Effects Of 220 MHz Radiofrequency Radiation On Sperm Quality Of Rats

Background In recent years,male sperm quality has generally declined worldwide,which resulted in male reproductive problems and aroused public attention.In China,the incidence of infertility in 2015 was 15%,and 50%cases were ascribed to male factors.It was reported that from the 1980s to the beginning of this century,the sperm density,total sperm count and the percentage of normal sperm decreased by 23.84%,30.07%and14.15%respectively in Chinese adult males,which indicated that the sperm quality was not optimistic.Previous studies have shown that environmental factors were the main cause of the decline of sperm quality in male.Meanwhile,with the rapid development of science and technology,radiofrequency radiation(RFR)has been widely used in various fields,accordingly,the intensity and the complexity of exposure to RFR also increased,which has triggered the widespread concern about the effects of RFR exposure on sperm quality.It was reported that testis was one of the sensitive target organs to RFR.The changes of structure and function of testis will directly or indirectly affect the sperm quality.At present,most studies focus on the effect of RFR on sperm quality in living environment,but less on RFR in occupational environment.Therefore,it is necessary to explore the effect of RFR in occupational environment on sperm quality.In this study,220 MHz RFR,which is commonly used in radio communication,was selected as the exposure signal.The effects of 220 MHz RFR on sperm quality in rats were detected by the number,abnormality and survival rate of sperm.The related mechanism was explored from the aspects of testicular morphology,Leydig cells(LCs)and Sertoli cell(SCs)secreting function and testicular cell apoptosis.Objective To clarify the effect of 220 MHz RFR exposure on sperm quality in rats and elucidate its mechanism.Part Ⅰ:The effects of 220 MHz radiofrequency radiation on sperm quality in rats Materials and Methods 1.RFR equipment:RFR exposure system consists of signal generator,amplifier,coupler,power sensor and radiation antenna,which generates modulated pulse wave with adjustable output power.2.Animal groups and exposure parameters:a total of 60 healthy adult male Sprague-Dawley(SD)rats(body weight 237.1±8.1 g)were randomly divided into two RFR groups(50 W/m~2 group and 100 W/m~2 group)and Sham group with 20 animals in each group.RFR exposure parameters were as follows:1)50 W/m~2 group:average power density 50 W/m~2,specific absorption rates(SARs)of whole body and testis were 0.030W/kg and 0.014 W/kg respectively.2)100 W/m~2 group:average power density 100 W/m~2,SARs of whole body and testis were 0.061 W/kg and 0.029 W/kg respectively.3)Sham group:animals were treated under the same experimental condition without RFR exposure.Rats in two exposure groups were individually placed in plexiglass boxes,and were then placed on the shelf in the RFR chamber,with head facing antenna.The rats were exposed or sham exposed to 220 MHz RFR for 30 d(1 h/day).3.Detection methods:1)Anal thermometer was used to measure the changes of anal temperature in rats before and after exposure to 220 MHz RFR.2)During 30 days of exposure,the changes of body mass of rats were recorded every 3 days.Besides,growth curve of body weight and weight gain of rats in three groups were calculated.3)Bilateral testes were weighted and testis index was calculated.4)Sperm suspension was obtained from isolated bilateral cauda epididymites,and then used to observe under microscope and analyze the number,the abnormality and the survival rate of sperms.Results 1.Effect of 220 MHz RFR on anal temperature of rats The results of anal temperature showed that the anal temperature of rats in the three groups changed less than 1℃before and after exposure to 220 MHz RFR for 1 h.There was no significant difference in the anal temperatures among the three groups(P>0.05).It suggested that 220 MHz RFR exposure did not cause significant change in body temperature.2.Effect of 220 MHz RFR on general conditions of rats During the whole experiment,eating,drinking,hair,beard and mental state as well as body weight of rats showed no significant changes in 50 W/m~2 group and 100 W/m~2group,compared with Sham group.After exposure to 220 MHz RFR for 30 d,there were no significant differences in body weight gain,bilateral testicular weight and testis index among three groups(P>0.05).It suggested that 220 MHz RFR exposure did not affect the general health status of rats.3.Effect of 220 MHz RFR on sperm quality of rats3.1 The results of sperm count showed that compared with Sham group,the number of sperms decreased significantly in 50 W/m~2 group(P<0.05)and 100 W/m~2 group(P<0.01),and the effect was more obvious with the increase of exposure dose.3.2 The results of abnormality of sperm showed that compared with Sham group,the abnormality of sperm