Effects of gamma rays on rat testis tissue according to the morphological parameters and immunohistochemistry: radioprotective role of silymarin

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1 Electronic Physicin (ISSN: ) June 2017, Volume: 9, Issue: 6, Pges: , DOI: Effects of gmm rys on rt testis tissue ccording to the morphologicl prmeters nd immunohistochemistry: rdioprotective role of silymrin Mohsen Mrzbn1, Mrym Anjmsho2, Prni Jfri3, Hossien Msoumi4, Rez Ahdi5, Dryoush Ftehi6 Ph.D. Student of Neuroscience, Fculty of Advnced Medicl Technologies, Tehrn University of Medicl Sciences, Tehrn, Irn 2 Ph.D., Assistnt Professor of Antomicl Sciences, Fculty of Medicine, Shhrekord University of Medicl Sciences, Shhrekord, Irn 3 M.D., Resercher in Cellulr Oncology, Fculty of Medicine, Shhrekord University of Medicl Sciences, Shhrekord, Irn 4 Ph.D., Assistnt Professor of Medicl Physics, Fculty of Medicine, Shhrekord University of Medicl Sciences, Shhrekord, Irn 5 Ph.D. of Neuroscience, Fculty of Prmedicine, Irn University of Medicl Sciences, Tehrn, Irn 6 Ph.D., Associte Professor of Medicl Physics, Fculty of Medicine, Shhrekord University of Medicl Sciences, Shhrekord, Irn 1 Type of rticle: Originl Abstrct Objective: To determine the rdioprotective effects of Silymrin in dult mle Sprgue-Dwley rts irrdited with γ-rys. Methods: The present experimentl study ws performed in Tehrn University of Medicl Sciences, Tehrn, Irn from December 2009 to Mrch The study ws performed on 40 rts, which were rndomly nd eqully divided into four groups: 1) control group: neither received Silymrin nor irrdited with γ-rys; 2) γ-irrdition group: testis region exposed to 2Gy of γ-rys; 3) Silymrin & γ-irrdition: rts received 100 mg/kg of Silymrin 24hrs before exposure to 2Gy of γ-rys; 4) Silymrin & γ-irrdition: rts received 200 mg/kg of Silymrin 24hrs before exposure to 2Gy of γ-rys. After niml experiments nd prepring the tissue sections, different histologicl nd histomorphologicl prmeters of seminiferous tubules nd the biologicl chrcteristics of Leydig cells were evluted pplying quntittive ssessment, Johnson scoring, nd Leydig cell poptosis ssy by TUNEL method. The dt were nlyzed pplying ANOVA nd Tukey's post hoc test, using SPSS softwre (V.19). Results: Irrdition of 2 Gy γ-rys to the testis of the rts significntly ffected the frequency of spermtogoni, primry spermtocyte, round spermtid, spermtozo, seminiferous tube nd lumen dimeters, thickness of the epithelium, Leydig cell nucler dimeter nd volume, epithelium height, nd poptotic cells (p<0.05). However, dministrtion of Silymrin improved the mentioned prmeters specificlly in 200 mg/kg of dosge. Conclusion: Silymrin could ct s potent rdioprotector nd it cn be used in modultion s well s improvement to rdition therpy to prevent mle reproductive function, specificlly seminiferous tubules in n niml model; however, its moleculr mechnism is still not cler nd needs more moleculr reserches. Keywords: Spermtogenesis, Rdiotherpy, Silymrin, Rdioprotector, Antioxidnt 1. Introduction Prostte cncer is the second leding cncer nd the fifth leding cuse of deth in men worldwide. In 2012, 1.1 million men were dignosed with prostte cncer, of which 307,000 died (1). This cncer is the most common mong men in 84 countries, most of which re developed (1). In ddition, the incidence of prostte cncer is Corresponding uthor: Dryoush Ftehi, Deprtment of Medicl Physics, Fculty of Medicine, Shhrekord University of Medicl Sciences, Shhrekord, Irn. Tel: Emil: d.ftehi@gmil.com. Fx: Received: September 27, 2016, Accepted: Jnury 15, 2017, Published: June 2017 ithenticte screening: Jnury 07, 2017, English editing: Mrch 01, 2017, Qulity control: April 26, The Authors. This is n open ccess rticle under the terms of the Cretive Commons Attribution-NonCommercilNoDerivs License, which permits use nd distribution in ny medium, provided the originl work is properly cited, the use is non-commercil nd no modifictions or dpttions re mde. Pge 4524

