| Objective: To construct a smoking model of male rats with different time and different doses,to evaluate the reproductive toxicity of smoking in male rats,and to further explore the molecular biological mechanism of reproductive toxicity caused by smoking;to elucidate learning and memory ability of offspring by Morris water maze test,and the possible mechanism of smoking in the generation of learning and memory ability of the offspring was explored in molecular level.Methods: 300 SD rats(200 males and100 females)were randomly divided into 2,4,6,8,and 12 weeks according to the time of smoking.The weekly group included blank control group(fresh air),low dose group(10cigarettes/day),middle dose group(20 cigarettes/day)and high dose group(30cigarettes/day /day),10 animals in each group.In the exposed group of cigarettes,5 male rats were mated with the female rats(unstained for 1 week)by1: 1 in the same cage before the execution.The pregnant female rats were continuously observed for growth and development after the natural delivery,and the offspring were weaned after 21 days.Randomly grouped into groups of 10 animals,half male and half female.All animal experiment was terminated after 49 days.Morris water maze test was used for evaluate the offspring,learning and memory ability,HE staining was used to observe the structure of the fathers,testis tissue and offspring,hippocampus;sperm smear method,calculate the sperm count and sperm deformity rate;observe and record the birth number and survival rate of the offspring;ELISA method was used to determine levels of IGF-1 and leptin in the serum of father and progeny.The SOD activity,MDA content and GSH-Px activity in the fathers,testis tissue and offspring hippocampus were measured.The NO content and NOS activity in the hippocampus of the offspring were determined.The immunohistochemical method was used to detect the parent.Caspase-8 protein expression in rat testis tissue and Caspase-3 protein expression in progeny hippocampus;RT-PCR and Western Blot assay for the related levels of genes and proteins in wnt/?-catenin pathway,Fas/FasL apoptosis pathway.The apoptosis was detected in the hippocampus of the offspring by TUNEL method.Results:(1)Rats exposed to cigarettes developed slow weight gain(P<0.05).The sperm count and the rate of deformity were different between different exposure times and exposure doses(P<0.05).Compared with the control group,the pathological changes of testis were more obvious in the exposed group of cigarettes with the increase of the time and dose,the testicular cells atrophied,the space of the seminiferous tubules gradually became larger,and the arrangement of spermatogonial cells was disordered,even a large area of seminiferous cells and spermatozoa of seminiferous tubules began to appear,interstitial cell proliferation was obvious,interstitial vascular hyperplasia,dilatation,and hyperemia.The female pregnancy rate and the number of births of the offspring significantly decreased(P<0.05).IGF-1and leptin in the serum of the parental rats exposed to cigarettes in different exposure periods(P<0.05).(2)The activity of SOD,GSH-Px and MDA contents in testis tissue was significantly increased in exposed groups(P < 0.05).The mRNA and protein expression of Fas,FasL,FADD,Caspase-3 and Caspase-8 in the testis of rats exposed to cigarettes increased with the prolongation of exposure time and the exposure dose(P<0.05).The expression of wnt-2b,?-catenin and GSK-3? in the testis of exposed rats were down-regulated in both mRNA and protein expression levels(P<0.05).(3)The growth of the weight of the offspring rats slowed down with the prolongation of the exposure time and dose of the fathers(P<0.05).There was an interaction between exposure dose and gender(P<0.05).IGF-1 protein content significant decreased in female offspring in all exposure groups except high dose group in 2th week and low dose group in 12 th week(P< 0.05);In male offspring,it was decreased in all exposed groups except the low,high-dose group in 8th week and low-dose group in 12 th week(P<0.05).The Leptin level in female and male offspring were both significantly decreased(P < 0.05),while increased significantly in 2nd week in female offspring and midium and high dose group in female and male offspring(P<0.05).In the water maze test of the offspring rat,the change of latency and total swimming distance in the positioning experiment of female rats were not significant;In the male offspring,the overall increase in latency and total swimming distance,and the number of crossings was also reduced(P<0.05).(4)In the female hippocampus of the offspring: the total NOS and NO content increased(P<0.05);iNOS content was decreased(P<0.05);The level of BDNF protein was down-regulated(P<0.05);The activity of SOD and GSH-Px were decreased and increased respectively(P<0.05);The number of Tunel positive cells and Caspase-3 positive cells both increased(P<0.05);The ?-catenin and GSK-3? mRNA level were both down-regulated(P<0.05);The protein level of wnt-2b,?-catenin,GSK-3? and phosphorylated GSK-3? protein were all increased(P<0.05).In the hippocampus of male offspring: the total NOS activity and iNOS content were both increased while NO content decreased(P<0.05);The level of BDNF protein decreased(P<0.05);The activity of SOD and GSH-Px and MDA contents in male offspring were all increased significantly(P<0.05);The number of Tunel positive cells and Caspase-3 positive cells were both increased(P < 0.05);The wnt-2b and?-catenin mRNA level were both up-regulated while GSK-3? was down-regulated(P<0.05);The protein level of wnt-2b,?-catenin,phosphorylated ?-catenin and phosphorylated GSK-3? were all increased while the GSK-3? was down-regulated(P<0.05).Conclusion: smoking caused damage to testicular tissue structure in male rats,decreased sperm production,and decreased birth number.The molecular biological mechanism may be smoking caused an imbalance in the oxidation and antioxidant system in testis tissue,and activates death receptor Fas/FasL apoptosis signaling pathway and inhibition of testicular cell wnt/?-catenin proliferation pathway,leading to irreversible damage and apoptosis of testicular tissue.The smoking of the father can cause the spatial learning and memory ability of the pups to be impaired,and the impact on male offspring is more obvious.The possible reason for this performance is that the smoking of the father causes the imbalance of oxidation and antioxidant system in the hippocampus of male rats and down-regulation of BDNF protein expression.smoking of the fathers increased the apoptotic rate in the hippocampus of the offspring,and up-regulated the key genes in the wnt/?-catenin proliferative pathway.The counterbalance between apoptosis and proliferation made the impairment in offspring not more obvious in Learning memory. |
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