RESUMEN
This article summarizes the available evidence on the efficacy of gangliosides to reduce the degree of reactive oxygen species (ROS)-mediated damage. The antioxidative efficacy of exogenous gangliosides in protecting different cells encouraged us to examine their ability to protect human spermatozoa. Gangliosides are sialic acid-containing glycosphingolipids with strong amphiphilic character due to the bulky headgroup made of several sugar rings with sialic acid residues and the double-tailed hydrophobic lipid moiety. The amphiphilicity of gangliosides allows them to exist as micelles in aqueous media when they are present at a concentration above their critical micellar concentration. The protective effect of ganglioside micelles on spermatozoa is believed to stem from their ability to scavenge free radicals and prevent their damaging effects. In our study, we particularly focused our attention on the protective effect of ganglioside micelles on DNA in human spermatozoa exposed to cryopreservation. The results indicate that ganglioside micelles can modulate the hydrophobic properties of the sperm membrane to increase tolerance to DNA fragmentation, thus protecting the DNA from cryopreservation-induced damage. Further actions of ganglioside micelles, which were documented by biochemical and biophysical studies, included (i) the modulation of superoxide anion generation by increasing the diffusion barrier for membrane events responsible for signal translocation to the interior of the cell; (ii) the inhibition of iron-catalysed hydroxyl radical formation due to the iron chelation potential of gangliosides; and (iii) inhibition of hydrogen peroxide diffusion across the sperm membrane.
Asunto(s)
Antioxidantes/farmacología , Daño del ADN/efectos de los fármacos , Gangliósidos/farmacología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/efectos adversos , Espermatozoides/efectos de los fármacos , Humanos , Peroxidación de Lípido/efectos de los fármacos , MasculinoRESUMEN
Gangliosides, the sialic acid-containing glycosphyngolipids, are amphiphilic compounds which in micellar form affect the properties and functions of a cellular membrane. The aim of this study was to test whether exogenous gangliosides supplied to cryopreservation media before freezing could protect sperm cells from cryopreservation-induced DNA damage assessed by Comet assay. Additionally, to investigate whether gangliosides were also able to reduce membrane integrity damage, malonaldialdehyde as a measure of lipid peroxidation and sperm-specific lactate dehydrogenase-C4 activity as an enzyme marker of sperm membrane leakage were determined. The monosialogangliosides (GM1) and trisialogangliosides (GT1b) were examined at a concentration of 100 µM, which was above their respective critical micellar concentrations. Exogenous gangliosides were not found to protect sperm membrane from lipid peroxidation. However, a freezing-/thawing-induced increase in Comet parameters was equally significantly prevented by the presence of both GM1 and GT1b (P < .05), indicating that the ceramide moiety, rather than the polar groups, is involved in the protective ability of gangliosides. The observed phenomena suggest that ganglioside micelles could modulate hydrophobic properties of the sperm membrane responsible for better tolerance to DNA fragmentation, thus protecting DNA integrity from cryopreservation-induced damage.
Asunto(s)
Criopreservación , Crioprotectores/farmacología , Fragmentación del ADN , Gangliósido G(M1)/análogos & derivados , Preservación de Semen/métodos , Espermatozoides/efectos de los fármacos , Adulto , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/patología , Ensayo Cometa , Citoprotección , Relación Dosis-Respuesta a Droga , Gangliósido G(M1)/farmacología , Humanos , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Malondialdehído/metabolismo , Micelas , Recuento de Espermatozoides , Motilidad Espermática , Recuperación de la Esperma , Espermatozoides/metabolismo , Espermatozoides/patologíaRESUMEN
We have reported previously that various gangliosides, the sialic acid containing glycosphingolipids, provide protection against sperm injury caused by reactive oxygen species (ROS). In this study, we investigated the effect of treatment of human spermatozoa with ganglioside GT1b on hydrogen peroxide (H(2)O(2))-induced DNA fragmentation and plasma membrane damage. Single-cell gel electrophoresis (Comet assay) used in the assessment of sperm DNA integrity showed that in vitro supplemented GT1b (100 microm) significantly reduced DNA damage induced by H(2)O(2) (200 microm) (p < 0.05). Measurements of Annexin V binding in combination with the propidium iodide vital dye labelling demonstrated that the spermatozoa pre-treated with GT1b exhibited a significant increase (p < 0.05) in the percentage of live cells with intact membrane and decreased phosphatidylserine translocation after exposure to H(2)O(2). Flow cytometry using the intracellular ROS-sensitive fluorescence dichlorodihydrofluorescein diacetate dye employed to investigate the transport of the extracellularly supplied H(2)O(2) into the cell interior revealed that ganglioside GT1b completely inhibited the passage of H(2)O(2) through the sperm membrane. These results suggest that ganglioside GT1b may protect human spermatozoa from H(2)O(2)-induced damage by rendering sperm membrane more hydrophobic, thus inhibiting the diffusion of H(2)O(2) across the membrane.
Asunto(s)
Gangliósidos/farmacología , Peróxido de Hidrógeno/farmacología , Espermatozoides/efectos de los fármacos , Anexina A5/metabolismo , Anexina A5/farmacología , Membrana Celular/metabolismo , Estructuras Celulares/metabolismo , Ensayo Cometa , ADN , Daño del ADN , Fragmentación del ADN/efectos de los fármacos , Gangliósidos/metabolismo , Humanos , Hidrógeno/metabolismo , Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Masculino , Peróxidos , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacologíaRESUMEN
Antioxidant activity of gangliosides GM1 and GT1b in the Fenton type of reaction was investigated by EPR spectroscopy using DMPO as a spin trap. Hydroxyl radical spin adduct signal intensity was significantly reduced in the presence of gangliosides at their micellar concentrations. Mean micellar hydrodynamic diameter was not changed, whereas significant changes in negative Zeta potential values were observed as evidenced by Zetasizer Nano ZS. This study showed that the primary mode of ganglioside action was not due to direct scavenging of OH., but rather to the inhibition of hydroxyl radical formation. This phenomenon is related to the ability of ganglioside micelles to bind oppositely charged ferrous ions, thus reducing their concentration and consequently inhibiting OH. formation.
