SummaryBackgroundInfertility is an important worldwide problem which affects 10–15% of couples globally. Altered NO production has also been implicated in the pathogenesis of the male infertility. The present study was designed to evaluate the changes in the activity of NO-synthase (NOS) and arginase in spermatozoa of patients with infertility.MethodsThe total NOS, Ca2+-dependent constitutive (cNOS) and Ca2+-independent inducible (iNOS) activity and arginase activity were assessed in sperm cells of patients with different forms of pathospermia.ResultsWe found a significant increase in iNOS activity, but significantly decreased cNOS and arginase activity in sperm cells of infertile men vs fertile, normozoospermic men (p<0.001). The arginase/NOS ratio significantly decreased compared to control group. The iNOS/cNOS ratio was drastically increased in patients with decreased fertility potential indicating predominance of iNOS. Men with leuko cytospermia were distinguished to have the most express iNOS activity.ConclusionsThese observations provide evidence for a disturbed balance between the L-arginine metabolic pathways in sperm cells of infertile men. This imbalance includes the considerable activation of the inducible isoform of NO-synthase accompanied by significant inhibition of its constitutive isoform which indicates disturbances in NO production. In patients with decreased fertility potential the arginase/NOS was shifted towards predominance of iNOS-derived NO production.

Hyperproduction of reactive oxygen species can damage sperm cells and is considered to be one of the mechanisms of male infertility. Cell protection from the damaging effects of free radicals and lipid peroxidation products is generally determined by the degree of antioxidant protection. Glutathione is non-enzymatic antioxidant which plays an important protective role against oxidative damages and lipid peroxidation. The aim of the present work is to determine the content of reduced and oxidized glutathione in sperm cells of infertile men. Semen samples from 20 fertile men (normozoospermics) and 72 infertile patients (12 oligozoospermics, 17 asthenozoospermics, 10 oligoasthenozoosper­mics and 33 leucocytospermic) were used. The total, oxidized (GSSG) and reduced (GSH) glutathione levels were measured spectrophotometrically. The levels of total glutathione were significantly lower in the spermatozoa of patients with oligozoo-, asthenozoo- and oligoasthenozoospermia than in the control. Infertile groups showed significantly decreased values of reduced glutathione in sperm cells vs. fertile men, indicating an alteration of oxidative status. The oxidized glutathione levels in sperm cells of infertile men did not differ from those of normozoospermic men with proven fertility. The GSH/GSSG ratio was significantly decreased in the oligo-, astheno- and oligoasthenozoospermic groups compared to the normozoospermic group. In patients with leucocytospermia the GSH/GSSG ratio was lower but these changes were not significant. In addition, glutathione peroxidase activity in sperm cells was decreased in patients with oligozoo-, astenozoo-, oligoastenozoospermia and with leucocytospermia. The most significant changes in glutathione peroxidase activity were observed in infertile men with leucocytospermia. Decreased GSH/GSSG ratio indicates a decline in redox-potential of the glutathione system in sperm cells of men with decreased fertilizing potential. Redistribution between oxidized and reduced forms of glutathione can be caused by depletion of intracellular stores of glutathione and intensification of lipid peroxidation processes. This leads to increased production of reactive oxygen species, further depletion of antioxidant pools and disturbances of structure and function of spermatozoa. Our results indicate that the evaluation of reduced glutathione level and GSH/GSSG ratio in sperm cells of infertile men can be helpful in fertility assessment.

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Na+,K+-ATPase plays an essential role in sperm motility, hyperactivation, chemotaxis, acrosome reaction etc. Na+,K+-ATPase is sensitive to ROS insult. Apart from production of highly reactive molecules, H2O2 can exert a number of direct effects on cells, their metabolism and enzymes. In the present study, exposure to exogenous H2O2 was used to characterize the effects of H2O2 on Na+,K+-ATPase activity in spermatozoa of infertile men with different forms of pathospermia. It was shown that Na+,K+-ATPase activities in spermatozoa of infertile men with different forms of pathospermia were inhibited by exposure to H2O2 (50−500 μM). H2O2, one of the most toxic oxygen species, has the ability to depress Na+,K+-ATPase activity in a dose-dependent manner. Severe inhibition of the hydrolytic activity was observed when higher H2O2 were used. The time course of incubation with 100 μM H2O2 showed a sharp decrease in the enzyme activity during the first 5 min of incubation for both normozoospermic and pathozoospermic men. The enzymatic activity of Na+,K+-ATPase in the sperm was completely destroyed at 20 min for asthenozoospermic men and 30 min for normozoospermic men. We show that an administation of H2O2 inhibited Na+,K+-ATPase activity in normozoospermic samples with IC50 of 106.6 ± 7.9 μM. IC50 for patients with asthenozoospermia was two times less than for healthy men with preserved fertility. For other studied groups, the differences in IC50 were not significant. These observations suggest that Na+,K+-ATPase in pathozoospermic samples is more vulnerable to H2O2-induced damage than in normozoospermic men. The Hill coefficient was significantly increased only for patients with asthenozoospermia, indicating increased positively cooperative binding. The decreases in Na+,K+-ATPase hydrolase activity in H2O2-treated sperm cells in men with normozoospermia were largely attenuated by exogenous GSH at 5 mM. This suggests that GSH partially protects the Na+,K+-ATPase from inhibition under experimental oxidative stress. However, treatment of oligo-, astheno- and oligoasthenozoospermic samples with 100 μM H2O2 and 5 mM GSH did not result in protection of Na+,K+-ATPase against induced oxidation, suggesting that the impaired Na+,K+-ATPase in pathozoospermic samples appears to be an irreversible event. In contrast, presence of GSH only after H2O2 treatment does not reverse Na+,K+-ATPase inhibition. This study has provided a deeper insight into the role Na+,K+-ATPase plays in sperm cells,it also could offer clues to the clinical application of antioxidant therapy in male infertility therapy.