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Programme for Developmental and Reproductive Biology, Biomedicum Helsinki, and Hospital for Children and Adolescents, University of Helsinki (L.S., V.P., K.E., M.O., L.D.), FIN-00029 Helsinki, Finland; and Wihuri Research Institute (J.K.H., M.O.P.), FIN-00140 Helsinki, Finland
Address all correspondence and requests for reprints to: Laura Suomalainen, M.D., Hospital for Children and Adolescents, University of Helsinki, Biomedicum, Helsinki, Haartmaninkatu 8, B529b, P.O. Box 700, FIN-00029, Helsinki, Finland. E-mail: laura.suomalainen{at}hus.fi.
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Introduction |
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Growing evidence indicates that the balance between intracellular levels of two sphingolipids, sphingosine-1-phosphate (S1P) and ceramide, is important in determining whether the cell will survive or die (6). Ceramide is formed from cell membrane sphingomyelin by sphingomyelinases, which are activated by a variety of stress factors such as anticancer drugs, ionizing radiation, TNF
, and Fas ligand, and also by growth factor withdrawal and oxidative stress (7). Ceramide acts as a mediator of cell-growth arrest and apoptosis in many tissues and cell lines. S1P, in turn, can be synthesized from sphingosine by sphingosine kinase, and it regulates a variety of proliferative cellular processes including cell growth and differentiation (8). It stimulates growth and opposes the apoptosis resulting from elevated levels of ceramide (6). However, S1P has also recently been shown to trigger apoptosis in various cells (9, 10, 11). In ovarian cancer cells, low levels of exogenous S1P (0.5–2 µmol/liter) stimulate growth, whereas higher levels (10 µmol/liter) induce apoptosis (12). Similarly, in neurons, S1P at 2 µmol/liter has no effect, whereas prolonged exposure to 10 µmol/liter S1P triggers apoptosis (13). One explanation for this perplexing effect of S1P may be the conversion of intracellular S1P to sphingosine or ceramide when S1P is present in large amounts (14). Furthermore, the cellular effects of S1P have been suggested to differ depending on the basal levels of ceramide, on the induction of ceramide generation levels by external stimuli, and on the rate of S1P degradation (6). Finally, S1P has recently been identified as the ligand for the EDG-1, -3, -5, -6, and -8 receptors (S1P1–5) (15, 16). Activation of EDG-1 (S1P1) regulates chemotaxis and angiogenesis in vitro (17), whereas EDG-5 (S1P2) and perhaps also EDG-3 (S1P3) are responsible for cell rounding and neurite retractions (18). Thus, it appears that S1P is a novel lipid mediator with a signaling function both inside and outside the cell (16).
Recent studies have demonstrated the ability of sphingolipids to regulate the survival of female mouse germ cells. S1P has been shown to have the ability to prevent doxorubicin-induced death of cultured mouse oocytes (19) and also chemotherapy- and irradiation-induced apoptosis of the oocytes in vivo (20). Moreover, mice with disruption of the acid sphingomyelinase gene have been shown to have a decreased level of oocyte apoptosis and subsequent oocyte hyperplasia. These findings have raised the interesting possibility that sphingolipids could be used to prevent excessive apoptosis of oocytes in women treated for cancer. Because cancer therapy also causes infertility through germ cell apoptosis in the male (21, 22, 23), a similar approach might be applied to prevent germ cell loss in male cancer patients. Therefore, the aim of the present study was to investigate the effects of S1P and ceramide on germ cell apoptosis in human seminiferous tubules in vitro.
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Patients and Methods |
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Fresh testis tissue was obtained from 23 men undergoing orchidectomy as a treatment for prostate cancer. The operations were performed between October 2000 and November 2002 at the Department of Urology, Helsinki University Central Hospital, Finland. The patients had received no hormonal, chemotherapeutic, or radiotherapeutic treatment before the orchidectomy. Furthermore, none suffered from cryptorchidism or any endocrinological disease. The Ethics Committees of the Hospital for Children and Adolescents and the Department of Urology, University of Helsinki, approved the study protocol.
