Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa
Generation of reactive oxygen species by equine neutrophils and their effect on motility of equine spermatozoa
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DOI：
10.1016/s0093-691x(01)00710-5
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发表时间：
2002-02-01
期刊：
影响因子：
2.8
通讯作者：
Ball, BA
中科院分区：
文献类型：
--
作者：
Baumber, J;Vo, A;Ball, BA
精液中污染的白细胞是人类精液中活性氧（ROS）的重要来源。当数量足够多时。它们可能对人类的精子功能产生有害影响。不幸的是，很少有关于白细胞在种马精液中的重要性发表的信息。本研究的目的是确定过氧化氢（H2 O2）的生产激活马中性粒细胞，并检查这种ROS生产对马精子活力的影响在体外，运动的马精子（两个射精，每个从四个种马）和外周血中性粒细胞分离的不连续Percoll梯度，洗涤和重悬在一个修改的台氏培养基。精子（25 × 10（6）/ml）与中性粒细胞在38 ℃孵育30分钟（0，0.5 × 10（6）、1 × 10（6）、5 × 10（6）和10 × 10（6）/ml），由蛋白激酶C激动剂、12-肉豆蔻酸酯、13-乙酸佛波酯（PMA; 100 nM）或白细胞趋化肽，甲酰-甲硫氨酰-亮氨酰-苯丙氨酸（FMLP; 0.1 mM）。在时间0 min（T0）和时间30 min（T30）通过计算机辅助精液分析（CASA）测定精子活力，并在T30使用Amplex Red（TM）测定试剂盒测定H2 O2。在T30时，（P < 0.01）H2 O2增加，添加5 x 10和10 x 10（6）粒中性粒细胞/ml，由FMLP激活（分别为0.76 +/- 0.3和0.99 +/- 0.4 μ M，单独精子中为0.0024 +/- 0.002 μ M），这种增加与总活动力的显著（P < 0.001）降低有关（分别为52 +/- 5.1和48 +/-6.0%，而单独精子为80 +/- 4.7%）。在T30时，加入5 × 10（6）和10 × 10（6）PMA激活的中性粒细胞/ml（1.88 +/- 0.2和2.07 +/- 0.3 μ M），H2 O2也显著增加（P < 0.001）。0009 +/- 0.0006 μ M）。本研究的结果表明，5 × 106活化中性粒细胞/ml足以在体外损害马精子活力。(C)2002年爱思唯尔科技有限公司All rights reserved.
Contaminating leukocytes in the ejaculate are an important source of reactive oxygen species (ROS) in human semen. When present in sufficient numbers. they can have a detrimental influence on sperm function in humans. Unfortunately, there is little published information regarding the importance of leukocytes in stallion semen. The objectives of this study were to determine the production of hydrogen peroxide (H2O2) by activated equine neutrophils and to examine the effect of this ROS production on equine sperm motility in vitro, Motile equine spermatozoa (two ejaculates each from four stallions) and peripheral blood neutrophils were isolated on discontinuous Percoll gradients, washed and resuspended in a modified Tyrode's medium. Spermatozoa (25 x 10(6)/ml) were incubated for 30 min at 38 degreesC with neutrophils (0,0.5 x 10(6).1 x 10(6), 5 x 10(6) and 10 x 10(6)/ml) activated by either the protein kinase C agonist, 12-myristate, 13-acetate phorbol ester (PMA; 100 nM) or the leukocyte chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP; 0.1 mM). Sperm motility was determined by computer-assisted semen analysis (CASA) at time 0 min (T0) and time 30 min (T30), and H2O2 was measured at T30 with the Amplex Red(TM) assay kit. At T30, there was a significant (P < 0.01) increase in H2O2, with the addition of 5 x 10 and 10 x 10(6) neutrophils/ml activated by FMLP (0.76 +/- 0.3 and 0.99 +/- 0.4 mu M, respectively, versus 0.0024 +/- 0.002 mu M in sperm alone), and this increase was associated with a significant (P < 0.001) decrease in total motility (52 +/- 5.1 and 48 +/- 6.0%, respectively, versus 80 +/- 4.7% in sperm alone). At T30, there was also a significant (P < 0.001) increase in H2O2 with the addition of 5 x 10(6) and 10 x 10(6) neutrophils/ml activated by PMA (1.88 +/- 0.2 and 2.07 +/- 0.3 mu M. respectively, versus 0,0009 +/- 0.0006 mu M in sperm alone). The results of this study demonstrate that 5 x 106 activated neutrophils/ml are sufficient to impair equine sperm motility in vitro. (C) 2002 Elsevier Science Inc. All rights reserved.