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Testis |
Program for Developmental and Reproductive Biology,3 Biomedicum Helsinki, and Hospital for Children and Adolescents, University of Helsinki, FIN-00029 HUS Helsinki, Finland
Helsinki Bioenergetics Group,4 Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland
Department of Anatomy,5 University of Turku, FIN-20520 Turku, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, male reproductive tract, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The regulation of cell death involves the participation of the different components of cellular energy metabolism, such as adenine nucleotides (ANs: ATP, ADP, and AMP) [7–13]. The ANs appear to be simultaneously the targets and modulators of cell death events. The apoptotic process changes the levels of ANs, and ANs themselves control the events of cell death [8, 10, 11, 13–15]. ATP especially may play an important, although contradictory, role in regulating cell death. In certain nontesticular cells, suppression of ATP production leads to induction, delay, or inhibition of cell death [16–23]. In certain other cell types, depletion of ATP switches the type of cell death from apoptosis to necrosis or cytotoxicity [8, 12, 21, 22, 24–28]. In these cells, ATP may be necessary for the apoptotic program, whereas in some other cell types death pathways independent of ATP production have been described [10–12, 16, 29].
In human testicular cells, the effects of the apoptotic process on AN concentrations and of manipulation of ATP production on cell death have remained unclear. Hypoxia suppresses human male germ cell apoptosis [30]. Because oxygen is a substrate for mitochondrial respiration, which in turn is the primary ATP production pathway in male germ cells [31–33], the antiapoptotic effect of hypoxia may be mediated via changes in ATP levels. Other situations that may indicate a role for ATP in the regulation of testicular cell death are testicular torsion/reperfusion and administration of fluoroacetate, both of which change ATP levels and affect germ cell death [34, 35]. However, because changes in oxygen tension, besides affecting ATP levels, also have other consequences such as modulation of production of reactive oxygen species (ROS), the effects of ATP depletion on germ cell death are not exactly known. In addition to hypoxia/anoxia, mitochondrial ATP production can be suppressed chemically by cyanide, which inhibits mitochondrial respiration at the level of cytochrome c oxidase (mitochondrial complex IV) [36]. As with other modulators of ATP production, the effects of cyanide on cell death have been controversial. In nontesticular cells, cyanide induces, inhibits, or delays apoptosis or switches the type of cell death from apoptosis to necrosis, irrespective of its concentration [22, 24, 26, 37, 38]. In the testis, its effect(s) on the regulation of apoptosis is completely unknown.
In the present study, we characterized the changes in the levels of ATP, ADP, and AMP during the process of male germ cell apoptosis and evaluated the effects of chemical anoxia, induced with potassium cyanide (KCN), on testicular cell death. Apoptosis was induced in human male germ cells in an in vitro tissue culture by incubating segments of seminiferous tubules under serum-free conditions, and the levels of ANs were studied during the process of cell death. The death-modulating effects of KCN, an inhibitor of mitochondrial respiration, also were investigated. Because the testis also contains somatic Sertoli cells, which mainly produce ATP via cytosolic glycolysis [39], 2-deoxyglucose (2-DG), an antimetabolite of glucose, was added to the cultures. In addition, we assessed the ability of pyruvate, which has been suggested to oppose the effects of cyanide [24, 40], and of a toxic concentration of hydrogen peroxide to modulate the effects of KCN on testicular cell death.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testicular tissue was obtained from 22 adult men 52–88 yr of age that were undergoing orchidectomy as treatment for prostate cancer. They had not received hormonal, chemotherapeutic, or radiotherapeutic treatments before the operation, and none of them had suffered from endocrinological disease or cryptorchidism. The operations were performed between October 1999 and September 2001 at the Department of Urology, Helsinki University Central Hospital (Helsinki, Finland). The ethics committees of the Departments of Children and Adolescents and of Urology, University of Helsinki, approved the study protocol (no. 14/95).