Tissue culture
In the present model, germ cell apoptosis was induced by in vitro incubation of human seminiferous tubules (24, 25, 26, 27, 28, 29, 30, 31). The segments of seminiferous tubules, rather than isolated germ cells, were cultured to maintain the germ cells in their physiological environment. For the cultures, the testis tissue was microdissected on a petri dish containing culture medium (nutrient mixture, Ham’s F10; Life Technologies, Inc., Paisley, UK) supplemented with 0.1% human albumin (Sigma Chemical Co., St. Louis, MO) and 10 µg/ml gentamicin (Life Technologies, Inc.). After microdissection, segments of seminiferous tubules (
2 mm in length) were incubated for 5 h at 34 C in a humidified atmosphere containing 5% CO2. After the incubation, the segments of seminiferous tubules were snap frozen and stored at -80 C or squashed under coverslips for in situ end labeling (ISEL) of DNA.
Detection of apoptosis
The caspase 3 activation. The activity of caspase 3 in time-course analysis of apoptosis induction and S1P (Biomol Research laboratories, Inc., Plymouth Meeting, PA) was measured by a caspase 3 fluorometric assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Briefly, samples of human testis tissue were homogenized in lysis buffer (R&D Systems) and centrifuged at 17,000 x g for 20 min, and the supernatants were collected for determination of protein concentration by the DC protein assay (Bio-Rad laboratories, Inc., Hercules, CA). Thereafter, 100 µg of protein homogenate in 50 µl of lysis buffer (R&D Systems), 50 µl of reaction buffer 3 (R&D Systems), and 5 µL of caspase 3 fluorogenic substrate (DEVD-AFC, R&D Systems) were added to 96-well plates, and the plates were incubated at +37 C for 2 h. Finally, fluorescence was measured on a fluorescent microplate reader (PerkinElmer HTS 7000 Plus Bio Assay Reader; PerkinElmer, Norwalk, CT) using 405-nm excitation and 505-nm emission filters. For negative controls, fluorescence was measured from wells with no substrate or no protein homogenate.
Southern blot analysis of apoptotic DNA fragmentation. DNA was extracted with the apoptotic DNA Ladder Kit (Roche Molecular Biochemicals, Mannheim, Germany) as described (25). DNA was quantified spectrophotometrically (absorbance at 260 nm), and 1 µg of the total DNA from each sample was subjected to 3'end-labeling with digoxigenin-dideoxy-UTP (Dig-dd-UTP; Roche) by terminal transferase (Roche) reaction. The DNA samples were electrophoresed on 2% agarose gels, blotted onto nylon membranes, and cross-linked to the membranes by UV irradiation. The membranes were then washed and blocked with 1% blocking reagent (Roche) in maleic buffer [100 mmol/liter maleic acid, 150 mmol/liter NaCl (pH 7.5)] for 30 min at room temperature. The 3'end-labeled DNA in the membranes was localized with alkaline phosphatase-conjugated antidigoxigenin antibody (Anti-Digoxigenin-AP; Roche), and the bound antibody was detected by the luminescence reaction (CSPD; Roche). The x-ray films exposed to chemiluminescence were scanned with a tabletop scanner (Hewlett-Packard Scanjet 6300C; Hewlett-Packard, Palo Alto, CA), and the digital image was analyzed with Gel Plot 2 macro for Scion image beta 4.0.2. (Scion Corp., Frederick, MD) analysis software. The amounts of apoptotic low-molecular-weight DNA fragments (<1.3 kb) in various samples were expressed in relation to low-molecular-weight DNA in the sample cultured for 5 h without survival factors, which was taken as 1.0 (100% apoptosis).
Nonradioactive ISEL of DNA.
