| Background:In the past decades,more and more infertility has been well recognized.Of all the couples trying to conceive,about 15 percent have infertility,male factors have a significant impact on infertility,accounting for approximately 40%of all cases.Maintenance of normal male fertility depends on normal sperm generation.Semen quality is an important factor that reflects male reproductive function.The decrease in sperm count and sperm quality are the main cause of male infertility.Unfortunately,in the past few decades,semen quality have been seen a marked decline even in healthy men.In addition,World Health Organization(WHO)has lowered the accepted values of normal sperm parameter,including count,motility,and morphology,because over the past decades,these parameters have a significant decline in healthy men.The sperm production of male animals mainly depends on the high self-renewal and differentiation potential of spermatogonial stem cells.Spermatogonial stem cells have the attributes of stem cells,which can self-renew and maintain the constant number of spermatogonial stem cell populations,and can also be directed to differentiate into primary spermatogenic cells,secondary spermatocytes,spermatids.Studies found that?6-integrin and?1-integrin are marker molecules on spermatogonialstem cells.Air pollution has become a global hot issue on account of the adverse influence on health.Many countries,particularly for developing countries,are experiencing the deterioration of the air.In recent decades,human fertility has shown a clear decline trend,and air pollution may be one of the reasons.In the past decades,there has been a rapid pace of economic development and urbanization in big cities,with the increase of air pollution.Air pollution has become a common problem by people all over the world.It is well known that air pollution has adverse effects on human health.Many epidemiological investigations and toxicological studies have shown that air pollution has a definite damage to the cardiovascular system and respiratory system;The male reproductive system,as an important human organ tissue,is also damaged by air pollution.In recent years,the influence of air pollution on human reproductive system have been aroused great interest.Domestic and foreign authors have published research reports on air pollution and male reproductive system damage,most of which are based on epidemiological survey data.Air pollution has been considered to bring about reduced sperm quality of males.However,the underlying mechanisms of decreased sperm quality caused by air pollution are still not fully elucidated.ObjectiveIn this study,we developed Sprague Dwaley male rat model of exposure to gasoline exhaust,observation and analysis of the impact of gasoline exhaust on male rat reproductive system function and clarified its potential mechanisms.To provide a reliable experimental basis for human reproductive health promotion and protection.Materials and Methods1.Twenty male Sprague Dawley rats(body weight 180–200g,6–8 weeks old)were providied by Guangdong Medical Laboratory Animal Center.The rats were randomly divided into two groups:a gasoline exhaust exposure group(n=10)and a clean air control group(n=10).All rats were given free access to water and food in this study.The animal room was kept for 12 hours light/dark cycle and maintained at 20±2°C with 45–65%relative humidity.Before beginning exposure,all the rats were adapted for one week.The gasoline exhaust group rats were exposed to gasoline exhaust for 6h,5 days per week,for 6 months.The rats in clean air control group were exposed to fresh air.Twenty-four hours after the last exposure,the rats were killed,body weights,tissue weights of testis and seminal vesicles were record.2.Animals were sacrificed after anaesthesia and the cauda epididymidis was quickly removed to perform analysis of sperm count and sperm deformity rate analysis.The testicle tissue section to perform analysis of morphological changes of seminiferous tubules.The total numbers of spermatogonia,primary spermatocytes,secondary spermatocytes,spermatids cells per seminiferous tubule were counted under a microscope.We randomly selected 50 seminiferous tubules of each rat and counted the number of cells in each seminiferous tubule.Then we calculated the average number of cells per seminiferous tubule per rat.Finally,we compared the statistical differences in the total number of cells per seminiferous tubule between the gasoline exhaust exposure group and control group.Immunohistochemical staining,quantitative reverse transcriptase-polymerase chain(RT-PCR)and Western Blot was used to detect expression of?6-integrin and?1-integrin.Immunohistochemical sections were observed under a microscope.First used low magnification,then used high magnification,and no less than 5 high magnification fields or 1000 cells.The percentage of positive cells is divided into 5 levels.Positive cells 0-5%were 0 points,positive cells 6%-25%were 1 point,26%-50%were 2point,51%-75%were 3 point,>75%were 4 point.Staining intensities score is divided into four grade,grade 0 is negative,grade 1 is weakly positive,grade 2 is moderately positive,grade 3 is strongly positive.Each case of the final immunoreactivity score(IRS)was multiply by the percentage and the staining intensity score.Results1.Body weights,tissue weights of testis and seminal vesiclesAfter 6 months exposure,the means weights of body,testis and seminal vesicles were record.No significant difference were detected in these data between gasoline exhaust exposure group and control group(P>0.05,for all.)2.Gasoline exhaust exposure harmed sperm qualityTo assess the degree of testicular injury,sperm quality was examined.Compared with control group(60.28±6.73*10~6/ml),sperm counts in the gasoline exhaust group(26.46±3.05*10~6/ml)were significantly lower(P<0.05).Abnormal sperm morphology rate in the exposure group(7.92±0.90%)were significantly higher compared with control group(3.90±0.33%)(P<0.05).3.Histomorphology change of testis after gasoline exhaust exposureIn control group,Cell layers of spermatogonia,primary spermatocytes,secondary spermatocytes,spermatids,spermatozoids were also normal.In the gasoline exhaust exposure group,intercellular substance became loose,amount of sperms in the lumen clearly reduced,and seminiferous epithelium had fewer cells than the control group.Fifty seminiferous tubules in each group were counted,there were 200.30±11.95 cells and 285.30±10.07 cells in the exposure group and the control group,respectively(P<0.05).4.Gasoline exhaust exposure led to spermatogonial stem cells impairmentThe results of qRT-PCR and western blotting demonstrated that compared with the control group the expression levels of?6-integrin and?1-integrin mRNA and protein in the exposure group were significantly downregulated(P<0.05,for all).Immunohistochemistry analysis corroborated these results.Conclusion1.Our study showed that male rats exposure to gasoline exhaust lead to reduce of spermatozoa in the epididymis,increase of sperm deformity,and change of morphology of seminiferous tubules in testicular tissue,and then impairment of reproductive system function.2.The results showed that exposure to gasoline exhaust caused impairment to spermatogonial stem cells through down-regulating?6-integrin and?1-integrin. |
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