increased slightly in 50 W/m~2 group(P>0.05),and increased significantly in 100 W/m~2 group(P<0.01).3.3 The results of survival rate of sperm showed that compared with Sham group,the survival rate of sperm decreased significantly in 50 W/m~2 group(P<0.05)and 100W/m~2 group(P<0.01),and the effect was more obvious with the increase of exposure dose.Part Ⅱ:The mechanism study of 220 MHz radiofrequency radiation induced sperm quality degradation in rats1.RFR equipment:Same as the Part I.2.Animal groups and parameters:Same as the Part I.3.Detection methods:1)Hematoxylin and Eosin(HE)staining was used to observe the morphological changes in testis,and the diameter of seminiferous tubules was measured by the cross method.2)Immunohistochemical staining was used to immunolocalize the expression of OCT4,a specific marker of spermatogonial stem cells in testis.3)Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of testosterone(T)in serum and the level of SCs secretory factors in testis,including glial cell-derived neurotrophic factor(GDNF),stem cell factor(SCF),transferrin(TRF),and androgen binding protein(ABP).4)The expression levels of GDNF,SCF,and apoptotic related proteins(Csapase 3,BAX and BCL 2)in rat testis were detected by western blot(WB).5)The mRNA levels of GDNF and its receptors GFRa1,SCF and its receptors C kit,17β-HSD3,and Caspase 3 in testicular tissue were evaluated using quantitative real time-polymerase chain reaction(qRT-PCR).6)Immunofluorescence staining was applied to immunolocalize the expression of Cleaved-Caspase 3 protein in testis.Results 1.Effects of 220 MHz RFR on testis histomorphology of rats The results of HE staining showed that the layers in the seminiferous tubules of three groups were well organized from external to internal as basal lamina,spermatogonia,spermatocyte,and spermatid.No statistically significant difference was observed in testicular morphology and diameters of seminiferous tubules(P>0.05)among three groups.It suggested that 220 MHz RFR exposure had no obvious effect on testicular morphology and structure in rats.2.Effects of 220 MHz RFR on spermatogonial stem cells of rats Immunohistochemical results showed that the protein level of OCT4 in the testis of rats exposed to 220 MHz RFR for 30 d did not change significantly,compared with Sham group(P>0.05).It suggested that 220 MHz RFR exposure had no obvious effect on spermatogonial stem cells.3.Effects of 220 MHz RFR on secreting function of LCs in rats The result of ELISA showed that compared with Sham group,the levels of serum T decreased statistically in 50 W/m~2 group(P<0.05)and increased significantly in 100W/m~2 group(P<0.01).The result of qRT-PCR showed that the expression of 17β-HSD3(T synthesis rate-limiting enzyme)did not change significantly in 50 W/m~2 group(P>0.05),but increased significantly in 100 W/m~2 group(P<0.01).These results suggested that 220 MHz RFR could affect the synthesis and secretion of LCs.4.Effects of 220 MHz RFR on secreting function of SCs in rats ELISA results showed that compared with Sham group,the concentration of GDNF,SCF,TRF and ABP in testicular tissue of rats in two exposure groups had no significant change(P>0.05).Western Blot results showed that there were no significant changes in the protein levels of GDNF and SCF in testicular tissues among three groups(P>0.05).qRT-PCR results showed that there was no significant difference in the mRNA levels of GDNF and its receptors GFRa1,SCF and its receptors C-kit in testicular tissue among three groups(P>0.05).These results suggested that 220 MHz RFR exposure could not affect the secreting function of SCs.5.Effects of 220 MHz RFR on testicular cell apoptosis Immunofluorescence staining of Cleaved-Caspase 3 protein in testis showed that compared with sham group,the level of Cleaved-Caspase 3 increased obviously in 50W/m~2 group(P<0.05)and 100 W/m~2 group(P<0.01).In addition,Cleaved-Caspase 3positive cells were mainly distributed in the inner of seminiferous tubules.WB results showed that the protein levels of Caspase 3 and the apoptotic index(BAX/BCL 2 ratio)increased significantly(P<0.01)in two exposure groups compared with the sham group.The results of qRT-PCR showed that the expression of Caspase 3 mRNA in testis increased slightly in 50 W/m~2 group(P>0.05)and 100 W/m~2 group(P>0.05).These results suggested that 220 MHz RFR exposure could induce testicular cell apoptosis,and the effect was more obvious with the increase of exposure dose.ConclusionsUnder the experimental conditions:1.220 MHz RFR exposure could decrease the sperm quality in adult male rats,and the effect was more obvious with the increase of exposure dose.2.The secreting function of LCs could be affected by 220 MHz RFR exposure.3.220 MHz RFR could induce the apoptosis of testicular tissue cells.4.The impairment of sperm quality may be related to the change of T level and the apoptosis of testicular cells induced by RFR.