Background: It has been reported that cellphone radiation (CR) is related to higher risk of many health problems, but whether CR can impair the expression of rate-limiting enzymes of testosterone synthesis has seldom been studied. Objective: To evaluate the effects of CR on the expression of steroidogenic acute regulatory protein (StAR) in the testes tissue and the sperm quality of adult male mice. Methods: Forty 3-month-old male mice, 22 - 26 g, were randomly assigned into four equal groups (n = 10 per group): the control group and three CR exposure groups including 8-hour group, 16-hour group and 24-hour group. Each mouse received different dosages of CR exposure for seven consecutive weeks. Semen in the epididymis, intratesticular testosterone (ITT) concentrations, and the expression of StAR were measured at the end of experiment. Results: The sperm number and motility, and the ITT concentrations in 24-h group were significant lower than those in the control group (P < 0.05, P < 0.01, and P < 0.05 respectively). No obvious changes were seen in 8-hour group and 16-hour group compared with the control (both P > 0.05). Similarly, only the expression of StAR in the 24-h group was significantly decreased after the exposure of CR (P < 0.05). Both the 8-h group and the 16-h group did not change much in the expression of StAR compared with the control group (P > 0.05). Conclusions: High dose exposure of CR can reduce the expression of StAR and ITT concentration, and then suppress the serum quality.
Nowadays most men own cellphones, and this phenomenon arouse the concern over the potential effects of cellphone radiation (CR) exposure on human health, including human fertility. In fact, it has been proved that cellphone can emit electromagnetic radiation at a frequency of between 800 and 2200 MHz and can be absorbed by the body [
Forty 3-month-old male C57BL/6 mice (weighted 22 g ± 3 g) were purchased from the Animal Center of Sun Yat-sen University (Guangzhou, China) and were kept on a 12 h-day/12 h-night schedule (lights on from 19:00 to 07:00 h) at constant temperature (22 ± 1˚C) and humidity (60%). The present experiment was supervised by the Sun Yat-sen University Institutional Animal Care and Use Committee.
All of the male mice were randomly arranged into four experimental groups (10 mice per group): one control group and three CR-exposure groups receiving different dosage of CR (8 hours, 16 hours or 24 hours per day) following to the method mentioned in former articles [
After the experiment, the excised epididymides were cut into small pieces and placed in 1 ml F12 media containing 0.1% bovine serum albumin prewarmed to 34˚C and incubated for 15 min to facilitate sperm transmigration from the epididymis. The number of sperm was counted using a hemocytometer. Each specimen was counted twice and averaged. Fresh sperm was loaded on a prewarmed glass slide to examine sperm motility by microscopy. Four randomly chosen fields of each sample were selected to assess sperm motility. The percentage of sperm with forward and progressive activity was counted to assay sperm motility.
The ITT was measured according to the following method: The right testis of each mouse was harvested and decapsulated. Two pieces weighted 50 mg each were picked out and were placed separately into two 1.5 ml microfuge tubes containing 1.0 ml Medium 199 (Corning Cellgro, VA, USA). Then, the testis tissue was incubated for 2 h at 34˚C, and then centrifuged for 5 min at a speed of 10,000×g. The supernatant was collected and frozen at −80˚C for future testosterone assay. Testosterone assay was conducted using a commercially available enzyme-linked immunosorbent assay kit (R & D Systems, MN, USA) following the manufacturer’s instructions.
Testis tissue fixed with 4% paraformaldehyde werecryo-embedded in an optimal cutting temperature medium (Sakura Finetek, Torrance, CA, USA) and were sectioned at a thickness of 4 μm. The sections were blocked by incubation in 5% normal serum and 0.1% Triton X-100 (Hyclone, Logan, Utah, USA) in PBS for 1 h at room temperature, and then incubated with primary antibody (Mouse polyclonal to StAR (D-2), 1:200, Santa Cruz, catalog number: sc-166821) overnight at 4˚C. The sections were incubated with secondary antibody (Goat anti-mouse IgGAlexa594, 1:1000 Invitrogen, Carlsbad, CA, USA, catalog number: A11032) for 30 min at room temperature followed by DAPI (blue, 1:1 Millipore, Darmstadt, Germany, catalog number: S7113) staining of nuclei next day. PBS replaced the primary antibody as a negative control. All the images were captured using an LSM710 confocal microscope (Zeiss, Jena, Germany) and were analyzed using the Image J software (University Health Network, USA).