To confirm the results of Southern blot analysis and to obtain information on the morphology of the cultured testis tissue, we performed ISEL of squash preparations of seminiferous tubules. Small segments of seminiferous tubules (1 mm in length) were squashed under coverslips to produce a monolayer of cells, and the preparations were fixed as previously described (32). These preparations were then rehydrated and washed twice for 5 min in distilled water. After incubation for 10 min with terminal transferase reaction buffer [1 mol/liter potassium cacodylate, 125 mmol/liter Tris-HCl, and 1.25 mg/ml BSA (pH 6.6)], the apoptotic DNA was 3'end-labeled with Dig-dd-UTP (Roche) by the terminal transferase reaction for 1 h at 37 C. For the negative controls, the terminal transferase enzyme was replaced with the same volume of distilled water. Dig-dd-UTP was detected with the antidigoxigenin antibody conjugated to horseradish peroxidase (Anti-Digoxigenin-POD; Roche). For location of the antibody, 0.05% diaminobenzidine substrate (Sigma) was added. Light counterstaining was performed with hematoxylin, and the samples were dehydrated and mounted. For quantitation of the ISEL data, nuclei per x200 visual field from three parallel slides representing each sample were counted under a light microscope. The average of ISEL-positive nuclei from each S1P-treated and control samples were compared with the total amount of nuclei.
Analysis of testicular levels of ceramide and sphingomyelin
We first wanted to study whether changes in the levels of ceramide and sphingomyelin are related to the induction of germ cell apoptosis in the human testis. The levels of these sphingolipids were measured from seminiferous tubules after induction of apoptosis by 5 h. Small samples of human testis tissue (0.031–1.008 g) were homogenized in 1030 µl of homogenization buffer [1% Triton X-100, 150 mmol/liter NaCl, 10 mmol/liter Tris (pH 7.4), 1 mmol/liter EDTA, 0.2 mM phenylmethylsulfonylfluoride, 0.5% Nonidet P-40, and 1 µg/ml leupeptin]. Samples (30 µl) of these homogenates were centrifuged at 17,000 x g, and the supernatants were collected for determination of protein concentration by the DC protein assay (Bio-Rad Laboratories, Inc.). Lipids of human testis tissue preparations were extracted by the modified method of Bligh and Dyer (33). Briefly, 1 ml of tissue homogenate was suspended with 1.25 ml of chloroform and 2.5 ml of methanol, vortexed, and incubated overnight at +4 C. After centrifugation for 5 min at 1700 x g, 1.25 ml of chloroform and 1.25 ml of 0.88% KCl were added, and the tubes were vortexed and centrifuged for 5 min at 1700 x g. The organic lower phase was transferred into a new tube and evaporated under nitrogen. The lipids were dissolved into chloroform-methanol (2:1, vol/vol) and analyzed by high-performance thin-layer chromatography (HPTLC) on a HPTLC silica gel 60 plate (Merck, Darmstadt, Germany) using dichloromethane-methanol-glacial acetic acid (100:2:5, vol/vol/vol) for ceramides and chloroform-methanol-glacial acetic acid-water (100:60:20:5, vol/vol/vol/vol) for sphingomyelin. Individual lipid classes were visualized by dipping each plate into CuSO4 (3%)-H3PO4 (8%) and then charring the plate on a heat block at 180 C for 10 min. Densitometric scanning of the bands and evaluation of the data were done with an automatic plate scanner (CAMAG TLC Scanner no. 3, Camag, Muttenz, Switzerland) and CAMAG TLC Software (Camag), respectively. In addition to three replicates of samples, at least six different amounts of mixes of lipid standards were added to each plate. The scanner software automatically corrects background in the HPTLC plate for both the standards and the samples, fits the regression curve based on the lipid standards, and calculates the amounts of lipids in the samples. The amounts of ceramide and sphingomyelin were normalized to the total cell protein, and the ceramide to sphingomyelin weight ratio was calculated. All the lipid standards were from Sigma. The independent cultures for time-course analyses of ceramide and sphingomyelin levels were repeated three times (n = 3 patients). In addition, the effects of fumonisin B1 (FB1; Sigma), n-acetyl-L-cysteine (NAC; Sigma), and potassium cyanide (KCN; Sigma) on the levels of ceramide and sphingomyelin were studied in three independent cultures (n = 3 patients).
