The effect of Laminaria japonica polysaccharides on therecovery of the male rat reproductive system

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y31(2011)286–294The effect of Laminaria japonica polysaccharides on the recovery of the male rat reproductive system and mating function damaged by multiple mini-doses of ionizing radiationsLuo Qiong a,∗,1,Liu Jun a,b,1,Yan Jun a,Zhou Yinzhu a,Cui Xiaoyan a,Yang Mingliang aa School of Public Health,Wuhan University,Wuhan,Hubei430071,Chinab College of Life Science and Technology,Xinjiang University,Urumqi,Xinjiang830046,Chinaa r t i c l e i n f oArticle history:Received13April2010Received in revised form31October2010Accepted21November2010Available online27November2010Keywords:Laminaria japonica polysaccharidesIonizing radiationSex hormoneOxidative damageMating functionDamage recoverya b s t r a c tTo evaluate the radiation-protect effects of Laminaria japonica polysaccharides(LJP)on malereproductive system damage and mating dysfunction induced by multiple mini-dose ioniz-ing radiations,male rats were administrated with radiation and/or LJP.Results showed thatmating function(such as erection,mount and ejaculation),sperm count and survival rate inLJP group were significantly better than the corresponding model group after the radiation.The testis organ coefficient,GSH(glutathione)content,serum sex hormones(luteinizinghormone,follicle-stimulating hormone,testosterone and estradiol)levels improved whileMDA(malondialdehyde)content decreased.In addition,SOD(superoxide dismutase),GSH-PX(glutathione peroxidase),LDH(lactate dehydrogenase)activities were enhanced whiletesticular tissue damage was reduced,14days after the cessation of radiation;all indicatorsin the LJP group were similar to the control group.Our results suggest that,LJP has some pro-moting effects on the recovery of the reproductive system and mating dysfunction inducedby radiation.©2010Elsevier B.V.All rights reserved.1.IntroductionAs a potential health risk to human,ionizing radiation havebeen paid much attention(Alonzo et al.,2008)and studiesAbbreviations:LJP,Laminaria japonica polysaccharides;MDA,malondialdehyde;SOD,superoxide dismutase;GSH,glutathione;GSH-PX, glutathione peroxidase;LDH,lactate dehydrogenase;LH,luteinizing hormone;FSH,follicle-stimulating hormone;Ts,testosterone;E2, estradiol;DNA,deoxyribonucleic acid;NMP,normal melting point;LMP,low melting point;RIA,radioimmunoassay;DMSO,dimethyl sulphoxide;PBS,phosphate buffered saline.∗Corresponding author at:Department of Nutrition and Food Hygiene,School of Public Health,Wuhan University,Donghu Road115, Wuhan,Hubei430071,China.Tel.:+862768758809;fax:+862768758648.E-mail addresses:luoqiong7@,drluoqiong@(L.Qiong).1These authors contributed equally to this work.on anti-radiation drugs are becoming increasingly impor-tant.Several remarkable materials have been proved to haveradiation-protect effects,such as the sulfhydryl-ammoniacompounds,cytokine and herb.However,the severe toxicityand side effects of these drugs limit their application(Jagetia1382-6689/$–see front matter©2010Elsevier B.V.All rights reserved.doi:10.1016/j.etap.2010.11.006e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y31(2011)286–294287Fig.1–Theflowchart of treatment in each group.and Baliga,2003).Therefore,searching anti-radiation drugs from traditional food sources is considered to be a good alter-native.Laminaria japonica,a large edible-medicinal seaweed(Wang et al.,2008),has nutritional value and many health functions (Jurkovi´c et al.,1995;Jane and Montse,2006).It has also been used for weight loss,detumescence and elimination phlegm in traditional Chinese medicine(Li et al.,2005;Wang et al., 2008).Recent studies showed that polysaccharides are the bio-logical components of L.japonica(Zhao et al.,2004;Wang et al.,2008).L.japonica polysaccharides(LJP)are natural macromolecular substances found in the intercellular or intra-cellular environment,composed of algin,laminarin,fucoidan and different proportions of galactose,xylose,glucuronic acid and little protein(Zvyagintseva et al.,2005;Deng et al., 2006).Several studies showed that besides anticoagulant, anti-arteriosclerosis,anti-tumor,anti-virus properties,could improve the quality of freezing spermatozoa and other biolog-ical properties,LJP also have anti-radiation effect(Tomohiro et al.,2006;Niu et al.,2008a,b;Zhao et al.,2008;Hu et al., 2009).Over the past years,our group has conducted several studies on LJP.Our former results showed that LJP had an anti-fatigue effect which can significantly enhance the oxygen use, reduce peroxide content and antioxidant enzyme activities in hypoxia mice and prolong swimming time in weight-bearing mice.Moreover,fucoidan can regulate the immune function by inhibiting apoptosis in lymphatic cells in rats after exposure to9.0Gy rays(Yan et al.,2002;Wu et al.,2003;Luo et