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Biology of Reproduction 61, 393-399 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Role of Radical Oxygen Species in Rat Testicular Germ Cell Apoptosis Induced by Heat Stress

Masato Ikedaa, Hideya Kodamab, Jun Fukudaa, Yasushi Shimizua, Masanori Murataa, Jin Kumagaia, and Toshinobu Tanakaa

a Department of Obstetrics and Gynecology, Akita University School of Medicine, and b Akita University College of Allied Medical Science, Akita city, 010-0041 Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was designed to clarify the role of radical oxygen species in testicular germ cell apoptosis induced by heat stress. Testicular cells isolated from immature rats were cultured with or without elevated temperature, and occurrence of apoptosis in these cells was defined by the appearance of DNA fragmentation following agarose gel electrophoresis and by flow cytometric quantification of apoptotic cells. At 32.5°C, < 1% of cells showed signs of apoptosis throughout the culture period, whereas under heat stress, the proportion of apoptotic cells increased to 5% at 37°C after 24 h of culture, or to 14% after 1-h exposure at 43°C followed by 23-h culture at 32.5°C. Similar to the effect of heat stress, exogenously supplied oxygen free radicals also induced apoptosis. In contrast, treatment with catalase significantly attenuated heat stress-induced apoptosis. Furthermore, heat stress of testicular cells was associated with an increased intracellular peroxide level as measured by a fluorescent probe, 2',7'-dichlorofluorescin diacetate. In conclusion, our data indicate the involvement of radical oxygen species during testicular germ cell apoptosis induced by heat stress. This study provides a useful in vitro model for the study of testicular germ cell apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In most mammalian species, the temperature of the testis is a few degrees lower than that of the abdomen [1], and this cooler temperature has been shown to be essential in order for the testis to maintain an optimal environment for spermatogenesis. Exposure of the testis to body temperature or above by local heating [2, 3], experimentally induced cryptorchidism [4–6], or varicocele [7, 8] causes disruption of spermatogenesis, resulting in temporary or permanent infertility. Recently, it has been suggested that this degenerative process of testicular germ cells by heat stress associated with cryptorchidism involves apoptosis, a form of programmed cell death [9–12]. However, the cellular mechanisms whereby heat stress induces apoptosis remain largely unknown.

During recent years, a large body of evidence has accumulated to indicate that oxidative stress may play a fundamental role in the regulation of apoptosis [13]. The bcl-2 protooncogene, which inhibits apoptotic cell death in various cell types, also may act as a natural antioxidant [14]. A variety of tumor cell lines [15, 16] and normal cell types, including endothelium [17], vascular smooth muscle cells [18], and renal epithelial cells [19], underwent apoptosis when exposed to oxidative stress in vitro. Antioxidant treatment, which enhances the endogenous antioxidant defense systems within cells, can inhibit a variety of apoptotic pathways [20–22].

The generation of oxidative free radicals occurs constantly in all living cells [23], and the rate of generation of free radicals in testis or spermatozoa appears to be temperature-dependent. For example, the levels of spontaneous lipid peroxidation of cultured mouse spermatozoa, measured by generation of malondialdehyde, has been reported to increase with temperature elevation [24]. In addition, activities of several scavenging enzymes in testes of rats with experimentally induced cryptorchidism were impaired, accompanied by increased peroxidation of cellular lipids [25]. However, a role of oxygen free radicals in the induction of testicular germ cell apoptosis by heat stress remains a matter of speculation.

In the present study, we investigated whether 1) isolated testicular cells undergo apoptosis in vitro under heat stress, 2) exogenously supplied oxygen free radicals induce apoptosis in isolated testicular cells, 3) antioxidant treatments confer protection against heat stress-induced apoptosis, and 4) heat stress increases intracellular peroxide levels in testicular cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media and Chemicals

F12-L15 medium (1:1 mixture of Ham's F12 and Lebovitz's L15) and fetal bovine serum (FBS) used for testicular cell culture were purchased from Lifeteck Oriental Co. (Tokyo, Japan). A DNA extraction kit (ApopLadder EXTM) was purchased from Takara Syuzou Co. (Tokyo, Japan). Apo 2.7 monoclonal antibodies labeled with phycoerythrin and 500 µg/ml of digitonin were purchased from Funakoshi Co. (Tokyo, Japan). Other chemicals used were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals and Preparation of Testicular Cells