2 Electronic physicin incresing in developing countries (2). By mens of prostte-specific ntigen test, dignosis of prostte cncer incresed incredibly during the 80s nd 90s (2). Prostte cncer hs been reported to be the cuse of deth in 30%70% of men ged over 60 yers who died becuse of unrelted cuses (1). The tretment of this cncer is typiclly performed in the light of different fctors such s tumor growth rte, generl helth, s well s the benefits nd side effects of the tretment. The current prostte cncer tretments re rdiotherpy, hormonotherpy, chemotherpy, nd cryotherpy (3). Rdiotherpy is the most importnt nd common method to tret cncer. Mostly rdiotherpy is pplied through two procedures: externl bem rdition therpy (teletherpy), nd internl rdition therpy (brchytherpy) (4). In prostte cncer, both of the methods cn be prescribed depending on the generl helth conditions of the ptient, plnning, nd the physicin s opinion. Rdiotherpy is imed t removing tumor cells nd preserving surrounding norml cells in the tretment field. However, some prts of orgns or tissues might receive notble dosge, which is reveled s either cute or chronic side effects (5). Using protecting medictions or rdioprotectors could reduce the side effects of rdiotherpy. These medictions in fct, reduce indirect effects of the rdition (6). Rdioprotectors decrese stble hydroxyl rdicls in the exposed re, by bsorbing rective oxygen species (ROS) (6, 7). Mny studies hve been performed to determine the effects of such compounds. Some of the studies hve confirmed the protective effects of the compounds; while, others hve reported uncler or no effects for them (7). Milk thistle (Silybum mrinum) ws first discovered s mteril with ntitumor effects, nd is being used to tret immune system dysfunctions nd lcoholic liver diseses in clinics (8). Milk thistle contins different ntioxidnts such s Silymrin, which is potent cell protective gent tht reduces oxidtive stress nd protects the cells ginst poptosis. It contins six flvonolignns consisting of 40%-50% silibinin, responsible for most biologicl ctivities (8, 9). Bsed on some studies, Silymrin hs rdioprotective effect ginst the rdition induced heptotoxicity in rts (10). Adhikri M. et l. showed tht specific doses of 25μg/ml Silymrin cn meliorte the deleterious effects of γ rdition on humn embryonic kidney cells in-vitro (11). Deep G. nd Agrwl R. showed tht Silymrin, with trgeting signling molecules, could djust the process of epithelil mesenchyml trnsition, protese ctivtion, dhesion, nd prevention of metstsis (9). Muduit C. et l. found tht by 2-4 Gy of ionizing rdition to dult rts testes, the poptotic cells incresed fter 24 hours nd reched to their mximum number fter 48 hours (12). Apoptotic cells were observed 72 hours fter 2 Gy of rdition, nd incresed s the dosge incresed from 1 Gy to 4 Gy. Growth fctor in sensitive testis cells, drops fter ionizing rdition (12). Mydlo JH. et l. exmined the effects of brchytherpy nd externl bem rdition therpy on prostte cncer tretment, sperm qulity, nd fertility in young men. Rdiotherpy reduces spermtogenesis by dischrging Leydig cells nd reduction of testosterone level. It lso hs direct effects on spermtogenesis with mitosis nd bnorml cell division (13). Otl M. et l. used sphingosine-1-phosphte (S1P) to protect sperm ginst ionizing rdition to rt s testes. They injected S1P to rt s testes 1-2 hours before rdition. Their results showed tht use of S1P (16%-47%) decresed germ cells deth nd protected them (14). Khn S. et l. used meltonin in order to protect mice testes ginst γ-irrdition induced dmge. Their results showed tht meltonin, s physiologicl rdioprotector, meliorted the rdition induced dmges of epididyml sperm bnormlities such s motility nd vibility (15). Koruji et l. exmined the morphologic chnges in dult rt s testes fter rditing 14 Gy of γ- rys. Their results showed tht rdition could cuse temporry zoospermi potentilly reversible fter eight weeks (16). Considering the role of rdiotherpy in treting different tumors, its induced effects on norml tissues, nd the importnce of rdioprotective substnces to reduce the side effects of this method on the region surrounding the tumor, the purpose of this study ws to evlute nd determine rdioprotective effects of Silymrin on Johnson scoring, histomorphologicl prmeters of seminiferous tubules nd biologicl chrcteristics of Leydig cells in SprgueDwley mle rts irrdited by the sme frction dose of γ- rys in prostte cncer rdiotherpy. 