The Effects Of Extremely Low Frequency Electromagnetic Fields On Male Reproductive Toxicity In In Vitro And In Vivo Study

Background:The widely exposure of modern society to extremely low frequency eletromagnetic fields(ELF-EMF) generated from the production, transmission and use of electricity has raised considerable scientific and public concerns about the potential risks to human health. Due to male genital organs very sensitive to environmental hazards and the rate of male infertility dramatically increasing these years, the impacts of ELF-EMF on male reproduction have become the focus of concern. However, the results of epidemiological and experimental studies remain contradictory and whether ELF-EMF exposure contributes to male reproductive toxicity still inconclusive. Previous studies indicate that the DNA damage of male germ cells might be one of causes of male reproductive disorder. Spermatogenesis is a process of spermatogonia differentiate to spermatids and plays an essential role in male reproduction. However, there is little study of ELF-EMF exposure on spermatogenesis.Methods:① A mouse spermatocyte-derived GC-2 cell line was intermittently(5 min on and 10 min off) exposed to 50 Hz ELF-EMF at an intensity of 1, 2 or 3 mT. For each exposure group, a corresponding sham exposure was conducted. The effects of ELF-EMF exposure on cell viability and its prolonged effects were measured by Cell Counting Kit-8(CCK-8). Through the use of an alkaline comet assay and immunofluorescence against γH2AX foci, we detected DNA strands breaks. Using a formamidopyrimidine DNA glycosylase(FPG)-modified alkaline comet assay, we measured oxidative DNA base damage. In order to validate the protocol, hydrogen peroxide was used as a positive control in the viability and the comet assay and etoposide was used as a positve control in the detection of gamma-H2 AX foci. In addition, cells cultured in an incubator without ELF-EMF signal were served as negative control. All experiments were performed at least in triplicates.② Adult male Sprague-Dawley(SD) about 8 weeks were exposed to 50 Hz ELF-EMF of 500 μT 4 h/day, 7 days/week for 4 and 8 weeks. The sham-exposed rats were placed on the same exposure system with switch turned off. During the exposure, we measured the body weight of rats once per week. After the last exposure, we measured the mass of testes and epididymes, detected serum testosterone level and analyzed the total number of cauda epididymis sperm and abnormal sperm ratio. H&E staining sections of testes were used to evaluate the histological change of testes and tubular diameters and the percentage of damaged tubules. PAS sections with hematoxylin counterstaining were performed to aid in the identification of the 14 different stages of the seminiferous epithelium cycle. Through with meiotic chromosomal spreads and immunofluorescence for SCP3 and γH2AX, we classified each spermatocytes as leptotene, zygotene, pachytene and diplotene and analyzed the percentage of each stage in meiosis I. Germ cells undergoing apoptosis in the seminiferous epithelium were detected by TUNEL assay. The oxidative stress level of testes were evaluated the contents of MDA and the activities of SOD and CAT.Results:① We found that the cell viability and the prolonged effects of ELF-EMF exposure in GC-2 cells were not significantly changed compared with the sham exposure. In addition, we found that ELF-EMF exposure resulted in significant increases of DNA damage parameters at 3 mT, including tail DNA%, tail length, tail moment and olive tail moment. ELF-EMF exposure at 3 mT also significantly increased γH2AX foci formation. However, we observed that ELF-EMF exposure did not significantly induced oxidative DNA base damage at any intensity.② After male SD rats were exposed to ELF-EMF for 4 and 8 weeks, several biological endpoints related to testicular function and spermatogenesis were measured, including the following: body mass, masses of testes and epididymis, sperm count and abnormal sperm ratio in the caudal epididymis, serum testosterone level, testicular histology, frequency of 14 stages of the cycle of the seminiferous epithelium and of 4 stages of meiosis I, germ cell apoptosis and testicular oxidative status. No significant differences were found in these biological endpoints between the sham control and the exposed rats in either the 4-week or 8-week exposure period. ELF-EMF exposure had no effects on spermatogenesis and reproductive function in adult male rats.Conclusion:This study investigated the toxic effects of ELF-EMF exposure on male reproduction in vitro and in vivo focused on the DNA damage in male germ cells and spermatogenesis in rats, respectively. In in vitro study, we found ELF-EMF exposure at 3 m T could induce DNA strand breaks but it could not induce oxidative DNA base damage. Moreover, the cell viability was not altered by ELF-EMF exposure. These in vitro results suggest that ELF-EMF might induce male reproductive toxicity. In in vivo study, following male adult rats exposed to ELF-EMF at 500μT for 4 and 8 weeks, the sperm quality and the process of spermatogenesis were not significantly changed. In addition, we found ELF-EMF exposure could not induce germ cell apoptosis and oxidative stress in testis tissue. These results suggest that ELF-EMF exposure at low intensity could not produce reproductive toxicity in vivo. These results may provide new experimental data for the male reproductive effects of ELF-EMF.