Tissue Preparation and Induction of Apoptosis
Apoptosis of human male germ cells was induced by culturing segments of seminiferous tubules under serum-free conditions [41–44]. Testis tissues derived from the operations were immediately microdissected on Petri dishes containing tissue culture medium (Nutrient mixture Hams F10, containing 6.1 mmol/L of D-glucose and 1.0 mmol/L of sodium pyruvate; Gibco Europe, Paisley, U.K.), supplemented with 0.1% human serum albumin (Sigma Chemical Co., St. Louis, MO) and 10 µl/ml gentamicin (Gibco). Segments of seminiferous tubules (3–5 mm in length) were isolated and transferred to culture plates containing the same tissue culture medium and incubated at 34°C in a humidified atmosphere containing 5% CO2. In all experiments, the pH was neutralized prior to culture.
Induction of Chemical Anoxia by Blockade of Mitochondrial Respiration and Inhibition of Glycolysis
The role of chemical anoxia and blockade of mitochondrial respiration in human testicular apoptosis was studied by adding KCN (Sigma), which inhibits cytochrome c oxidase (complex IV), to the culture medium at final concentrations of 5 mmol/L and 10 mmol/L. Cytosolic ATP production was inhibited by blocking glycolysis with 2-DG (Sigma) concentrations of 5 mmol/L or 10 mmol/L. High concentrations of KCN and 2-DG were used to ensure total inhibition of mitochondrial respiration and glycolysis, respectively. Stock solutions of the reagents were prepared in Krebs-Henseleit buffer (115 mmol/L NaCl, 3.6 mmol/L KCl, 1.3 mmol/L KH2PO4, 25 mmol/L NaHCO3, 1 mmol/L CaCl2, 1 mmol/L MgCl2, pH 7.2), and the pH was neutralized prior to culture.
Exposure to Hydrogen Peroxide
To determine whether a high (toxic) concentration of the oxidant H2O2 would change the type of human male germ cell death from apoptosis to necrosis, segments of seminiferous tubules were exposed to 10 mmol/L H2O2 (Merck, Darmstadt, Germany).
Treatment with Extra Pyruvate
To find out whether concentrations of pyruvate exceeding the basal concentration in the culture medium would oppose the effects of KCN, extra sodium pyruvate (Sigma) was added to the medium at final concentrations of 5, 10, and 50 mmol/L.
Determination of ATP, ADP, and AMP Concentrations
Samples of testicular tissue were snap-frozen in liquid nitrogen. To extract ANs (ATP, ADP, and AMP), the samples were homogenized in 0.42 N ice-cold perchloric acid. The homogenates were then neutralized with 4.42 N KOH and centrifuged. The ATP, ADP, and AMP concentrations of 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 (UV) detector set at 254 nm. The published method [45] was modified as follows. Buffer A (0.1 M KH2PO4, 8.0 mmol/L tetrabutylammonium hydrogen sulfate, pH 6.0) was run at 1.5 ml/min for 2.5 min followed by a linear increase over 10 min to 100% buffer B (buffer A with 30% methanol), which was maintained for 2.5 min, and then by a linear increase over 1 min back to 100% buffer A, which was run for 4 min. The compounds were identified and quantified by the retention times and peak areas of known standards, as calibrated by spectrophotometry. The AN concentrations were expressed in relation to wet testis tissue weight (µmol/g of testis). The adenylate energy charges (ECs) were calculated from the ATP, ADP, and AMP concentrations, according to the following formula [46]: ([ATP] + 1/2[ADP]) ÷ ([ATP] + [ADP] + [AMP]).
