de Lamirande E, Gagnon C
Urology Research Laboratory, Royal Victoria Hospital, Montréal, Québec, Canada.
Free Radic Biol Med. 1995 Mar;18(3):487-95. doi: 10.1016/0891-5849(94)00169-k.
Previous results demonstrating the role of the superoxide anion in human sperm capacitation indirectly suggest that the superoxide anion is not only essential to this process, but is also generated by spermatozoa themselves. Our aim was to provide direct evidence for production of superoxide anion by capacitating spermatozoa. Three chemiluminescence probes were tested to detect the superoxide anion. Luminol- and lucigenin-amplified chemiluminescence of capacitating spermatozoa did not differ from that of background level. However, significant SOD-inhibitable chemiluminescence was observed when the Cypridina luciferin analog, MCLA, was used as probe. The chemiluminescence associated to capacitation of spermatozoa incubated in Ham's F-10 supplemented with fetal cord serum ultrafiltrate was 1270 +/- 77 mV/10 s (with 8 x 10(6) cells/ml; n = 37), and corresponded to levels of sperm hyperactivation (12 +/- 1%) and capacitation (17 +/- 2%) that were significantly different from those of control spermatozoa (4.9 +/- 0.8% and 6 +/- 1%, respectively). MCLA was cell impermeant and superoxide specific. The level of capacitation-associated chemiluminescence was directly related to sperm concentration up to 30 x 10(6) cells/ml. Sperm production of superoxide anion started at the beginning of incubation in capacitating conditions and was sustained over more than 4 h. This is the first direct evidence for the generation, by human spermatozoa, of a sustained level of superoxide anion that is associated with the progressive development of hyperactivation and capacitation.
先前的研究结果表明超氧阴离子在人类精子获能过程中发挥作用，这间接提示超氧阴离子不仅对该过程至关重要，而且由精子自身产生。我们的目的是为获能精子产生超氧阴离子提供直接证据。测试了三种化学发光探针来检测超氧阴离子。获能精子的鲁米诺和光泽精增强化学发光与背景水平无差异。然而，当使用海萤荧光素类似物MCLA作为探针时，观察到显著的超氧化物歧化酶（SOD）抑制性化学发光。在补充有胎牛血清超滤物的哈姆F-10培养基中孵育的精子获能相关化学发光为1270±77 mV/10 s（细胞浓度为8×10⁶个细胞/ml；n = 37），并且对应于精子超活化（12±1%）和获能（17±2%）水平，这与对照精子（分别为4.9±0.8%和6±1%）显著不同。MCLA不能透过细胞且具有超氧化物特异性。获能相关化学发光水平与精子浓度直接相关，直至30×10⁶个细胞/ml。在获能条件下孵育开始时精子就开始产生超氧阴离子，并持续超过4小时。这是人类精子产生与超活化和获能的逐步发展相关的持续水平超氧阴离子的首个直接证据。
