Study Of The Mechanism Of Spermatogenic Cells Apoptosis In Rats Exposed To 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/kgMnCl₂,30mg/kg MnCl₂),8 rats/group.15mg/kg MnCl₂ and 30mg/kg MnCl₂ groups were contaminated manganese(MnCl₂·4H₂O) 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 MnCl₂ exposure for 4w could lead to the quantity of sperm decreased.There existed time-effects relationship between them.2.15mg/kg MnCl₂ exposure for 4w could induce the apoptosis of rat spermatogenic cells,there existed dose-effects and time-effects relationship between them.3.15mg/kg MnCl₂ 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 MnCl₂ 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.