al.,2004; Ren et al.,2007).In this study,we evaluated the effects of LJP on the damages to male rats’reproductive system and mating function caused by multiple mini-doses of partially ionizing radiation.2.Materials and methods2.1.Experimental animals and diets564-week-old male Wistar rats were obtained from the CDC Animal Center in Hubei Province(Wuhan,China). Rats were housed in groups of four in rectangular cages (40cm×25cm×20cm)with wire mesh lids under standard-ized animal room conditions(12h light/dark photoperiod at 23◦C and60%humidity).Food in pellets and tap water were available ad libitum.Animal experiments were performed according to the guidelines for care and use of laboratory ani-mals established by Wuhan University(Wuhan,China),which in accordance with the Declaration of Helsinki of the World Medical Association(Wang et al.,2002).2.2.Drugs and chemicalsDried L.japonica was obtained from Lianjiang county, Fujian province,China.The LJP were prepared ourselves. Malondialdehyde(MDA),superoxide dismutase(SOD),glu-tathione(GSH),glutathione peroxidase(GSH-PX)and lactate dehydrogenase(LDH)kits were purchased from Jiancheng Bio-engineering Institute(Nanjing,China).Estradiol benzoate and progesterone injection were purchased from Jinyao Company (Tianjin,China).Olive oil was obtained from the Products of Shanghai Chemical Reagent of China Medicine Group(Shang-hai,China).Luteinizing hormone(LH),follicle-stimulating hormone(FSH),testosterone(Ts),estradiol(E2)RIA kits were from Jiuding Medical and Biological Engineering Co.,Ltd. (Tianjin,China).Other reagents were purchased from Sigma (St.Louis,MO,USA).2.3.Preparation of L.japonica polysaccharidesThe L.japonica polysaccharide was extracted according to Li’s methods(Li et al.,2000).With accordance of Li’s procedures, we extracted sodium alginate67.20g,crude fucoidan2.73g and laminarin0.48g from300g dried L.japonica.These prod-ucts were mixed and stored at4◦C in refrigerator for further use.2.4.Experimental grouping and treatmentAfter7days adaptive feeding,56rats were randomly divided into7groups:the normal control group,3model groups and 3LJP groups.Theflowchart of treatment in every group is shown in Fig.1.The model and LJP groups were adminis-trated with60Co␥partial irradiation on abdomen with the places above the anterior superior iliac crest parts covered with3mm thick grid.Radiation source-target distance was 80cm;with total dose was2.3Gy rat−1.The absorbed dose rate was0.925Gy min−1(GWXJ8060Co teletherapy machine, Zhongnan Hospital,Wuhan University).1d,7d and14d after the cessation of irradiation,the rats in model and LJP groups were killed by severing the femoral artery.Groups were named as control group,model1d group,model7d group,model14d288e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y31(2011)286–294group and LJP1d group,LJP7d group,LJP14d group,corre-spondingly.Rats in the control group were killed14d after the cessation of irradiation in the same manner.2.5.Mating and erectile experimentTo ensure the mating test proceeded successfully,healthy female rats were conducted bilateral ovariectomy in advance. 48h and4h before the mating test;female rats were injected with estradiol benzoate(200␮g kg−1)and proges-terone(2mg kg−1),respectively.Observations were carried out from19:00to24:00under dim lighting.The observation lasted 20min.The mating experiment parameters in this study included:(1)mount latency,length of time prior to mount-ing or intromission(in s);(2)mount frequency,number of mounting events(during the20min observation period);(3) ejaculation frequency,number of licking the penis within the 20min period following a success of mounting event.The erectile experiment parameters:erection latency,the length of time(in sec)from thefirst recorded penis erection. The tested male rats were recorded using a BL-420E biological function system to give local electrical stimulation of4mA, 50Hz,2ms(Taimong Co.,Chengdu,China).2.6.Determination of reproductive organ coefficient, sperm count and survival rate and serum sex hormoneRats of in each group were weighed by PG2002.5electronic bal-ance(Mettler,Toledo Instruments,China).Testis,epididymis, seminal vesicles and prostate of the sacrificed rats were weighed by Precision Electronic Analytical Balance(AG204 Mettler,Toledo Instruments,China).The organ coefficient was calculated by the following formula:organ coefficient (%)=organ weight(g)/animal body weight(g)×100.Shredded epididymis was transferred into6ml tube pre-pared with saline,water bath at37◦C for10–15min.The resulting solution was poured into blood count panels.Sperm count(N)was performed in5boxes under microscope(200×). The amount of the sperm was diluted to N×5×104times per milliliter.Sperm survival rate was determined by using one-step eosin–nigrosin staining technique(Bjorndahl et al., 2003).Then samples of200sperms were