In vitro acid sphingomyelinase (ASMase) activity assay
Ceramide generated by degradation of sphingomyelin is due to activation of the neutral sphingomyelinase (NSMase) or ASMase (34). Because no appropriate NSMase inhibitors could be obtained, we studied the role of ASMase in the generation of ceramide with two different inhibitors of ASMase, Desipramine (Sigma) and imipramine (Imipramine HCL; Calbiochem, San Diego, CA). To confirm the potency of the inhibitors, the ASMase activity from samples treated with desipramine and imipramine was measured in samples obtained from two patients. Small samples of human testis tissue were homogenized in 1000 µl of homogenization buffer [1% Triton X-100, 150 mmol/liter NaCl, 10 mmol/liter Tris (pH 7.4), 1 mmol/liter EDTA, 0.2 mmol/liter phenylmethylsulfonylfluoride, 0.5% Nonidet P-40, and 1 µg/ml leupeptin]. Samples (30 µl) of these homogenates were centrifuged at 17,000 x g, and the supernatants were collected for determination of protein concentration by the DC protein assay (Bio-Rad Laboratories, Inc.). Protein homogenate (100 µg) was mixed with 100 µl of ASMase buffer [250 mmol/liter sodium acetate (pH 5.0)] containing approximately 40,000 cpm [14C] sphingomyelin (CFA 566, Amersham Biosciences, Piscataway, NJ) and incubated for 1 h at +37 C. The reactions were stopped by the addition of 1.5 ml chloroform-methanol (2:1, vol/vol) and 200 µl of distilled water and vortexing. After centrifuging at 1800 x g for 5 min, 300 µl of upper aqueous phase containing the released radioactive phosphorylcholine was transferred to scintillation vials for determination of radioactivity in a liquid scintillation counter (1209 Rackbeta, LKB; Wallac, Turku, Finland). Negative control containing no enzyme was assayed concomitantly.
Treatments
The effect of S1P on human male germ cell apoptosis was studied by adding exogenous S1P to the culture medium and by determining the amount of low-molecular-weight DNA fragmentation (n = 8 patients) or caspase 3 activation (n = 3 patients) from S1P-treated vs. nontreated seminiferous tubules. For the experiments, S1P was first dissolved in methanol (0.5 mg/ml). The methanol stock was aliquoted, and the solvent was evaporated with a stream of nitrogen. Immediately before use, S1P was dissolved in culture medium to prepare a 125-µM stock and used at final concentrations of 1, 10, and 20 µmol/liter.
In addition to the degradation of sphingomyelin by sphingomyelinases, ceramide can be formed at the initial step of signaling pathway by enhanced de novo synthesis from sphingosine or palmitoyl-coenzyme A and serine by the enzyme ceramide synthetase (35). To test whether this pathway contributes to the observed elevation of intracellular ceramide, we studied the effect of the ceramide synthetase inhibitor FB1 on testicular apoptosis (n = 3 patients). For this purpose, FB1 was dissolved in culture medium to final concentrations of 100 and 250 µmol/liter immediately before use.
To further study the role of ceramide in the process of germ cell apoptosis, we tested the effects of NAC, a thiol antioxidant and glutathione precursor, on testicular ceramide levels and apoptosis (n = 3 patients). For this purpose, NAC was prepared as a 1-M stock in distilled water, with the pH adjusted to 7.5 with NaOH, and used at 100 mmol/liter.
Chemical anoxia was induced by exposing the testicular tissue to KCN, which acts at the mitochondrial level by inhibiting cytochrome oxidase (complex IV) (n = 3 patients). KCN was freshly prepared in Krebs-Henseleit buffer [115 mmol/liter NaCl, 3.6 mmol/liter KCl, 1.3 mmol/liter KH2PO4, 25 mmol/liter NaHCO3, 1 mmol/liter CaCl2, and 1 mmol/liter MgCl2 (pH 7.2)] at a final concentration of 50 mmol/liter.
