MnTBAP对精子超快速冷冻后动力、凋亡及DNA碎片的影响

Effect of MnTBAP onmotility,apoptosis and DNA fragmentation of spermatozoa after ultra-rapid cryopreservation

[目的]探究锰卟啉(MnTBAP)对精子超快速冷冻后动力学参数、细胞凋亡及DNA碎片的影响。[方法]选择35名体检健康男性精液进行超快速冷冻,以甘油-蛋黄-枸橼酸(GYC)作为基础冷冻剂,随机分为Control组(冷冻前)、M0组(GYC)、M10组(GYC+10μmol/L MnTBAP)、M20组(GYC+20μmol/L MnTBAP)、M40组(GYC+40μmol/L MnTBAP),在液氮中保存72 h后复苏,CASA系统检测精子动力学参数[曲线速度(VCL),平均路径速度(VAP),侧摆幅度(ALH)],流式细胞术检测细胞凋亡率和DNA碎片指数(DFI),并进行对比分析。[结果]与Control组比较,M0组精子总活力、顶体完整性、VCL、VAP、ALH降低,畸形率、细胞凋亡率、DFI升高;与M0组比较,M10组、M20组、M40组精子总活力、顶体完整性、VCL(50.16±8.12 vs 35.67±7.16/38.13±8.05/44.92±7.63)μm/s、VAP(29.51±6.11 vs 19.63±4.36/19.87±5.2/22.57±5.84)μm/s、ALH(3.24±2.16 vs 2.51±1.26/2.96±1.59/3.25±1.35)μm/s升高,畸形率、细胞凋亡率(16.95±6.37 vs 12.62±4.05/9.38±3.12/6.03±2.39)%、DFI(22.15±4.12 vs 18.01±3.05/17.21±3.24/13.56±2.14)%降低,且M40组上述指标改善效果最好(P<0.05)。[结论]MnTBAP能提高精子超快速冷冻后运动能力,降低细胞凋亡率和DNA碎片性,且添加40μmol/L剂量效果最好,可作为保护剂应用于精液冻存中。

[Objective]To explore the effect of manganese(Ⅲ)tetrakis-(4-benzoic acid)porphyrin(MnTBAP)on sperm kinematic parameters,apoptosis and DNA fragmentation of spermatozoa during ultra-rapid cryopreservation.[Method]Semen samples collected from 35 healthy men undergoing medical examination were subjected to ultra-rapid cryopreservation with glycerol-egg yolk-sodium citrate(GYC)as a protective medium for the spermatozoa during freezing.The samples were randomly divided into Control group(before freezing),M0 group(GYC),M10 group(GYC+10μmol/L MnTBAP),M20 group(GYC+20μmol/L MnTBAP),and M40 group(GYC+40μmol/L MnTBAP).The spermatozoa were stored in liquid nitrogen for 72 h,and recovered.The Computer-Assisted Sperm Analyzing(CASA)system was utilized to detect sperm kinetic parameters[velocity of curve line(VCL),velocity of average path(VAP),amplitude of lateral head displacement(ALH)].The apoptosis rate and DNA fragment index(DFI)was measured by flow cytometry,Then the comparison was conducted among groups.[Result]Compared with Control group,total sperm motility,acrosome integrity,VCL,VAP,and ALH were decreased,and abnormality rate,apoptosis rate,and DFI were increased in M0 group.Compared with M0 group,total sperm motility,acrosome integrity,VCL(50.16±8.12 vs 35.67±7.16/38.13±8.05/44.92±7.63)μm/s,VAP(29.51±6.11 vs 19.63±4.36/19.87±5.2/22.57±5.84)μm/s,and ALH(3.24±2.16 vs 2.51±1.26/2.96±1.59/3.25±1.35)μm/s were elevated,and the abnormality rate,apoptosis rate(16.95±6.37 vs 12.62±4.05/9.38±3.12/6.03±2.39)%,and DFI(22.15±4.12 vs 18.01±3.05/17.21±3.24/13.56±2.14)%were decreased in M10,M20 and M40 groups,furthermore,the overall improvement effect of the above indexes was the best in M40 group(P<0.05).[Conclusion]MnTBAP improves sperm motility and reduces apoptosis and DNA fragmentation after ultra-rapid cryopreservation and is most effective at a dose of 40μmol/L,which can be used as a protective agent in semen freezing.