Immature male Wistar rats (40 days of age) were purchased from Nihon SLC Co. (Sizuoka, Japan). The animals were killed by ether anesthesia, and testicular cells were isolated by the method of Nagao [26] with modifications. Briefly, testes were removed and decapsulated mechanically. Seminiferous tubules were gently expressed and incubated in PBS containing 0.25% collagenase (type 1) for 15 min at 32.5°C with occasional shaking. The seminiferous tubules were then washed and were incubated again in PBS containing 0.25% trypsin (Difco, Detroit, MI) for 15 min at 32.5°C with gentle pipetting. After incubation, the trypsin treatment was terminated by adding FBS to a level of 10% (v:v). The resulting cell suspension was filtered through 108-µm nylon mesh to remove cell aggregates and tissue debris; the cells were then collected by centrifugation. The cells recovered were resuspended in F12-L15 medium supplemented with 1 mg/ml sodium bicarbonate, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 15 mM Hepes (pH 7.3), 14 ng/ml phenol red, and 10% FBS. The final concentration of testicular cells in the medium was adjusted to approximately 5 x 106/ml. The cell suspension (1 ml) was plated in a six-well C-1 plate (Sumitomo Verkleit Co., Tokyo, Japan). The cells were incubated in a humidified atmosphere of 5% CO2 in air with or without heat stress. For the control group, cells were cultured at 32.5°C, an optimal temperature for testicular germ cells. For heat-stressed conditions, cells were cultured either at abdominal temperature (37°C) throughout the culture period, or at a higher level of heat stress (43°C) for 1 h followed by incubation at 32.5°C. All procedures were performed under sterile conditions, and after incubation, viability of cultured testicular cells was assessed using trypan blue staining. The protocol for animal experimentation described in this paper was approved by the Animal Research Center Committee, Akita University School of Medicine. All experiments adhered to Guidelines for Animal Experimentation of the university.

DNA Electrophoresis

For analysis of DNA fragmentation by agarose gel electrophoresis, total DNA was isolated from testicular cells using a DNA extraction kit (ApopLadder EXTM). The isolated DNA was suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and quantified by absorbance at 260 nm. The DNA samples, 200 µg/ml per lane, were loaded onto 2.0% agarose gel (NuSieveR 3:1 Agarose, FMC BioProducts, Rockland, ME) containing single-strength TBE buffer (89 mM Tris-borate, 2 mM EDTA, pH 8.0) and ethidium bromide and were separated by electrophoresis for 30 min at 100 V. The gels were dried for 3 h at room temperature. DNA bands were visualized by a UV transilluminator (TM-15; Funakoshi Co.) before being photographed with a Polaroid (Cambridge, MA) camera.

Quantification of Apoptotic Cells by Flow Cytometry

Incidence of apoptotic cells was quantified by flow cytometry using Apo 2.7 monoclonal antibodies, which reacted with a mitochondrial membrane protein exposed in cells undergoing apoptosis, according to the methods of Zhang et al. [27]. Briefly, the cultured testicular cells were fixed in PBS containing 0.25% paraformaldehyde for 2 min at 4°C. After fixation, the cells were washed by centrifugation and then incubated in 20 µl of Apo 2.7 monoclonal antibodies labeled with phycoerythrin and 500 µg/ml of digitonin for 30 min at 4°C. Samples were then washed twice in PBS, filtered through 45-µm nylon mesh, and applied to a FACScan (Epics Elite; Coulter Co., Tokyo, Japan) with excitation and emission settings of 488 nm and 615 nm, respectively.

For analysis of the percentage of apoptotic cells by flow cytometry in most experiments, the testicular cells after 24 h of culture were gently pipetted, and the cells present in the supernatant were used for analysis. In this condition, the proportion of somatic cells (such as peritubular and Sertoli cells) of total cells examined was reduced because a large number of such somatic cells adhered to the plate after 24 h of culture. According to DNA histograms determined by flow cytometry using propidium iodide staining, the proportions of meiotic-phase cells (4s), diploid cells (2s), and haploid cells (s) in isolated testicular cells recovered from the supernatant of 24-h culture were 13%, 15%, and 72%, respectively (data not shown). Because of the proportion of diploid cells, which contained mostly somatic cells and germ cells at G-1 stage, > 80% of testicular cells as analyzed by flow cytometry were considered germ cells.

