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Regular Article |
a Department of Histology and Cell Biology, Nagasaki University School of Medicine, Nagasaki 852-8523, Japan
b Faculty of Pharmaceutical Sciences, Kanazawa University, Ishikawa 920-0934, Japan
c Department of Clinical Pharmaceutics, Nagasaki University School of Pharmaceutical Sciences, Nagasaki 852-8521, Japan
ABSTRACT
Apoptosis of germ cells is very common in normal and injured mammalian testes. The aim of this study was to examine the possible involvement of the Fas and Fas ligand (FasL) system in the induction of germ cell apoptosis in normal and ischemia-reperfusion testes of adult mice. Apoptosis was assessed by the TUNEL method and by DNA gel electrophoresis. Fas and FasL mRNAs were detected by Northern blotting and reverse transcription polymerase chain reaction techniques, and proteins were analyzed by Western blotting and immunohistochemistry. Apoptosis of germ cells was identified in the normal testis especially around stages XI and XII, whereas the expression of Fas and FasL was largely confined to Leydig cells and Sertoli cells, respectively. However, in the testes reperfused after 1 h of ischemia, a high number of TUNEL-positive cells were identified in parallel with increased Fas-positive germ cells, whereas FasL expression in Sertoli cells was almost constant irrespective of the duration of reperfusion. Moreover, i.p. injection of anti-Fas antibody, which blocks the interaction between Fas and FasL, inhibited apoptosis, as indicated by the reduced number of TUNEL-positive cells, except for apoptosis at stages XI and XII. Our results indicate that the Fas/FasL system mediates apoptosis of spermatogenic cells in the injured testis but not spontaneous apoptosis in the normal testis.
apoptosis, cytokines, spermatogenesis, stress, testes
INTRODUCTION
In mammalian spermatogenesis, 25%–75% of the expected sperm are lost naturally [1–3]. In addition, during developmental and neonatal stages of mice, significant numbers of primordial germ cells and spermatogonia disappear eventually [4, 5]. Among the causes of germ cell loss, cell death through apoptosis [6] has been identified [7–10]. Apoptosis of germ cells, especially of spermatocytes, is known to be induced in hypophysectomized [11, 12] or GnRH antagonist-treated rats [13, 14]. Moreover, cryptorchidism of immature [15] and mature [16, 17] rats increased the frequency of germ cell apoptosis. Exposure of testis to toxic compounds [18–20] and estrogenic steroids [21] also results in atrophy of the testes in rodents through apoptosis. These findings indicate that apoptosis may be a common pathway in the induction of testicular germ cell death in normal as well as injured testes. However, our knowledge of the molecular mechanisms underlying the induction of germ cell apoptosis is very limited.
Apoptosis is an active mode of cell death, first characterized by Kerr et al. [6]. Apoptosis can be discriminated from necrosis based on particular morphological features, including cell shrinkage, chromatin condensation to nuclear periphery, and formation of apoptotic bodies. Internucleosomal DNA fragmentation occurs prior to the appearance of the morphological changes of apoptosis, which can be detected as a ladder formation on DNA gel electrophoresis [22] and as nuclear staining by the TUNEL method [23].
Fas antigen (APO-1/CD95) [24, 25] was isolated as type I transmembranous glycoprotein, a member of the tumor necrosis factor/nerve growth factor receptor family. Fas is known to mediate apoptosis in a variety of immune and nonimmune tissues, including liver [26], ovary [27], vagina [28], intestine [29], and kidney [30]. As a natural ligand for Fas, Fas ligand (FasL) was discovered and identified as a type II transmembranous protein that initiates apoptosis in activated T cells by binding to Fas expressed on these cells [31]. Recent studies have demonstrated that the expression of FasL is not confined to immune cells but is also present in the testis [20, 32, 33], ovary [27], and thyroid [34] under various pathological conditions [29, 30, 35]. In the case of adult testis, FasL is expressed predominantly in Sertoli cells and is thought to inhibit the invasion of lymphoid cells into seminiferous tubules [32]. However, the relationship between Fas system and germ cell apoptosis is largely unknown.
