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Testis |
Programme for Developmental and Reproductive Biology,3 Biomedicum Helsinki and Hospital for Children and Adolescents, University of Helsinki, FIN-00029 HUS, Helsinki, Finland
Wihuri Research Institute,4 FIN-00140, Helsinki, Finland
Departments of Pediatrics and Physiology,5 University of Turku, FIN-20520, Turku, Finland
Department of Oncology,6 Helsinki University Hospital, FIN-00029, Helsinki, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, male reproductive tract, spermatogenesis, stress, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability to control early intracellular events that signal the activation of cell death is a potentially important target in protection of radiation-induced germ cell loss. Sphingolipids, which are widely distributed in cell membranes, have been implicated in the regulation of cell growth, differentiation, and apoptosis [9]. Ceramide is metabolized from cell membrane sphingomyelin by sphingomyelinases, which are activated by a variety of stress factors such as anticancer drugs, ionizing radiation, tumor necrosis factor 
, Fas ligand, and also by growth factor withdrawal and oxidative stress [10]. Additionally, ceramide can be synthetized de novo by ceramide synthetase [10]. Ceramide acts as a mediator of cell-growth arrest and apoptosis in many tissues and cell lines. Sphingosine-1-phosphate (S1P), in turn, is formed by phosphorylation of a metabolite of ceramide, sphingosine, by sphingosine kinase [11]. In contrast with the growth-inhibitory and proapoptotic actions of ceramide and sphingosine, S1P regulates diverse processes such as cellular growth, inhibition of ceramide-mediated apoptosis, and cell migration [12].
Morita et al. have reported that radiation-induced premature ovarian failure can be prevented in female mice by protecting the ovaries by means of S1P before irradiation. S1P-treated ovaries retained a normal distribution of follicles as well as overall tissue mass [13]. No genetic anomalies in progeny of the irradiated mothers were found [14]. This encouraging result in female mice prompted us to study whether S1P would also be able to protect male germ cells from irradiation-induced cell death.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal experiments were approved by the Institutional Animal Care and Use Committee of the Wihuri Research Institute, Helsinki, Finland. Wild-type C57BL/6 male mice were obtained from the University of Helsinki experimental animal facilities. All mice were young male adults, 8–10 wk of age.
Study Design
In the irradiation and S1P experiments, the mice were anesthetized by intraperitoneal injection of 600–800 µl 1.25% Avertin (2,2,2-tribromoethanol in tert-amyl-alcohol; 1600 mg/ml; Sigma-Aldrich, St. Louis, MO) 1–2 h before irradiation. The mice received a dose of 0, 0.1, 0.5, 1.0, or 2.0 Gy total-body irradiation at the Department of Oncology, Helsinki University Hospital, Helsinki, Finland. Irradiation was performed with a Varian Clinac 600C linear accelerator (Varian Medical Systems, Palo Alto, CA) using a 6-MV photon beam and a dose rate of 2 Gy/min. The mice were placed in a prone position in a plastic box and irradiated by means of a single posterior field covering the whole box + 2 cm margin to achieve maximum uniformity of dose distribution. The absorbed dose was calculated at a depth of 2 cm. In addition, a 1.5-cm-thick Plexiglas absorber was added at the entrance side of the field to obtain a full-dose build-up. The effects of irradiation and S1P was studied from two time points. The first time point (16 h) was chosen because apoptosis of the rat testicular germ cells, which starts approximately 8 h after irradiation, increases to maximum effect at about 16 h [15]. The second time point (21 days) was selected because differentiation of mouse testicular germ cells from early spermatogonia to spermatocytes and to spermatids lasts approximately 9 and 18 days, respectively (see Fig. 3). Thus, 21 days after irradiation, apoptosis of the spermatogonia that had taken place at the time of irradiation can be observed as a reduction in the 4C population of spermatocytes and the 1C population of spermatids by DNA flow cytometry.
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Irradiation Experiments
Sixteen hours after 0-, 0.5-, or 1.0-Gy irradiation, the mice (n = 6) were anesthetized by CO2 and killed by cervical dislocation, and tissue samples were collected in 2.5% glutaraldehyde for electron microscopic studies. Twenty-one days after 0-, 0.1-, 0.5-, 1.0-, or 2.0-Gy irradiation, the mice (n = 12) were anesthetized by CO2 and killed by cervical dislocation; then the testes were weighed and, after stage-specific preparations of seminiferous tubules, DNA flow cytometric analyses were performed.
