染锰大鼠生精细胞凋亡机理的研究
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摘要
目的:研究染锰大鼠睾丸精子数量,生精细胞凋亡、caspase-3mRNA、caspase-3及支持细胞vimentin表达的影响,探讨染锰大鼠生精细胞凋亡的机理。方法:雄性SD大鼠(体重120±10g)48只随机分为6组:空白对照组,15mg/kg和30mg/kg MnCl_2组。8只/组。15mg/kg MnCl_2组和30mg/kg MnCl_2组分别染锰(MnCl_2·4H_2O)4w和6w,空白对照组给予等容NS,给锰及生理盐水途径均为腹腔注射,5d/w,1次/d,大鼠分别于第4w末和第6w末处死,取睾丸和附睾作以下检测:1.检测精子数量;2.TUNEL法检测睾丸生精细胞凋亡指数;3.原位杂交法检测生精细胞caspase-3mRNA表达;4.免疫组化(SABC法)法检测生精细胞caspase-3及支持细胞vimentin表达。结果:1.锰对大鼠精子数量的影响:与空白对照组比较,各染锰组精子数量均显著降低(P＜0.01)。染锰剂量相同,染锰6w组与染锰4w组比较,精子数量均显著降低(P＜0.01)。各组大鼠精子数量和AI呈负相关(r=-0.700,P＜0.01)。精子数量和生精细胞caspase-3mRNA阳性细胞率呈负相关(r=-0.892,P＜0.01)。精子数量和生精细胞caspase-3阳性细胞率呈负相关(r=-0.870,P＜0.01)。精子数量和支持细胞vimentin阳性细胞率呈正相关(r=0.895,P＜0.01)。2.锰对大鼠生精细胞AI的影响:与空白对照组比较,各染锰组AI均升高(P＜0.05)。染锰剂量相同,染锰6w组与染锰4w组比较,AI均显著升高(P＜0.01)。染锰时间相同,30mg/kg MnCl_2组与15mg/kg MnCl_2组比较,AI均显著升高(P＜0.01)。各组大鼠生精细胞AI和caspase-3mRNA阳性细胞率呈正相关(r=0.842,P＜0.01)。各组大鼠生精细胞AI和caspase-3阳性细胞率呈正相关(r=0.855,P＜0.01)。各组大鼠生精细胞AI和支持细胞vimentin阳性细胞率呈负相关(r=-0.859,P＜0.01)。3.锰对大鼠生精细胞caspase-3mRNA表达的影响:与空白对照组比较,各染锰组caspase-3mRNA阳性细胞率均显著升高(P＜0.01)。染锰剂量相同,染锰6w组与染锰4w组比较,caspase-3mRNA阳性细胞率均显著升高(P＜0.01)。染锰时间相同,30mg/kg MnCl_2组与15mg/kg MnCl_2组比较,caspase-3mRNA阳性细胞率均显著升高(P＜0.01)。各组大鼠caspase-3mRNA阳性细胞率和caspase-3阳性细胞率呈正相关(r=0.976,P＜0.01)。各组大鼠生精细胞caspase-3mRNA阳性细胞率和支持细胞vimentin阳性细胞率呈负相关(r=-0.975,P＜0.01)。4.锰对大鼠生精细胞caspase-3表达的影响:与空白对照组比较,各染锰组caspase-3阳性细胞率均显著升高(P＜0.01)。染锰剂量相同,染锰6w组与染锰4w组比较,caspase-3阳性细胞率均显著升高(P＜0.01)。染锰时间相同,30mg/kgMnCl_2组与15mg/kgMnCl_2组比较,caspase-3阳性细胞率均显著升高(P＜0.01)。各组大鼠生精细胞caspase-3阳性细胞率和支持细胞vimentin阳性细胞率呈负相关(r=-0.959,P＜0.01)5.锰对大鼠睾丸支持细胞vimentin表达的影响:与空白对照组比较,各染锰组vimentin阳性细胞率均显著降低(P＜0.01)。染锰剂量相同,染锰6w组与染锰4w组比较,vimentin阳性细胞率均显著降低(P＜0.01)。染锰时间相同,30mg/kg MnCl_2组与15mg/kg MnCl_2组比较,vimentin阳性细胞率均显著降低(P＜0.01)。结论:1.染锰15mg/kg 4w即可诱发大鼠睾丸精子数量减少,两者存在一定的时间-效应关系。2.染锰15mg/kg 4w即可诱导大鼠生精细胞凋亡,且生精细胞AI随染锰时间的延长和剂量的增加而增加,存在一定的时间-效应关系和剂量-效应关系。3.染锰15mg/kg 4w即可诱发大鼠生精细胞caspase-3mRNA及caspase-3表达,并且caspase-3mRNA及caspase-3的表达随染锰时间和剂量的增加而增加,二者均存在一定的时间-效应关系和剂量-效应关系。4.染锰15mg/kg 4w即可抑制大鼠睾丸支持细胞vimentin表达,vimentin表达随染锰时间和剂量的增加而降低,二者存在一定的时间-效应关系和剂量-效应关系。5.染锰大鼠睾丸生精细胞caspase-3mRNA及caspase-3选择性高表达和支持细胞vimentin选择性低表达可能是锰诱发大鼠生精细胞凋亡的主要机理,也是导致大鼠生精障碍的主要原因。
Objective:Our purpose was to study the effects of manganese on the quantity of sperm apoptosis,caspase-3mRNA,caspase-3 and vimentin expression in the rats testis,and to discuss the mechanism of spermatogenic cell apoptosis of male rats induced by manganese. Methods:48 male rats(weighting 110-130g) were randomly divided into 6 groups(blank control,15mg/kgMnCl_2,30mg/kg MnCl_2),8 rats/group.15mg/kg MnCl_2 and 30mg/kg MnCl_2 groups were contaminated manganese(MnCl_2·4H_2O) by I.P.for 4 and 6 weeks respectively and blank control groups was injected with NS.All rats were injected once everyday,five times per week.In the 4~(th) and 6~(th) weekend,their testis and epididymis were collected to do following examinations:1.The quantity of sperm was examined;2.The spermatogenic cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) technique;3.The expression of caspase-3mRNA in testis were investigated by in situ hybridization;4.The expression of caspase-3 and vimentin in testis were investigated by immunohisto chemical methods (SABC).Results:1.The effects of manganese on the quantity sperm:Compared with blank control groups,the quantity of sperm was decreased significantly in every manganese exposure groups(P＜0.01).Among manganese exposure groups of same dose,the quantity of sperm was decreased significantly in 6w manganese exposure groups than in 4w manganese exposure groups(P＜0.01).There existed a negative correlation between the quantity of sperm and AI(r=-0.700,P＜0.01).There existed a negative correlation between the quantity of sperm and the