【摘要】 目的:探讨线粒体靶向抗氧化剂Mitoquinone(Mito Q)对冻融人精子的保护作用。方法:选取60份健康生育男性精液标本,每份精液一式6份,不含Mito Q者设为对照组(G0),而G1、G2、G3、G4、G5实验组混合液中分别含有2 nmol/L、20 nmol/L、200 nmol/L、2μmol/L、20μmol/L Mito Q,37℃孵育1 h后检测活性氧(ROS)、丙二醛(MDA)和线粒体膜电位(MMP)变化。选取合适Mito Q浓度B1、B2组用于精子冷冻保存,B0组未添加Mito Q,B1、B2组在精子冷冻保护液中分别含有200 nmol/L和2μmol/L Mito Q,进行冷冻保存,检测冷冻复苏后的ROS水平、MDA含量和MMP改变。结果:新鲜精液添加Mito Q孵育后,G3组和G4组前向运动精子百分率[(30.8±10.2)%和(32.7±13.5)%]和总活动率[(70.6±9.0)%和(70.3±11.9)%]显著高于G0组[(17.6±5.0)%、(54.9±11.5)%](P<0.05);随着Mito Q浓度的增加,ROS水平呈下降趋势,G3、G4、G5组(分别为86.5±31.6、93.6±42.0、45.1±15.0)显著低于G0组(160.8±39.7)(P<0.05);MDA含量G3、G4组[分别为(0.9±0.5)、(0.9±0.5)μmol/mg]明显低于G0组[(1.9±1.1)μmol/mg](P<0.05),而G5组[(1.7±0.7)μmol/mg]不但没有降低,反而显著高于G3、G4组(P<0.05);与G0组MMP(1 701±251)相比,G5组(1 156±216)显著降低(P<0.05),而G1、G2、G3、G4组(分别为1 810±298、1 995±437、1 950±334、1 582±314)无明显变化。冷冻复苏后各组前向运动精子百分率和总活动率均较新鲜精液明显下降(P<0.01),B1组前向运动精子百分率[(3.2±2.3)%]较B0组[(0.8±0.6)%]明显改善(P<0.05);B1组精子总活动率[(43.0±9.5)%]较B0组[(26.5±11.4)%]明显改善(P<0.05);B1组ROS[(34.6±12.3)]和B2组ROS[(37.0±10.5)]均较B0组[(56.9±14.3)]显著下降(P<0.05),B1组MDA[(1.4±0.5)μmol/mg]和B2组MDA[(1.4±0.6)μmol/mg]均较B0组[(2.6±1.0)μmol/mg]显著下降(P<0.05),B1组MMP[(1 010.0±131.5)]和B2组MMP[(880.6±128.6)]均显著高于B0组[(721.1±24.8)](P<0.05)。结论:在精液冻存液中添加200 nmol/L的Mito Q能有效提高人精子质量,可作为精液冷冻保护添加剂用于精液的冷冻保存。更多还原

【Abstract】 Objective: To investigate the potential protective effect of the mitochondria-targeted antioxidant Mitoquinone( Mito Q) on post-thaw human sperm. Methods: Semen samples were collected from 60 normal fertile men,each divided into six parts of equal volume to be incubated at 37 ℃ in normal saline( G0,control) or in the extender with 2 nmol / L( G1),20 nmol / L( G2),200 nmol / L( G3),2 μmol / L( G4),and 20 μmol / L of Mito Q( G5). After one hour of incubation,the samples were subjected to computer-assisted semen analysis( CASA) for sperm motility,flow cytometry for reactive oxygen species( ROS),thiobarbituric acid assay for the concentration of malondialdehyde( MDA),and Mito Tracker fluorescent staining and flow cytometry for the sperm mitochondrial membrane potential( MMP). Then,the semen were cryopreserved with none( B0),200 nmol / L( B1),and 2 μmol / L of Mito Q( B2),followed by detection of the changes in the ROS,MDA,and MMP of the post-thaw sperm. Results: The percentage of progressively motile sperm and total rate of sperm motility were significantly higher in G3( [30. 8 ± 10. 2]% and [70. 6 ± 9. 0]%)and G4( [32. 7 ± 13. 5]% and [70. 3 ± 11. 9]%) than in G0( [17. 6 ± 5. 0]% and [54. 9 ± 11. 5]%)( P < 0. 05). The level of ROS dropped markedly with the increased concentration of Mito Q,86. 5 ± 31. 6 in G3,93. 6 ± 42. 0 in G4,and 45. 1 ±15. 0 in G5,as compared with 160. 8 ± 39. 7 in G0( P < 0. 05). The content of MDA was remarkably lower in G3( [0. 9 ± 0. 5]μmol /mg) and G4( [0. 9 ± 0. 5]μmol /mg) than in G0( [1. 9 ± 1. 1] μmol /mg)( P < 0. 05),but not in G5( [1. 7 ± 0. 7]μmol /mg),which was even higher than in G3 and G4( P < 0. 05). The MMP showed a significant reduction in G5( 1 156 ± 216)in comparison with G0( 1 701 ± 251)( P < 0. 05) but exhibited no remarkable difference between G0 and G1( 1 810 ± 298),G2( 1 995 ± 437),G3( 1 950 ± 334),or G4( 1 582 ± 314). The percentage of progressively motile sperm and total rate of sperm motility after freezing-thawing were significantly decreased as compared with those of the fresh semen( P < 0. 01),but both were remarkably higher in B1( [3. 2 ± 2. 3]% and [ 43. 0 ± 9. 5]%) than in B0( [0. 8 ± 0. 6]% and [26. 5 ± 11. 4]%)( P <0. 05). The ROS level was significantly lower in B1 and B2 than in B0( 34. 6 ± 12. 3 and 37. 0 ± 10. 5 vs 56. 9 ± 14. 3,P <0. 05),and so was the MDA content( [1. 4 ± 0. 5] and [1. 4 ± 0. 6] μmol / mg vs [2. 6 ± 1. 0] μmol / mg,P < 0. 05),but the MMP was markedly higher in B1 and B2 than in B0( 1 010. 0 ± 130. 5 and 880. 6 ± 128. 6 vs 721. 1 ± 24. 8,P < 0. 05). Conclusion: Addition of Mito Q to the freezing extender at 200 nmol / L may effectively improve the quality of human sperm and Mito Q is a good protective addictive for human sperm cryopreservation.更多还原