The role of ASMase in the generation of ceramide was studied with two different inhibitors of ASMase, desipramine (n = 3 patients) and imipramine (n = 2 patients). For this purpose, desipramine was prepared as a 5-mmol/liter stock in distilled water and used at 10 and 50 µmol/liter. Imipramine was prepared as a 100-mmol/liter stock in distilled water and used at 10 and 30 µmol/liter.
Statistics
The cultures for studying Southern blot analysis of the effects of S1P on low molecular DNA fragmentation were repeated on eight independent occasions, and the effects of FB1, NAC, KCN, and desipramine were repeated on three independent occasions. Quantitative data represent integrated optical density from scanned x-ray films. The effect of S1P on caspase 3 was measured from samples of three independent patients. Data obtained from three to eight cultures (mean ± SEM) were analyzed by one-way ANOVA, and if significant differences existed, this was followed by comparison of the groups with the two-tailed unpaired Student’s t test. P < 0.05 was considered statistically significant. The cultures for time-course analyses of testicular lipid levels, caspase 3 activation, and Southern blot analysis of apoptotic DNA fragmentation as a function of time were repeated three times independently. The cultures for the effects of desipramine and imipramine on testicular ceramide levels and the acid sphingomyelinase assay were performed twice, and the assays were from two parallel samples. The relative number of ISEL-positive nuclei represents the mean of three parallel slides from two independent cultures.
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Results |
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During the culture, the level of ceramide increased rapidly (within 1 h) (Fig. 1
, A and B). At 2.5 h, the level of ceramide had almost reached its maximum. To further clarify the kinetics of apoptosis induction, we measured the time-course activation of caspase 3 during human testicular apoptosis from samples incubated in serum-free conditions for 15 min, 30 min, 1 h, 5 h, and 10 h. Caspase 3 activity started to increase after 1 h (Fig. 1B). Culturing the segments of seminiferous tubules under serum-free conditions resulted in induction of apoptotic DNA fragmentation within 5 h, which further increased at 48 h (Fig. 1C).
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In the samples treated with the inhibitors, the level of ceramide did not differ from those of controls. Moreover, neither desipramine at 10 µM (P = 0.06) and 30 µM (P = 0.83) nor imipramine at 10 µM (mean = 1.22) and 30 µM (mean = 0.97) had significant effects on testicular germ cell apoptosis as measured by Southern blot analysis of DNA fragmentation. The ASMase activity was suppressed by 38.5% and 17.6% with desipramine and imipramine, respectively. Importantly, both inhibitors decreased the ASMase activity to the same level as was detected in controls, i.e. noncultured or treated samples (data not shown).
Effect of FB1 on ceramide levels and germ cell apoptosis
The level of ceramide and sphingomyelin in FB1-treated seminiferous tubules was decreased by 25% and 24%, respectively (P < 0.01). However, the ceramide to sphingomyelin ratio did not differ from that of control samples cultured in serum-free conditions without any treatments (Fig. 2B). Furthermore, no significant effects of FB1 (100 or 250 µM) on germ cell apoptosis were observed by Southern blot analysis of DNA fragmentation (Fig. 2A).
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After 5-h culture, NAC (100 mmol/liter) suppressed low molecular DNA fragmentation by 78% (P < 0.05) relative to 5-h control culture (Fig. 3A) but had no significant effect on testicular ceramide levels (Fig. 3B).
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KCN suppressed low molecular DNA fragmentation by 64% after 5-h culture (P < 0.05) compared with control (Fig. 4A) but had no significant effect on ceramide levels (Fig. 4B).
