REACTIVE OXYGEN SPECIES AND HUMAN SPERMATOZOA - ANALYSIS OF THE CELLULAR MECHANISMS INVOLVED IN LUMINOL-DEPENDENT AND LUCIGENIN-DEPENDENT CHEMILUMINESCENCE
REACTIVE OXYGEN SPECIES AND HUMAN SPERMATOZOA - ANALYSIS OF THE CELLULAR MECHANISMS INVOLVED IN LUMINOL-DEPENDENT AND LUCIGENIN-DEPENDENT CHEMILUMINESCENCE
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DOI：
10.1002/jcp.1041510305
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发表时间：
1992-06-01
影响因子：
5.6
通讯作者：
WEST, KM
中科院分区：
文献类型：
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作者：
AITKEN, RJ;BUCKINGHAM, DW;WEST, KM
我们已经表明，人类精子产生和释放活性氧，可以检测到的化学发光技术。负责这种活动的细胞机制的分析表明，探针，鲁米诺，经历过氧化氢和精子过氧化物酶位于顶体内介导的细胞内的双加氧反应。支持这一模型的观察结果包括：（1）鲁米诺依赖的信号可被过氧化物酶抑制剂苯肼和叠氮化钠抑制;（2）这种抑制可被添加对叠氮不敏感的过氧化物酶辣根过氧化物酶（HRP）逆转;（3）用氰化钾（KCN）抑制细胞内超氧化物歧化酶（SOD）可抑制鲁米诺信号;（4）在用3，3 '5，5'四甲基联苯胺（TMB）纯化的人精子群中可检测到过氧化物酶活性;（5）这种过氧化物酶在顶体囊泡内的pH值下具有活性;（6）在表现出先天性顶体缺失的精子中，过氧化物酶活性和鲁米诺依赖的化学发光最小。人类精子也可以产生光泽精依赖的荧光信号，既不能抑制与过氧化物酶抑制剂，也不能通过添加过氧化物酶增强。然而，这些信号可以通过抑制细胞内SOD与KCN或抑制外源性SOD增强，表明光泽精响应超氧阴离子释放到细胞外空间。荧光技术的能力，以检测和区分生产的超氧化物和过氧化氢的精子应促进进一步分析活性氧作为介质的正常和异常的人类精子功能。
We have shown that human spermatozoa generate and release reactive oxygen species that can be detected by chemiluminescence techniques. Analysis of the cellular mechanisms responsible for this activity suggests that the probe, luminol, undergoes an intracellular dioxygenation reaction mediated by hydrogen peroxide and a sperm peroxidase located within the acrosome. Support for this model included the following observations: (1) the luminol-dependent signal could be suppressed with peroxidase inhibitors, phenylhydrazine and sodium azide; (2) this suppression could be reversed by the addition of an azide-insensitive peroxidase, horse radish peroxidase (HRP); (3) inhibition of intracellular superoxide dismutase (SOD) with potassium cyanide (KCN) suppressed the luminol signal; (4) peroxidase activity could be detected in purified populations of human spermatozoa with 3,3'5,5' tetramethylbenzidine (TMB); (5) this peroxidase was active at the pH prevailing within the acrosomal vesicle; and (6) peroxidase activity and luminol-dependent chemiluminescence were minimal in spermatozoa exhibiting a congenital absence of acrosomes. Human spermatozoa could also generate lucigenin-dependent chemiluminescent signals that could neither be suppressed with peroxidase inhibitors nor enhanced by the addition of peroxidase. However, these signals could be enhanced by suppression of intracellular SOD with KCN or inhibited by exogenous SOD, suggesting that lucigenin was responding to superoxide anion released into the extracellular space. The ability of chemiluminescent techniques to detect and discriminate the production of superoxide and hydrogen peroxide by spermatozoa should facilitate the further analysis of reactive oxygen species as mediators of normal and abnormal human sperm function.