Effects of 60 Hz electromagnetic field exposure on testicular germ cell apoptosis in mice
Jin Sang Lee1,4, Sang Seok Ahn 2, Kyeong Cheon Jung 3, Yoon-Won Kim1,4 , Sang Kon Lee 1,2
1Institute of Medical Science, 2Department of Urology, 3Department of Pathology, 4Department of Microbiology, Hallym University School of Medicine, Chunchon, 200-060, Korea
Asian J Androl 2004 Mar; 6: 29-34
Keywords: electromagnetic field; testis; apoptosis; TUNEL; flow cytometry; 7-aminoactinomycin D
Aim: To evaluate the effects of 60 Hz extremely low frequency (ELF) elelctromagnetic field (EMF) exposure on germ cell apoptosis in the testis of mice. Methods: Adult male BALB/c mice (7 weeks of age) were exposed to a 60 Hz EMF of 0.1 mT or 0.5 mT for 24 h/day. A sham-exposed group served as the control. After 8 weeks of exposure, the mice were sacrificed. Germ cell apoptosis in the testis was assessed by histopathological examination, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) and flow cytometric examination of isolated spermatogenic cells stained with 7 aminoactinomycin D (7-AAD). Results: EMF exposure did not significantly affect the body and testis weights, but significantly increased the incidence of germ cell death. The distinguishing morphological feature of EMF exposure was a decrement in the number of well organized seminiferous tubules. Quantitative analysis of TUNEL-positive germ cells showed a significantly higher apoptotic rate in the 0.5 mT exposed mice than that in the sham controls (P<0.05), while the difference between the two exposed groups was insignificant. The TUNEL-positive cells were mainly spermatogonia. In flow cytometry analysis, the percentage of live cells [forward scatter count (FSC)high7-AAD-] was lower in the exposed groups than that in the controls (Figure 5A), but the decrease in viability was not statistically significant. Conclusion: Continuous exposure to ELF EMF may induce testicular germ cell apoptosis in mice.
Extremely low frequency (ELF) fields are defined as those having frequencies up to 300 Hz, which is a non-ionizing radiation having photon energy too weak to break the atomic bonds. Sixty Hz electromagnetic fields (EMF) were generated from human-made sources such as domestic electric devices, electric transport system, etc . There is little confirmed evidence about the effect of ELF EMF on human health and fertility . Exposure to ELF EMF have not had significant risk on implantation and developing fetus in animal studies [3-5], but may cause minor skeletal anomalies and a decrease in the number of female rats impregnated by exposed males. A short term but significant decrease in elongated spermatids was found in exposed mice . However, exposure to ELF EMF did not appear to induce dominant mutation in the germ cells of male mice . Our multi-generation study showed a consistent decrease in testicular weight in the second-generation mice exposed to ELF EMF without significant effects on implantation . Those results suggest that exposure to MF produces possible cytogenetic effect on spermatogenic cells. The present study was designed to investigate the effect of continuous exposure to ELF EMF on apoptosis of spermatogenic cells in mice.
2 Materials and methods
Seven week-old adult male BALB/c mice weighing 25-30 g were purchased from Daehan Biolink (Chung-buk, Korea) and acclimatized at the mouse facility at the Hallym University for 1 week before the experiments. The mice were housed under a 12 h light/12 h dark cycle, constant temperature (19-22 ) and 30 % - 70 % humidity and were fed distilled water and lab chow ad libitum. Experiments were performed in accordance with the Animal Experimentation Committee Regulation.
2.2 EMF generating device
The device was designed and constructed by the Department of Industrial Electronic Engineering, Dankook University (Figure 1) . It was adjusted to generate a ELF EMF of 60 Hz by three parallel rectangular coils in acryl frame. The International Radiation Protection Association (IRPA) guideline indicated that the permissible maximum magnetic flux density for occupational exposure is 0.5 mT (5 G) for 24 hours per day . Thus the magnetic field intensity was adjusted to 0.1 mT and 0.5 mT with a variation of less than 5 % as measured with a HI-3604 system manufactured by the Holaday Industries Inc (Cedar Park, TX, USA). Measurement of the EMF was performed at three locations in the chamber every week. The mice were housed in specially designed non-metallic polycarbonate cages fitted with non-metallic water bottles and placed on the tray of EMF-exposure device.
Figure 1. Magnetic field exposure system.
2.3 Experimental design
Fifteen mice were divided at random into 3 groups of 5 animals each. Two experimental groups were exposed to a 60 Hz magnetic field at 0.1 mT (1 G) or 0.5 mT (5 G) for 24 hours/day for 8 weeks and another group exposed to sham conditions served as the controls. The experiment was repeated three times. Each group of five mice was placed at the center of the exposure chamber in one cage. The body weight was recorded every week. After the end of the exposure the mice were sacrificed by cervical dislocation and both testes were excised and their weights recorded.