observed.The live ones were colorless while the dead ones were red.Sperm survival rate=unstained sperm count/(stained+unstained sperm count)×100%(BX51microscope,Olympus,Japan).Serum was collected by centrifuging blood samples obtained from artery blooding for10min at1000×g.The collected serum was stored at−20◦C refrigerators.Radioim-munoassay(GC-1200,Science and Technology Dazhongjia Optoelectronic Devices Branch,China)were used in serum sex hormone measurements,including luteinizing hormone (LH),follicle-stimulating hormone(FSH),testosterone(Ts)and estradiol(E2).2.7.Determination of testicular tissue MDA,SOD, GSH,GSH-PX,LDH and the HE stain of testicular tissueAfter weighing,half of one testicle was thoroughly rinsed under saline to be made into10%homogenate.The homogenate was centrifuged at4◦C for10min at1000×g.Oxidative indicators,such as MDA,SOD,GSH,GSH-PX and LDH were determined using a spectrophotometer(722,Shang-hai Precision Scientific Instrument Ltd.,China).Another half of the testicle was sectioned into4␮m sections to be per-formed by HE staining afterfixed by10%neutral formaldehyde solution for24h.Then morphological changes of testicular tis-sue were observed using BX51microscope camera(Olympus, Japan).2.8.Statistical analysisAll data were expressed as mean±standard deviation.All calculations and statistical analyses were done with SPSS soft-ware for Windows version17.0(SPSS Inc.,Chicago,IL).We could see that all data was normally distribution by explore analysis.Statistical significance in the difference between the means of the control group to model and LJP groups was eval-uated by one-way analysis of variance(ANOVA),and between LJP groups and corresponding model groups was evaluated by independent-sample t-test.Significance was set as P<0.05.3.Results3.1.Effect of LJP on mating function damage and the recovery in irradiated ratsAs shown in Fig.2A,penile erection latency in all model groups and the LJP1d group significantly increased compared with the control group(P<0.05).But latency in LJP7d,14d groups were shortened remarkably compared with the correspond-ing model groups(P<0.05).Similarly,except mount latency of LJP14d group was nearly to the control group,all other groups were significantly prolonged.However,mount latency of the LJP7d,14d groups was shortened greatly compared with that of the corresponding model groups(Fig.2B).Frequencies of mount and ejaculation changes were similar to the penile erection latency.There were no significant reductions in7d, 14d LJP groups while frequencies in other groups were con-siderably lower than the control group(P<0.05)(Fig.2C and D).3.2.Effect of LJP on reproductive system damage andthe recovery in irradiated ratsCompared with the control group,organ coefficients of testi-cles,epididymis,seminal vesicles and prostate decreased in all other groups.But except testicular organ coefficient,there were no significant differences existed among each group (P>0.05)(Fig.3B and C).Testicular organ coefficient in the7d, 14d model and LJP groups was significantly lower than the con-trol group(P<0.05)(Fig.3A).However,the reduction extent of LJP groups was much lighter than corresponding model groups (P<0.05).Except serum LH levels of model14d group,the other model groups and all LJP groups have no significant differ-ences(P>0.05),LH levels in LJP groups also decreased but the LJP14d group was significantly higher than the model 14d group(P<0.05)(Fig.4A).The changes in serum FSH lev-els for the model and LJP groups were similar to the changese n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y31(2011)286–294289Fig.2–Effect of LJP on sexual function in irradiated rats (n=8);the groups:control,model1d,model7d,model14d, LJP1d,LJP7d,LJP14d;*P<0.05compared with the control group;#P<0.05compared with corresponding model group.in LH levels.Differences between the all model groups and the control group were significant(P<0.05).Notably,FSH levels in the LJP groups did not differ from that of the correspond-ing model group but was close to the control group(Fig.4B). The serum Ts levels in all model groups and LJP1d,7d groups were significantly reduced compared with the control group (P<0.05),Ts levels in the LJP14d group was close to the con-trol group and significantly different from the model14d group (P<0.05)(Fig.4C).E2contents in model and LJP groups changed similar to the Ts in the contrary way.Significantdifferences Fig.3–Effect of LJP on coefficient of reproductive organ in irradiated rats(n=8);the groups:control,model1d,model 7d,model14d,LJP1d,LJP7d,LJP14d;*P<0.05compared with the control group;#P<0.05compared with corresponding model group.existed between the1d,7d model groups and the control group (P<0.05).Ts levels of the LJP14d group were similar to the con-trol group14days after the cessation of the radiation(P>0.05) (Fig.4D).From Fig.5A