Exogenous gangliosides modulate cellular free radical production
Mirjana Gavella1, Vaskresenija Lipovac1, Mariastefania Antica2 & Verica Garaj-Vrhovac3
Recent studies have reported that gangliosides, the sialic acid containing glycosphingolipids, are able to modulate many cellular functions. As reactive oxygen species (ROS) in human semen originate from both spermatozoa and infiltrating leukocytes, we aimed to investigate i) the ability of gangliosides to protect human spermatozoa from hydrogen peroxide (H2O2)-induced DNA and membrane damage and ii) the effect of gangliosides on the production of ROS by PMA-stimulated human polymorphonuclear neutrophils (PMN). Single-cell gel electrophoresis (Comet assay) was used in the assessment of sperm DNA integrity, Annexin V/PI staining was used to evaluate apoptosis after exposure to H2O2, while flow cytometry using dichlorodihydrofluorescein diacetate dye (DCFH-DA) was employed to measure intracellular ROS. Extracellular superoxide production was investigated by SOD-inhibitable cytochrome c reduction and EPR spectroscopy using DEPMPO spin trap. In vitro supplemented GT1b (100 μM) to the sperm suspension significantly reduced DNA damage induced by 200 μM H2O2 (P<0.05), increased the percentage of live cells with intact membrane (P<0.01) and decreased phosphatidylserine translocation after exposure to H2O2. Flow cytometry analysis revealed that GT1b completely inhibited the passage of H2O2 through the sperm membrane. Experiments with PMA-activated PMN showed that exogenously added mono- and trisialoganglioside GM1 and GT1b in a concentration of 100 μM, which was above their respective critical micellar concentrations, induced a significant delay (P<0.05) in extracellular superoxide anion production. In addition, intracellular ROS production was shown to be inhibited in the neutrophils pretreated with both gangliosides. The results gave evidence that the attachment of gangliosides at the surface of the cell protects human spermatozoa from DNA fragmentation and increased apoptosis induced by exposure to H2O2. Furthermore, ganglioside micelles added to neutrophils slowed down the uptake of PMA and consequently activation of cells for generation. The observed phenomena can be attributed to the ability of gangliosides to increase membrane diffusion barrier for toxic oxidative agents and for stimulants responsible for triggering membrane events leading to ROS production.