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, Mannheim, Germany) as recently described [44]. DNA was quantified by spectrophotometry (absorbance at 260 nm), after which the DNA samples (1 µg) were 3' end-labeled with digoxigenin-dideoxy-UTP (Dig-dd-UTP; Roche) using the terminal transferase (Roche) reaction, subjected to electrophoresis on 2% agarose gels, and blotted onto nylon membranes. DNA was cross-linked to the membranes by UV irradiation. The membranes were washed and blocked with 1% blocking reagent (Roche) in maleic buffer (100 mmol/L maleic acid, 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 previously described [41]. The bound antibody was detected by the chemiluminescence reaction (CSPD; Roche) at room temperature for 5 min and was enhanced at 37°C for 15 min as previously described [41]. X-ray films were exposed to chemiluminescence and then scanned with a table-top scanner (Microtec ScanMaker; Microtec International, Taipei, Taiwan), and the digitized information (optical density) was analyzed with NIH-Image 1.61 software (National Institutes of Health, Bethesda, MD). Low-molecular-weight DNA fractions (<1.3 kilobases) of the 0-h sample were set at 1.0 (100%) for comparisons with other settings. Thus, the results are expressed in relation to the starting (0 h) time point.
In Situ End-Labeling of DNA
Short (1–3 mm) segments of seminiferous tubules were gently squashed under a coverslip to enable the cells to move out from the tubules and produce a monolayer on the microscope slide. These squash preparations were fixed as previously described [41]. The slides were frozen in liquid nitrogen, the coverslips were removed, and the frozen slides were dipped into 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, washed in PBS, dehydrated, and stored at -20°C until stained. DNA in situ 3' end-labeling (ISEL) was performed as described previously [44] with modifications. 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, 1.25 mg/ml BSA, pH 6.6). The DNA in the samples was 3' end-labeled with Dig-dd-UTP by the terminal transferase (TdT; Roche) reaction for 1 h at 37°C. For the negative controls, the TdT enzyme was replaced with the same volume of distilled water. The samples were then treated with blocking solution (2% blocking reagent in 150 mmol/L NaCl, 100 mmol/L Tris-HCl, pH 7.5). Anti-digoxigenin 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), and the slides were weakly counterstained with hematoxylin and then dehydrated.
Electron Microscopy
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. Segments 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 an EM stain apparatus (Leica, Vienna, Austria) according to the instructions provided by the manufacturer, using a program with 30' stain I (containing uranyl acetate) and 1'30'' stain II (containing lead citrate), both at room temperature. Observations were made with a JEM 1200 EX transmission electron microscope (JEOL, Tokyo, Japan). Classification of germ cell types was based on their characteristic morphology, and ultrastructural changes were used to identify the cells as apoptotic or necrotic.
Statistical Analysis
The experiments were repeated on at least three independent occasions. For statistical comparisons, data obtained from the replicate experiments (mean ± SEM) were analyzed by one-way ANOVA, followed by the post hoc test with a Bonferroni correction. Results were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testicular apoptosis was induced by culturing segments of seminiferous tubules under serum-free conditions. Four and 24 h of incubation resulted in 
10-fold (P < 0.001) and 12-fold (P < 0.001) increases in apoptotic DNA fragmentation, respectively, relative to that at the beginning of the culture (0 h) (Fig. 1A). During culture, the concentrations of ATP, ADP, and AMP decreased (Fig. 1B). Relative to 0 h, after 4 and 24 h of culture, the ATP concentrations decreased by 62% (P < 0.01) and 93% (P < 0.01), the ADP concentrations decreased by 53% (P < 0.01) and 78% (P < 0.01), and the AMP concentrations decreased by 31% (P < 0.1) and 56% (P < 0.1), respectively (Fig. 1B). After 4 h of incubation, there was only a moderate fall in the ECs, whereas after 24 h of culture the ECs had decreased by 33% (P < 0.1) (Fig. 1C).
