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1Program for Developmental and Reproductive Biology, Biomedicum Helsinki, and Hospital for Children and Adolescents, 3Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki; 4Department 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
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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testis; spermatogenesis; apoptosis; mitochondria; oxidative phosphorylation
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 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 ATP-producing 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.
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MATERIALS AND METHODS |
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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 µg/ 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 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 µM, of TTFA 100 and 200 µM, of antimycin A 10 and 20 µM, 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 µM. Uncoupling of OXPHOS was achieved with 2,4-dinitrophenol (DNP; Sigma) at final concentrations of 200 and 400 µM. 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 µg/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 µg/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 µg) 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·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 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·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·HCl, pH 7.5]. Antidigoxigenin antibody conjugated with horseradish peroxidase (Anti-Digoxigenin-POD; Roche) was used to detect the Dig-dd-UTP-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 µg) 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 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 (µmol/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.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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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).
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 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. Moreover, 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 DNP were similar with the lower concentration of inhibitor (see MATERIALS AND METHODS).
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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 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.
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DISCUSSION |
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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 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 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 different types of apoptotic cascades within certain cell types (39, 67).
Because cell-to-cell interactions play an important role in germ cell survival, it is possible that the antiapoptotic compounds act on the Sertoli cells rather than the germ cells by modulating the supply of pro- or antiapoptotic paracrine factors. The present in vitro model, having the advantage of maintaining physiological contacts between the different cell types, allows investigation of paracrine systems. One such system, which has been suggested to regulate germ cell death in the testis and in our in vitro model, is the Fas-Fas-L system (12, 13, 24, 34, 48, 49). The proapoptotic Fas-L expressed by the Sertoli cells, and perhaps also by the germ cells, has been suggested to activate the Fas receptors and consequently the apoptotic cascade in the germ cells (12, 13, 24, 34, 48). Here we aimed at investigating whether additional Fas activation would be able to modulate the effects of the mitochondrial inhibitors. Another aspect in terms of adding Fas activators to the culture was based on the literature, in which uncouplers of OXPHOS have been shown to presensitize certain nontesticular cells to the Fas death signal (35). Moreover, it has been suggested that cross talk between the Fas receptor and mitochondrial signaling can occur when Fas is activated by increased amounts of the agonistic Fas antibody (35, 63). In our study, the antiapoptotic role of the mitochondrial inhibitors was not modified by the activating anti-Fas antibody or human recombinant Fas-L (Fig. 5). That the activation of the Fas receptors did not induce apoptosis in the presence of the mitochondrial inhibitors may have several explanations. It may suggest that 1) these compounds inhibit the particular apoptotic pathway that is triggered by Fas or a pathway that has steps common in with it and 2) that the inhibitory actions of the compounds take place downstream of the Fas receptors in the germ cells (i.e., not e.g., via the action of Sertoli cells). However, 3) that these compounds would act primarily on the Sertoli cells or 4) that the Fas receptors would not activate germ cell death pathways are not totally excluded. The first of these possibilities (i.e., suggestion 3) could be explained by induction of Sertoli cell production of antiapoptotic factor(s), which could act on germ cells downstream of the Fas receptors. Whichever the explanation, the results show that activators of the Fas system failed to induce apoptosis when the mitochondrial ATP production was impaired and that the mitochondrial inhibitors or uncouplers did not presensitize the testicular cells to Fas-induced death.
From the present study, we conclude that the mitochondrial ATP production machinery plays an important role in regulating primary pathways of human male germ apoptosis, triggered by the hormone- and serum-free culture conditions. The results indicate that it is unlikely that any of the complexes (I-IV) of the mitochondrial respiration alone, the functional electron transport chain, or the membrane potential is/are important. Rather, a straightforward conclusion from our experiments is that ATP synthesized by the F0F1-ATPase is crucial for the primary pathways of testicular cell apoptosis. We also conclude that there seem to be secondary pathways of human testicular apoptosis that do not require mitochondrial ATP production. The present study increases the understanding of the role of the mitochondrial catabolic pathways in the complex network of regulatory events of male germ cell life and death.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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, Seminiferous tubule; 
, 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 hormone- and 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).
