收稿日期: 2021-07-30
修回日期: 2021-11-19
网络出版日期: 2022-04-13
基金资助
国家自然科学基金(81802028);河南省科技攻关计划项目(212102310716);河南省本科高校省级大学生创新创业训练项目(S202110472024);新乡市科技攻关计划项目(GG2019034);新乡市科技攻关计划项目(GG2021011)
Affect of Trichomonas vaginalis excretory secretory protein on sperm quality of mice
Received date: 2021-07-30
Revised date: 2021-11-19
Online published: 2022-04-13
Supported by
National Natural Science Foundation of China(81802028);Science and Technology Project of Henan Province(212102310716);Innovation and entrepreneurship training program for College Students of Henan Province(S202110472024);Science and Technology Project of Xinxiang City(GG2019034);Science and Technology Project of Xinxiang City(GG2021011)
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 × 107/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。
罗澄扬 , 郝利霞 , 郭丽华 , 王兵立 , 纪思帆 , 连安娜 , 朱晓慧 , 梅雪芳 , 田晓薇 , 王帅 , 张振超 . 阴道毛滴虫分泌蛋白对小鼠精子质量的影响[J]. 中国寄生虫学与寄生虫病杂志, 2022 , 40(2) : 236 -241 . DOI: 10.12140/j.issn.1000-7423.2022.02.016
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 × 107/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.
Key words： Trichomonas vaginalis; Secretory protein; Mouse; Sperm quality
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