| Background:With increasing utilisation of nuclear technologies in the energy production, medicaland industrial applications, the likelihood of radiation exposure to occupational workers,patients and public have been increasingly highlighted for special attention. The directinteractions of ionizing radiation (IR) with target biomolecules, the socalled ionizationprocess, which occurs on a time scale under10–14second, can only be prevented byphysical shielding during IR. In major part, detrimental effects of IR on biological tissueare mediated via indirect interactions, which increased production of hydroxyl radicals(OH) by radiolysis of H2O. It has been demonstrated by Ohsawa et al. hydrogen (H2)could selectively reduce cytotoxic reactive oxygen species (ROS), such as OH andperoxynitrite (ONOO) in vitro, and exert therapeutic antioxidant activity in a rat middlecerebral artery occlusion model for the first time. Recently, our department demonstratedthat H2pretreatment could protect cultured cells, intestine and heart in mice from ionizingradiation, and improve the30-day-survival rate in irradiated mice. Besides, Terasaki et al.suggested that H2therapy could be a useful way for protection in radiation lung injury inmice. Results with these show the potential of H2as an effective radioprotectant withoutknown toxic side effects, but the underlying mechanism involved in the radioprotectionrole of H2in vivo is not reported as yet. Spermatogenesis is a compound process of thegerm cell of male proliferation and maturation from spermatogonia to spermatozoa.Spermatogenesis is especially sensitive to ionizing radiation (IR); doses as low as0.1Gyare known to cause damage to spermatogonia. Testis was chosen to study the underlyingradioprotective mechanism and role of H2, because of the availability of assays in themouse to detect radiation damage and because testis is one of the most sensitive targets inman that is damaged during irradiation.Contents：1. Radioprotective role of H2on irradiated germ cells in mice1.1Radioprotective role of H2on irradiated spermatogonia1.1.1Radioprotective role of H2on early apoptosis of spermatogenic cells1.1.2Radioprotective role of H2on viability of spermatogenic cells1.2Radioprotective role of H2on spermatogenesis1.2.1Radioprotective role of H2on daily sperm production 1.2.2Radioprotective role of H2on sperm count, motility and morphology1.3Radioprotective role of H2on irradiated testicular tissues in mice1.3.1Radioprotective role of H2on testis index1.3.2Radioprotective role of H2on morphologic observation in testis1.3.3Radioprotective role of H2on tubule differentiation index2. Radioprotective mechanism of H2on irradiated germ cells in mice2.1Identification of OH that is reduced by H22.1.1Identification of OH that is reduced by H2by the Fenton reaction2.1.2Identification of OH that is reduced by H2by the radiolysis of H2O2.1.3Identification of OH that is reduced by H2in vivo2.2Radioprotective role of H2on oxidative stress in testis2.2.1Radioprotective role of H2on testicular antioxidant status2.2.2Radioprotective role of H2on testicular oxidative damage2.2.3Determination of H2concentration in vivoMaterial/Methods:1. H2was dissolved in physiological saline for2h under high pressure (0.4MPa) to asupersaturated level using hydrogen-rich water-producing apparatus produced by ourfaculty.2. Animal protocols were approved by the Animal Care and Use Committee of SecondMilitary Medical University. Eight-week old male C57BL/6or BALB/c mice wererandomly divided into non-irradiated control, H2only, radiation+saline andradiation+H2groups. The mice received total-body irradiation in a holder designed toimmobilize unanaesthetized mice such that the abdomens were presented to the beam.3. At12h after exposure, testes were removed, and the testes of each mouse were fixed inBouin’s fluid, dehydrated in a graded ethanol series and embedded in paraffin, thensectioned to5-μm-thick and mounted on glass slides. These sections from every blockwere both used for hematoxylin&eosin staining and terminal transferase mediated endlabeling (TUNEL).4. mice were sacrificed by cervical dislocation under isoflurane anaesthesia at29d afterirradiation. Body weight and testis weight were recorded. The left testis was stored at–20°C for spermatid head counts. The right testis was fixed in Bouin’s fluid forhistological analysis.5. mice were sacrificed by cervical dislocation under isoflurane anaesthesia at35d and56d after irradiation. Tubule differentiation index (TDI), daily sperm production, andepididymal sperm quality were evaluated.6. To identify OH that H2reduces, we produced OH by the Fenton reaction which is aniron-salt-dependent decomposition of hydrogen peroxide (H2O2), generating the highlyreactive OH, and semiquantified the levels of OH by spin-trapping using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the free radical trapper.7. We also produced OH by radiolysis of H2O, the process produces the free radicalssuch as OH, atoms or molecules containing unpaired electrons, which tend to behighly chemically reactive, and semiquantified the levels of OH using2-[6-(4’-hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoate (HPF).8. HPF probe (15μl,25μM) was given intratesticularly in anesthetized mice20minbefore IR. Testes were quickly dissected from the anesthetized mice after IR andprepared for cryosection.9. The testicular superoxide dismutase (SOD) activity, concentrations of glutathione(GSH), and the levels of malondialdehyde (MDA), protein carbonyl and8-hydroxy-2’-deoxyguanosine (8-OHdG) were assessed by the commercial kits.10. A H2microelectrode (Unisense) was penetrated