LonP1表达与线粒体稳态以及精子活力的关联性研究

Relationship between LonP1 expression and sperm mitochondrial homeostasis &sperm motility

目的探究线粒体lon蛋白水解酶(LonP1)对精子线粒体稳态的维持调节作用及其可能存在的调节机制。方法收集2016年6月至2017年6月于温州医科大学附属第一医院生殖中心进行精液常规检查的120名男性精液标本,根据精子活力分为正常活力精子(NS)和弱精子症精子(AS)两组,采用计算机辅助精液分析系统(CASA)检测各组精子运动参数,通过免疫荧光法和免疫蛋白印迹法检测精子中LonP1的蛋白表达水平,流式细胞术检测各组精子的线粒体膜电位强度(MMP,以绿色荧光强度/红色荧光强度比值表示,比值越小代表膜电位越高)和线粒体内活性氧(mROS)水平,比较两组各指标参数差异,并分析LonP1表达水平和精子活力及精子线粒体功能相关指标(MMP,mROS)的关联性。另外,NS组精液经密度梯度离心法处理后,沉淀精子加入培养液重悬,以LonP1功能抑制剂CDDO-ME 2.5μmoL/L和5μmol/L为终浓度进行体外培养,同时设置空白对照组(0μmol/L),比较各组精子运动参数、LonP1表达水平以及精子线粒体功能相关指标。结果 (1)人类成熟精子中存在LonP1的表达,且稳定表达于精子中段的线粒体鞘部;(2)NS组和AS组精子LonP1表达水平(0.52vs.0.22)与精子活力(69.89%vs.26.10%)、MMP(4.72%vs.12.73%)呈正相关(P<0.05),与mROS含量呈负相关(418.85vs.460.52)(P<0.05);(3)经LonP1功能抑制剂CDDO-ME处理后,与空白对照组比较,CDDO-ME药物处理组的LonP1蛋白表达水平随CDDO-ME作用浓度增加而显著降低(P<0.05),5μmol/L浓度组的MMP水平显著下降(78.81%vs.7.34%)、mROS含量增加(640.68vs.462.26),差异均有统计学意义(P<0.05);(4)CDDO-ME处理后,除精子活率(PR+NP)外,精子运动参数平均路径(VAP)和曲线速率(VSL)均随CDDO-ME作用浓度增加而下降(P<0.05),与空白对照组相比,5μmol/L浓度组的精子活力(PR)(52.08%vs.65.15%)、曲线速率(VCL)(48.28μm/s vs.78.78μm/s)、侧摆幅度(ALH)(1.28μm vs.1.93μm)、直线性(LIN)(38.83%vs.56.05%)和前向性(STR)(45.53%vs.81.25%)均显著下降,差异有统计学意义(P<0.05)。结论精子线粒体鞘部存在LonP1的稳定表达,LonP1能够通过精子线粒体稳态维持改善精子活力。

Objective：To investigate the role of mitochondrial lon protein hydrolase（LonP1）in the maintenance of sperm mitochondrial homeostasis and its possible regulatory mechanisms.Methods：The semen specimens of 120 male donors from Reproductive Center of First Affiliated Hospital of Wenzhou Medical University during Jun.2016 to Jun.2017 were collected,and then the semenspecimens were divided into two groups：normal viable spermatozoa（NS）and asthenospermia（AS）.The semen parameters of each group were detected by computer assisted semen analysis system（CASA）.The expression of LonP1 was determined by Western blotting and immunofluorescence.The mitochondrial membrane potential（MMP）（In terms of the ratio of green/red fluorescence intensity,the smaller the ratio,the higher the membrane potential）and mitochondrial reactive oxygen species（mROS）of sperm were determined by flow cytometry.Then the correlation between LonP1 expression,sperm motility and sperm mitochondrial functionrelated indicators（MMP,mROS）were analyzed.In addition,semen in NS group was treated by density gradient centrifugation method,and the precipitated sperms were re-suspended in culture medium.Sperms were in vitro culture treated with the final concentrations（2.5μmol/L and 5μmol/L）of LonP1 inhibitor CDDO-ME.Meanwhile,the blank group（0μmol/L）was set.The sperm motility parameters,LonP1 expression level and sperm mitochondrial function-related indexes were compared among the groups.Results：LonP1 was stably expressed in the mitochondrial sheath in the middle part of sperm in human mature sperm.The expression level of LonP1 in NS group and AS group（0.52 vs.0.22）was positively correlated with MMP（4.72% vs.12.73% ）and sperm motility（69.89% vs.26.10% ）（P〈0.05）,and negatively correlated with the mROS levels（418.85 vs.460.52）（P〈0.05）.The expression of LonP1 protein was significantly decreased after the treatment of CDDO-ME（P〈0.05）.The expression of LonP1 protein in CDDO-ME treatment group was significantly decreased with the increase of CDDO-ME concentration（P〈0.05）.Meanwhile,MMP was significantly decreased（78.81% vs.7.34% ）,and the mROS level was significantly increased（640.68 vs.462.26）in 5μmol/L group compared with the blank group（P〈0.05）.After CDDO-ME treatment,the semen parameters VAP and VSL were significantly decreased with the concentrations of CDDO-ME（P〈0.05）.The PR（52.08% vs.65.15% ）,VCL（48.28μm/s vs.78.78μm/s）,ALH（1.28μm vs.1.93μm）,LIN（38.83% vs.56.05% ）and STR（45.53% vs. 81.25% ）were significantly decreased in 5μmol/L group（P〈0.05）.Conclusions：LonP1 was stably expressed in the mitochondrial sheath in the middle part of human mature sperm.LonP1 can improve sperm motility by maintaining sperm mitochondrial homeostasis.