Ganglioside GT1b protects human spermatozoa from hydrogen peroxide‐induced DNA and membrane damage

Abstract

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.

Modulation of ROS production in human leukocytes by ganglioside micelles

“…The idea that ganglioside micelles could be responsible for the increased diffusion barrier across the cell membrane is in agreement with our previous findings that exogenous GT1b acts as an inhibitor of hydrogen peroxide diffusion across the sperm membrane (33).…”

Protective effects of exogenous gangliosides on ROS-induced changes in human spermatozoa

“…This is in agreement with our previous findings that exogenous GT1b acts as an inhibitor of H 2 O 2 diffusion across the sperm membrane. 21 We established that 43% and 73% of ganglioside micelles are removed in two washes, respectively, 64 and they can therefore be assumed to simply adhere to the membrane surface through ion or hydrogen bonds. 83 It appears that ganglioside micelles, which are loosely attached to the sperm cell membrane, increase membrane hydrophobicity during cryopreservation.…”

“…Our biochemical and biophysical studies have provided evidence of the ability of gangliosides to protect human sperm against LPO, superoxide and hydroxyl formation and ROS-induced DNA damage. [17][18][19][20][21][22] The protective effect of gangliosides on the lipid peroxidation level As a model for studying the ability of different gangliosides to counteract lipid peroxidation in human spermatozoa, we used ferrous/ascorbate-induced LPO. In this system, LPO is not initiated through hydroxyl radical generation, but rather through the ferrous ion-catalysed breakdown of pre-existing lipid hydroperoxide in the sperm plasma membrane and the subsequent propagation of the lipid peroxidation chain reaction via alkoxyl radical generation.…”

“…Flow cytometric analyses revealed that the pre-treatment of spermatozoa with GT1b micelles almost completely abolished H 2 O 2 passage through the cell membrane ( Figure 4). 21 Reduced DNA fragmentation as a result of decreased cellular H 2 O 2 suggests that the ganglioside GT1b acts as an inhibitor of H 2 O 2 diffusion across the sperm membrane. DNA integrity is also compromised during human sperm cryopreservation, which is routinely used in the management of human male infertility.…”

“…Of particular interest are differences between the effects of various types of gangliosides on the lipid peroxidation level, superoxide anion production [17][18][19][20] and H 2 O 2 and cryopreservation-induced DNA damage of human spermatozoa. 21,22 GANGLIOSIDES Gangliosides, which are sialic acid-containing glycosphingolipids, are expressed in the outer leaflet of the plasma membrane of all mammalian cell membranes, including spermatozoa, 23,24 and are particularly abundant in neuronal cell membranes. 25 A large number of gangliosides have been isolated from the brains of various organisms, but only four structures, GM1, GD1a, GD1b and GT1b, constitute more than 90% of gangliosides in the brains of various mammalian species.…”