2.4 Testicular histology and scoring
One testis of each mouse was fixed in Bouin's solution. Five mm sections were stained with hematoxylin & eosin (H & E). The number of tubules per section was estimated to be between 120 to 160. A total of at least 100 tubules in each section were evaluated. They were scored from 1 to 10 according to the criteria described by Johnsen . The tubules without lumen are not included for analysis. In order to calculate a mean score, the number of tubules observed at a score is multiplied by the score number and the sum of all multiplications is divided by the total number of tubules scored.
2.5 In situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling staining (TUNEL)
For assessment of apoptosis in the testis tissue, the in situ Apoptosis Detection Kit (Takara, Shiga, Japan) was used. The paraffin-embedded tissues were cut into 5 mm serial sections, attached to silane-coated slides, deparaffinized in xylene and redehydrated in phosphate-buffered saline (PBS, pH 7.4). The prepared sections were then digested by incubation with 5 mg/mL proteinase K for 20 min at room temperature and washed three times for 5 min each in double-distilled water. Peroxidase and DNases were inactivated by incubating the sections in 2 % H2O2 for 5 min at room temperature. Sections were then rinsed three times with distilled water, treated with terminal deoxynucleotidyl transferase (TdT) reaction mixture containing TdT and FITC-conjugated dUDP and incubated in humidified atmosphere at 37 for 60 min. The reaction was stopped by transferring the slides to 2 side scatter count (SSC) buffer for 10 min (twice) and TB buffer (300 mmol/L sodium chloride, 30 mmol/L sodium citrate) for 10 min (twice) and were then incubated for 30 min in blocking reagent at room temperature, followed by washing in phosphate-buffered saline twice for 5 min. They were covered with peroxi-dased-conjugated anti-fluorescein isothiocyanate (FITC) antibody for 30 min at room temperature. The enzyme reaction was developed with 0.03 % 3'-diaminoben-zidine tetrahydrochloride containing 0.006 % hydrogen peroxide. As a negative control, TdT enzyme was excluded from the TdT reaction mix. The substrate reaction was stopped by rinsing the slides in cold tap water and the sections were finally counterstained with haematoxylin and mounted. On each slide, TUNEL positive cells were identified in 100 seminiferous tubules under a light microscope. Cells were scored as TUNEL positive when they showed intensely dark brown staining.
2.6 Preparation of germ cell-rich fraction and flow cytometry
The germ cell rich fractions isolated from the testis were stained with 7-aminoactinomycin for the identification of early and late apoptotic cells. Germ cells were isolated enzymatically. The mice were killed by cervical dislocation and the testes excised and placed in precooled PBS. The tunica albuguinia was removed and segments (1.5 mm or 2 mm in length) of seminiferous tubules were isolated. The fleshly isolated segment was pipetting vigorously and rinsed two times in PBS. After centrifugation at 1,500 rpm for 10 min, the supernatant was removed. The tubular fragments were treated with a solution containing 0.2 % trypsin (Sigma, St Louis, USA) and 2 g/mL DNase (Sigma) in a shaking bath at 800 oscillation/min for 15 min at 37 . The supernatant (containing germ cells) was passed through a nylon mesh. The germ cell rich pellet was washed twice in PBS and prepared for flow cytometry. Cell viability was approximately 75 % - 85 % as judged by trypan dye exclusion.
The cells were washed three times with PBS and incubated for 30 min at room temperature in the dark in PBS containing 7-aminoactinomycin D (7-AAD; Sigma). Samples were acquired on a fluorescence activated cell sorter (FACS) Calibre cytometer (Beckton Dickinson, USA) and emissions from 7-AAD were detected in the channels. The combined analysis of cell size forward scatter count (FSC) and 7-AAD fluorescence revealed the existence of four populations : living lymphocytes with a normal morphology (unmodified FSC/SSC) and a normal membrane integrity (7-AAD-); early apoptotic cells with a modified morphology (lower FSC and higher SSC) and a moderate membrane alteration (7-AADlow); late apoptotic cells, exhibiting a cell shrinkage comparable to early apoptotic lymphocytes (low FSC and high SSC), but showing a complete loss of membrane integrity (7-AADhigh); the fourth subset, characterized by extremely small size FSC and weak 7-AAD staining, corresponds to cell debris or apoptotic bodies. The latter was excluded in this study.
2.7 Statistical analysis
Data were expressed as meanSD. The GraphPad program (ISI Software, Philadelphia, PA, USA, version 3.01) was used. Comparisons were done using Kruskal-Wallis one way ANOVA and Dunn's multiple comparison test and P<0.05 was set as significant.
3.1 Body and testis weights
Figure 2. Effect of exposure to 60 Hz EMF on body weight (A) and testis weight (B). Data represent results from three separate experiments. Values in meanSD. No significant difference between different groups.
3.2 Testicular biopsy score
In the control group, the average testicular biopsy score was 9.740.09, whereas in the 0.1 mT and 0.5 mT exposed groups, they were significantly lower (8.860.23 and 8.760.05, respectively, P<0.05, Figure 3). The incidence of tubules at score 10 was significantly lower in the 0.5 mT than that in the 0.1 mT exposed mice (12.6 %3.8 % and 29.7 %8.4 %, respectively, P<0.05).