Exposure of Cells to Reactive Oxygen Species

A xanthine oxidase system has been used to generate oxygen-free radicals in various studies [28, 29]. It was reported previously that in vitro generation of superoxide anion (·O2-) was dependent on the concentration of xanthine, rather than that of xanthine oxidase [29]. In preliminary experiments, we measured levels of the superoxide anion in the medium by using nitro blue tetrazolium [30] and obtained the same results. When indicated concentrations of xanthine reacted with a fixed concentration of xanthine oxidase in vitro, the superoxide anion was generated continuously for approximately 30 min in a xanthine concentration-dependent manner (data not shown).

Antioxidants

A series of antioxidants, including 200 U/ml superoxide dismutase (SOD), 200 U/ml catalase, 25 µM O-phenanthroline, and 140 mM dimethyl sulfoxide (DMSO), were used to determine potential roles of the radical oxygen species in the induction of apoptosis. These antioxidants were applied 5 min before the cells were exposed to heat or oxidative stress.

Measurement of Intracellular Peroxides

Peroxide levels within the testicular cells before or after heat stress were assessed using the previously described method [31]. Briefly, testicular cells at a concentration of 5 x 105/ml were loaded with a fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFH/DA; 5 µM), for 30 min at 37°C. After loading, the cells were washed twice in PBS and filtered through 45-µm nylon mesh. Intracellular peroxide levels were measured using a FACScan flow cytometer with excitation and emission settings of 488 nm and 530 nm, respectively.

Data Analysis

The data were expressed as mean ± SEM of at least three independent experiments. Comparison of the proportion of apoptotic cells or cell viability between groups was performed using one-way ANOVA, followed by the Fisher's protected least-significant-difference post hoc test. Differences in the proportion of apoptotic cells or cell viability over time within each group were determined using ANOVA with repeated measure. Statistical analysis was performed using StatView (Abacus Concepts, Berkeley, CA). Difference was considered significant at a 5% level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Heat Stress on Apoptotic DNA Fragmentation in Cultured Rat Testicular Cells

Total DNA was isolated from the testicular cells 4 h after the beginning of incubation with or without heat stress, and the occurrence of apoptosis was defined by gel-fractionation analysis of DNA fragmentation (Fig. 1). The cells cultured at 32.5°C demonstrated minimal DNA fragmentation consistent with low levels of apoptosis (lane 1). However, the cells under heat-stressed conditions demonstrated discrete bands of fragmented DNA (lanes 2 and 3).



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  		FIG. 1. Effects of heat stress on apoptotic DNA fragmentation in cultured rat testicular cells. Testicular cells were isolated from immature male Wistar rats (40 days of age) and cultured with or without heat stress. Total DNA was isolated from the testicular cells 4 h after the beginning of incubation, and the occurrence of apoptosis was defined by gel-fractionation analysis of DNA fragmentation. The DNA samples, 200 µg/ml per lane, were loaded onto 2.0% agarose gel. Left lane contains molecular weight markers. Lane 1 represents control cells incubated at 32.5°C; lane 2, at 37°C for 4 h; lane 3, at 43°C for 1 h followed by incubation at 32.5°C for 3 h.

Time-Dependent Changes of Testicular Cell Viability and Percentage of Apoptotic Cells during Culture

After we confirmed the occurrence of apoptosis in the isolated testicular cells by gel-fractionation analysis, proportions of apoptotic cells under different conditions were quantified by flow cytometry using Apo 2.7 monoclonal antibodies (Fig. 2). At 32.5°C, more than 90% of cells survived for 3 days, and the proportion of apoptotic cells throughout the culture period was < 1%. When testicular cells were cultured at 37°C, the proportion of apoptotic cells increased in a time-dependent manner, reaching 15% on Day 3. In the meantime, the cell viability decreased to 40% on Day 3. When exposed to 43°C for 1 h followed by incubation at 32.5°C, the proportion of apoptotic cells increased to 14% on Day 1, and a further increase in the proportion of apoptotic cells was not observed on Day 2. Phase-contrast micrographs showing testicular cells at the end of 3 days of culture at 32.5°C or 37°C are presented in Figure 3.



