Department of Biological Chemistry, University of Padova, Viale G. Colombo 3, 35131 Padova, Italy.
Hum Reprod. 2011 Dec;26(12):3264-73. doi: 10.1093/humrep/der321. Epub 2011 Sep 22.
Generation of controlled amounts of reactive oxygen species (ROS) and phosphorylation of protein tyrosine residues (Tyr) are two closely related changes involved in sperm capacitation. This study investigated the effect of altered endogenous ROS production on Tyr-phosphorylation (Tyr-P), acrosome reaction (AR) and cell viability during sperm capacitation. The possible origin of the altered ROS production was also evaluated by apocynin (APO) or oligomycin (Oligo) addition.
A total of 63 samples of purified sperm were analysed for ROS production by enhanced chemiluminescence, Tyr-P pattern by immunocytochemistry, and AR and viability by fluorochrome fluorescein isothiocyanate (FITC)-labelled peanut (Arachis hypogaea) agglutinin and propidium iodide positivity, respectively.
Samples were divided into four categories depending on the ability of sperm to produce ROS, expressed as Relative Luminescence Units (RLU), in capacitating conditions: low ROS production (LRP), range about 0.0-0.05 RLU; normal (NRP), 0.05-0.1 RLU; high (HRP), 0.1-0.4 RLU; very high (VHRP) 0.4-2.0 RLU. In NRP sperm heads, capacitation induced Tyr-P in 87.9 ± 4.3%, and the AR occurred in 62.5 ± 5.4% of cells; in LRP, HRP and VHRP Tyr-P labelling rarely spread over the head, acrosome-reacted cells only accounted for a small number of sperm, and the non-viable cells (NVC) were increased. The addition of APO, but not Oligo, drastically decreased ROS production in analysed samples.
This study proposes the optimal threshold for endogenous ROS production for correct sperm viability and functioning, and indicates the direct involvement of APO-sensitive NADPH oxidase in ROS production.
活性氧（ROS）的受控生成和蛋白质酪氨酸残基（Tyr）的磷酸化是与精子获能相关的两种密切相关的变化。本研究调查了改变内源性 ROS 生成对 Tyr 磷酸化（Tyr-P）、顶体反应（AR）和精子获能过程中细胞活力的影响。还通过添加 apocynin（APO）或寡霉素（Oligo）评估了改变的 ROS 生成的可能来源。
通过增强化学发光分析了总共 63 个纯化精子样本的 ROS 生成，通过免疫细胞化学分析 Tyr-P 模式，通过荧光素异硫氰酸酯（FITC）标记的花生（Arachis hypogaea）凝集素和碘化丙啶阳性分别分析 AR 和活力。
根据精子在获能条件下产生 ROS 的能力（表示为相对发光单位（RLU）），将样本分为四组：低 ROS 产生（LRP），范围约为 0.0-0.05 RLU；正常（NRP），0.05-0.1 RLU；高（HRP），0.1-0.4 RLU；非常高（VHRP）0.4-2.0 RLU。在 NRP 精子头部，诱导获能诱导 Tyr-P 在 87.9±4.3%的细胞中发生，并且 AR 发生在 62.5±5.4%的细胞中；在 LRP、HRP 和 VHRP 中，Tyr-P 标记很少扩散到头，顶体反应细胞仅占精子的一小部分，并且非存活细胞（NVC）增加。添加 APO，但不添加 Oligo，会大大降低分析样本中的 ROS 生成。
本研究提出了内源性 ROS 产生的最佳阈值，以确保精子活力和功能正常，并表明 APO 敏感的 NADPH 氧化酶直接参与 ROS 生成。