Ischemia-reperfusion induces acute injury in various tissues such as the heart, brain, kidney, and testis and is often used as an experimental model of infarct or hyperoxygenation. In addition, the role of apoptosis has been highlighted in ischemia-reperfusion tissue dysfunction, suggesting that apoptosis can potentially influence the extent of tissue injury. Specifically in the testis, analysis of tissue atrophy following ischemia-reperfusion is important for our understanding of germ cell injury in patients with testicular torsion [36].
In the present study, we first examined the expressions of Fas and FasL in the testes of normal adult mice at both mRNA and protein levels, and compared the levels of such expression with the appearance of apoptosis, using TUNEL staining. Second, we used a similar approach to analyze testicular atrophy after ischemia-reperfusion. To obtain further evidence of the involvement of Fas in apoptosis, we examined the inhibitory effect of anti-Fas serum, which can block the interaction between Fas and FasL, on the induction of germ cell apoptosis in the ischemia-reperfusion testes.
MATERIALS AND METHODS
Chemicals and Biochemicals
Paraformaldehyde (PFA) was purchased from Merck (Darmstadt, Germany), and 3,3'-diaminobenzidine-4HCl (DAB) was purchased from Dojin Chemical Co. (Kumamoto, Japan). BSA, proteinase K, and Brij 35 were purchased from Sigma Chemical Co. (St. Louis, MO). Biotin-16-dUTP and terminal deoxynucleotidyl transferase (TdT) were from Boehringer Mannheim (Elkhart, IN). All other reagents used in this study were from Wako Pure Chemicals (Osaka, Japan) and were of analytical grade.
Antibodies
Anti-Fas sera were prepared by immunization of rabbits against synthetic oligopeptides corresponding to the extracellular domain (P2; amino acids 127–137) and the intracellular domain (P4; amino acids 292–306) of mouse Fas [37], as described previously [27, 38]. Anti-FasL serum was generated with a synthetic peptide corresponding to the intracellular domain (P5; amino acids 41–55) of rat FasL [31]. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG F(ab)'2 (1:200) was purchased from MBL (Nagoya, Japan). HRP-conjugated goat anti-biotin (1:100) was from Vector Laboratories (Burlingame, CA).
Animals
Male adult ICR mice (5–7 wk) weighing 30–40 g were used in the present study. The experimental protocol was approved by the Animal Ethics Review Committees at our institutions.
Ischemia-Reperfusion of Mouse Testis
Under anesthesia induced by i.p. injection of pentobarbital (65 mg/kg body weight), an abdominal incision was made. The testicular artery and vein of the left testis were occluded with a vascular clamp for 60 min, after which time the clamp was removed and the organ was allowed to reperfuse. At various time points after reperfusion (0 h, 12 h, 24 h, and 48 h), the mice (n = 5 per time point) were anesthetized again, and the testes were removed. The right testis was used as a control in each animal. In another group of mice, sham operations were performed in a similar fashion, except the vessels were not clamped.
Tissue Preparation
The testes and livers were cut into several small pieces and divided into two groups. In the first group, the pieces were quickly frozen in liquid nitrogen and later used for the extraction of DNA or RNA. In the second group, tissue samples were fixed in 4% PFA in PBS at 4°C overnight and embedded in paraffin using standard procedures. The sections were later stained with hematoxylin and eosin (H&E) and used for histological evaluation of tissue damage.
Gel Electrophoresis of DNA
High-molecular-weight DNA was extracted from frozen tissue samples using the DNA extractor WB kit (Wako Pure Chemicals). The concentration of DNA was measured spectrophotometrically at 260 nm. Aliquots of DNA samples (20 µg/lane) were separated on a 2% agarose gel and stained with ethidium bromide. Takara pHY (Tokyo, Japan) was used as a molecular size marker.