caspase-3mRNA-positive-cell rate(r=-0.892,P＜0.01).There existed a negative correlation between the quantity of sperm and the caspase-3-positive-cell rate (r=-0.870,P＜0.01).There existed a positive correlation between the quantity of sperm and the vimentin-positive-cell rate(r=0.895,P＜0.01).2.The effects of manganese on the spermatogenic cell apoptosis index(AI):Compared with blank control groups,AI was increased in every manganese exposure groups(P＜0.05).Among manganese exposure groups of same dose,AI was increased in 6w manganese exposure groups than it in 4w manganese exposure groups(P＜0.01).Among manganese exposure groups of same time,AI was increased significantly in 30mg/kg manganese exposure groups than it in 15mg/kg manganese exposure groups(P＜0.01).There existed a positive correlation between AI and the caspase-3mRNA-positive-cell rate(r=0.842,P＜0.01).There existed a positive correlation between AI and the caspase-3-positive-cell rate(r=0.855,P＜0.01).There existed a negative correlation between AI and vimentin-positive-cell rate(r=-0.859,P＜0.05).3.The effects of manganese on the expression of caspase-3mRNA in the spermatogenic cells:Compared with blank control groups,the caspase-3mRNA-positive-cell rate was increased in every manganese exposure groups(P＜0.01).Among manganese exposure groups of same dose,the caspase-3mRNA-positive-cell rate was increased in 6w manganese exposure groups than it in 4w manganese exposure groups(P＜0.01).Among manganese exposure groups of same time,the caspase-3mRNA-positive-cell rate was increased significantly in 30mg/kg manganese exposure groups than it in 15mg/kg manganese exposure groups(P＜0.01).There existed a positive correlation between the caspase-3mRNA-positive-cell rate and the caspase-3-positive-cell rate(r=0.976,P＜0.01).There existed a negative correlation between the caspase-3mRNA-positive-cell rate and the vimentin-positive-cell rate(r=-0.975,P＜0.01). 4.The effects of manganese on the expression of caspase-3 in the spermatogenic cells: Compared with blank control groups,the caspase-3-positive-cell rate was increased in every manganese exposure groups(P＜0.01).Among manganese exposure groups of same dose,the caspase-3-positive-cell rate was increased in 6w manganese exposure groups than it in 4w manganese exposure groups(P＜0.01).Among manganese exposure groups of same time,the caspase-3-positive-cell rate was increased significantly in 30mg/kg manganese exposure groups than it in 15mg/kg manganese exposure groups(P＜0.01).There existed a negative correlation between the caspase-3-positive-cell rate and the vimentin-positive-cell rate (r=-0.959,P＜0.01).5.The effects of manganese on the expression of vimentin in the sertoli cells:Compared with blank control groups,the vimentin-positive-cell rate was decreased in every manganese exposure groups(P＜0.01).Among manganese exposure groups of same dose,the vimentin-positive-cell rate was decreased in 6w manganese exposure groups than it in 4w manganese exposure groups(P＜0.01).Among manganese exposure groups of same time,the vimentin-positive-cell rate was decreased significantly in 30mg/kg manganese exposure groups than it in 15mg/kg manganese exposure groups(P＜0.01).Conclusions:1. 