Oehninger S, Blackmore P, Mahony M, Hodgen G
Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Norfolk, Virginia 23507, USA.
J Assist Reprod Genet. 1995 Jan;12(1):41-7. doi: 10.1007/BF02214128.
Reactive oxygen species (ROS) have been reported widely to cause deleterious effects on sperm viability and function due to peroxidation of membrane lipids. However, their action appears more selective at low concentrations; recent evidence indicates that the superoxide anion can promote capacitation and induce hyperactivated motility (HA) in human spermatozoa and that hydrogen peroxide (H2O2) may participate in capacitation of hamster spermatozoa. The objective of these studies was to investigate the direct effects of H2O2 on functions crucial to fertilization in human spermatozoa.
In these prospective studies, we examined the dose- and time-dependent effects of H2O2 on sperm membrane-mediated events (binding to the zona pellucida and changes in intracellular calcium concentration [Ca2+]i, motility patterns, and acrosome reaction). Sperm from fertile donors were used in the experiments under capacitating conditions after separation of the motile fraction by wash/swim-up. [Ca2+]i was measured by the fluorescent fura-2 indicator, and sperm-zona pellucida binding was assessed with the hemizona assay (HZA). Hyperactivated motility was evaluated by computerized analysis, and the percentage of acrosome reacted sperm was detected by FITC-Pisum sativum lectin and indirect immunofluorescence.
In the HZA, H2O2 did not influence sperm-zona pellucida binding at low concentrations (0.05 mM and 0.1 mM), but significantly reduced binding at 0.2 mM (P < 0.004 vs controls). H2O2 significantly decreased HA in a dose-dependent manner (P < 0.0001) and had a significant effect (P < 0.01) on acrosome reaction (stimulatory effect at 0.01 mM). H2O2 did not affect basal [Ca2+]i; however, H2O2 (0.1 mM through 10 mM) decreased the initial phase of progesterone-induced (P4: 1 microM) enhancement of [Ca2+]i in a dose- and time-dependent fashion. Preincubation of sperm with catalase (20 micrograms/ml) potentiated the P4-induced increase of [Ca2+]i. H2O2 did not significantly modify [Ca2+]i increase in response to inomycin (10 microM).
These experiments show that H2O2 directly affects sperm functions crucial to fertilization in a dose- and time-dependent fashion. Low concentrations maintain capacitation, whereas higher concentrations have deleterious effects, as determined by the end points of the capacitation process. The latter effects are probably dependent on modifications of plasma membrane and intracellular homeostasis by the oxidative process.
活性氧（ROS）已被广泛报道可因膜脂质过氧化而对精子活力和功能产生有害影响。然而，它们在低浓度时的作用似乎更具选择性；最近的证据表明，超氧阴离子可促进人精子的获能并诱导超活化运动（HA），而过氧化氢（H2O2）可能参与仓鼠精子的获能。这些研究的目的是研究H2O2对人精子受精关键功能的直接影响。
在这些前瞻性研究中，我们研究了H2O2对精子膜介导事件（与透明带结合以及细胞内钙浓度[Ca2+]i变化、运动模式和顶体反应）的剂量和时间依赖性影响。在通过洗涤/上浮分离活动部分后，在获能条件下使用来自可育供体的精子进行实验。[Ca2+]i通过荧光fura-2指示剂测量，精子-透明带结合通过半透明带试验（HZA）评估。通过计算机分析评估超活化运动，通过FITC-豌豆凝集素和间接免疫荧光检测顶体反应精子的百分比。
在HZA中，低浓度（0.05 mM和0.1 mM）的H2O2不影响精子与透明带的结合，但在0.2 mM时显著降低结合（与对照组相比，P < 0.004）。H2O2以剂量依赖性方式显著降低HA（P < 0.0001），并对顶体反应有显著影响（P < 0.01，在0.01 mM时有刺激作用）。H2O2不影响基础[Ca2+]i；然而，H2O2（0.1 mM至10 mM）以剂量和时间依赖性方式降低孕酮诱导（P4：1 microM）的[Ca2+]i增强的初始阶段。用过氧化氢酶（20微克/毫升）预孵育精子可增强P4诱导的[Ca2+]i增加。H2O2对离子霉素（10 microM）诱导的[Ca2+]i增加没有显著影响。
这些实验表明，H2O2以剂量和时间依赖性方式直接影响精子对受精至关重要的功能。低浓度维持获能，而较高浓度具有有害影响，这由获能过程的终点确定。后者的影响可能取决于氧化过程对质膜和细胞内稳态的改变。