TUNEL Staining
To identify nuclei with DNA strand breaks at a cellular level, TUNEL was performed according to the method of Gavrieli et al. [23], with a slight modification. Paraffin sections (5–6 µm) were cut onto silane-coated glass slides, dewaxed with toluene, and rehydrated in an ethanol series. After washing with PBS, the sections were treated with 5 µg/ml of proteinase K in PBS at 37°C for 15 min. The sections were then rinsed once with deionized distilled water and incubated with TdT buffer (25 mM Tris/HCl buffer, pH 6.6, containing 0.2 M potassium cacodylate and 0.25 mg/ml BSA) alone at room temperature for 30 min. After incubation, the slides were reacted with 200 U/ml TdT dissolved in TdT buffer supplemented with 5 µM biotin-16-dUTP, 20 µM dATP, 1.5 mM CoCl2, and 0.1 mM dithiothreitol at 37°C for 1 h. The reaction was terminated by washing with 50 mM Tris/HCl buffer (pH 7.4) for 15 min. Endogenous peroxidase activity was inhibited by immersing the slides in 0.3% H2O2 in methanol at room temperature for 15 min. The signals were detected immunohistochemically with HRP-conjugated goat anti-biotin antibody, as described previously [27, 35].
For statistical analysis, more than 10,000 germ cells/animal were counted, and the number of TUNEL-positive cells was expressed per 1000 of the total germ cells (mean ± SEM). Data for different groups were compared for statistical difference using Student t-test. A P value of <0.05 denoted the presence of a significant difference.
Northern Blotting
Total RNA was extracted from mouse tissues using the method described by Chomczynski and Sacchi [39]. Poly(A)-containing RNA was isolated by oligo(dT)-cellulose chromatography, and 10 µg was separated on an agarose gel containing formaldehyde. The region between nucleotide positions 28 and 1056 of the mouse Fas cDNA [37] was amplified by reverse transcription-mediated polymerase chain reaction (RT-PCR) using poly(A)-containing RNA from the mouse liver. The region between nucleotide positions 116 and 400 of the rat FasL cDNA [31] was obtained by PCR using rat liver DNA as a template. Part of the human ß-actin pseudogene was used as a probe for ß-actin mRNA. These DNAs were labeled with 32P by random priming and used as hybridization probes for Northern blot analysis of Fas and FasL mRNA. Hybridization signals were detected by autoradiography as described previously [40].
RT-PCR
Poly(A)-containing RNA from testes or livers of mice was subjected to RT-PCR. Oligonucleotide primers were designed to amplify the region between nucleotide positions 724 and 1057 of the mouse Fas cDNA and the region between positions 542 and 745 of the rat FasL cDNA. PCR proceeded in the presence of [
-32P]dCTP, and the products were separated on a polyacrylamide gel followed by autoradiography.
Isolation of Protein and Western Blotting
Testes (30–70 mg for each testis) were homogenized in the lysis buffer consisting of 10 mM phosphate buffer (pH 7.2), 0.1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml leupeptin, and 1 µg/ml chymostatin. Protein concentration was determined by the method of Bradford [41] using BSA as a standard. Five micrograms of the lysate was mixed with the loading solution consisting of 62.5 mM dithiothreitol, 5% SDS, and 10% glycerol, boiled for 5 min, separated by SDS-PAGE with a 10%–20% gradient gel according to the method of Laemmli [42], and electrophoretically transferred onto polyvinylidene difluoride membranes (Immobilon, Millipore Corp., Bedford, MA). The membranes were blocked for 1 h with PBS containing 5% nonfat dry milk and incubated for 2 h with rabbit anti-Fas (P4) and anti-FasL (P5) serum, diluted at 1:800 and 1;rc200 with 0.5% nonfat dry milk in PBS, respectively. As the second antibody, HRP-conjugated goat anti-rabbit IgG, F(ab')2 was reacted at 1:200 with 0.5% nonfat dry milk in PBS for 1 h, and the bands were visualized with H2O2 and DAB in the presence of nickel and cobalt ions, according to the method of Adams [43].