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Chemical Anoxia Induced by KCN ± 2-DG Delayed Apoptotic DNA Fragmentation and Further Reduced Testicular Concentrations of ANs
Chemical anoxia was induced by exposure to KCN, an inhibitor of mitochondrial respiration, and glycolysis was blocked by 2-DG, an antimetabolite of glucose. After culture for 4 h, human testicular apoptosis induced by culturing testicular tissue under serum-free conditions (4 h control) was inhibited by KCN (Fig. 2). Low-molecular-weight DNA fragmentation was suppressed by 78% (P < 0.001) after 4 h of exposure to KCN (10 mmol/L) relative to the 4-h control (Fig. 2, A and B). After 24 h of incubation, the antiapoptotic effect of KCN was still significant, but clear DNA laddering indicating delayed apoptosis was occurring (Fig. 2, A and B). In addition to laddering, some nonspecific smearing of DNA, indicating necrotic death, was seen in occasional experiments (data not shown). Because ATP, in addition to mitochondrial respiration, may also be produced by cytosolic glycolysis, 2-DG was added to the culture. However, the effects of KCN on germ cell death were not affected by a 2-DG concentration of 10 mmol/L (Fig. 2, A and B), and 2-DG by itself did not inhibit testicular apoptosis (data not shown). Compared with the control cultures, the testicular ATP levels were further decreased by KCN (Fig. 2C). In these KCN-treated samples, the ATP was usually totally depleted. The mean values are not 0 because in sporadic samples the levels of ATP were detectable. ATP concentration and the occurrence of nonspecific necrotic smearing in Southern blot analyses were not correlated, i.e., the level of ATP was detectable in one sample showing smearing, whereas in another ATP was undetectable. Combining 2-DG with KCN did not further reduce the ATP concentrations significantly (Fig. 2C). In the presence of KCN, the ADP and AMP concentrations were diminished in parallel fashion with the ATP concentrations (data not shown). Neither the ATP:ADP ratios nor the ECs of the samples treated with KCN differed significantly from those of the control samples (data not shown). Lower concentrations of KCN (5 mmol/L) and 2-DG (5 mmol/L) did not significantly alter the results.
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Death-Suppressing Effect of Chemical Anoxia as Confirmed by ISEL
The findings of the Southern blot analysis of DNA fragmentation were confirmed by ISEL of the seminiferous tubule samples (Fig. 3). Representative samples of cells from human seminiferous tubules were obtained by squashing segments of seminiferous tubules under coverslips. With this method, cells from seminiferous epithelium produce a monolayer on the slide (Fig. 3A). Consistent with the results of Southern blot analysis, we observed induction of germ cell death (4 h control) and its inhibition by chemical anoxia, i.e., by KCN ± 2-DG (Fig. 3). There was no staining when the TdT enzyme was replaced by the same volume of distilled water as a negative control (data not shown).
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Morphological Changes in Dying Germ Cells under the Electron Microscope
Dying cells were identified by electron microscopy. After 4 h of incubation under serum-free culture conditions (4 h control), different phases of apoptotic change, including nuclear and/or cytoplasmic condensation, were observed in germ cells. The dying cells were mainly spermatocytes (Fig. 4, A–C) and spermatids, consistent with our previous studies [41, 42]. In the very late phases of cell death, the specific cell types could not be identified.
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When seminiferous tubules were cultured for 4 h in the presence of KCN, different types of effects on morphology occurred. Most of the testicular cells showed no specific morphological changes (consistently with the Southern blot analysis). A few cells were apoptotic. As in the control cultures, the apoptotic cells in the KCN-treated samples were mainly germ cells, i.e., spermatids and spermatocytes. After exposure to KCN, however, sporadic somatic Sertoli cells undergoing apoptosis were also visible (Fig. 4G). In these dying Sertoli cells, the chromatin clumped at the nuclear envelope and cytoplasm appeared condensed (Fig. 4G). Some spermatocytes and spermatids also appeared necrotic. These necrotic cells had swollen unrecognizable cytoplasmic organelles, nondetectable plasma membranes, and irregular condensation of chromatin (Figs. 4E). In some spermatocytes and occasional spermatids, atypical structural changes were visible, which could be toxic or abnormal necrotic effects. In otherwise normal-looking round spermatids, mild alterations were detected, including swollen mitochondria and small condensed chromatin granules in the nuclei (Fig. 5B). In some spermatocytes, more extensive alterations were visible. In these spermatocytes, electron-dense nuclei consisted of compact clumps of chromatin, and in the cytoplasm many large unidentifiable vacuoles were visible in otherwise electron-dense surroundings (Fig. 4D).
