas-induced death. B: quantification
of low-molecular-weight DNA (⬍1.3 kb) fragmentation. Each value represents
a mean of 3 replicate experiments ⫾ SE.
2,4-DNP would all block apoptosis, unless their action would
lead to a common effect. Indeed, the obvious common feature
is blockade of the F0F1-ATP synthase activity. This occurs
specifically with oligomycin and indirectly by either blocking
the respiratory chain (at any respiratory chain complex and
including anoxia) or by uncoupling the OXPHOS. Furthermore, the results specifically exclude other potentially important mitochondrial parameters, such as the membrane potential,
as being involved in germ cell apoptosis. Although the ATP
synthase itself has been suggested to be important for the death
control (39, 40, 57), the most obvious explanation for the
observed effects is the lowered concentration of mitochondrial
ATP. In some nontesticular cells, ATP is, indeed, proposed to
be necessary for the apoptotic program, which is an active
process that consequently may require energy in the form of
ATP (9, 33, 52). Of note, apoptotic pathways independent of
ATP production have also been described, and the role of the
ATP production machinery in controlling cell death appears to
depend on the cell type and on the inducer of apoptosis, as
seems to be the case with most of the regulators of apoptosis
(2, 10, 17, 23, 36, 44, 59, 66).
In the present study, 2-DG, an antimetabolite of glucose,
decreased the ATP levels significantly less than inhibition of
the OXPHOS, and apoptosis was unaffected (Fig. 4). Hence
the first possibility is that ATP concentration may have to be
decreased under a critical threshold to achieve the antiapoptotic
effect. Here it must be emphasized that the concentrations of
ATP were determined from total samples of segments of
seminiferous tubules. Thus the determination neither distinguishes between mitochondrial and cytoplasmic ATP nor
makes a distinction between the cell types. Therefore, the
second possibility is that the observed ATP concentration
decrease (mediated by 2-DG) reflects the decrease taking place
in other cells than those undergoing apoptosis. Supportive of
this notion is the demonstration that these other nondying cell
types, such as Sertoli cells and spermatogonia, are known to
use glycolysis for their energy production (whereas the dying
cell types prefer OXPHOS; see Refs. 3, 28, 45, 53, 64). Of
note, when using OXPHOS inhibitors, we cannot exclude the
possibility that the ATP decrease would occur in different cells
than the death process. However, this seems unlikely, and we
rather think that, with these mitochondrial inhibitors, the detectable ATP (because the ATP levels were not totally depleted) may well be derived from other cells than the rescued
ones, and the rescued spermatids and spermatocytes, in turn,
may have been in total ATP depletion.
The antiapoptotic effects of the mitochondrial inhibitors
(⫾2-DG) were no longer significant after 24 h of incubation,
and clear DNA laddering indicating delayed testicular cell
apoptosis was observed (Fig. 4, D and E). EM further confirmed that the type of death was mainly apoptotic. That the
germ cells were able to die despite the inhibitors of the ATP
production machinery indicates activation of secondary apoptotic pathways within these cells. These secondary pathways
appear not to be regulated by the OXPHOS-associated factors.
Furthermore, that the germ cells were able to die by apoptosis
when ATP was depleted indicates that these secondary pathways may not require ATP either. This is supported by findings
with nontesticular cells showing, on one hand, the existence of
pathways that appear not to require mitochondrial functions or
ATP (2, 10, 17, 23, 36, 44, 66) and, on the other, activation of
HUMAN MALE GERM CELL DEATH AND ATP PRODUCTION
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ACKNOWLEDGMENTS
We acknowledge the skillful technical assistance of Sari Linden. We also
thank the staff of the Department of Urology, Helsinki University Central
Hospital (Finland) for very pleasant cooperation.
GRANTS
The work was supported by the Sigrid Juselius Foundation (Finland), the
Finnish Medical Foundation, the Finnish Medical Society Duodecim, the
Cancer Society of Finland, the Research and Science Foundation of Farmos
(Finland), the Helsingin Sanomain 100-vuotis juhlasaatio (Finland), and the
Jalmari and Rauha Ahokas Foundation (Finland).
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different types of apoptotic cascades within certain cell types
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death.
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