S1P Experiments
For the control group of animals (n = 3), 30 µl of vehicle containing 5% polyethylene glycol, 2.5% ethanol, and 0.8% Tween-80 in phosphate-buffered saline (PET-PBS) was injected once intratesticularly into the right testis while the left one remained as an untreated control. For S1P-treated mice (n = 8), 30 µl of 50 µM S1P in PET-PBS (Biomol, Plymouth Meeting, PA) was injected once into the right testis and 30 µl of 200 µM S1P in PET-PBS once into the left testis.
At the 16-h time point, the effect of S1P on 0.5-Gy irradiation-induced rapid germ cell demise was studied from stage-specific squash preparations by in situ end-labeling (ISEL) analysis of DNA fragmentation. Also, after irradiation doses of 0.5 and 1.0 Gy, tissue samples were collected in 2.5% glutaraldehyde for electron microscopic studies.
At the 21-day time point, after stage-specific preparation of seminiferous tubules, the long-term effects of S1P on 0.5-Gy irradiation-induced apoptosis were studied by DNA flow cytometry.
Seminiferous Tubule Preparations
Sixteen hours or 21 days after total-body irradiation, the testes were decapsulated in phosphate-buffered saline in a Petri dish, the seminiferous tubules were gently teased apart, and three 1-mm-long segments of seminiferous tubules at each of the stages II–V, VI–VIII, and IX–XII per mouse were prepared under a transillumination stereomicroscope (MS 5; Leica, Wetzlar, Germany) [16–18].
In Situ End-Labeling of Apoptotic DNA
Isolated 1-mm segments of seminiferous tubules were transferred in 10 µl of PBS on silan-coated microscope slides and the samples then squashed by placing a coverslip on top so that the cells within the tubule segments produced a monolayer around both ends of the tubule segments. The samples were fixed, dehydrated, and stored as described previously [19]. DNA 3' end labeling was performed as described previously [20, 21]. In brief, after rehydration, the samples were microwaved for 5 min in 10 mM citric acid (pH 6) for antigen retrieval, preincubated with terminal transferase reaction buffer (potassium cacodylate, 1 mol/L; Tris-HCl, 125 mmol/L; BSA, 1.25 mg/ml; pH 6.6) and 3' end labeled with digoxigenin-dideoxy-UTP (Dig-dd-UTP; Roche Molecular Biochemicals, Indianapolis, IN) by means of the terminal transferase (Tdt; Roche) reaction at 37°C for 1 h. Dig-dd-UTP-labeled DNA was visualized by antidigoxigenin antibody conjugated to horseradish peroxidase (Anti-Digoxigenin-POD; Roche) and diaminobenzidine (Sigma). The slides were counterstained lightly with hematoxylin before dehydration and mounting.
Electron Microscopy
Stage-specific segments of the seminiferous tubules were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer and embedded in epoxy resin. The tissue blocks were sectioned at 50 nm with an ultramicrotome (Reichert Jung, Vienna, Austria) and stained with uranyl acetate and lead citrate using Leica EMstain apparatus (Leica). The samples were examined with a JEOL JEM 1200 EX transmission electron microscope (JEOL, Tokyo, Japan) at the Institute of Biotechnology, Electron Microscopy Unit.
DNA Flow Cytometry
DNA flow cytometric analyses after irradiation were performed as described previously, with some modifications [18, 22]. Stage-specific 1-mm-long single segments of seminiferous tubules were treated with a detergent (0.3% Nonidet P-40; BDH, Poole, England) in PBS containing 0.2% bovine serum albumin (Sigma, St. Louis, MO) and ribonuclease A (5 µg/ml; Sigma) for 10–15 min at 4°C, followed by a 10-sec vortex mix and incubation for 10 min at 37°C. Propidium iodide (25 µg/ml; Sigma) was added to the samples for staining of the nuclei, and 10 µl of diluted fluorescent particle solution (500 beads/µl, TrueCount Beads; Becton Dickinson, Mountain View, CA) was added to each sample as an internal volume standard for the quantification of absolute cell numbers. The samples were filtered through 45-µm metal strainers (Retsch, Haan, Germany) and 200 µl of PBS was added to each sample to increase the volume. The samples were analyzed by means of a FACSCalibur flow cytometer (Becton Dickinson) using an excitation wavelength of 488 nm. A total of 5000 fluorescent impulses were counted excluding the beads that were gated out of the rest of the sample. The number of nuclei in each peak of the DNA histograms was calculated using CellQuest Pro software (Becton Dickinson) and converted to absolute numbers using the internal standard. The 1C population consists of step 1–12 spermatids. The 1C' population consists of step 13–16 spermatids, which bind less propidium iodide than step 1–12 spermatids and therefore form a hypohaploid peak. The 4C population consists mainly of primary spermatocytes (except for preleptotene spermatocytes that are dispersed in the S-phase and the 2C peak, depicting chromatin synthesis and diploid chromatids, respectively) but also of spermatogonia (G2/M) [18].