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S1P was able to inhibit caspase 3 activation by 15% relative to 5-h control culture (P < 0.05) (Fig. 5A). Furthermore, as assayed by low molecular DNA fragmentation, S1P (1–10 µmol/liter) inhibited apoptosis induced in germ cells by the culture of seminiferous tubules (Fig. 5A). The most effective antiapoptotic concentration was 10 µmol/liter. Low-molecular-weight DNA fragmentation was suppressed by 8% (P = 0.09) and 30% (P < 0.001) with S1P concentrations of 1 and 10 µmol/liter, respectively. By ISEL, only a few apoptotic cells were present in control tubules not exposed to apoptosis-inducing conditions (Fig. 5Ba). The number of apoptotic germ cells was greatly increased when the tubules were cultured for 5 h in serum-free conditions (Fig. 5Ba). Consistent with the results of Southern blots, a reduced number of apoptotic cells was observed in the samples incubated with 1 and 10 µmol/liter of S1P (Fig. 5Ba), with no apparent abnormalities in the morphology of the germ cells. It has to be noted that human seminiferous tubules contain several spirally oriented stages of spermatogenesis in which the number of apoptotic cells vary considerably. Therefore, the number of ISEL-positive cells in individual squash preparations also showed variation, which was independent of the treatments. However, the result shown in Fig. 5 is based on examination of eight to 12 slide preparations and represents the most typical finding. No staining was observed when terminal transferase enzyme was replaced with distilled water (negative control, data not shown). For quantification of the data shown in the experiment shown in Fig. 5Bb, the relative numbers of ISEL-positive nuclei was calculated from three parallel slides of each sample (n = 2 patients). In control samples (cultured in serum-free conditions for 5 h), the percentage of ISEL-positive nuclei was 52% (Fig. 5Bb), whereas the percentages of positive nuclei in samples treated with 1 and 10 µmol/liter of S1P were 32% and 17%, respectively (Fig. 5Bb).
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Discussion |
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The ceramide pathway activated (i.e. degradation of sphingomyelin by sphingomyelinases or de novo synthesis by ceramide synthetase) depends on the cell type and the inducing stimulus (36). In our study, the ceramide synthetase inhibitor FB1 had no significant effects on male germ cell apoptosis, which is consistent with the similar inability of FB1 to suppress apoptosis in mouse oocytes (20). Two inhibitors of ASMase, desipramine and imipramine, affected neither testicular ceramide levels nor apoptosis when added to the culture of seminiferous tubules. These results suggest that activation of NSMase instead of ASMase contributes to the observed increase in the ceramide levels during culture of the seminiferous tubules. This conclusion is supported by a number of observations showing a critical role for NSMase instead of ASMase in cell death (37, 38, 39, 40). Consistently, spermatozoa from ASMase knock-out mice were found to exhibit a similar degree of sphingomyelin degradation to wild-type mice (41).
To further study the intracellular target of ceramide in the apoptotic cascades, we tested the ability of NAC and KCN to affect testicular ceramide levels. NAC is a thiol antioxidant and glutathione precursor that has been previously shown to inhibit male germ cell apoptosis (30). Somewhat surprisingly, NAC did not affect ceramide levels, although it is a glutathione precursor, and glutathione has been shown to be an inhibitor of NSMase (38). This is likely to be due to the inability of the exogenous NAC to restore appropriate glutathione levels during in vitro culture conditions. Moreover, we found NAC to inhibit germ cell apoptosis but to cause no significant changes in ceramide levels, suggesting that NAC inhibits apoptosis downstream of ceramide or acts on a different but parallel signaling pathway. Finally, increased ceramide levels and exogenously added cell-permeable ceramide analogs have been reported to affect mitochondrial functions (42, 43). Indeed, ceramide has been shown to form large stable pores in mitochondrial membranes, allowing cytochrome c to be released in cytosol (44). Based on these data, KCN, a specific inhibitor of oxidative phosphorylation (complex IV) and a mitochondrial poison (45), could have an effect on testicular ceramide levels and apoptosis. However, we found that although KCN inhibited germ cell apoptosis effectively, it had no effect on the level of ceramide. This finding suggests that in human testicular germ cells, ceramide functions either upstream or in parallel to mitochondrial complex IV and most likely upstream of ATP production, thus serving as an early intracellular effector of apoptosis.
