| Objective To establish the male reproductive toxicity models on rats,analysis the sperm quality and explore the possible mechanisms on oxidative stress,ASK1-JNK/p38 signaling pathway and apoptosis.Methods Sixty adult SD male rats were randomly divided into 5 groups(12 rats in each group): control group treated with corn oil intragastrically.ACN groups treated with ACN(intragastrically)dissolved in corn oil at doses of 11.5,23.0,46.0 mg/kg bw,respectively.NAC group treated with 300.0 mg/kg NAC before exposed to 46.0 mg/kg ACN.All animals were administrated at a dose of 5.0 ml/kg.Administration was conducted 6 days per week for 28 days.Twenty-four hours after the last treatment,all rats were anesthetized by ether,and then blood samples were directly taken from the heart.(1)The testis and epididymis were removed and weighed.(2)Testis of 3 rats in each group were prepared for histopathological examination.(3)Testicular tissues were homogenized for measuring the activities of ACP,AKP,LDH and SDH.(4)Left epididymal sperms were collected for evaluating sperm motility through a CAS A system,and right epididymal sperms were used to determine sperm morphology and sperm DNA damage.(5)Testosterone and follicle stimulating hormone were measured in serum using ELISA kits.(6)Testicular tissues were homogenized for assessing oxidative stress parameters,including SOD,CAT,GSH-Px,MDA,GSH and T-AOC.(7)TUNEL assay was used to detect the apoptosis of testicular cells.(8)The expressions of ASK1,MKK7,JNK,p38,Caspase-9,Bax,Bcl-2,and Caspase-3 mRNA in testis were detected by RT-PCR.(9)Protein expressions of ASK1,p-AS K1,MKK7,JNK,p-JNK,p38,p-p38,Caspase-9,Cyt c,Bax,Bcl-2,and Caspase-3 were measured by Western blot.Results(1)There was no significant difference in body weights,absolute weight of testis and organ coefficients of animals treated with ACN(P>0.05),the absolute weight of epididymics signif icantly decreased(P<0.05).(2)The testis tissue architecture in rats of control group showed normal spermatogenesis with normal architecture and cells,whereas shrinkage and sparse of seminiferous,disorganization of germ cells,lower cells maturity,sperms count decreased were observed in rats treated with ACN at doses of 23.0 and 46.0 mg/kg.After intervention with NAC,the injury of testis reversed,the seminiferous tubules arranged tightly and the lumen was enlarged in rats of NAC group than in ACN group treated at a dose of 46.0 mg/kg.(3)Testicular activity of ACP was increased after treatd with 46.0 mg/kg ACN,LDH increased after treated with 23.0 mg/kg ACN,meanwhile the activity of AKP decreased after treated with 11.5 and 23.0 mg/kg as compared to control(P<0.05).(4)The percent of grade “a” sperm increased,while the percent of grade “d” sperm decreased in ACN groups as compared to control(P<0.05).The sperm parameters signif icantly increased including VCL,VS L,VAP and MAD,but BCF decreased signif icantly after treated with ACN(P<0.05).The VCL,VSL,VAP and MAD of the sperms were decreased,and the BCF was increased after intervention with NAC compared to rats treated with 46.0 mg/kg ACN(P<0.05).ACN groups represented the increase in sperm abnormalities and DFI index as compared to control(P<0.05).NAC significantly decreased the sperm DFI index in rats than the rats treated with 46.0 mg/kg ACN(P<0.05).(5)Animals treated with ACN showed a reduction in testosterone while follicle stimulating hormone increased compared to control group(P<0.05).(6)11.5 mg/kg ACN signif icantly decreased the activity of SOD and T-AOC in testes,and 23.0 mg/kg ACN significantly decreased the activity of CAT and SOD,meanwhile increased the level of MDA compared to control rats(P<0.05).Animals treated with 46.0 mg/kg ACN showed signif icant increase in CAT and SOD activities,and decrease in GSH-Px,T-AOC activities and GS H level compared to control animals.Moreover,NAC reduced MDA level and CAT activity compared to rats treated with 46.0 mg/kg ACN.(7)TUNEL assay results showed that the apoptosis of germ cells was obvious and apoptosis cells in per seminiferous tubule signif icantly increased in rats treated with 23.0 and 46.0 mg/kg ACN compared to co ntrol group(P<0.05);(8)RT-PCR results showed that compared to the control group,the testicular expressions of p38,JNK,Bax and Caspase-9 mRNA were up-regulated in rats treated with 11.5 mg/kg ACN,the expressions of ASK,MKK,p38,JNK mRNA were up-regulated in rats treated with 23.0 mg/kg ACN,and ASK,MKK7,p38,JNK,Bax and Caspase-3 m RNA were up-gulated and Bcl-2 mRNA was down-regulated in rats after administrated with 46.0 mg/kg ACN.After the intervention of NAC,the expressions of ASK1,MKK7 and p38 mRNA were lower than rats treated with 46.0mg/kg ACN(P<0.05).(9)Western Blot showed that the testicular protein expressions of p-p38 and Bcl-2 were decreased and Caspase-9 was increased in ACN group at a dose of 11.5 mg/kg compared to control group,the expressions of p-ASK1,MKK7,p-JNK,p38,Cyt c,Caspase-9,Bax and cleaved-Caspase-3 were increased in rats after administrated with 23.0 mg/kg ACN,and the expressions of p-ASK1,MKK7,p-JNK,p38,p-p38,Cyt c,Bax,Caspase-9 and cleaved-Caspase-3 were increased in 46.0 mg/kg ACN group while the expression of Bcl-2 was increased(P<0.05).Moreover,the expressions of p-ASK1,MKK7,p-JNK,Caspase-9 and cleaved-Caspase-3 were lower in rats testis of NAC group than in rats treated with 46.0 mg/kg ACN(P<0.05).Conclusions(1)Overproduction of ROS is the main cause of ACN-induced oxidative damage in testis of rats.(2)Activation of ASK1-JNK/p38 signaling pathway and mitochondrial apoptotic pathway mediated via ROS is one of the mechanisms refer to ACN-induced testicular cell damage. |
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