15mg/kg MnCl_2 exposure for 4w could lead to the quantity of sperm decreased.There existed time-effects relationship between them.2.15mg/kg MnCl_2 exposure for 4w could induce the apoptosis of rat spermatogenic cells,there existed dose-effects and time-effects relationship between them.3.15mg/kg MnCl_2 exposure for 4w could induce the expression of caspase-3mRNA and caspase-3 in rat spermatogenic cells,the expression of caspase-3mRNA and caspase-3 of all manganese exposure groups were increased with elongation of time and increase of dose after manganese exposure,there existed dose-effects and time-effects relationship between them.4.15mg/kg MnCl_2 exposure for 4w could inhibit the expression of vimentin in rat spermatogenic cells,vimentin-positive-cell rate was decreased with elongation of time and increase of dose after manganese exposure.There existed dose-effects and time-effects relationships between them.5.The up-regulated expression of caspase-3 mRNA,caspase-3 and the down-regulated expression of vimentin could be an important mechanism in the apoptosis of rat spermatogenic cells and spermatogenic disturbance induced by manganese.
Objective:Our purpose was to study the effects of manganese on the quantity of sperm apoptosis,caspase-3mRNA,caspase-3 and vimentin expression in the rats testis,and to discuss the mechanism of spermatogenic cell apoptosis of male rats induced by manganese. Methods:48 male rats(weighting 110-130g) were randomly divided into 6 groups(blank control,15mg/kgMnCl_2,30mg/kg MnCl_2),8 rats/group.15mg/kg MnCl_2 and 30mg/kg MnCl_2 groups were contaminated manganese(MnCl_2·4H_2O) by I.P.for 4 and 6 weeks respectively and blank control groups was injected with NS.All rats were injected once everyday,five times per week.In the 4~(th) and 6~(th) weekend,their testis and epididymis were collected to do following examinations:1.The quantity of sperm was examined;2.The spermatogenic cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) technique;3.The expression of caspase-3mRNA in testis were investigated by in situ hybridization;4.The expression of caspase-3 and vimentin in testis were investigated by immunohisto chemical methods (SABC).Results:1.The effects of manganese on the quantity sperm:Compared with blank control groups,the quantity of sperm was decreased significantly in every manganese exposure groups(P＜0.01).Among manganese exposure groups of same dose,the quantity of sperm was decreased significantly in 6w manganese exposure groups than in 4w manganese exposure groups(P＜0.01).There existed a negative correlation between the quantity of sperm and AI(r=-0.700,P＜0.01).There existed a negative correlation between the quantity of sperm and the caspase-3mRNA-positive-cell rate(r=-0.892,P＜0.01).There existed a negative correlation between the quantity of sperm and the caspase-3-positive-cell rate (r=-0.870,P＜0.01).There existed a positive correlation between the quantity of sperm and the vimentin-positive-cell rate(r=0.895,P＜0.01).2.The effects of manganese on the spermatogenic cell apoptosis index(AI):Compared with blank control groups,AI was increased in every manganese exposure groups(P＜0.05).Among manganese exposure groups of same dose,AI was increased in 6w manganese exposure groups than it in 4w manganese exposure groups(P＜0.01).Among manganese exposure groups of same time,AI was increased significantly in 30mg/kg manganese exposure groups than it in 15mg/kg manganese exposure groups(P＜0.01).There existed a positive correlation between AI and the caspase-3mRNA-positive-cell rate(r=0.842,P＜0.01).There existed a positive correlation between AI and the caspase-3-positive-cell rate(r=0.855,P＜0.01).There existed a negative correlation between AI and vimentin-positive-cell rate(r=-0.859,P＜0.05).3.The effects of manganese on the expression of caspase-3mRNA in the spermatogenic cells:Compared with blank control groups,the caspase-3mRNA-positive-cell rate was increased in every manganese exposure groups(P＜0.01).Among manganese exposure groups of same dose,the caspase-3mRNA-positive-cell rate was increased in 6w manganese exposure groups than it in 4w manganese exposure groups(P＜0.01).Among manganese exposure groups of same time,the caspase-3mRNA-positive-cell rate was increased significantly in 30mg/kg manganese exposure groups than it in 15mg/kg manganese exposure groups(P＜0.01).There existed a positive correlation between the caspase-3mRNA-positive-cell rate and the caspase-3-positive-cell rate(r=0.976,P＜0.01).There existed a negative correlation between the caspase-3mRNA-positive-cell rate and the vimentin-positive-cell rate(r=-0.975,P＜0.01). 