2. Mteril nd Methods 2.1. Animls nd ethic This experimentl study ws performed in Tehrn University of Medicl Sciences, Tehrn, Irn from December 2009 to Mrch Forty 9-week-old Sprgue-Dwley mle rts, weighing ± 0.2g, were purchsed from the Psteur Institute (Tehrn, Irn). Animls were llowed to hve esy ccess to food nd wter nd were exposed to control conditions of constnt illumintion (12:12 hours, light from 8 to 20), temperture (23±2 C) nd humidity (55-65%); ll nimls were mintined in cges for one week prior to the strt of experiments for cclimtiztion, nd they were eligible to be prt of the experiments when they hd pssed the two consecutive sex cycles. All procedures were crried out in ccordnce with the Guide for the Cre nd Use of Lbortory Animls prepred by the Ntionl Acdemy of Sciences, nd published by the Ntionl Institutes of Helth (NIH publiction revised 1985), nd lso the guidelines of Tehrn University of Medicl Sciences (Tehrn, Irn). Pge 4525

3 Gmm irrdition Gmm irrdition ws performed pplying Coblt-60 source (Thertron 780C, Atomic Energy of Cnd Limited, Knt, ON, Cnd). The chrcteristics of the source were: hlf-life: 5.27 yers, dose rte: 0.5 Gymin-1, medium energy: 1.25 MeV. The source hd dimeter of 20 mm. The SSD (source to skin distnce) ws 80 cm nd the CID (collimtor to isocenter distnce) ws 30 cm. Rts were plced in ventilted Plexigls cge nd irrdited in groups of four, simultneously, t room temperture of 23±2 C. The prostte region (pelvic re nd reproductive system) of the rts ws irrdited with totl dose of 2 Gy γ-rys Grouping nd Silymrin dministrtion The rts were grouped in 4 groups of 10. The first group, the control group, were neither treted with Silymrin nor irrdited by γ-rys; but received intrperitonel injection of 10 ml/kg norml sline. The second group were only irrdited with 2 Gy of γ-rys. The third group ws initilly treted with 100 mg/kg of Silymrin (Sigm-Aldrich, S G, Itly) nd then they were irrdited with 2 Gy of γ-rys 24 hours. lter. Similrly, the fourth group ws initilly treted with 200 mg/kg of Silymrin nd then they were irrdited with 2 Gy of γ-rys 24 hours lter. The solution of Silymrin ws dministered directly into the stomch of the rts vi orl gvge 24 hours before the γirrdition Histology At completion of the experiments, the rts were deeply nesthetized with chloroform nd their testes were removed from bdominl cvity. The testes were fixed with 10% formldehyde for 72 hrs. For light microscopy, the sections were dehydrted by successive series of ethnol clered by xylene nd impregnted by melted prffin. Then, the smples were embedded in prffin wx. Tissues were sectioned t 5µm nd stined with hemtoxylin nd eosin (H&E) to observe the structure. Then, the slides were exmined t 400x mgnifiction Leydig cell poptosis ssy by TUNEL To detect poptotic cells, TUNEL stining ws performed using n poptosis kit (Roche, Germny) by TUNEL stining ccording to the mnufcturer s instruction. The smples, in prffin block, were cut into 5 µm sections by rotry microtome, nd then plced on glss slides. The sections were deprffinized in xylene nd plced in 700w microwve for 10 minutes. Then, the sections were incubted with 3% H2O2 in PBS for 10 minutes in the drk. Next, the sections were incubted in the TUNEL rection mixture tht contined terminl deoxynucleotidyl trnsferse nd fluorescein-dutp for 60 minutes t 37ºC in humidified tmosphere. Then, the smples were rinsed with PBS three times, the sections were visulized by converter-pod (Roche Applied Science, Mnnheim, Germny) for 30 minutes t 37ºC in humidified tmosphere in the drk, nd then rinsed with PBS, followed by the ddition of µl diminobenzidine (DAB, Ct No: D8001, Sigm-Aldrich) substrte nd n dditionl PBS rinse. The slides were