and B,the sperm count in the model and LJP groups all decreased compared with the control group (P<0.05).The sperm survival rate changed alike the former indicators.But sperm count and survival rate in LJP groups was higher than the corresponding model groups(P<0.05).3.3.Effect of LJP on testicular tissue injury and the recovery in irradiated ratsAs shown in Fig.6A,the content of MDA in model groups and 1d,7d LJP groups was obviously higher than the control group (P<0.05);and it obviously decreased more in LJP groups than corresponding model groups(P<0.05).Compared with control group,the activity of SOD in model groups decreased obviously (P<0.05),but in LJP1d group,the activity of SOD increased (P<0.05),and in LJP7d,14d groups,SOD activities of decreased obviously(P<0.05).The activities of SOD increased in LJP1d,290e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y31(2011)286–294Fig.4–Effect of LJP on serum sex hormone in irradiated rats(n=8);the groups:control,model1d,model7d,model 14d,LJP1d,LJP7d,LJP14d;*P<0.05compared with the control group;#P<0.05compared with corresponding model group.14d groups compared to corresponding model groups(P<0.05) (Fig.6B).As it was shown in Fig.7A,compared with the control group,GSH levels in model groups and LJP7d,14d groups decreased significantly(P<0.05).But it in LJP1d,14d groups were obviously higher than the corresponding each model groups(P<0.05).GSH-PX activities changed the same to the GSH(Fig.7B).The activities of GSH-PX in model groups and LJP groups also significantly decreased compared with control group(P<0.05);but were obviously higher in LJP groupscom-Fig.5–Effect of LJP on sperm count and survival rate in irradiated rats(n=8);the groups:control,model1d,model 7d,model14d,LJP1d,LJP7d,LJP14d;*P<0.05compared with the control group;#P<0.05compared with corresponding modelgroup.Fig.6–Effect of LJP on MDA and SOD of testicular tissue in irradiated rats(n=8);the groups:control,model1d,model 7d,model14d,LJP1d,LJP7d,LJP14d;*P<0.05compared with the control group;#P<0.05compared with corresponding model group.e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y31(2011)286–294291Fig.7–Effect of LJP on GSH,GSH-PX and LDH of testicular tissue in irradiated rats(n=8);the groups:control,model1d,model7d,model14d,LJP1d,LJP7d,LJP14d;*P<0.05 compared with the control group;#P<0.05compared with the corresponding model group.pared to the corresponding model groups(P<0.05).The LDH activities in the model groups significantly decreased(P<0.05) compared with the control group.Differences between the LJP groups and the control group were not significant(P>0.05)but significant compared with the corresponding model groups (P<0.05)(Fig.7C).In concern with the organ coefficient changes,seminifer-ous tubule damages were observed in model and LJP groups (Fig.8).Unlike seminiferous tubules well organized by sper-matogonia,spermatocytes and sperm cells regularly in the control group,the basement membrane of model groups shrinked with numbers of spermotagenic cells reduced.There were even some puffed spermatogenic cells and exfoli-ated sperms showed somewhere of the tubules.Damages intensified over time and14d after the radiation,basement membrane disorder occurred.Changes of the LJP groups were similar to model groups,but arrangements of spermatogenic cells were better than the corresponding model groups,as well as the number of mature sperms were better than the corresponding model groups.4.DiscussionWell known as Chinese traditional herbal medicine,L.japonica has been widely used as a popular functional food nowadays. LJP are the most important functional components in L.japon-icafirst extracted by Skoryna in1964(Zhang and Zhang,1999). Early in1987,L.japonica polysaccharides had been proved to have anti-radiation effects(Deng et al.,1987).In the following decades,more and more studies indicated the radiation-protect effects of LJP(Li et al.,2002;Albertus,2004).However, few literatures have reported the intervention of LJP on mul-tiple mini-dose of partially ionizing radiation damage so far. This study explored the effects of LJP on multiple mini-doses of partially ionizing radiation-induced reproductive system damage,mating dysfunction and the radiation-protect mech-anism tofind a low toxicity,effective radiation protection resource.As main organs of reproductive system,testicles are more sensitive to harmful irradiation and the injuries are more severe than any other organs(Zeng et al.,2004;Niu et al.,2008a,b).Former study showed that when organisms were injured by radiation,the spontaneous recovery occurred meanwhile(Xiong et al.,2001).However,if the dose of radi-ation is much higher than the threshold value,damage occurs irreversibly.In this study,testicles and somniferous tubule damage aggravated severely over time with scarcely self-recovery in all groups administrated