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  		FIG. 2. Time-dependent changes of cell viability and percentage of apoptotic cells in cultured testicular cells. Testicular cells were isolated from rat testis and cultured in vitro with or without heat stress. Viability of cells and incidence of apoptotic cells in the cultured testicular cells were evaluated for 3 days. Cell viability was measured by trypan blue staining (A), and incidence of apoptotic cells was measured by flow cytometry using Apo 2.7 monoclonal antibodies (B). The control group was cultured at 32.5°C; heat-stressed groups were cultured at 37°C for 3 days or 43°C for 1 h, followed by 32.5°C. Each point represents the mean ± SEM of four experiments. *p < 0.05 versus control; **p < 0.01 versus control.



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  		FIG. 3. Phase-contrast micrographs showing the testicular cells at the end of 3 days of culture with or without heat stress. Morphology of the cells at 32.5°C (A) was normal, whereas a number of agglutinated cells, presumably due to degeneration, appeared in the cells at 37°C (B). x200 (reproduced at 100%).

Effect of Reactive Oxygen Species on the Apoptosis of Cultured Rat Testicular Cells

To examine whether reactive oxygen species could directly induce apoptosis of testicular cells, cells were exposed to indicated concentrations of xanthine (0.2–5.0 mM) and a fixed concentration of xanthine oxidase (20 mU/ml) in the absence of heat stress. DNA gel electrophoresis of testicular cells exposed to xanthine oxidase and xanthine demonstrated DNA fragmentation characteristic of apoptosis (Fig. 4A, lanes 2–4). As evidenced by flow cytometric analysis after 24 h of culture, increasing concentrations of xanthine resulted in a dose-dependent increase in the proportion of apoptotic cells (Fig. 4B). In contrast, treatment with xanthine oxidase alone or xanthine alone did not influence the incidence of apoptotic cells (data not shown).



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  		FIG. 4. Influence of reactive oxygen species on the apoptosis of cultured rat testicular cells, and the protective effect of antioxidant treatment. Indicated concentrations of xanthine (0.2–5.0 mM) and a fixed concentration of xanthine oxidase (20 mU/ml) were added to cultured testicular cells in the absence of heat stress (32.5°C). Occurrence of apoptosis in these cells after 4 h of culture with or without oxidative stress was defined by the appearance of DNA fragmentation following agarose gel electrophoresis (A). Left lane, molecular weight markers. Lane 1 represents the control cells incubated at 32.5°C; lane 2, incubated with 0.2 mM xanthine; lane 3, 1.0 mM xanthine; and lane 4, 5.0 mM xanthine. After 24 h of culture, the proportions of apoptotic cells were quantified by flow cytometry using Apo 2.7 monoclonal antibodies (B). To investigate which reactive oxygen species were responsible for apoptosis, a series of antioxidants, including 200 U/ml SOD, 200 U/ml catalase, 25 µM O-phenanthroline, and 140 mM DMSO, were added before the cells were exposed to oxidative stress (1.0 mM xanthine and 20 mU/ml xanthine oxidase), and the proportion of apoptotic cells after 24 h of culture was measured by flow cytometry (C). Values are mean ± SEM of four experiments. X, Xanthine; *p < 0.05 versus control; **p < 0.01 versus control.

To investigate which reactive oxygen species were most responsible for apoptosis induced by cotreatment with xanthine and xanthine oxidase, several antioxidants were added before the cells were exposed to oxidative stress (Fig. 4C). Among antioxidants studied, catalase and SOD conferred significant protection against apoptosis, although the effect of SOD was weaker than that of catalase.

Influence of Antioxidants on Heat Stress-Induced Apoptosis

To determine potential roles of the radical oxygen species in the process of heat stress-induced apoptosis, a series of antioxidants were administered to cultured cells before heat stress, and incidence of apoptotic cells was quantified after 24 h of culture (Fig. 5A). Among antioxidants studied, catalase conferred significant protection, whereas other antioxidants, including SOD, O-phenanthroline, and DMSO, were ineffective.



