| Objective: Microwave Radiation (MWR) is an important environmentalelectromagnetic pollutant with the rapid development of wireless communicationtechnology and mobile phone users in the world. It has been reported that MWRexposure may cause damage on male reproduction system, and it is therefore importantto clarify the route and mechanism of the MWR damage. Circadian rhythms of thephysiological and behavioral functions have been widely demonstrated in mammals,which result in different sensitivities of animals to toxicants and MWR at different timesof a day. This project is to study the circadian rhythm of male reproduction system andmelatonin (melatonin, MEL) toxicity induced by MWR with the combination ofchronobiology and reproductive endocrine toxicology on the basis of established animalmodel of microwave radiation, and to ellucidate the mechanism underlyingGATA-4/SF-1signal pathway that inhibits testosterone secretion in TM3Leydig cells,so that to provide with new ways in prevention and treatment of the male reproductionmalfunction caused by MWR.Methods:(1) Chronotoxicity of reproductive system in male rats exposed to MWR.Animals in circadian rhythm (as indicated by melatonin measurements) were dividedinto several groups and exposed to1800MHz RF at205μw/cm2power density for2h/dfor32days at different zeitgeber time (ZT) points, viz., ZT0, ZT4, ZT8, ZT12, ZT16and ZT20. Sham-exposed animals were used as controls in the study. From each rat,0.1ml blood samples were colleted from the caudal vein every4hours (ZT0, ZT4, ZT8,ZT12, ZT6and ZT20) after MWR exprosure to determine the concentration oftestosterone using the ELISA KIT. Testicular and epididymis tissues were collected forHE pathological section and assessed for testosterone levels, daily sperm productionand sperm motility, testis marker enzymes γ-GT and ACP, cytochrome P450side-chaincleavage (p450cc) mRNA expression, and steroidogenic acute regulatory protein (StAR)mRNA expression. The data obtained were then fitted for cosinor analysis as expressed in Eq.F(t)=M+Acos(w t+j).(2) Circadian alterations of melatonin secretion in male ratsexposed to MWR. The MWR and blood samples celection were the same as the firstsection. The concentration of MEL was determined by the ELISA KIT. Pineals werecollected after MWR from Sham and MWR groups at the end of exposure timepointsrespectively to determine melatonin synthetase NAT mRNA by Real-time PCR.(3)Signal pathway of testosterone secretion inhibition by melatonin and effects of MWRon testosterone secretion in TM3cells. TM3Cells were plated in the presence orabsence of melatonin (0,10-6,10-8,10-10and10-12mol/L) and melatonin receptorantagonist Luzindole (1μmol/L) for3h in multiwell-plates (six wells). Thenintracellular cAMP and testosterone levels in the supernatant were measured by ELISA.Gene and protein expression of the pathway foctors (Mel1a，GATA-4，SF-1，StAR，P450cc and3β-HSD) were determined by Real-qPCR and Western blot.Immunohistochemical (GATA-4) and immunofluorescence (SF-1and Mel1a) analysiswere used to verifiy the signal pathway in vivo model by intra-testicular injection ofmelatonin in C57BL/6J mice. The TM3Cells were exposed to1800MHz MWR (205μw/cm2) for2h to determine mitochondrial membrane potential by flow cytometry. Theintracellular cAMP and testosterone levels in the supernatant were measured by ELISA.Gene and protein expression of SF-1, StAR and P450cc were determined by Real-qPCR.Results:(1) MWR exposure led to histopathologic changes in testicles, lower dailysperm production and sperm motility, down-regulated activity of γ-GT and ACP, withthe most pronounced change in the MWR0group. Rats exposed to MWR exhibited analteration in circadian rhythms of plasma testosterone, which were absent in MWR0,MWR8, MWR12and MWR20groups. At the same time, the daily average testosteronelevel was most reduced in the MWR0group. Two testosterone synthesis regulatoryfactors of P450cc and StAR mRNA lost their circadian rhythms after MWR exposureand changed most dramatically in the MWR0group. These results suggest that the malereproduction system in rat was most sensitive to MWR exposure at ZT0.(2) MWRexposure decreased daily average testosterone levels, especially in the MWR0group.MWR exposure disturbed the circadian rhythms of plasma melatonin in MWR8, MWR16and MWR20groups, with the greatest amplitude reduction in MWR16group. These findings indicate that the biggest change in plasma melatonin occured at ZT16:00. Thecircadian rhythm of the NAT gene exprsession in pineal changed after MWR exprosure,with the most down-regulation at ZT16:00timepoint. Simultaneously, MWR exposurealtered the mean values and amplitudes of the NAT gene exprsession rhythm, with adelayed peak phase.(3) By measurements in vitro, it was shown that melatonininhibited cell proliferation, lowered the testosterone levels in the supernatant, decreasedintracellular cAMP, and down-regulated mRNA expression of nuclear transcriptionfactor GATA-4and its target genes SF-1(NR5A1), StAR, P450scc (CYP11A1) and3β-HSD, as well as their protein expression. However, the effects of melatonin wereblocked by Luzindole, especially in high melatonin dose level. At0.5h,1h and2h afterintra-testicular injection of melatonin, plasma testosterone level was reduced, Mel1a,GATA-4, SF-1(NR5A1) expression was downregulated, but these effects disappeared at4h. MWR exposure decreased testosterone levels in the culture supernatant of TM3cells, induced reduction of intracellular cAMP and mitochondrial membrane potentiallevels, down-regulated mRNA expression of testosterone synthesis regulatory factor ofSF-1, StAR and P450scc.Conclusions:(1) Animal and cell models of microwave radiation were established, andchronotoxicity of reproductive function and testosteron secretion exposed to MWR wasrevealed.(2) MWR altered circadian rhythms of melatonin secretion and melatoninsynthetase NAT mRNA, and melatonin mediated the reproductive chronotoxicity in ratsby MWR.(3) Melatonin inhibited testosterone secretion via the GATA-4/SF-1signalingpathway in vitro and vivo, with the GATA-4as a key regulatory factor. |
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