Data Presentation and Statistical Analysis
At the 16-h time point, the Mann-Whitney test was used to test differences in apoptotic cell counts between the S1P-treated and nontreated groups. Average counts from three to five squash preparations per testis were used and the amounts of apoptotic cells were expressed as percentages of the total numbers of cells in the samples.
At the 21-day time point, differences in cell numbers between the radiation dose groups within the three stages among the 1C and 4C populations were tested by one-way analysis of variance followed by Dunnett test. The same method was used when testing the effect of S1P on cell numbers as well as for testing the effect of dose of radiation on testicular weight. One to three tubule segment measurements per stage per testis were used in the statistical analyses of cell numbers. Results are expressed as mean ± SEM. Values of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The weights of the testes decreased 21 days after irradiation (n = 3) (Fig. 1). A significant decrease in testis weight was already seen after 0.5-Gy irradiation, the weight being 77% (P < 0.05) of the nonirradiated testes. After 2.0 Gy, the decrease in testis weight was 44% (P < 0.05).
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Morphological Changes in Testicular Tissue 16 h after Irradiation
Using electron microscopic analysis, the most common type of cell death caused by irradiation was found to be apoptosis. All spermatogenic stages had some apoptotic germ cells in nonirradiated samples (Fig. 2). In both nonirradiated and irradiated samples, the morphological signs of apoptosis were most frequently found in germ cells, particularly in spermatogonia. Occasional apoptotic germ cells other than spermatogonia, i.e., spermatocytes and spermatids, were also seen. Apoptosis of the germ cells ranged from early apoptosis, in which the nuclear chromatin had started clumping, to later phases of apoptosis, in which, in addition to the chromatin, the cytoplasm had become condensed and no cytoplasmic organelles could be discerned. In very late phases of cell death, neither the specific cell type nor the type of cell death could be identified (Fig. 2, A–C). In S1P-treated segments of seminiferous tubules, occasional apoptotic cells (mainly spermatogonia) were observed, whereas most of the germ cells had retained their normal appearance 16 h after irradiation, thus indicating that S1P in itself did not induce morphological changes (Fig. 2, D–E).
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Irradiation Increases the Amount of Testicular Germ Cell Apoptosis Dose Dependently
In the mouse, differentiation of germ cells from early spermatogonia to spermatocytes and to spermatids lasts approximately 9 and 18 days, respectively (Fig. 3). Twenty-one days after irradiation, apoptosis of the spermatogonia that had taken place at the time of irradiation was observed as a reduction in the 4C population of spermatocytes and the 1C population of spermatids by DNA flow cytometry (Fig. 4). The amount of cells in the 1C and 4C populations markedly decreased with increasing dose of irradiation (n = 3) (Fig. 4 and Table 1). This was seen more clearly in the 1C than in the 4C cell population and the effects of radiation were most prominent in stages II–V and VII–VIII (Table 1). In stages II–V, the decrease in the 1C cell population was already statistically significant after 0.1-Gy irradiation, the decrease being 31% vs. nonirradiated testes (n = 3) (P < 0.05). After 2.0 Gy, the decrease in the 1C cell population was 92%. In stages VII–VIII, the decrease in the 1C cell population was significant after 0.5-Gy irradiation, the loss of cells being 75% vs. nonirradiated testes (n = 3). After 2.0 Gy, the decrease in the cell population was 97% (n = 3). In the 4C population, the effect of irradiation on germ cell population was statistically significant after 1.0 Gy in stages II–V and VII–VIII, the decreases being 74% and 73%, respectively (n = 3). In stages IX–XII, a statistically significant decrease of 77% was observed after 2.0-Gy irradiation (n = 3). In nonirradiated testes, which were treated with vehicle alone (n = 3) or with 200 µM S1P (n = 4), the 1C and 4C populations did not differ from those in nonirradiated, nontreated testes (Table 1).