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  		FIG. 5. Influence of antioxidants on heat stress-induced apoptosis. A series of antioxidants, as described for Figure 3, were added to isolated testicular cells before heat stress (43°C for 1 h), and incidence of apoptotic cells was quantified by flow cytometry after 24 h of culture (A). In order to eliminate the influence of Hepes, this experiment was repeated using Hepes-free medium (B). Values are mean ± SEM of four experiments. **p < 0.01 versus control (43°C).

Because Hepes has been reported to have radical-scavenging ability and may influence the Haber-Weiss reaction [32, 33], we repeated this experiment using medium without Hepes. Culture at 32.5°C using Hepes-free medium did not increase the percentage of apoptotic cells (Fig. 5B, left bar), and the results of experiment described above (Fig. 5A) were almost completely reproducible in the Hepes-free medium (Fig. 5B), indicating that the influence of Hepes could be neglected.

Although catalase (200 U/ml) significantly inhibited apoptosis induced by heat or oxidative stress, catalase-treated cells exhibited a higher level of apoptosis than cells cultured in the absence of xanthine or heat stress. We examined the dose response for catalase treatment and found that different concentrations of catalase (> 200 U/ml) did not confer a higher level of protection (data not shown). There was a small fraction that was resistant to catalase treatment.

Intracellular Peroxide Levels in Testicular Cells following Heat Stress

The possibility that heat stress increases intracellular peroxide levels in testicular cells was assessed using fluorescence based on oxidation of DCFH/DA. Heat stress caused a temperature-dependent increase in the intracellular peroxide production by testicular cells, as indicated by the right-side shift of the fluorescence peak of the cells under heat stress (Fig. 6A). As shown in Figure 6B, addition of catalase significantly decreased the levels of intracellular peroxide levels of testicular cells following heat stress, whereas treatment with SOD was minimally effective. Elevation of the levels of intracellular peroxide was detected as early as 5 min after exposure to heat stress at 43°C (data not shown).



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  		FIG. 6. Intracellular peroxide levels in testicular cells following heat stress. After 15 min of culture with or without heat stress, the testicular cells were loaded with the fluorescent probes DCFH/DA (5 µM) for 30 min at 37°C, and intracellular peroxide levels were measured using a FACScan flow cytometer (A). Catalase or SOD was added to cultured testicular cells, and intracellular peroxide levels were measured after 15 min of incubation under heat stress (43°C) (B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Surgical induction of cryptorchidism has been frequently used as a model for the study of defective spermatogenesis due to heat stress. Shikone et al. [9] have reported that in surgically induced cryptorchid testis of immature rats, DNA cleavage into low-molecular-weight ladders characteristic of apoptosis was increased 4.2-fold at 7 days after the operation, and that the germ cells that underwent apoptosis were mainly primary spermatocytes as evidenced by in situ analysis. Similar findings were obtained also in adult mice, although the response to cryptorchidism was more vigorous in the adult mice [11]. Allan et al. [34] have also reported that, after immersion of adult rat testis into hot water (43°C) for 30 min, spermatogonia and preleptotene primary spermatocytes developed morphologic features characteristic of apoptosis, whereas other spermatocytes demonstrated necrotic changes. Extending these in vivo studies, the present study demonstrated heat stress-induced apoptosis in cultured testicular cells.

In the present in vitro model, testicular germ cells underwent apoptosis when exposed to exogenously supplied oxygen free radicals, and the observed increase in apoptosis was inhibited by cotreatment with antioxidants. Heat stress-induced apoptosis was also attenuated significantly by catalase treatment. Furthermore, heat stress significantly increased the intracellular peroxide levels. These results are consistent with the hypothesis that oxidative stress may play a fundamental role in the induction of apoptosis under heat-stressed conditions.

A combination of xanthine oxidase and xanthine, an enzymatic free radical-generating system [28, 29], primarily generates the superoxide anion, which is immediately dismuted into hydrogen peroxide (H2O2). Among antioxidants tested, only catalase, a scavenger of hydrogen peroxide, provided a significant protective effect against either heat or oxidative stress-induced apoptosis. The results may indicate that hydrogen peroxide is a contributing radical oxygen species for testicular cell apoptosis, although the presence of a small catalase-resistant fraction suggested that a small percentage of cells may undergo apoptosis through different mechanisms. Consistent with our findings, catalase has been reported to confer protection against various apoptosis-inducing stimuli, such as UV irradiation and cytotoxic drugs [21], and an appropriate concentration of hydrogen peroxide has been shown to induce apoptosis in a variety of cell types [15, 35, 36].