Asunto(s)
Antioxidantes/metabolismo , Gangliósido G(M1)/química , Gangliósido G(M2)/química , Gangliósidos/metabolismo , Micelas , Antioxidantes/química , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Depuradores de Radicales Libres/química , Radicales Libres , Gangliósidos/química , Peróxido de Hidrógeno , Radical Hidroxilo/química , Hierro , Modelos Biológicos , Modelos Químicos , Conformación Molecular , Fosfatos/químicaRESUMEN
The effect of various types of gangliosides, the sialic acid-containing glycosphingolipids, on human sperm membrane during lipid peroxidation induced by Fe(2+)/ascorbate ions was investigated. The monosialoganglioside (GM1), disialogangliosides (GD1a and GD1b), and trisialoganglioside (GT1b) were examined at a concentration of 100 microM, which was above their respective critical micellar concentrations. Lipid peroxidation was determined by quantification of malondialdehyde (MDA) concentration. The molecular orientational order in the membrane was assessed by fluorescence spectroscopy and electron paramagnetic resonance spectroscopy. Both approaches revealed a significant increase in membrane rigidity following oxidation, which correlated with an increase in the MDA level. The preincubation of spermatozoa with GM1 and GD1a did not have any effect on induced lipid peroxidation. In the presence of GD1b and GT1b, a reduced formation of MDA and a decrease in membrane rigidity was detected. The inhibitory effect of GT1b micelles toward membrane oxidation damage was found to be greater than that of GD1b. In conclusion, a direct relationship between the reduced content of the accumulated MDA and the longer preservation of the native-like membrane molecular ordering during sperm oxidation in the presence of GT1b suggests its protective effect. This phenomenon could be due to the specific GT1b conformation and its negative surface potential.
Asunto(s)
Membrana Celular/efectos de los fármacos , Gangliósidos/farmacología , Peroxidación de Lípido , Espermatozoides/efectos de los fármacos , Membrana Celular/ultraestructura , Difenilhexatrieno/análogos & derivados , Espectroscopía de Resonancia por Spin del Electrón , Polarización de Fluorescencia , Gangliósido G(M1)/farmacología , Humanos , Masculino , Malondialdehído/análisis , Espectrometría de Fluorescencia , Espermatozoides/ultraestructuraRESUMEN
The role of gangliosides in the copper-induced oxidative modification of human low-density lipoprotein (LDL) was studied focusing on the early stage of LDL oxidation in which the concentration of conjugated dienes increases only weakly. The changes in the protein and lipid component were followed using fluorescence spectroscopy. The results indicate that binding of gangliosides to LDL causes slower destruction of tryptophan fluorescence and suppresses cross-linking between the reactive groups of the protein and the products of lipid peroxidation. The protective role of gangliosides could be assigned to their interference with the lipid-protein interaction in the LDL particle, which might be important for the maintenance of the native plasma antioxidant status in vivo.
Asunto(s)
Cobre , Gangliósidos/química , Lipoproteínas LDL/química , Polarización de Fluorescencia , Gangliósidos/farmacología , Humanos , Lipoproteínas LDL/aislamiento & purificación , Oxidación-Reducción , Triptófano/químicaRESUMEN
Oxidative modification of lipids, proteins and DNA by reactive oxygen species in the organism and imbalance between the concentrations of free radicals and the antioxidant defenses may be related to processes such as aging and diseases (cardiovascular, neurodegenerative, cancer, etc.). Although the relationship between oxidant status and antioxidant defence in aging of different species, organs or sexes has been investigated extensively, the studies have produced conflicting results. In order to determine the extent of age-associated alteration, oxidant production and antioxidant status were measured in tissues of CBA and AKR mice of both sexes. At the same time we will focus on lipid peroxidation (LPO) process and superoxide dismutase activity (SOD) of AKR mice related to ontogeny of thymic lymphoma in mice of different age and sex. Male and female CBA and AKR mice aged 3, 6, 12 or 18 months were used. Lipid-bound sialic acid (LSA) content was determined as a malignancy marker. LPO processes of CBA and AKR mice were monitored according to the presence of thiobarbituric acid reactive substances (TBARS) in liver and thymus, and antioxidant status as SOD activity in whole blood. TBARS concentration increased significantly with age in the liver of CBA and AKR mice of both sexes, but only in male thymuses of both strains. TBARS concentration in female thymuses of both strains was unchanged during aging. Thus, age-associated LPO processes of tumor-free mice of both strains were tissue-dependent. In the liver of tumor-bearing CBA and AKR mice as well as in thymuses of AKR mice, TBARS concentration significantly decreased and was neither sex nor tissue related. SOD activity was strain-dependent but independent of sex. However, SOD activity in mice with developed thymomas was drastically reduced in comparison to tumor-free mice. Our data indicate that age associated LPO processes in both strains are only tissue-dependent and SOD activity mainly strain-dependent in tumor-free mice. In tumor-bearing mice LPO processes and SOD activity were not tissue, sex or strain dependent.