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The Effects of S1P on Male Germ Cell Death 16 h after Irradiation
Sixteen hours after irradiation, in stage-specific squash preparations for in situ end labeling of DNA fragmentation (ISEL), no statistically significant differences were found between nontreated (n = 4) and 50 µM or 200 µM S1P-treated groups (n = 4), although a statistically nonsignificant trend toward lower amounts of ISEL-positive cells was detected in stages II–V and IX–XII (Fig. 5A). Consistent with the EM analysis, no additional morphological changes induced by S1P in itself were observed in the squash preparations (Fig. 5B).
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The Effects of S1P on Testicular Germ Cell Number 21 Days after Irradiation
Twenty-one days after 0.5-Gy irradiation, a significant reduction in the number of spermatids in the 1C population (i.e., that were spermatogonia at the time of irradiation) was seen at stages II–V and VII–VIII when compared with the nonirradiated samples (Table 1). At stages IX–XII, in turn, irradiation did not cause a statistically significant reduction in the number of any cells in the 1C population (Table 1). Thus, in the 1C population, S1P could not prevent germ cell loss caused by 0.5-Gy irradiation.
In the 4C population of spermatocytes (i.e., which had been spermatogonia at the time of irradiation) 21 days after irradiation, a significant decrease in the cell numbers were seen in vehicle-treated control mice at every stage. In contrast, 21 days after S1P treatment and 0.5-Gy irradiation, the number of cells in the irradiated plus S1P-treated mice (at every stage) did not significantly differ from the number of cells in nonirradiated control mice (Table 1 and Fig. 6). At stages II–V, the cell numbers from S1P-treated animals were 16% and 34% higher after those treatments with 50 µM and 200 µM S1P, respectively, than in irradiated and vehicle-treated animals (Table 1 and Fig. 6). At stages VII–VIII, this effect of S1P treatment was clearer, total cell numbers being 35% and 47% higher than in the irradiated, vehicle treated animals (Table 1 and Fig. 6). Finally, at stages IX–XII, the cell numbers in the S1P-treated animals were 40% and 38% higher than in the irradiated, vehicle-treated animals (Table 1 and Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exposure of mice to irradiation doses of up to 5 Gy has previously been demonstrated to increase germ cell death significantly, the changes being most prominent among the B spermatogonia, as examined by electron microscopy and TUNEL analysis [23]. Also recently, irradiation of mouse spermatogonia with 0.25 and 4 Gy produced a dose-related increase in DNA damage in sperm collected 45 days later as measured by the Comet assay [24]. The epithelial cycle has been previously shown to divide into two parts with different responses to 1-Gy irradiation [25]. Stages III–VII were demonstrated to correspond to an area with high sensitivity to irradiation. In stages VII–VIII, in turn, the sensitivity to irradiation was reduced, suggesting this radiosensitivity to be dependent on the proliferative activity of the stem cells [25]. Accordingly, the radiosensitivity of the undifferentiated spermatogonia, which are committed to differentiate, varied according to the activity of the cell cycle; in stages III–VII, the D0 value was 0.4–0.7 Gy; in stages XII/I, the D0 was 1.0 Gy; and in stages IX–X, 2.2. Gy [26]. These results are in agreement with our findings, in which the number of differentiating spermatogonia reduces significantly after 0.1 Gy in stages II–V and after 0.5 Gy in stages II–V and VII–VIII, whereas in stages IX–XII, a statistically significant decrease is detected only after 2.0 Gy, as measured by DNA flow cytometry 21 days after irradiation. Here, the model for DNA flow cytometric analyses of mouse spermatogenesis 21 days after irradiation were based on studies made in rat [22]. In the present work, we show that, despite differences between species in stage-specific characters in DNA histograms of flow cytometry [18], flow cytometry is a valid method to quantify irradiation-induced testicular apoptosis in mice.
