The Mina & Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
Hum Reprod. 2011 Sep;26(9):2274-82. doi: 10.1093/humrep/der232. Epub 2011 Jul 19.
Hyper-activated motility (HAM) is part of the sperm capacitation process, which is necessary for fertilization. In this study, we investigated the effect of visible light on sperm motility and hyperactivation and evaluated pathways mediating these effects.
Human sperm (1 × 10⁷ cells/ml) in capacitation media were irradiated for 3 min with 40 mW/cm² visible light (400-800 nm with maximum energy at 600 nm). Sperm motility was assessed and analyzed by computer-assisted sperm analysis. The involvement of sperm capacitation factors was investigated as follows. The generation of reactive oxygen species (ROS) was measured using 20,70-dichlorofluorescein diacetate. Protein kinase A (PKA) and sarcoma protein kinase (Src) activity were measured using western blot analysis and inhibited using 50 µM H89 and 10 µM PP2, respectively. Soluble adenlyl cyclase was inhibited using 20 µM 2-OH-Estradiol. The intracellular concentration of free Ca(2+) was assessed using the fluorescent calcium indicator, Fluo-4/AM. Sperm DNA fragmentation was determined using the sperm chromatin dispersion test.
Light irradiation of human sperm caused a significant increase in hyper-HAM but not total motility. The production of ROS and activation of soluble adenylyl cyclase and PKA mediated the effect of light on HAM. Light irradiation also activated Src, and inhibition of Src significantly reduced the effect of light on HAM. Light irradiation caused a rapid increase in intracellular Ca²⁺ concentration and the increase in HAM was significantly reduced when voltage-dependent-Ca²⁺-channel activity was blocked or when Ca²⁺-deficient medium was used.
Light irradiation of human sperm for a short time causes a significant increase in HAM in a mechanism mediated by ROS production, activation of PKA, Src and Ca²⁺ influx.
超激活运动（HAM）是精子获能过程的一部分，而获能对于受精是必需的。在这项研究中，我们研究了可见光对精子运动和超激活的影响，并评估了介导这些影响的途径。
在获能培养基中，将 1×107 个/ml 的人精子用 40 mW/cm2 的可见光（400-800nm，最大能量在 600nm）照射 3 分钟。通过计算机辅助精子分析评估和分析精子运动。如下所述，研究了精子获能因子的参与情况。使用 20,70-二氯荧光素二乙酸酯测量活性氧（ROS）的产生。使用蛋白质激酶 A（PKA）和肉瘤蛋白激酶（Src）活性的 Western blot 分析测量蛋白激酶 A（PKA）和肉瘤蛋白激酶（Src）活性，并分别使用 50μM H89 和 10μM PP2 抑制。使用 20μM 2-OH-雌二醇抑制可溶性腺苷酸环化酶。使用荧光钙指示剂 Fluo-4/AM 评估细胞内游离 Ca（2+）浓度。使用精子染色质弥散试验确定精子 DNA 碎片化。
人精子的光照射导致超-HAM 显著增加，但总运动没有增加。ROS 的产生以及可溶性腺苷酸环化酶和 PKA 的激活介导了光对 HAM 的影响。光照射还激活了 Src，而 Src 的抑制显著降低了光对 HAM 的影响。光照射导致细胞内 Ca²⁺浓度的快速增加，当电压依赖性 Ca²⁺通道活性被阻断或使用缺乏 Ca²⁺的培养基时，HAM 的增加显著减少。
人精子的短时间光照射会导致 HAM 显著增加，其机制是通过 ROS 产生、PKA、Src 和 Ca²⁺内流的激活介导的。