Immunohistochemistry
Immunohistochemical staining for Fas and FasL proteins was performed according to the method described previously [27, 38]. Paraffin sections (5–6 µm) of testes were cut onto silane-coated glass slides, dewaxed with toluene, and rehydrated with serial ethanol solutions. The sections were preincubated with 5% BSA in PBS for 1 h and reacted with the anti-Fas (P4; 1:800) and anti-FasL (P5; 1:100) antisera for 2 h. After washing with 0.075% Brij in PBS, HRP-labeled goat anti-rabbit IgG F(ab')2 was reacted for 1 h, and the sites of HRP were visualized with H2O2 and DAB. The sections were counterstained with methyl green. As a negative control, some sections were reacted with normal rabbit serum instead of the specific antiserum.
Intraperitoneal Injection of Anti-Fas Serum
The testis was subjected to ischemia for 1 h, followed by 2 h of reperfusion and then i.p. injection of an aliquot (100 µl) of anti-P2 antiserum (n = 5). The mice were killed for examination of apoptosis of testicular germ cells 22 h after the injection. As a control (n = 3), the same volume of normal rabbit serum was injected instead of anti-P2 antiserum. In addition, anti-P2 antiserum was injected in normal mice (n = 5) using the above dose. To confirm the influx of injected rabbit antibody into the seminiferous tubules, sections from some specimens were reacted with HRP-anti-rabbit F(ab)2, as described above.
RESULTS
TUNEL Staining in Normal Mouse Testis
To assess the presence of germ cell apoptosis in normal spermatogenesis, we conducted TUNEL staining on paraffin-embedded sections of adult mouse testis. As shown in Figure 1, autonomous germ cell death was clearly seen among spermatogonia, spermatocytes, and spermatids. However, among seminiferous tubules of different stages of the seminiferous epithelial cycle, the distribution of TUNEL-positive cells was not even; TUNEL-positive cells seemed to be most abundant at stages XI–XII, when the meiotic cell division of spermatocytes is ready or just in process. In seminiferous tubules of other stages, TUNEL-positive cells were scarce. On average, 7.70 ± 0.63 cells per 1000 germ cells (mean ± SEM, n = 5) were TUNEL positive. Essentially no TUNEL-positive cells were seen in the interstitial tissue of normal testis.
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Expression of Fas and FasL in Normal Mouse Testis
In the next step, we examined the expression of Fas and FasL in the testis, at both mRNA and protein levels. Northern blot analysis revealed that the expression of Fas mRNA was very weak in the normal mouse testis (almost undetectable in Fig. 2). In contrast, a high level of FasL mRNA expression was present in this tissue. The low level of Fas mRNA expression as compared with that of liver was also confirmed by RT-PCR. To determine the expression of Fas and FasL proteins in normal testis, Western blotting was performed using antisera raised against Fas (P4) and FasL (P5) peptides. As shown in Figure 3, two bands of 31 kDa and 58 kDa for Fas [35] and a single band for FasL were detected. Moreover, immunohistochemical staining for Fas and FasL conducted on paraffin-embedded sections of testis revealed a signal for Fas (Fig. 4A) predominantly in Leydig cells, whereas FasL expression was restricted to Sertoli cells and its intensity varied among seminiferous tubules (Fig. 5A). When the first antibody was replaced with normal rabbit serum as a control, no staining was observed (data not shown).