In the rat testis, germ cell apoptosis starts within minutes after irradiation and the number of apoptotic cells increases until about 16 h [15]. In the present study on the mouse, only trends toward lower amounts of ISEL-positive germ cells in S1P-protected testes could be seen at the 16-h time point when compared with the nontreated testes. Because physiological death of selected spermatocytes and spermatids is a common feature of normal spermatogenesis [2, 3], the total amount of dying germ cells may have masked the protective effect of S1P on spermatogonia at this time point. Probably therefore, the protection of differentiating spermatogonia by S1P could be seen only 21 days after irradiation as a decrease in number of 4C cells in irradiated but not in irradiated plus S1P-treated testes.
The sphingomyelin pathway seems to be important in somatic testicular cells. In porcine Sertoli cells, sphingomyelin hydrolysis pathway was related to production of lactate, a crucial energetic metabolite for germ cells [27]. Further, in rat Leydig cells, ceramide-dependent pathway regulated hCG-stimulated Leydig cell steroidogenesis at the level of cAMP production and at post-cAMP events [28]. Ceramide generation was also completely blocked by ceramide synthetase inhibitor Fumonisin B1 and exogenous ceramide itself was shown to directly induce apoptosis of rat Leydig cells in vitro [29]. It is becoming clear that the specialized functions required for proper proliferation and differentiation of the spermatogonial stem cells are mainly provided by the neighboring differentiated Sertoli cells [30–34]. Concomitantly, fine architecture of the seminiferous epithelium and the interactions between different types of seminiferous epithelial cells, e.g., Sertoli and Leydig cells, are crucial for spermatogenesis. The sphingomyelin pathway may thus have a role in paracrine control of germ cell proliferation, differentiation, and survival provided by the Sertoli cells. Further, acid sphingomyelinase knockout mice that lack one of the enzymes that converts sphingomyelin to ceramide was found to have pathological testicular tissue and sperm due to lipid accumulation, but the quantitative process of sperm formation was not impaired [35]. We, on the other hand, demonstrated recently that ceramide levels increase during serum withdrawal-induced human male germ cell apoptosis in vitro and that this apoptosis is partly (30%) inhibited by S1P [36]. Thus, in male germ cells, the sphingomyelin pathway may not be the primary initial transduction pathway of apoptosis but may serve as an alternative route of the programmed cell death.
The intracellular balance between the sphingolipid second messengers S1P and ceramide may determine whether the cell will survive or die [11]. Even though, in several cell lines, the inhibitory effect of S1P on apoptosis is related to caspase 3, inhibition of human male germ cell apoptosis has been suggested to be caspase 3 independent [36, 37]. Moreover, S1P has been identified as a ligand for the G-protein coupled EDG-1–3, -5, -6 (S1P1–5) receptors [38]. At present, their possible roles in the regulation of male germ cell apoptosis, however, is unclear. Thus, regulation of male germ cell death seems to involve several parallel and even uncharacterized apoptotic pathways.
Recently, mating trials revealed that S1P-based protection of female germ cells from irradiation was not associated with genomic damage at anatomical, histological, biochemical, or cytogenetic levels [14]. Our present results suggest that, also in males, S1P may have some role in protection of germ cells against irradiation, although the number of animals used in this study was limited. Moreover, although it acts upstream in apoptosis cascades, whether S1P protects male germ cells upstream or downstream of DNA damage caused by irradiation remains unclear. More work needs to be done before sphingolipid pathways of testicular germ cells could be considered as therapeutic targets in protecting male gonads from apoptosis induced by external stress such as cancer therapy. Therefore, the discussion about its therapeutic potential in the males should be only speculative.
In conclusion, we have shown that S1P moderately protects the very early stages of spermatogenesis from irradiation-induced apoptosis. This effect is seen in the A-paired spermatogonia that develop into the 4C population, which consists mainly of spermatocytes 3 wk after irradiation. This may reflect that apoptosis at these very early developmental stages may be more dependent on the sphingomyelin pathway than apoptosis of germ cells in the later steps of spermatogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Laura Suomalainen, Hospital for Children and Adolescents, Programme for Developmental and Reproductive Biology (5th Floor, Room B529b), Biomedicum Helsinki, University of Helsinki, P.O. Box 700, FIN-00029 HUS, Helsinki, Finland. FAX: 358 9 4717 1947; laura.suomalainen{at}hus.fi
Received: 1 August 2003.
First decision: 28 August 2003.
Accepted: 10 November 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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