with60Co␥par-tially radiation.Sperm count and motility rate decreased continuously.These results were in accordance with former researches.But the extent of damage in LJP groups was much lighter than corresponding model groups.It indicated that LJP could effectively reduce spermatogenic cell injury in testic-ular seminiferous tubules,alleviate sperm damage extent to increase sperm quantity.The pathways that ionizing radia-tion damages organism are various.Free radicals induced by radiation directly or indirectly are one of most important ones (Chen and Mao,2002;Li and Zhang,2003).Content of lipid peroxidation product or antioxidant and antioxidase activities can reflect the degree of oxidative damage.There were no sig-nificant improvements observed in antioxidase or antioxidant activities in model groups over time,such as SOD,GSH-PX and GSH.Lipid peroxidation product MDA didn’t change remarkable as well.But in LJP groups,MDA content decreased continuously and was nearly to the control group14d after the cessation of radiation while GSH content and SOD,GSH-PX activities began to rise then.It is in accordance with Yan’s researches that LJP can effectively reduce lipid peroxidation (Yan et al.,2003;Wu et al.,2005;Zhu et al.,2008).Through these ways it alleviated testicular germ cells oxidative dam-age and promoted the spontaneous recovery of spermatogenic cells and testicular seminiferous tubules.LDH,as the main enzyme for glucose metabolism in testis spermatogenic cells, its activity is related to the mature of seminiferous epithelium and sperm activity and could be used for an important indi-cator of spermatogenesis(Li et al.,1988).In this study,LDH activity in model groups decreased continuously compared292e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 31(2011)286–294Fig.8–T ypical photomicrographs of the testicular seminiferous tubules of rat in each group (H&E staining,200×):(A)model 1d group;(B)model 7d group;(C)model 14d group;(D)LJP 1d group;(E)LJP 7d group;(F)LJP 14d group;(G)the control group.with the control group.After 14d of the cessation of radiation,the reproductive system damage of model group continued and the self-repair was slow.But it in LJP groups was nearly to the control group.It showed that through increasing antiox-idase and decreasing peroxidation product,LJP can alleviate the radiation damage and promote the defense system recov-ery to alleviate testicles and somniferous tubules oxidative damage and improve various spermatogenic cells recovery.Meanwhile,LJP can increase LDH activity in testis tissue to further increase the sperm quantity,quality and reproductive function.Various forms of radiation could cause sex hormones dis-order (Grajewski et al.,2000).The results showed that serum LH,FSH,Ts levels decreased significantly after 14d of ces-sation of radiation while E2levels increased but still lower than the control group.It indicated that the multiple mini-dose of partially irradiation caused rats’sex hormone disorder.Serum sex hormone (LH,FSH,Ts,E2)in all LJP groups were close to the control group.14d after the cessation of radia-tion,LH and Ts levels in LJP group were significantly different from the corresponding model group.Probably,LJP gener-ated the hormone-like effects to keep balance of serum sex hormones and then maintain the spermation functions of tes-ticles.According “Procedures of Assessment and Methods of Examination on the Function of Health Foods”published byChina’s Ministry of Health,reproductive function test were carried to evaluate the radiation-protect effects of LJP (Bureau of Health Supervision,1998).Results showed that erection and mount latency in each model groups extended with the mount and ejaculation frequency tency and fre-quency in LJP groups changed in the opposite way and 14d after radiation they were nearly equal to the control group.It further proved that multiple mini-dose radiation damaged the reproductive system.But through adjust the functions of hypothalamus–pituitary–testicular gonadal axis (Dou and Jiang,2007),LJP produced significant improvement effects on rats’reproductive dysfunction and sex behavior disorder.5.ConclusionLJP has the effect of reducing the male reproductive system injury and mating function damage induced by multiple mini-dose of partially radiation and promoting the self-repair.The mechanism of such reports not only related to Wu et al.’s (2003)report that the LJP can enhance the immune function to promote its anti-radiation,but also relate to the effect of LJP on increasing the antioxidant enzyme activity,reducing oxidative damage in testis germ cells and the tissue injury of rats and promoting their antioxidante n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y31(2011)286–294293defense system resume(Yan et al.,2003;Zhu et al.,2008). 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