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The different categories of morphological changes (i.e., apoptosis, necrosis, and atypical changes) had some overlapping features. For example, similar types of small condensed chromatin granules were observed in the nuclei of abnormally necrotic/toxic round spermatids (Fig. 5B) and in the nuclei of apoptotic round spermatids. The type of irregular clumping of nuclear chromatin detected in necrotic round spermatids (Fig. 5C) resembled that of apoptotic round spermatids. In late phases of cell death, neither the type of cell death nor the specific cell type could be identified. Twenty-four hours of incubation with KCN increased the relative number of cells showing necrotic (Fig. 4E) and toxic changes compared with the number of apoptotic and nondying cells. Spermatozoa with swollen mitochondria also were visible (Fig. 5E). We found structural alterations caused by KCN only in sporadic cells, whereas after exposure to a high concentration of H2O2 most cells appeared necrotic (Fig. 5C).
Death-Suppressing Role of KCN Was Not Significantly Modulated by Extra Pyruvate, Whereas Exposure to a Toxic Concentration of H2O2 Switched Apoptosis to Necrosis
Because pyruvate, at least at concentrations higher than the basal concentration in the culture medium, inhibits the effects of KCN on mitochondrial respiration [24, 40], we added extra pyruvate to the cultures. In Southern blot analysis of DNA fragmentation, pyruvate at concentrations of 5 and 10 mmol/L did not modulate the antiapoptotic role of KCN. At a concentration of 50 mmol/L, pyruvate in the presence of KCN resulted in no DNA laddering in two experiments, whereas in two other experiments hardly detectable laddering was observed. In quantitative analysis, the samples exposed to KCN + pyruvate did not differ significantly from the samples treated with KCN alone (data not shown). Pyruvate by itself did not have an effect on testicular apoptosis.
Because testicular cells were able to undergo apoptosis despite ATP depletion and only sporadic necrotic cells were observed, we tested whether the type of testicular cell death could be switched from apoptosis to necrosis in this human tissue culture model. Therefore, we exposed the tissue to 10 mmol/L H2O2, which in nontesticular cells induces acute necrosis [47]. We also induced necrosis to validate the Southern blot analysis of DNA fragmentation, because DNA laddering indistinguishable from that seen in apoptosis has been associated with necrosis [48]. In the present study, 10 mmol/L H2O2 induced morphologically typical necrosis in practically all the germ cell types of the testis. In Southern blot analysis, H2O2 produced a nonspecific smear, indicating necrotic cell death, and no laddering was seen (Fig. 6A). Thus, Southern blot analysis appears to be a valid method for studying apoptosis and its regulation and for reliably distinguishing between apoptotic and necrotic cell death in human testicular tissue. ISEL however, with the present fixation and staining protocols (used for squash preparations), failed to distinguish between these two types of cell death; positive ISEL results for the human germ cells were seen in H2O2-treated samples (Fig. 6B) even though the type of cell death was shown by Southern blot analysis and electron microscopy to be necrotic. In rodents, ISEL (also called TUNEL) is specific for male germ cell apoptosis [1 49, 50]. In those experiments, male germ cell death was induced in rats in vivo, and testicular sections (rather than squash preparations) were fixed in glutaraldehyde [1, 49, 50]. In the present experiments, exposure to H2O2 resulted in ATP depletion that did not differ significantly from that caused by KCN ± 2-DG treatment (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Incubation of human seminiferous tubules under serum-free culture conditions induced germ cell apoptosis and concomitantly decreased the concentrations of ANs (ATP, ADP, and AMP) (Fig. 1). The finding of a decline in ATP stores is in agreement with findings in nontesticular cells, in which apoptosis also resulted in a decrease in ATP [13, 14]. The cessation of ATP production may result from H+ gradient collapse caused by mitochondrial permeability transition (PT) [15, 29, 