阴道毛滴虫分泌蛋白对小鼠精子质量的影响

Affect of Trichomonas vaginalis excretory secretory protein on sperm quality of mice

收集阴道毛滴虫分泌蛋白(TvESP),用蛋白浓度测定试剂盒测定TvESP浓度,并进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定。从昆明小鼠的附睾收集精子并调整精子密度为1×10^(7)/ml。将小鼠精子分为0、5、10、20、40、80 ng/μl浓度组(每组设3个平行孔),分别与终浓度为0、5、10、20、40、80 ng/μl的TvESP共培养1 h,倒置显微镜下观察精子活力,细胞流式检测精子凋亡情况;小鼠精子与TvESP作用0.5、1、2 h后,台盼蓝染液染色,计算精子死亡率;小鼠精子分别与终浓度为0和40 ng/μl的TvESP共培养1 h,荧光染料染色,荧光显微镜下观察精子顶体结构。组间比较采用单因素方差分析。结果显示,TvESP作用1 h后,5、10、20、40、80 ng/μl浓度组活力精子占比分别为(31.90±4.10)%、(23.00±4.90)%、(18.80±3.50)%、(13.60±1.20)%和(8.60±1.20)%,均低于0 ng/μl浓度组的(44.50±3.20)%(F=30.76,P<0.01);5、10、20、40、80 ng/μl浓度组精子的晚期凋亡率分别为(17.99±1.73)%、(22.43±1.53)%、(26.76±2.20)%、(32.05±1.68)%和(41.37±2.28)%,均高于0 ng/μl浓度组的(15.42±1.10)%(F=256.79,P<0.01)。TvESP作用0.5 h后,10、20、40和80 ng/μl浓度组精子的死亡率分别为(11.90±1.70)%、(14.10±1.40)%、(16.30±1.20)%、(17.90±1.30)%,均高于0 ng/μl浓度组的(7.80±1.10)%(F=15.72,P<0.05);TvESP作用1 h后,5、10、20、40、80 ng/μl浓度组精子的死亡率分别为(12.90±0.90)%、(14.60±0.90)%、(16.20±1.30)%、(18.10±1.10)%、(20.00±1.40)%,均高于0 ng/μl浓度组的(9.90±1.90)%(F=15.76,P<0.01);TvESP作用2 h后,10、20、40、80 ng/μl浓度组精子的死亡率分别为(17.00±1.20)%、(18.30±1.20)%、(20.50±1.20)%、(22.80±1.50)%,均高于0 ng/μl浓度组的(12.90±0.50)%(F=22.22,P<0.01)。TvESP作用1 h后,40 ng/μl浓度组精子的顶体轮廓不清、结构完整性被破坏。TvESP可降低精子质量。

Trichomonas vaginalis excretory secretory protein(TvESP)was collected for determining the concentration using Bradford method kit and further analyzed of TvESP by thesodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Mouse sperm were collected from the epididymis of Kunming mice,and the sperm density was adjusted to 1×10^(7)/ml.Sperm were divided into 6 groups and co-cultured with TvESP at final concentrations of 0,5,10,20,40 and 80 ng/μl,respectively.After co-culturing with TvESP for 1 h,sperm motility was observed using an inverted microscope,and sperm apoptosis was detected by flow cytometry.After co-culturing for 0.5,1 and 2 h,the sperm viability was analyzed by trypan blue staining.After sperm were incubated with TvESP at final concentrations of 0 and 40 ng/μl for 1 h,sperm were stained with a fluorescent dye and the acrosome was observed under a fluorescence microscope.One-way ANOVA was used for comparing between groups.The results showed that the percentage of active sperm in the 5,10,20,40 and 80 ng/μl groups were(31.90±4.10)%,(23.00±4.90)%,(18.80±3.50)%,(13.60±1.20)%and(8.60±1.20)%,respectively,which were significantly lower than that of the 0 ng/μl concentration group(44.50±3.20)%(F=30.76,P<0.01).The apoptosis rates of sperm in the 5,10,20,40 and 80 ng/μl groups were(17.99±1.73)%,(22.43±1.53)%,(26.76±2.20)%,(32.05±1.68)%and(41.37±2.28)%,respectively,which were significantly higher than that of the 0 ng/μl group(15.42±1.10)%(F=256.79,P<0.01).After treatment with TvESP for 0.5 h,the mortality rates of sperm treated with 10 ng/μl(11.90±1.70)%,20 ng/μl(14.10±1.40)%,40 ng/μl(16.30±1.20)%,80 ng/μl(17.90±1.30)%of TvESP were significantly higher than that of the 0 ng/μl group(7.80±1.10)%(F=15.72,P<0.05).The sperm mortality rate in the 5 ng/μl group(10.10±1.40)%was not statistically different from that of the 0 ng/μl group.After treatment with TvESP for 1 h,the sperm mortality rates in the 5,10,20,40 and 80 ng/μl groups were(12.90±0.90)%,(14.60±0.90)%,(16.20±1.30)%,(18.10±1.10)%,(20.00±1.40)%,which were significantly higher than that of the 0 ng/μl group(9.90±1.90)%(F=15.76,P<0.01).After 2 h treatment,the mortality rates in the 10,20,40 and 80 ng/μl groups were(17.00±1.20)%,(18.30±1.20)%,(20.50±1.20)%and(22.80±1.50)%,respectively,which were significantly higher than that of the 0 ng/μl group(12.90±0.50)%(F=22.22,P<0.01),and the mortality rate in the 5 ng/μl group(15.00±0.40)%was not statistically different from that of the 0 ng/μl group.After co-culturing the sperm with TvESP at a final concentration of 40 ng/μl for 1 h,the integrity of acrosome was destroyed.All results suggested that TvESP can reduce sperm quality.