into the testis, the negative currentobtained from the H2microelectrode was converted to regional H2concentration usinga calibration curve generated from known levels of H2saturated saline.Result：1. Radioprotective role of H2on irradiated germ cells in mice1.1Radioprotective role of H2on irradiated spermatogonia1.1.1Apoptotic cells were observed mainly in spermatogonia and spermatocytes, butnot in spermatids or sperm of all experimental groups. In H/E staining, the majority ofabnormal spermatogonia showed condensation of nuclear chromatin; however, the typicalmorphological characteristics of apoptosis, i.e. margination of chromatin and nuclearfragmentation, were rare.1.1.2Quantitative analysis for the spermatogonia and spermatocytes revealed that theapoptotic findings in the H2groups were significantly less than those of control groups.1.2Radioprotective role of H2on spermatogenesis1.2.1Testicular spermatid head counts were evaluated29d after irradiation toexamine the ability of A1through B spermatogonia to survive and differentiate into latespermatids. As predicted, after exposure of mice to0.5Gy,1.0Gy,2.0Gy and4.0Gyradiation, spermatid head counts were11.8×107/g,7.0×107/g,4.1×107/g and2.9×107/gin the H2groups, respectively. These were1.4-fold,1.5-fold,1.6-fold and1.7-fold higherthan those of the non-H2groups, which were77.7%,45.9%,27.1%and18.8%of normalnonirradiated control level.1.2.2We also evaluated cauda epididymal sperm morphology, which is a meaningfulparameter to assess the fertilizing ability of spermatozoa. Sperm quality in the H2groupswere significantly higher than those of control groups.1.3Radioprotective role of H2on irradiated testicular tissues in mice 1.3.1Body weight and organ weight were recorded at sacrifice, and organ index wascalculated (organ index=organ weight/body weight×100). Organ indices of testis in the H2groups were significantly higher than those of the non-H2groups.1.3.2At29d after irradiation, spermatozoa and spermatids were decreased or absentin some tubules, and disordered and shrinking tubules were observed. All these changeswere ameliorated by H2pre-treatmen. As shown, H2pre-treatment significantly reduced theseminiferous epithelium injury caused by IR.1.3.3At35days after exposure, survival of the stem spermatogonia were evaluatedusing the TDI (tubule differentiation index), which is the percentage of tubules thatcontained three or more differentiated spermatogenic cells derived from cells that werestem cells at the time of irradiation. TDI in the H2groups were significantly higher thanthose of control groups.2. Radioprotective mechanism of H2on irradiated germ cells in mice2.1Identification of OH that is reduced by H22.1.1Signals of the DMPO-OH radical were reduced by H2at different levels by theFenton reaction,0.8mM H2reduced levels of OH in71.2%by the Fenton reaction in themixture of0.1mM H2O2and0.03mM ferrous iron.2.1.2Fluorescence signals from the oxidized HPF were reduced by H2in differentlevels in radiolysis of H2O,0.8mM H2reduced levels of OH in88.7%in radiolysis ofH2O in5Gy of60Co γ-ray.2.1.3The testicular fluorescence in the H2group was significantly less than that ofcontrol group; however, when we treated the mice with H2after irradiation, the decreasewas not significant.2.2Radioprotective role of H2on oxidative stress in testis2.2.1H2pre-treatment seems to restore testicular antioxidant status, SOD is anenzymic scavenger of superoxide and GSH is a cellular nonenzymic tripeptide antioxidant.2.2.2H2seems to attenuate testicular oxidative damage caused by IR, it is wellestablished that oxidative stress damage usually leads to the accumulation of cytotoxicproducts such as MDA, protein carbonyl and8-OHdG.2.2.3Injection of hydrogen-rich saline increased H2concentrations in testes, and thelevels of H2peaked approximately5min following injection in testis, and returned tonormal levels15min later. Discussion:Signals of the DMPO-OH radical were reduced by H2in different levels in theFenton reaction. Fluorescence signals from the oxidized HPF were reduced by H2indifferent levels in radiolysis of H2O, but as we treated the system with H2after irradiation,the descent is not significant. Injection of hydrogen-rich saline increased H2concentrationsin testes, the levels of H2peaked approximately5min following injection in testis, andreturned to normal levels15min later. The testicular fluorescence in the H2group wassignificantly less than that of control group, however, when we treated the mice with H2after irradiation, the descent is not significant. The pH values have no significant differencebetween the two solutions. H2restored SOD and GSH, H2reduced MDA, protein carbonyland8-OHdG. Quantitative analysis for the spermatogonia and spermatocytes revealed thatthe apoptotic findings in the H2groups were significantly less than those of control groups.At29d after irradiation, organ indices of testis in the H2groups were significantly higherthan those of the non-H2groups, treating mice with H2before IR can increase the numberof spermatid head count and protect seminiferous epithelium from IR. At35d afterirradiation, TDI in the H2groups were significantly higher than those of control groups. At56d after irradiation, daily sperm production and sperm quality in the H2groups weresignificantly higher than those of control groups.Conclusion:We showed that H2protect spermatogenesis in irradiated mice. The effect of reducingtoxic OH in vivo plays an important role in the radioprotective effects of H2. Using H2may be a novel strategy of fertility preservation in irradiated males in the future. |
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