53], which in turn is associated with apoptosis in many cell types [8, 12, 15, 29, 53]. This explanation for the decline in ATP concentrations would be reasonable for the present findings also, because human testicular apoptosis appears to include PT [30]. However, the mechanism underlying the decline in the concentrations of ATP (and other ANs) needs further evaluation because there are other possible explanations, such as an increase in ATP hydrolysis or in ATP-consuming reactions, substrate limitation, or an efflux of ATP (and other ANs) from the dying cells. The apoptotic process itself results in perturbation of mitochondrial ATP/ADP exchange and thereafter in a decreased rate of oxidative phosphorylation (OXPHOS) [13]. This defect, suggested to precede other mitochondrial deficiencies such as PT, has been associated with a decline in ATP concentrations and an increase in cytoplasmic ADP concentrations [13]. The finding of decreased ADP and AMP concentrations in addition to decreased ATP concentrations is not necessarily at variance with the findings concerning the defect in mitochondrial ATP/ADP exchange. We measured total ADP instead of cytoplasmic ADP, and thus although it seems unlikely, an increase in cytoplasmic ADP and a decrease in some other cellular compartment cannot be excluded. In addition, the testicular cells in the present study may exhibit a more profound compromise in energy status, which in turn would explain the decrease at all AN concentrations. Thus, because of the significant fall in the concentrations of all ANs, degradation of ANs could be enhanced during the cultures and the lost pool could be found in their degradation products [54]. Although the mechanism remains unclear, the present findings clearly demonstrate that incubation of testis tissue under serum-free conditions results in induction of germ cell apoptosis, which is associated with a decline in testicular ATP, ADP, and AMP concentrations.
The apoptotic process itself changes the concentrations of ANs, and the ANs regulate the cascades of cell death [7, 9, 10, 12, 24]. In particular, ATP affects cell death [10, 12, 24]. However, its role in controlling the death pathways appears to depend on the cell type and the inducer of cell death. In the present study, we investigated the hitherto unclear role of ATP generation in testicular apoptosis. Blockade of mitochondrial respiration by KCN (with or without inhibition of glycolysis by 2-DG), although usually totally depleting ATP, effectively inhibited apoptotic DNA laddering after 4 h of culture (Fig. 2). Thus, ATP may play an important role in regulating the primary cell death cascades of male germ cells. However, because the antiapoptotic effect of KCN was observed even in the absence of 2-DG (i.e., in a situation in which some ATP production may have occurred via glycolysis), the death-suppressing role of KCN may not be mediated via ATP per se but rather via some other targets of KCN action. Exposure to KCN prevents the formation and maintenance of the mitochondrial membrane potential, which in turn results in cessation of ATP production by OXPHOS. Thus, the mitochondrial membrane potential as such may play an important role in controlling germ cell death, the fall in ATP being merely a simultaneous but nonregulative event. Mitochondrial membrane potential could, for example, be required for opening the mitochondrial PT pore complex, which in turn appears to regulate testicular cell death [30]. Other targets of action could be the effects of KCN on cytochrome c release or changes in the redox states of pyridine nucleotides (e.g., NADPH), which both are likely to be related to PT, on the concentrations of other ANs (because they were also decreased in the KCN-treated samples), or even on some nonmitochondrial events. ROS, in turn, are unlikely to explain the effects of KCN or gas hypoxia [30] on cell death, because these two treatments result in opposite changes in the levels of ROS, i.e., hypoxia lowers and chemical anoxia increases the concentration of ROS [37, 55]. Even though the exact site(s) of the antiapoptotic action of KCN in the present model remains to be clarified, the results indicate that functional mitochondrial respiration plays an essential role in human testicular cell death.