The intracellular pathway by which hydrogen peroxide induces apoptosis remains a matter of speculation. Hydrogen peroxide itself is a relatively unreactive molecule, and its peroxidative cellular toxicity is thought to be attributable to a highly reactive hydroxyl radical (·OH), which is produced as a result of metal-catalyzed Fenton or Harber-Weiss reaction [37]. However, in our experiment, scavenging of hydroxyl radical by DMSO or an iron chelator did not confer significant protection, suggesting that apoptosis-promoting pathway is not associated with the peroxidative damage of cellular components by hydroxyl radical.

Another free radical that may be involved in apoptosis is peroxynitrite. Peroxynitrite is generated by the interaction of nitric oxide (NO) and superoxide, and has been recently shown to induce apoptosis in several cell lines [38, 39]. It has been proposed that xanthine oxidase works with NO synthase to produce peroxynitrite in patients with varicocele [40]; and endothelial NO synthase, which was not detectable in normal germ cells, was detected in degenerating or apoptotic germ cells in human testis [41]. Because peroxynitrite serves as a substrate for catalase [42], and DCFH/DA can react with peroxynitrite [43] or NO [44], the results of present study do not rule out this possibility. Whether generation of peroxynitrite occurs in testis in a heat-stressed condition and is involved in the apoptotic process is an issue for further investigation.

Heat stress was shown to induce an elevation of intracellular peroxide levels, and recent studies have demonstrated the possibility that elevation of intracellular peroxide levels may be a signal triggering apoptosis. Several intracellular signals leading to cell apoptosis, such as activation of the nuclear regulatory protein NF{kappa}B [45], cross-linking of the Fas receptor [35], and activation of caspase [46], are associated with an increase of intracellular production of radical oxygen species, suggesting that these apoptosis-promoting pathways might involve the redox regulation system. Recently, the Fas system has been implicated as a key regulator of germ cell apoptosis in the testis [47]. Whether heat stress-induced apoptosis of testicular cells may be mediated by the Fas system remains to be determined.

Components of the antioxidant enzyme system have been investigated in the rat testis. As compared to the liver, the rat testis expressed equivalent levels of SOD activity but only 5% of the glutathione peroxidase activity and 2% of catalase activity [48]. As compared to somatic cells, such as Sertoli or peritubular cells, the testicular germ cells presented a different antioxidant composition characterized by a low glutathione-dependent enzyme activity [49]. These data indicated a low capacity for scavenging hydrogen peroxide (and peroxynitrite?) in the testicular germ cells. A considerable proportion of germ cells is known to be constantly deleted by apoptosis during normal spermatogenesis [34], and testicular germ cells may utilize intracellular peroxide levels as determinants for the extent of germ cell apoptosis. From the clinical standpoint, the present study may offer insight into the effectiveness of antioxidant treatments on male infertility [50–52], because the oligospermic condition of some patients with male infertility may be associated with constant exposure to increased oxidative stress in the testis as a consequence of temperature elevation following formation of varicocele [7, 8, 50, 53].

In conclusion, our data indicated the involvement of oxidative stress in the induction of apoptosis due to heat stress and suggested that oxidative stress itself is a direct inducer of apoptosis in the testicular cells. Hydrogen peroxide appears to be a contributing radical oxygen species responsible for the induction of testicular cell apoptosis, although there is a small fraction that is resistant to catalase treatment. This study presented a useful in vitro model for study of the mechanism of heat stress on testicular cell apoptosis.


   ACKNOWLEDGMENTS
 
The authors thank professor Aaron J.W. Hsueh at the division of Reproductive Biology of Stanford University School of Medicine for his help in preparation of this manuscript.


   FOOTNOTES
 
1 Correspondence: Hideya Kodama, Akita University College of Allied Medical Science, 1-1-1 Hondo, Akita city, 010-0041 Japan. FAX: 18 837-4396; kodamah{at}ams.akita-u.ac.jp Back

Accepted: March 12, 1999.

Received: June 16, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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