4.The effects of manganese on the expression of caspase-3 in the spermatogenic cells: Compared with blank control groups,the caspase-3-positive-cell rate was increased in every manganese exposure groups(P＜0.01).Among manganese exposure groups of same dose,the caspase-3-positive-cell rate was increased in 6w manganese exposure groups than it in 4w manganese exposure groups(P＜0.01).Among manganese exposure groups of same time,the caspase-3-positive-cell rate was increased significantly in 30mg/kg manganese exposure groups than it in 15mg/kg manganese exposure groups(P＜0.01).There existed a negative correlation between the caspase-3-positive-cell rate and the vimentin-positive-cell rate (r=-0.959,P＜0.01).5.The effects of manganese on the expression of vimentin in the sertoli cells:Compared with blank control groups,the vimentin-positive-cell rate was decreased in every manganese exposure groups(P＜0.01).Among manganese exposure groups of same dose,the vimentin-positive-cell rate was decreased in 6w manganese exposure groups than it in 4w manganese exposure groups(P＜0.01).Among manganese exposure groups of same time,the vimentin-positive-cell rate was decreased significantly in 30mg/kg manganese exposure groups than it in 15mg/kg manganese exposure groups(P＜0.01).Conclusions:1. 15mg/kg MnCl_2 exposure for 4w could lead to the quantity of sperm decreased.There existed time-effects relationship between them.2.15mg/kg MnCl_2 exposure for 4w could induce the apoptosis of rat spermatogenic cells,there existed dose-effects and time-effects relationship between them.3.15mg/kg MnCl_2 exposure for 4w could induce the expression of caspase-3mRNA and caspase-3 in rat spermatogenic cells,the expression of caspase-3mRNA and caspase-3 of all manganese exposure groups were increased with elongation of time and increase of dose after manganese exposure,there existed dose-effects and time-effects relationship between them.4.15mg/kg MnCl_2 exposure for 4w could inhibit the expression of vimentin in rat spermatogenic cells,vimentin-positive-cell rate was decreased with elongation of time and increase of dose after manganese exposure.There existed dose-effects and time-effects relationships between them.5.The up-regulated expression of caspase-3 mRNA,caspase-3 and the down-regulated expression of vimentin could be an important mechanism in the apoptosis of rat spermatogenic cells and spermatogenic disturbance induced by manganese.
引文
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[1]吕文,周玲(介评),(审校).锰元素体内平衡的营养状况.国外医学:医学地理分册,2007,28(1):36-38.
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[9]张玉敏,柏松,王薛君,等.氯化锰处理小鼠精子质量的变化.中国职业医学,2004,31(02):23-25.
[10]姜岳明,吕志光.锰对接触男工性腺激素水平的影响.广西医科大学学报,2000,17(1):20-22.
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[20]Wang R A,Nakane P K,Koji T.Autonomous cell death of mouse male germ cells during fetal and postnatal period[In Process Citation].1998,58(5):1250-1256.
[21]贾娜,高星,王瑞生.二乙氧基乙醇诱导大鼠生精细胞凋亡及其分子机制研究.毒理学杂志,2005,19(1):26-28.
[22]杨利丽,李如江,等.过量酒精对大鼠生精细胞凋亡的影响.中国公共卫生,2003,19(2):167-168.
[23]Vera,Yanira,Diaz-Romero M,et al.Mitochondria-Dependent Pathway Is Involved in Heat-Induced Male Germ Cell Death:Lessons from Mutant Mice.Biol Reprod,2004,70(5):1534-1540.
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