exmined by pthologist in blinded mnner. Only circulr tubulr cross-sections cut in boldfce were studied. The quntittive evlution of poptotic index ws performed by the mnul counting of the men number of TUNEL-positive cells per interstitil spce per group (four sections per rt nd 25 interstitil spces per section). The poptotic cells in the 100 tubules were recorded using light microscopy t 400x mgnifiction Quntittive ssessment of seminiferous tubules nd Johnson scoring An oculr grid ws used to mesure the prmeters of the seminiferous tubules. Twenty tubulr circulr or nerly circulr profiles were rndomly selected from ech niml. Then, two perpendiculr dimeters of ech cross-section of the seminiferous tubules were mesured nd the men vlue ws clculted t 400x mgnifiction. In ddition, the dimeters of ech lumen of the somniferous tubules nd epithelium height were mesured. In this study the spermtogenesis of control nd SCI groups were evluted ccording to Johnson Score (Tble 1) Clculting the volume of Leydig cells nd germ cell count Using liner grid nd light microscope (Olympus BX51, Germny) t 1000x mgnifiction, the volumes of the nuclei of the Leydig cells were clculted. Thus, per ech niml, 20 cells were studied. The number of spermtogoni, primry spermtocytes, round spermtids, nd spermtozo were counted. The loction nd morphology of the cells within the seminiferous tubules were used to identify them. Ech prmeter ws estimted by the exmintion of 20 histologicl sections of ech seminiferous tubule Sttisticl nlysis The ANOVA test ws pplied to compre the verges of the vlues within ech group of Silymrin or γirrdition. Additionlly, Tukey's post hoc test ws used to compre the four groups. The p-vlues re two-sided t Pge 4526

4 Electronic physicin significnce level of SPSS softwre (Version 19, SPSS Inc., Chicgo, Illinois, USA) ws used for the sttisticl nlysis. Tble 1. The clcultion criteri of fertility nd the durtion of spermtogenesis using Johnson scoring Seminiferous tubules fetures Full spermtogenesis, lrge number of sperm heds re locted on the sidelines of round nd regulr lumen. Although there re lrge number of sperms, no round nd regulr lumen cn be seen. Sperm count is very low. There is no sperm, however, lrge number of round spermtids re visible A few round spermtids cn be seen There is no sperm or round spermtid. There is lrge number of primry spermtocytes. Very few primry spermtocytes re seen. There re no primry spermtocytes. Only spermtogoni is observed There re no germ cells, only Sertoli cells cn be seen. Neither germ cells nor Sertoli cells re observed nd the tubules re trophic. Score Results Detils of different sperm prmeters evluted in this study re summrized in Tbles 2 nd 3. Moreover, sttus of spermtogenesis, histologicl chrcteristics of testis, nd immunohistochemistry of seminiferous tubules re illustrted in Figures 1 nd 2. Figure 1. Comprison of the effects of 2 Gy γ-irrdition on the testis tissue of Sprgue-Dwley rt in the bsence nd presence of Silymrin in comprison with the control group. A) Control group, B) γ-irrdition lone group, C) Silymrin (100 mg/kg) & γ-rys group nd D) Silymrin (200 mg/kg) & γ-rys group. Pge 4527

5 Figure 2. Illustrtions of immunohistochemicl stining of spermtogonil nd primry spermtocyte cells investigted in the present study, compring the effects of 2 Gy γ-irrdition on the testis tissue of the SprgueDwley rt in the bsence nd presence of Silymrin. A) Control group, B) γ-irrdition lone group, C) Silymrin (100 mg/kg) & γ-rys group nd D) Silymrin (200 mg/kg) & γ-rys group. Tble 2. The men ± SD* vlues of different histologicl prmeters of seminiferous tubules s found in the present study. Group 1: Control 2: γ-irrdition 3:Silymrin (100 4:Silymrin (200 lone mg/kg) & γ-rys mg/kg) & γ-rys Johnson clssifiction 9.67 ± ± ± ± 0.67b Number of spermtogoni 3.92 ± ± ± ± 0.35b Number of primry spermtocyte 65.8 ± ± ± ± 0.41b Number of round spermtid ± ± ± ± 1.47b,c Number of spermtozo ± ± ± ± 1.94c : significnt difference compred to the control group (the group 1 (p < 0.05); b: significnt