An important finding of the present study is that S1P, the plausible antagonist of ceramide, can prevent in vitro-induced male germ cell death. Inhibition of apoptosis by S1P has been related to caspase 3 in several cell lines. In our study, S1P only suppressed the caspase 3 activation by 15%, although it was able to inhibit apoptotic DNA laddering in testicular germ cells by 30%. This difference in the degree of inhibition of caspase activation and DNA laddering by S1P may reflect the presence of parallel apoptotic pathways in the present model. Thus, in the present culture model, one of the inductors of cell death is serum deprivation, but other simultaneous inductors, such as relative hyperoxia, may activate parallel apoptotic pathways, some of which are not blocked by S1P. Moreover, prolonged stress will most probably lead to activation of secondary apoptotic pathways, which lead to death of the S1P-treated cells with a delayed kinetics. Furthermore, although caspase 3 is the primary effector caspase in most apoptotic cascades, caspase 3-independent and even caspase-independent cell deaths have also been described (46, 47, 48, 49, 50). Interestingly, in a recent study on cultured human testicular cells, caspase 3 activation was related to apoptosis of Sertoli cells, whereas rapid apoptosis of germ cells occurred in a caspase-independent manner (51). Accordingly, in our model in which the majority of cells undergoing apoptosis are germ cells, the modest effect of S1P on caspase 3 activation may represent the presence of caspase 3-independent germ cell deaths that are not prevented by S1P.
In contrast to the partial inhibition of germ cell apoptosis in the testis by S1P, complete inhibition of apoptosis was recently observed in female germ cells both in vitro and ex vivo when apoptosis was stimulated by radiation or anticancer drugs, respectively (19, 20). Thus, the ability of S1P to inhibit apoptosis seems to be different in male and female gonadal tissues. Moreover, high concentrations of S1P, devoid of the antiapoptotic effect, may exceed the physiological levels of S1P and even be toxic (52). In our hands, higher concentrations of S1P (20 µmol/liter, data not shown) did not inhibit apoptosis. The similar ability of low (physiological) concentrations, but not higher concentrations, to block testicular apoptosis has been recently observed with the steroid hormones testosterone and estradiol (25, 29). It is also possible that the low concentrations of S1P have a specific antiapoptotic effect mediated by binding of S1P to one of its receptors (EDG), whereas the effects of high concentrations of S1P are due to a nonspecific effect of S1P on membrane function or to conversion of S1P to its metabolites. Furthermore, S1P in high concentrations has been proposed to be converted to ceramide (52).
Because dysregulation of apoptosis contributes to many disease processes (53, 54, 55), modulation of cell death for therapeutic purposes is an important goal for research. However, long-term effects of blocking the executioner steps of apoptosis on tissue homeostasis is largely unknown, and for instance, pharmacological inhibition of executioner caspases has been shown to lead to a condition resembling necrosis (56). Therefore, it appears that strategies aimed at cell protection should be directed at inhibiting early intracellular signaling events leading to apoptosis. Our findings that in cultured human seminiferous tubules, intracellular levels of ceramide increased considerably earlier than did nuclear apoptosis and that S1P blocked human male germ cell apoptosis, together with the previously documented finding that S1P blocked mouse oocyte apoptosis without interfering with cell structure (20), support the concept that certain sphingolipids are early mediators of apoptosis. Modulation of sphingolipid pathways may thus be reasonable when aiming at protecting germ cells from inappropriate cell death.
Taken together, the present results show that apoptosis of the male germ cells appears to involve sphingolipids and can partly be inhibited by S1P. Thus, in the future, modulation of the sphingolipid pathways of testicular germ cells may have a role in attempts to protect male gonads from apoptosis induced by external stress such as cancer therapy.
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Acknowledgments |
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Footnotes |
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Abbreviations: ASMase, Acid sphingomyelinase; Dig-dd-UTP, digoxigenin-dideoxy-UTP; FB1, fumonisin B1; HPTLC, high-performance thin-layer chromatography; ISEL, in situ end labeling; KCN, potassium cyanide; NAC, n-acetyl-L-cysteine; NSMase, neutral sphingomyelinase; S1P, sphingosine-1-phosphate.
Received April 30, 2003.
Accepted August 12, 2003.
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down-regulates the Fas ligand and inhibits germ cell apoptosis in the human testis. J Clin Endocrinol Metab 86:4480–4488This article has been cited by other articles:
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