Light-mediated activation reveals a key role for protein kinase A and sarcoma protein kinase in the development of sperm hyper-activated motility.

摘要

BACKGROUND: 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. METHODS: Human sperm (1 x 10 cells/ml) in capacitation media were irradiated for 3 min with 40 mW/cm(2) 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 microM H89 and 10 microM PP2, respectively. Soluble adenlyl cyclase was inhibited using 20 microM 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. RESULTS: 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 Ca(2) concentration and the increase in HAM was significantly reduced when voltage-dependent-Ca(2)-channel activity was blocked or when Ca(2)-deficient medium was used. CONCLUSIONS: 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 Ca(2) influx.