difference compred to the group 2 (p < 0.05); c: significnt difference compred to the group 3 (p < 0.05); *SD: stndrd devition Tble 3. The men ± SD* vlues of different histomorphologicl prmeters of seminiferous tubules nd the biologicl chrcteristics of Leydig cells s found in the present study. Group 1: Control 2: γ-irrdition 3:Silymrin (100 4:Silymrin (200 lone mg/kg) & γ-rys mg/kg) & γ-rys Tube dimeter (µm) ± ± ± ± 2.44b,c Lumen dimeter (µm) ± ± ± ± 0.62b Thickness of epithelium (µm) ± ± ± ± 0.6 Leydig cell nucler dimeter (µm) 6.39± ± ± ± 1.03 Leydig cell nucler volume (µm3) ± ± ± ± 2.27b,c Height of seminiferous epithelium ± ± ± ± 0.62b (μm) : significnt difference compred to the control group (the group 1, p < 0.05); b: significnt difference compred to the group 2 (p < 0.05); c: significnt difference compred to the group 3 (p < 0.05); *SD: stndrd devition 3.1. Histology The opticl photomicrogrphs of the cross-sections for the control group indicted the integrity of the tubules nd interstitil tissue. The seminiferous tubules were observed completely nd clerly. All sperm cell lines were seen Pge 4528

6 Electronic physicin nd the bsl membrne of the seminiferous tubules s well s the spermtogoni lines on it could be observed. The Leydig cells in the interstitil tissue were observed with cidophilic cytoplsm. Furthermore, ccording to Johnson scoring nd histologicl illustrtion spermtogenesis ws visible (96.7%). Microscopic imges showed some unusul spces nd diltions ssocited with lymphocytes nd plsm in group 2, which indicted inflmmtion in the interstitil tissue. Moreover, in this group, vcuoles were bundntly visible in the interstitil tissue nd spermtogoni; while significnt reduction ws found for the primry spermtocytes (Figure 1-A). The seminiferous tubules were degenerted, nd decresing ws seen in the dimeter of the seminiferous tubules. The seminiferous tubules were disordered nd the bsement membrne ws folded. Epithelium ws trophic, nd severe vcuoliztion could be observed with spermtogoni cells on the bsement membrne; however, spermtogenesis could not be observed in ll sections. Primry spermtocytes rrested in pchytene stge. More thn one third of the spermtogenesis (38.6%) could be seen. The Leydig cells in the interstitil tissue hd pyknotic nuclei, nd were widely distributed. Furthermore, destructive cells with certin fetures, such s declined cytoplsmic cidophilus nd incresed nucler stining, were considerble (Figure 1-B). Microscopic imge evlution of the slices of the 3 rd group illustrted tht lthough rte of spermtogenesis ws improved (59.2%); we found slight reduction in the edem nd inflmmtion of the tissue, s well s mild regenertive effects in the seminiferous tubules. The Leydig cells in the interstitil tissue incresed prtly with norml ppernce, nd their cytoplsm ws lmost cidophilic, which indictes slight return of necrotic effects t the cellulr level. More thn hlf of the sperm cells were seen; however, reduction in the tubulr dimeter nd thickness, disruption in germinl epithelium, nd n increse in the interstitil spce could still be seen (Figure 1-C). In group 4, microscopic imges demonstrted tht the seminiferous tubules could be clerly seen with ll sperm cell lines. The bsl membrne of the seminiferous tubules could be seen with spermtogoni lines. The Leydig cells observed in the interstitil tissue, hd completely cidophilic cytoplsm. Spermtogenesis ws improved (78.3%) nd the cells hd distinguishble order. Some minor effects of vcuoliztion were seen; lthough, edem ws still visible in the interstitil tissue (Figure 1-D) Quntittive ssessment of seminiferous tubules nd Johnson scoring The verge vlues of different histologicl prmeters of seminiferous tubules re presented in tble 2. As Tble 2 shows, Johnson score ws significntly decresed in the 2 nd group (γ-irrdition lone) in comprison to the control group (p=0.0097). In contrst, compring the 3 rd group [Silymrin (100 mg/kg) & γ-rys] with the 2nd group, remrkble, but not significnt, increse ws found for the Johnson score. Also, significnt increse