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DNA Ladder Formation and TUNEL Staining in Ischemia-Reperfusion Testis
To assess the involvement of apoptosis in the germ cell loss after ischemia-reperfusion of testis, DNA was extracted from mouse testes exposed to various intervals of reperfusion after 1 h of ischemia and was analyzed for the appearance of the DNA ladder on agarose gel electrophoresis (Fig. 6). Typical ladder bands appeared at 12–24 h after reperfusion. However, a significant level of smear staining was seen at both 12 and 48 h.
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For further histological examination, paraffin-embedded sections of mouse testes treated in a manner similar to that described above were analyzed for the presence of nuclei with DNA strand breaks by using TUNEL staining. As shown in Figure 7, in parallel with increased DNA ladder intensity, the density of TUNEL-positive cells increased significantly after 12 h after reperfusion, reaching a maximum at 24 h after reperfusion but diminishing thereafter. Quantitative analysis of the number of TUNEL-positive cells revealed that the ratio of TUNEL-positive cells per 1000 germ cells increased proportionately with the time of reperfusion and again reached a maximum at 24 h after reperfusion (Fig. 8).
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For the interstitial tissue, TUNEL-positive Leydig cells appeared at 12 h after reperfusion and seemed to increase, reaching a plateau at 24 h after reperfusion.
Expression of Fas and FasL in Ischemia-Reperfusion Mouse Testis
To investigate changes over time in the expression of Fas and FasL in the ischemia-reperfusion testis, we conducted Western blot analysis, similar to that displayed in Figure 3. Clearly, the intensity of both bands for Fas was increased after reperfusion, reaching a plateau at 24 h after reperfusion. However, the band for FasL seemed nearly similar irrespective of the duration of reperfusion, although a slight fluctuation was noted in the level of FasL expression.
When paraffin-embedded sections of testes exposed to various periods of reperfusion were stained for Fas immunohistochemically, both the staining intensity and number of positive cells were markedly increased after reperfusion (Fig. 4). At 24 h after reperfusion, many germ cells, most of which were spermatocytes and spermatids, became positive for Fas immunostaining. For FasL immunostaining, only Sertoli cells were positive at any time point, and the staining intensity seemed to be slightly elevated at 24 h after reperfusion (Fig. 5).
Inhibition of Induction of Germ Cell Apoptosis after Ischemia-Reperfusion by Anti-Fas
To examine the involvement of the Fas system in the induction of germ cell apoptosis by ischemia-reperfusion, each mouse was injected i.p. with a 100-µl aliquot of anti-Fas (P2) antiserum after 2 h of reperfusion, followed by examination of the appearance of TUNEL-positive cells after 24 h of reperfusion. As shown in Figure 9, injection of the antiserum markedly reduced the number of TUNEL-positive germ cells. In contrast, injection of normal rabbit serum instead of the anti-Fas serum but at the same volume failed to inhibit apoptosis of germ cells. TUNEL-positive cells in seminiferous tubules of stages XI–XII were still present even after injection of anti-P2 serum, a pattern similar to that seen in normal testis. The number of TUNEL-positive Leydig cells was also reduced by the injection of anti-P2 serum.
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Quantitative analysis revealed that the number of TUNEL-positive germ cells/1000 germ cells was 7.84 ± 1.05 in ischemia-reperfusion mice injected with the anti-P2 serum, significantly lower than the number (16.94 ± 1.61) in mice injected with normal rabbit serum (P < 0.01). Furthermore, injection of anti-P2 serum into normal mice did not significantly affect the number of apoptotic germ cells (6.92 ± 0.63). Immunohistochemical staining of testicular sections using HRP-conjugated anti-rabbit IgG after injection of anti-P2 serum confirmed the entry of i.p. injected rabbit IgG into seminiferous tubules (data not shown).
