弱精症精子SOD2和PRDX6的表达变化及其与精子运动能力的相关性分析  

Expression changes of superoxide dismutase 2 and peroxiredoxin 6 in asthenospermia and their correlation with sperm motility

作　　者：杨梓涵 郝小玲 王虹[2] 叶柳 黄润 孟江萍[1] YANG Zihan;HAO Xiaoling;WANG Hong;YE Liu;HUANG Run;MENG Jiangping(Center for Reproductive Medicine,the First Affiliated Hospital of Chongqing Medical University,Chongqing,400016;College of Laboratory Medicine,Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学附属第一医院生殖医学中心,重庆400016 [2]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《第三军医大学学报》2021年第20期2199-2207,共9页Journal of Third Military Medical University

摘　　要：目的探究活性氧(reactive oxygen species,ROS)对精子的损伤作用,分析抗氧化蛋白超氧化物歧化酶2(SOD2)和过氧化物酶6(PRDX6)在弱精症患者精子中的表达变化,并分析其与精子运动能力的相关性。方法选取2020年1-11月我中心收集的弱精症患者精液标本(PR<32%,PR+NP<40%,精子密度≥15×10^(6)/mL)84例,正常精液标本(PR≥32%,PR+NP≥40%,精子密度≥15×10^(6)/mL)55例。通过精液计算机辅助分析系统(computer-aided sperm analysis,CASA)检测精液常规参数及精子运动参数;不同浓度H_(2)O_(2)处理正常精子后通过CASA检测精子活力和运动参数变化,伊红染色检测精子存活率变化,Western blot检测精子SOD2和PRDX6表达变化;运用实时荧光定量PCR(RT-qPCR)和Western blot同时对弱精症组精子和正常组精子SOD2和PRDX6表达进行定量检测,进一步统计分析SOD2和PRDX6的表达变化及其与精液参数的相关性。结果CASA检测结果显示精子活力在0.10、0.35、1.00 mmol/L H_(2)O_(2)处理后显著降低;精子运动参数中曲线速率(VCL)、直线速率(VSL)、平均路径速率(VAP)和平均角位移(MAD)在0.05、0.10、0.35 mmol/L H_(2)O_(2)处理后剂量依赖式降低,侧摆幅度(ALH)、直线性(LIN)、摆动性(WOB)和前向性(STR)在0.10、0.35 mmol/L H_(2)O_(2)处理后降低;伊红染色结果显示精子存活率在0.35、1.00 mmol/L H_(2)O_(2)处理后降低,而Western blot检测结果显示精子SOD2和PRDX6表达呈H_(2)O_(2)(0.10、0.35和1.00 mmol/L)剂量依赖式增高。RT-qPCR检测结果显示弱精症组精子SOD2[(0.297±0.038)vs(0.895±0.140),P<0.01]与PRDX6[(0.743±0.107)vs(1.416±0.163),P<0.01]mRNA相对表达量低于正常组。Western blot结果显示弱精症组精子SOD2[(0.689±0.147)vs(1.466±0.212),P<0.01]与PRDX6[(0.726±0.084)vs(1.183±0.100),P<0.01]蛋白相对表达量低于正常组。相关性分析结果显示精子SOD2 mRNA相对表达量与前向运动精子百分率(r=0.267,P<0.01)和总活力(r=0.329,P<0.01)呈正相关Objective To explore the damaging effects of reactive oxygen species(ROS)on spermatozoa,and investigate the expression changes of superoxide dismutase 2(SOD2)and peroxiredoxin 6(PRDX6)in spermatozoa of asthenospermia patients,so as to analyze their correlation with sperm motility.Methods A total of 84 semen samples from asthenospermia patients[progressive motility(PR)<32%,PR+non-progressive(NP)<40%,sperm density≥15×10^(6)/mL]and 55 normal semen samples(PR≥32%,PR+NP≥40%,sperm density≥15×10^(6)/mL)were collected in our center from January to November 2020.The normal semen parameters were detected using a computer-aided sperm analysis(CASA)system.After the samples were treated with different concentrations of H_(2)O_(2),and the altered parameters were measured by CASA.The changes in sperm survival rate were determined by eosin staining,and the expression changes of SOD2 and PRDX6 were evaluated by Western blotting.Finally,RT-qPCR and Western blotting were adopted to compare the levels of SOD2 and PRDX6 in spermatozoa between the asthenospermia patients and normal controls,and the correlation of the levels with semen parameters were further analyzed.Results CASA results showed that sperm motility was significantly restrained after 0.10,0.35 and 1.00 mmol/L H_(2)O_(2) treatment;and the motility parameters like curvilinear velocity(VCL),straight line velocity(VSL),average path velocity(VAP)and mean angular displacement(MAD)were decreased in a dose-dependent manner with the increase of H_(2)O_(2) concentration,while the amplitude of lateral head displacement(ALH),linearity(LIN),wobble(WOB)and straightness(STR)were reduced at concentrations of 0.10 and 0.35 mmol/L.Eosin staining indicated that the sperm survival rate was decreased after 0.35 and 1.00 mmol/L H_(2)O_(2)treatment,and Western blotting showed the expression of SOD2 and PRDX6 in spermatozoa was up-regulated at a dose-dependent manner with the increase of H_(2)O_(2) concentration.In addition,both the mRNA levels of SOD2(0.297±0.038 vs 0.895±0.140,P<

关 键 词：弱精症 精子运动能力 ROS SOD2 PRDX6 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学] R345[医药卫生—基础医学]