ws seen when we compred the 4th group [Silymrin (200 mg/kg) & γ-rys] with the 2nd group (p=0.014). Respecting the Johnson score, we found remrkble, but not significnt, increse compring the 4 th group with the 3rd group. Similr results were found for the other prmeters, i.e. verge numbers of spermtogoni, primry spermtocyte, round spermtid, nd spermtozo (Tble 2). The verge vlues of different histomorphologicl prmeters of seminiferous tubules nd the biologicl chrcteristics of Leydig cells re shown in Tble 3. As Tble 3 demonstrtes, tube dimeter ws significntly decresed in the 2 nd group when we compred it with the control group (p=0.0078). Compring the 3 rd group with the 2nd group non-significnt increse ws found for the tube dimeter; however, significnt increse ws seen when we compred the 4 th group with the 2nd group (p=0.0094). Regrding the tube dimeter, significnt difference ws seen between the 4 th nd the 3rd groups (p=0.016). Similr results were observed for other prmeters of seminiferous tubules, i.e. lumen dimeter, primry spermtocyte, nd thickness of the epithelium. Additionlly, similr results were found for nucler dimeter nd nucler volume of the Leydig cells. Furthermore, similr results were seen for the height of the seminiferous epithelium (Tble 3) Leydig cell poptosis ssy by TUNEL Immunohistochemistry illustrtions show the verge of the poptotic cells which turned brown in group 2 ws significntly higher thn those of the control group (8.24±0.53) vs. 1.03±0.65); respectively (p=0.012). Also, quntittive nlysis of TUNEL ssy showed poptotic index in spermtocyte I cells were significntly incresing in group II. The frequency of poptotic cells per 100 tubules in groups 3 nd 4 ws 5.17±1.92 nd 3.96±1.04, respectively; which represented significnt decrese in the number of dmged cells in group 4 compred to group 2 (p=0.023). This lso showed tht nimls which received 200 mg/kg of Silymrin hd some improvements in support reproductive cells nd their testes (Figure 2). 4. Discussion The results of the present study demonstrted tht irrdition of 2 Gy γ-rys to the testis of the Sprgue-Dwley rt, significntly reduced different histologicl prmeters of seminiferous tubules (i.e. frequency of spermtogoni, primry spermtocyte, round spermtid, spermtozo. Furthermore, our results showed tht γ-irrdition could destruct nd induce roughly stble dmges of different histomorphologicl prmeters of seminiferous tubules nd Pge 4529

7 the biologicl chrcteristics of Leydig cells. However, the mentioned prmeters were modulted nd or significntly improved when we dministered 100 mg/kg (nd or 200 mg/kg) of Silymrin to the Sprgue-Dwley rt, 24 hours before 2 Gy γ-irrdition. Apoptosis is n event tht occurs in ssocition with extrcellulr molecules s well s intrcellulr messges, nd protects the cells ginst spontneous cell suicide. This process occurs in response to cytotoxic gents through spermtogenesis, mking blnce between helthy nd ded germ cells (17). Apoptosis in testis tissue usully occurs in A2, A3 nd A4 stges of spermtogoni; however, poptosis rrely occurs in the spermtocytes of the stges 1 nd 2 s well s spermtids; unless specific environmentl nd chemicl fctors induce this process (18). Testis tissue is highly ctive with poor cell renewl system nd ntioxidnt defense, which cn be n esy trget for the induction of rdition through the production of the free rdicls tht cuse indirect nd reversible dmges (19). Oxidtive stress in the testes cn disrupt the estrogen cpcity of the Leydig cells. The cpcity of germinl cells (germ) is different from norml spermtozo, leding to impired spermtogenesis nd infertility through the oxidtion of proteins, lipids, nd germ cells DNA (20). DNA bses re sensitive to oxidtive stress, nd the peroxidtion of structures cn cuse bsl modifictions, DNA chin breks, nd the exchnge of chromtin components. Free rdicls produced by ionizing rdition, re the cuse of diseses nd cell dmge, especilly widespred infections (21). Rdition-induced DNA dmges re very importnt cuse of deleterious effects on reproductive cells which my