Diaphorase activities were measured in spermatozoa from 104 infertile men. The relationships between enzyme activity, sperm characteristics (density, motility, viability), and distribution of electrophoretic patterns were examined. There was a highly significant difference @ < .001) between the sperm diaphorase activities in specimens with sperm concentration above 40 million per milliliter (0.685 f 0.5 mIU/106 sperm; n -57) when compared with the diaphorase values of the oligospermic specimens with sperm density below 40 million per milliliter (1.53 f 1.5 mIU/106 sperm; n -47). However, there was no evidence to support any relationship between sperm diaphorase activities, sperm motility, viability, and the distribution of three diaphorase electrophoretic patterns. This indicates that these parameters do not account for the differences between the diaphorase activity of spermatozoa of infertile men with sperm concentrations below and those above 40 million per milliliter.

No abstract

The in-vitro effect of pentoxifylline, a methylxanthine derivative, on superoxide anion generation by sperm was studied in 27 men with superoxide anion production ranging from 0.57 to 13.8 nmoles 02-. per 10(6) sperm. Superoxide anion release stimulated by phorbol myristate acetate, a potent activator of oxygen radical generation, was reduced by 29-72% following the addition of 10 mM pentoxifylline. The inhibitory effect of this millimolar concentration of the drug did not depend on the initial superoxide production in the presence of phorbol myristate acetate. These results point to a potential use for pentoxifylline in the treatment of male infertility in men with an increased superoxide anion generation capacity.

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.

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.

This study has investigated the antioxidant capacity of human seminal plasma due to the presence of both high and low molecular weight antioxidant factors. Methods for the measurement of superoxide dismutase-like activity (SOD-like) and total antioxidant status (TAS) were automated, and had a within-run coefficient of variation of 7.3% for SOD-like activity and 4.8% for TAS. In 69 semen samples from unselected infertile men, SOD-like activity in seminal plasma ranged from 2 to 16 U/ml, with a mean of 6.9 +/- 2.8 U/ml. As SOD-like activity was correlated positively with levels of citric acid (p < 0.0001), zinc (p < 0.0002) and acid phosphatase activity (p < 0.0005), and there was no correlation with fructose levels, our results suggest that prostatic secretions are an important source of superoxide anion scavengers. Evaluation of SOD-like activity in infertile men with accessory sex gland infections (n = 12) showed significantly lower activity (p < 0.003) compared to values found in 12 infertile men without signs of infection. The values obtained for total antioxidant status (equivalent to the antioxidant capacity of alpha-tocopherol analogue) ranged from 1.7 to 2.3 mmol/L, with a mean of 2.1 +/- 0.1 (n = 40), reflecting the protective activity of ascorbate, urate and albumin, and to a very low extent of glutathione and taurine. The data obtained by TAS assay correlated with fructose, a major marker of vesicular secretion (p < 0.005), suggesting that low molecular weight components with antioxidant capacity derive partly from the seminal vesicles. The results indicate that the relative contribution of antioxidant defence systems capable of counteracting the deleterious action of superoxide anions, depends on the secretory activity of accessory sex glands and is independent of excessive ROS production due to increased oxidative stress.

The effect of exogenous gangliosides, the sialic acid-containing glycosphingolipids, on oxidative changes in human spermatozoa was investigated. The incorporation of disialogangliosides or trisialogangliosides (GD1b and GT1b, respectively) into the iron/ascorbate promoter system for induction of lipid peroxidation decreased the release of malondialdehyde (MDA) from peroxidizing spermatozoa. The application of monosialogangliosides and disialogangliosides (GM1 and GD1a, respectively) did not have any effect under identical experimental conditions. GT1b, at a micromolar concentration, significantly inhibited the production of MDA, a breakdown product of lipid peroxide decomposition in spermatozoa of normozoospermic infertile men (P < 0.001; n = 51). An enhanced generation of MDA exhibited by the sperm population from the low-density Percoll fraction containing defective and/or immature spermatozoa was significantly reduced in the presence of GT1b. These results and the experiments on the influence of iron-chelating agent ethylenediamine tetraacetic acid (EDTA) as well as ferrous ion concentration itself on lipid peroxidation support the hypothesis that the protective effect of ganglioside against MDA generation could be the result of its chelating activity. Furthermore, superoxide anion release of phorbol myristate acetate-stimulated spermatozoa was significantly reduced in the presence of 50 and 100 micromol l(-1) GD1b (P < 0.05) and GT1b (P < 0.005). The inhibitory effect of 100 micromol l(-1) GT1b on spermatozoa from infertile normozoospermic men was statistically significant (P < 0.001; n = 21) and did not depend on the initial superoxide anion production. In conclusion, the protective action of GD1b and GT1b could be related to both scavenging of free radicals and metal-chelating properties, which might have relevance in the protection against oxidation-induced processes in human spermatozoa.

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.