Figure 3. Testicular biopsy score. bP<0.05, compared with control. Difference between two exposed groups insignificant.
3.3 Testicular histopathology
A significantly increased number of dying germ cells appeared at certain seminiferous tubules in mice exposed to 0.1 mT and 0.5 mT EMF, while they were rarely seen in unexposed mice (Figure 4A, left column). The degenerating cells became lost from the epithelium as shown by a decrease in the number of spermatogonia that were characterized by condensed chromatin and shrinkage of cytoplasm as well as the presence of apoptotic bodies, suggesting of apoptotic death (Figure 4A, left column, arrows).
Figure 4. Effect of EMF on apoptosis of testicular germ cells. Testicular tissues stained with H & E (A, left column) or TUNEL technique (A, right column). TUNEL-positive cells lost from epithelium (A) and were counted in 100 seminiferous tubules (B). Data represent results of 3 separate experiments. bP<0.05, compared with control.
As depicted in Figure 4A (right column) and 3B, the degenerating cells were stained by TUNEL. Exposure to EMF increased the number of TUNEL-positive cells 7-10 folds as compared with the controls (Figure 4B). Statistically significant differences in the number of TUNEL-positive cells per seminiferous tubule were found between the 0.5 mT exposed group and the sham controls (P<0.05, Figure 4B). TUNEL-positive cells lost from the epithelium were characterized by degraded nucleus and condensed chromatin. TUNEL-postive cells without distinct morphological changes were also found at the light microscopic level, which were mainly spermatogonia along the basement membrane.
3.5 Flow cytometry analysis
There was a decrease in the percentage of live cells (FSChigh7-AAD-) in the exposed groups than in the controls (Figure 5A), but this decrease in viability was not statistically significant (Figure 5B).
Figure 5. Effect of EMF on early and late apoptosis of testicular germ cells. Germ cell stained with 7-aminoactinomycin D, followed by flow cytometry. Representative data (A) and summary results of flow cytometry (B) are depicted.
We performed the investigation in three separate experiments in order to reduce the processing time in the preparation of germ cells to avoid cell death and to document the reproducibility of results.
Quantitative analysis of seminiferous tubules showed a significant decrease in spermatogenesis in EMF-exposed mice. Therefore, the decrease in testis weight in the ELF EMF-exposed mice observed in previous studies [8, 13] might be associated with an increased incidence of germ cell apoptosis.
Highly increased numbers of TUNEL-positive cells were found to be restricted to certain tubules. Stage specific activation of spontaneous germ cell apoptosis occurs during spermatogenesis . Interestingly, exposure of seminiferous tubules to various apoptosis inducers, as heat and certain chemicals, led to a similar morphological pattern [15, 16], indicating that the pathway leading to apoptosis is strictly regulated and certain spermatogenic stages or germ cell types might be more vulnerable to apoptotic insult.
The distinguishing feature of testicular biopsy analysis was that the number of normal seminiferous tubules was decreased with the increase in EMF intensity, even though the difference in the testicular biopsy score between the two exposed groups was not significant. These findings might be related to the occurrence of stage nonspecific activation of apoptosis or to the disruption of apoptotic control.
It has been shown that intermittent EMF stimulation is more harmful biologically than continuous stimulation[6,17] and in mice exposed to ELF EMF for 44 weeks, spermatogenesis may be normal , which may indicate that continuous exposure to ELF EMF might produce habituation to field effects. In in vitro study, intermittent field exposure resulted in an increase in DNA strand break . However, continuous ELF EMF exerts harmful effect on prenatal development in mice .
In mice, spontaneous apoptosis was most commonly observed in spermatocytes, less frequently in spermatogonia and seldom in spermatids [18, 19]. In the present study, most of the TUNEL-positive cells were spermatogonia. In the study of germ cell apoptosis induced by exogenous glucocorticoid, the TUNEL positive cells observed was also mostly spermatogonia . Flowcytometric analysis in mice demonstrated that long term exposure to ELF EMF had a possible effect on the proliferation and differentiation of spermatogonia . These facts suggest that apoptosis induced by ELF EMF occurred through different pathways
It is well known that germ cell death is triggered by deprivation of gonadotropin or testosterone . Pineal melatonin production is suppressed and catecholamine increased by exposure to ELF EMF . By reducing the melatonin, EMF could increase the circulating estrogen level and change the endocrinologic environment. Such a hormonal change seems to be inhibitory to the hypothalamic-pituitary-gonadal axis. In conclusion, the present study indicates that continuous exposure to 60 Hz EMF induced testicular germ cell apoptosis in BALB/c mice.
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Professor S. K. Lee, Department of Urology,
Sacred Heart Hospital, Hallym University, School of Medicine, 153 Kyodong,
Chunchon, 200-060, Korea.