DISCUSSION
In the present study, we confirmed the expression of Fas and FasL in the testis of normal adult mice, although there was no obvious association between the Fas system and the occurrence of germ cell apoptosis. We also used mice with experimentally induced ischemia-reperfusion testes as a model of testicular torsion or a temporary testicular ischemia model. In these mice, however, ischemia-reperfusion induced simultaneous expression of Fas and FasL within the seminiferous tubules, and this expression was topographically related to the appearance of TUNEL-positive apoptotic cells. We also demonstrated that i.p. injection of anti-Fas (P2) antiserum significantly inhibited apoptosis induced by ischemia-reperfusion but did not inhibit constitutive apoptosis, strongly indicating the involvement of the Fas-FasL system in the induction of germ cell apoptosis in nonphysiological conditions of the testis.
Considering that 25%–75% of germ cells are usually eliminated by apoptosis during normal mammalian spermatogenesis [1–3], it is important to pursue the regulatory mechanisms underlying the induction of germ cell apoptosis. In this regard, previous studies demonstrated that germ cell apoptosis can be induced by administration of toxic compounds such as mono-(2-ethylhexyl) phthalate [20], methoxyacetic acid [18], and cyclophosphamide [19] and by depletion of androgens by ethane dimethane sulphonate [44–46], hypophysectomy [47], GnRH antagonist [13, 14], excess amount of estrogen [21], and by cryptorchidism [15–17]. However, our knowledge on the molecular and cellular mechanisms that induce germ cell apoptosis is only limited.
The Fas system was originally characterized as a key mechanism for inducing apoptosis in immune cells [24, 25] and later in various normal tissues such as liver [26], vagina [28], and ovary [27] and in chronic inflammatory conditions such as ulcerative colitis [29] and Hashimoto's thyroiditis [34] or in cancerous tissues [37, 48]. In Fas-mediated apoptosis, the binding of FasL as a natural inducer to Fas antigen is a prerequisite. Therefore, the temporal and spatial association between Fas and FasL expressions should be demonstrated in tissues where the Fas system is implicated as a possible mediator of the induction of apoptosis.
As an immune privileged organ [32], the testis is known to express FasL at the highest level among tissues [31], whereas the level of Fas expression is very low. Moreover, FasL expression is exclusively restricted to Sertoli cells [33], and that of Fas is largely found in Leydig cells, in addition to a few germ cells [20]. These findings, together with the present results showing a spatial disconnection between TUNEL-positive cells and Fas-positive cells, indicate that the Fas system does not play a critical role in the induction of germ cell apoptosis in the testis of normal adult mice. Recently, Lee et al. [20] described the involvement of the Fas system in the regulation of apoptosis of normal germ cells, but their results did not demonstrate a close connection between Fas-positive cells and TUNEL-positive cells at a cellular level. Also, we found that there was no difference in the occurrence of TUNEL-positive germ cells between wild-type mice and Fas-deficient lpr/lpr mice or functional FasL-lacking gld/gld mice (data not shown). More importantly, however, expression of both Fas and FasL was markedly elevated after treatment with cytotoxic compounds [20], after androgen withdrawal [45, 46], or after ischemia-reperfusion stress (the present study), and these manipulations seemed to be associated with the induction of germ cell apoptosis. Thus, we propose that the Fas system plays an active role in the induction of germ cell apoptosis in damaged testes rather than in normal mature testis. In this context, Ogi et al. [49] reported that even in lpr/lpr mice germ cell apoptosis was induced by experimental cryptorchidism. Moreover, very recently Richburg et al. [50] showed that exposure of gld/gld mice to a Sertoli cell-specific toxicant caused only a minimal increase in germ cell apoptosis, whereas these mice were as sensitive as wild-type mice to radiation exposure. These findings seem to demonstrate strongly that the Fas system can be triggered to kill germ cells after certain types of testicular injury.
The question remains as to why germ cells at stages XI–XII are specifically targeted for apoptosis in normal adult mouse testis. Although we cannot fully understand the reason at present, we assume that some structural and functional genetic errors in the meiotic prophase undergoing genetic recombination may cause abnormalities in the process of cell division, leading to the induction of germ cell apoptosis, as in the case of radiation [50].