ccelerte the process of chromosoml berrtions, gene muttions, poptosis of germ cells, nd sustinble development of testes trophy; which ultimtely leds to longterm zoospermi nd infertility (15, 19). Rdition not only interferes with the cellulr ntioxidnt defense system, it lso contributes to ltertion of mitochondril electron trnsport chin which is one of its min cellulr trgets, nd destroys cell divisions in the germ cells, nd ultimtely cuses pyknosis nd kryolysis (19, 21, 22). Rdition lso increses germ cell deth in testes by ctivting cspse 3, which re the min meditors of poptotic pthwy (or ply essentil roles in poptosis), nd thereby reduces the thickness of germinl cell epithelium nd the trophy of the testes tissue (14, 22). Silymrin hs certin nti-inflmmtory effects cusing reduction in the inflmmtion nd edem in the testes tissues of groups 3 nd 4. This substnce probbly inhibits the migrtion of leukocytes nd increses neutrophil infiltrtion in the re of inflmmtion, which plys key role in the nti-inflmmtory ction (23). Silymrin lso stbilizes the membrne structures, nd inhibits the relese of inflmmtory meditors such s cyclooxygense nd lipoxygense. Silymrin decreses the expression of the growth fctor TGF-β1, prevents the ctivtion of NF-ƙB nd cell prolifertion-relted kinses such s TNF-α, nd decreses cytotoxicity to void poptosis (11, 24). Due to hving ntioxidnt property, Silymrin is ble to scvenge free rdicl nd ROS. It prevents the progression of cell dmge nd poptosis by inhibiting lipid peroxidtion (24). The results obtined in the present study, demonstrted certin chnges in spermtogoni, spermtocytes, spermtid, nd the number of spermtozo 24 hours fter γ-irrdition. This study lso showed direct effects of γ-rdition, shrp decline in ll the ctegories of Johnson s scoring, number of spermtogoni, spermtocytes, spermtid cycle, nd spermtozo, which confirms the results obtined by Muduit C. et l & Koruji M. et l. (12, 16). Similrly, Chui Y. et l reported tht the highest reduction (72.9%) in the number of spermtogoni occurred fter one-week irrdition with 2 Gy (25), while the corresponding reductions in our study were derived s 61.4%, 40.8%, nd 21.7% in the groups of γ-irrdition lone (II), Silymrin (100 mg/kg) (III), nd (200 mg/kg) (IV), respectively. They concluded tht their results represent rdioprotective property of Silymrin. Additionlly, due to nontoxicity nd noninvsive effects of Silymrin, its concentrtion cn increse its rdioprotective effects in externl rdition tretment (26). In this study, the optiml concentrtion of Silymrin to protect germ cells ginst ionizing rdition ws not investigted; however, we suggest performing it in the future. The results obtined in our study for these prmeters in groups 3 nd 4 confirm the rdioprotective effect of Silymrin on the germ cells. Antioxidnt molecules such s Silymrin cn modulte the oxidtive sttus of cell environment vi increse in cell glutthione, nd prevents lipid peroxidtion, resulting in improvement of the ntioxidnt defense system (24). Silymrin hs been ble to decrese the development of neurons in cerebrl ischemi s well s improvement of functionl recovery, s the results show tht the neuroprotective potentil of Silymrin is due to its ntioxidnt nd ntipoptotic properties (27). As recommendtions, the germ cell nd sperm production in the mmmlin testis depends on norml Leydig cell function nd its biochemicl interctions with germ cells which is pivotl mechnism in spermtogenesis process. Epithelium of seminiferous tubules in mmmls is very complex but well-orgnized cell popultion tht is mde up of germ cells with different levels of sensitivity to rdition. Leydig cells nd Sertoli cells, lthough closely relted to germ cells, hve been reported to be reltively sensitive to rdition. Rdition vi ROS production is cpble of disrupting the steroidogenic cpcity of Leydig cells, nd lso decrese in sperm count, thus resulting in germ cell poptosis, disruption of spermtogenesis nd fertility cpcittion. Silymrin trget mitochondril ROS, nd scvenge the free rdicls during the rdition exposure s prt of strtegy to mitigte cellulr stress nd poptosis. In conclusion, biologicl protection of mle fertility ginst rdition hzrd, is mjor concern bout ny other hrmful effects of rdition tht my be trnsmitted from one genertion to nother. Understnding the Pge 4530