Despite some inhibition of cell death, culture for 24 h in the presence of KCN (with or without 2-DG) resulted in some DNA fragmentation, indicating apoptotic cell death (Fig. 2). Thus, chemical anoxia (with or without inhibition of glycolysis) delayed apoptosis in many of the testicular cells instead, for example, of driving all the cells to necrosis. There may be several explanations for why the cells underwent apoptosis despite severe ATP depletion. First, because the ANs were measured in tissue rather than in specific cells, individual cells may have had ATP concentrations that were too low to be detected with HPLC but high enough for the apoptotic process to progress. However, we regard this explanation as unlikely because exposure to 10 mmol/L H2O2 induced testicular necrosis even though the ATP concentrations did not differ significantly from those of samples treated with KCN (with or without 2-DG). Second, the antiapoptotic effect of KCN, and therefore also the delay in cell death, may have been mediated by a mechanism that is not associated with ATP concentration. Third, the pyruvate in the culture medium may have affected the results, because pyruvate opposes the effects of cyanide [24, 40]. However, this explanation does not appear to fit because to alter the effects of KCN on testicular cell death, a very high concentration of extra pyruvate (50 mmol/L) was required (even then the effect was not significant). Fourth, longer incubations may have induced activation of secondary apoptotic pathways that possibly do not require ATP. Whatever the explanation, our findings clearly show that at least some testicular cells die by delayed apoptosis despite chemical anoxia caused by the inhibition of mitochondrial complex IV (with or without blockade of glycolysis).
The delayed apoptotic death of testicular cells indicates existence of secondary KCN-independent pathways. Because KCN at the concentrations used completely inhibited mitochondrial respiration, the secondary pathways appear not to require functional mitochondrial respiration. The secondary pathways may not require ATP either, because the dying cells were mainly germ cells, which produce ATP via OXPHOS (which was blocked by KCN), and because many of the cells died by delayed apoptosis even when ATP generation was completely blocked (i.e., OXPHOS by KCN and glycolysis by 2-DG). In support of the suggestion of activation of the secondary pathways, some findings suggest apoptotic cascades that appear to not require mitochondrial function or ATP [10–12, 16, 29, 56–61]. Induction of the mitochondrion- and/or ATP-independent cascades seem to be either cell or trigger specific. When taking place within the same cell system, the stimuli activating the mitochondrion- and/or ATP-independent cascades appear to differ from those activating the mitochondrial pathways. In the present tissue culture model, the inducers of primary and secondary pathways may also be different, because there probably are multiple triggers of death. Thus, although the main trigger of testicular cell death was withdrawal of serum (which in itself contains numerous factors), other moderators may also have played a role in the induction of apoptotic cascades. For example, the samples were exposed to relative hyperoxia and consequent oxidative stress, because the tissues were manipulated in conditions in which the partial oxygen pressure was 21%, which is higher than the oxygen pressure in testis in vivo. During prolonged stress (caused by the culture conditions), Sertoli cells may have expressed death-promoting factors, which may then have been additional inducers of germ cell death. Nevertheless, it seems reasonable to suppose that when continuously exposed to severe stress (such as the present culture conditions) the cells sooner or later die, if not via primary then via secondary pathways.