How can the injected anti-Fas antibody neutralize the interaction between Fas on germ cells and FasL on Sertoli cells? Testicular germ cells after meiotic recombination are usually protected by a blood-testis barrier consisting of the tight junction between Sertoli cells against the attack of immune cells. Therefore, it seems strange that i.p. injected antibody can reach the inside of the seminiferous tubules. However, it has been already reported that the injection of antibody against c-kit ligand effectively inhibits the interaction between c-kit on germ cells and c-kit ligand expressed on Sertoli cells [4]. In addition, we showed here that the injected rabbit IgG could be detected immunohistochemically. Therefore, we suspect that there are some routes for specific substances from systemic circulation to move into the inside of seminiferous tubules or that ischemia-reperfusion causes partial breakage of the tight junctions between Sertoli cells to enable the anti-Fas antibody to enter the tubule lumen. Alternatively, during the normal process of the entry of spermatogenic cells into the lumen through the blood-testis barrier, serum components may accompany these cells into the seminiferous tubules.
Clinically, testicular torsion is an ischemic disorder of the testis, which is followed by a serious loss of germ cells. Recent studies have shown that short-term ischemia causes permanent aspermatogenesis in rats [51] and that the loss of germ cells occurs through germ cell-specific apoptosis [36]. The present results further indicate that the Fas system may be involved in the induction of germ cell apoptosis by ischemia-reperfusion, although there is no sufficient explanation for the induction of Fas. Fas induction can be viewed as a cellular response to oxygen stress [30, 36, 52], but so far no responsive elements to oxygen stress have been identified in the promoter region of the Fas gene. Certain cytokines such as interferon-gamma may be involved in the induction of Fas, as found in the granulosa cells of rat ovary [27]. Further studies are necessary to characterize the molecular mechanisms involved in the induction of Fas expression in germ cells to allow therapeutic manipulation of germ cell damage after ischemia-reperfusion.
ACKNOWLEDGMENTS
The authors thank Dr. Shigeyoshi Oba and Mr. Haruyuki Ohta for their skillful technical help.
FOOTNOTES
First decision: 6 September 2000.
1 This study was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture (12470003 to T.K.) and a grant from the Japanese Environment Agency (to T.K.). 
2 Correspondence: Takehiko Koji, Department of Histology and Cell Biology, Nagasaki University School of Medicine, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan. FAX: 81 95 849 7028; tkoji{at}net.nagasaki-u.ac.jp
Accepted: October 26, 2000.
Received: August 7, 2000.
REFERENCES
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 |
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|
|
|
|
 |
|
 M. Okazaki, T. Matsuyama, T. Kohno, H. Shindo, T. Koji, Y. Morimoto, and T. Ishimaru Induction of Epithelial Cell Apoptosis in the Uterus by a Mouse Uterine Ischemia-Reperfusion Model: Possible Involvement of Tumor Necrosis Factor-{alpha} Biol Reprod, May 1, 2005; 72(5): 1282 - 1288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
 |
|
 M. Port, H.U. Schmelz, M. Stockinger, C. Sparwasser, P. Albers, T. Pottek, and M. Abend Gene Expression Profiling in Seminoma and Nonseminoma J. Clin. Oncol., January 1, 2005; 23(1): 58 - 69. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chandrasekaran and J. H. Richburg The p53 Protein Influences the Sensitivity of Testicular Germ Cells to Mono-(2-Ethylhexyl) Phthalate-Induced Apoptosis by Increasing the Membrane Levels of Fas and DR5 and Decreasing the Intracellular Amount of c-FLIP Biol Reprod, January 1, 2005; 72(1): 206 - 213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
 M. Embree-Ku and K. Boekelheide Absence of p53 and FasL Has Sexually Dimorphic Effects on Both Development and Reproduction Experimental Biology and Medicine, July 1, 2002; 227(7): 545 - 553. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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