8 Electronic physicin rdition sensitivity of germ cells, nd strengthening them, pves the wy for the development of rdition modertors s well s rdioprotective gents tht cn be effectively used for high-sensitivity protection of germ cells ginst the hrmful effects of ionizing rdition. In this study, there were limittions which prolonged the time of the study or stopped some of the stges including 1) lck of ccess to testing kits TUNEL nd lte rrivl to the site of the project 2) lck of dvnced mchines for precise imge nlysis nd lck of softwre for cell counts or cell morphometry 3) the unvilbility of irrdition devices for smll nimls nd preclinicl equipment for exctness nd similrity of rdition similr to humn rdition devices. 5. Conclusions In this study, we investigted the rdioprotective effect of Silymrin on the histologicl nd histomorphologicl prmeters of seminiferous tubules s well s the biologicl chrcteristics of Leydig cells of dult mle SprgueDwley rts irrdited with 2 Gy γ-rys. Our results showed tht Silymrin cn improve the histologicl nd histomorphologicl prmeters of seminiferous tubules such s frequency of spermtogoni, primry spermtocyte, round spermtid, spermtozo, tube dimeter, lumen dimeter, thickness of epithelium, Leydig cell nucler dimeter, Leydig cell nucler volume, nd height of seminiferous epithelium, s well s poptotic cells. Therefore, one cn conclude tht Silymrin could ct s potent rdioprotector nd it cn be used djcent to rdition therpy to prevent mle reproductive function, specificlly seminiferous tubules in n niml model; however, its moleculr mechnism is still not cler, nd needs more moleculr reserches. Acknowledgments: This study ws supported by grnt from the Reserch Institute for Islmic nd Complementry Medicine, No. p26/m/t/1088. Also, the uthors would like to thnk the lb stff of the Cellulr nd Moleculr Reserch Center in Tehrn University of Medicl Sciences for their kind coopertion. The uthors pprecite Dr. Mehdi Mehdizdeh for his technicl ssistnce. Conflict of Interest: There is no conflict of interest to be declred. Authors' contributions: All uthors contributed to this project nd rticle eqully. All uthors red nd pproved the finl mnuscript. References: 1) Ferly J, Soerjomtrm I, Dikshit R, Eser S, Mthers C, Rebelo M, et l. Cncer incidence nd mortlity worldwide: sources, methods nd mjor ptterns in GLOBOCAN Int J Cncer. 2015; 136(5): E doi: /ijc PMID: ) Bde PD, Youlden DR, Krnjcki LJ. Interntionl epidemiology of prostte cncer: geogrphicl distribution nd seculr trends. Mol Nutr Food Res. 2009; 53(2): doi: /mnfr PMID: ) Chen FZ, Zho XK. Prostte cncer: current tretment nd prevention strtegies. Irn Red Crescent Med J. 2013; 15(4): doi: /ircmj PMID: , PMCID: PMC ) Fitzgerld TJ, Wng T, Goel HL, Hung J, Stein G, Lin J, et l. Prostte crcinom nd rdition therpy: therpeutic tretment resistnce nd strtegies for trgeted therpeutic intervention. Expert Rev Anticncer Ther. 2008; 8(6): doi: / PMID: , PMCID: PMC ) Bskr R, Lee KA, Yeo R, Yeoh KW. Cncer nd rdition therpy: current dvnces nd future directions. Int J Med Sci. 2012; 9(3): doi: /ijms PMID: , PMCID: PMC ) Rosen EM, Dy R, Singh VK. New pproches to rdition protection. Front Oncol. 2015; 4: 381. doi: /fonc PMID: , PMCID: PMC ) Hzr B, Ghosh S, Kumr A, Pndey BN. The prospective role of plnt products in rdiotherpy of cncer: current overview. Front Phrmcol. 2012; 2: 94. doi: /fphr PMID: , PMCID: PMC ) Krimi G, Vhbzdeh M, Lri P, Rshedini M, Moshiri M. "Silymrin", promising phrmcologicl gent for tretment of diseses. Irn J Bsic Med Sci. 2011; 14(4): PMID: , PMCID: PMC ) Deep G, Agrwl R. Antimetsttic efficcy of silibinin: moleculr mechnisms nd therpeutic potentil ginst cncer. Cncer Metstsis Rev. 2010; 29(3): doi: /s PMID: , PMCID: PMC Pge 4531

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