Although chemical anoxia seemed to delay the death of most of the testicular cells, it also induced morphological alterations in some of the cells. Electron microscopy revealed that after exposure to KCN some spermatids and spermatocytes and occasional Sertoli cells underwent apoptosis. Changes in some other spermatocytes and spermatids were considered to be typically necrotic, and some other germ cells appeared abnormally necrotic or toxic (Figs. 4 and 5). The death of the Sertoli cells is interesting because even though there have been sporadic reports of dying Sertoli cells, these cells seem, generally and according to our previous observations, to be more resistant than germ cells to death-inducing stimuli [42–44, 62–64]. Even in the absence of 2-DG, KCN induced Sertoli cell apoptosis, which may indicate the importance of mitochondrial respiration in these cells even though they produce ATP mainly by glycolysis [39]. Alternatively, this Sertoli cell apoptosis may reflect the overall toxicity of KCN. The possibility of toxicity is supported by the finding of KCN-mediated necrotic or toxic changes in the germ cells. The reasons for the sporadic nature of the structural alterations in some cells is unclear. The possibility of uneven penetration of KCN into the tissue cannot be excluded, although it seems unlikely for several reasons. First, KCN exerts its effects via a gaseous form (HCN), which readily penetrates into the cells. Second, the neighboring cells showed normal morphology, and the possibility of such large local differences in the penetration of the gas seems unlikely. Third, the experimental findings were confirmed in repeated experiments. Fourth, by exposing the tissue to a high concentration of H2O2, we were able to induce necrotic alterations in practically all the germ cells. Thus, although the mechanism leading to the present morphological findings induced by KCN is not known, some individual testicular cells appear to be more sensitive to the toxic effects.
The results of the present study suggest an important role for mitochondrial respiration in the regulation of human testicular cell death. Incubation of segments of seminiferous tubules under serum-free conditions induced germ cell apoptosis and concomitantly decreased the concentrations of ATP, ADP, and AMP. Thus, in testicular cells, the apoptotic process appears to result in a decline in cellular ANs. Chemical anoxia induced by KCN further reduced the ATP concentrations and effectively inhibited apoptotic DNA laddering at 4 h. The effects of chemical anoxia and gas hypoxia on the concentrations of ROS are opposite, suggesting that the antiapoptotic roles of these two treatments are not explained by ROS, but rather in most of the testicular cells, mitochondrial respiration plays a crucial role in controlling primary cell death cascades. The reality of the antiapoptotic effect of KCN was supported by electron microscopy findings; most of the cells exposed to KCN for 4 h had normal morphology. However, in occasional germ cells, KCN induced atypical structural changes, suggesting higher sensitivity of individual spermatocytes and spermatids to the toxic effects of KCN. Although some germ cells showed signs of necrosis or toxicity, many of the testicular cells underwent delayed apoptosis despite the ATP depletion caused by 24 h of exposure to chemical anoxia, i.e., to KCN (with or without 2-DG). Thus, in the human testis, there seem to be secondary apoptotic pathways that do not require functional mitochondrial respiration (or ATP). 2-DG, an antimetabolite of cytosolic glycolysis, did not modify the results obtained with KCN alone, whereas a high concentration of H2O2 switched the type of cell death from apoptosis to necrosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Krista Erkkilä, Hospital for Children and Adolescents, Program for Developmental and Reproductive Biology, Room B529b, Biomedicum Helsinki, University of Helsinki, P.O. Box 700, FIN-00029 HUS Helsinki, Finland. FAX: 358 9 4717 1947; krista.erkkila{at}hus.fi
Received: 3 December 2002.
First decision: 13 January 2003.
Accepted: 27 March 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 L. Suomalainen, V. Pentikainen, and L. Dunkel Sphingosine-1-Phosphate Inhibits Nuclear Factor {kappa}B Activation and Germ Cell Apoptosis in the Human Testis Independently of Its Receptors Am. J. Pathol., March 1, 2005; 166(3): 773 - 781. [Abstract] [Full Text] [PDF]
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 M. Otala, M. O. Pentikainen, T. Matikainen, L. Suomalainen, J. K. Hakala, G. I. Perez, M. Tenhunen, K. Erkkila, P. Kovanen, M. Parvinen, et al. Effects of Acid Sphingomyelinase Deficiency on Male Germ Cell Development and Programmed Cell Death Biol Reprod, January 1, 2005; 72(1): 86 - 96. [Abstract] [Full Text] [PDF]
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