Total RNA from the testes of these mice was extracted using an RNeasy mini kit (Qiagen, Tokyo, Japan) following the manufacturer’s instruction. Reverse transcription reactions were performed by utilizing murine leukemia virus reverse transcriptase and oligo-dT primers (Fermentas, Lithuania). Real-time PCR was conducted using the ThunderbirdSYBR qPCR Mix (Toyobo, Osaka, Japan) according to the protocol of the manufacturer’s. β-actin mRNA was selected as an internal control. A Light Cycler 480 Detection System (Roche, Basel, Switzerland) was chosen to detect signals. The primer sequences are depicted as follows: 5-GGGCATACTCAACAACCAG-3 (upper primer) and 5-ACCTCCAGTCGGAACACC-3 (lower primer) for StAR, and 5-TCGTGCGTGACATTAACGAG-3 (upper primer) and 5-ATTGCCTATCGTGATGACCT-3 (lower primer) for β-actin.
Results were presented as mean ± s.e.m. One-way ANOVA was utilized to assess the significance of differences in each group. In all cases, P < 0.05 was considered to be statistically significant. The statistical data were analyzed by using SPSS, version 21.0 (SPSS Inc., Chicago, IL, USA). All analytic results were performed using the GraphPad Software package (GraphPad Software, La Jolla, CA, USA).
To determine whether sperm number and motility changed after the exposure of CR, epididymal sperm from the mice were analyzed. Results indicated that there was a significant decrease in sperm number and motility in the 24-h group compared with the control group (sperm number: [9.34 ± 3.87] × 106 cells/ml vs. [14.19 ± 4.85] × 106 cells/ml, P < 0.05; sperm motility: [27.73 ± 7.45]% vs. 41.21 ± 10.00%, P < 0.01) (
As shown in
The effects of CR at different exposure time on the expression of StAR are shown in
The effect of CR exposure on the expression levels of StAR genes was determined by quantitative real-time PCR (
1, P < 0.05). No statistical differences were seen between the other two experimental groups and the control group (both P > 0.05).
We have previously proven that exposure to CR could decrease the serum testosterone concentration and inhibit sexual behaviors of adult male mice. This study further verifies that CR exposure can also decrease the ITT and sperm quality and demonstrates for the first time that CR exposure can reduce the ex-
pression of one of the rate-limiting enzyme of testosterone synthesis, StAR.
Male infertility has been a public problem all over the world. Evaluating the possible side effects from use of some new technologies is critical to illuminatethis problem. With an increasing worldwide usage of cellphones, identifying the risks of cellphone is particularly crucial. Men always carry cellphones in their pants pockets which make them at a risk of exposure to harmful microwaves and might do harm to their fertility. Many researches have demonstrated that cellphone can affect the fertility of males. Ji-Geng Yan et al. has proved that carrying
cellphones near reproductive organs could negatively affect fertility of male rats [
In the present study, we examined the sperm in the epididymis and observed that the sperm number and motility decreased significantly after long period exposure of CR (24-h group), but didn’t change much after short or middle period exposure (8-h group and 16-h group). This result was similar with the outcomes of many other researches [
In order to explore the mechanism of it, we set up to investigate the effects of CR exposure on the expression of the rate limiting enzyme, StAR, in the testis of adult male mice. To the best of our knowledge, this is the first time for this topic conducted on adult male animal mode. The results showed that no statistically changes of the StAR expression in testis tissue were seen in the 8-hour group and 16-hour group. Only the StAR expression in mice’s testis in 24-hour group was statistically lower than that of the control group. These data showed that low and middle dosage exposure to CR did not affect the expression of StAR in the testis of adult male mice, but high dosage exposure to CR can reduce the expression of StAR in testis tissue, which indicated that CR might act in a dose-dependent manner.
Our study had several limitations. Further studies are needed to explore whether CR exposure affects rate-limiting enzymes of synthesis including cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) to make further efforts to elucidate the mechanism of it. Another limitation is that the number of mice in this study is relatively small, which may influence the outcomes of statistical analysis.
In conclusion, the present study demonstrated that high dose of CR exposure can suppress the express of StAR in the testis tissue, which lead to the decrease of the testosterone synthesis and the ITT concentration, and then inhibit spermatogenesis. This result indicates that reducing CR exposure time is very important to decrease the injury to male fertility.
This work was funded by the Fundamental Research Funds for the Central Universities (16ykpy44) and Natural Science Foundation of Guangdong Province (2016A030310142).
Zang, Z.-J., Gao, Y., Huang, S.-Z., Ji, S.-Y., Jiang, M.-H. and Shu, J. (2017) Effects of Cellphone Radiation on Sperm Quality and the Expression of StAR in the Testis of C57BL/6 Mice. Occupational Diseases and Environmental Medicine, 5, 79-87. https://doi.org/10.4236/odem.2017.54008