ABSTRACT
PURPOSE: NADPH oxidase 5 (NOX5), the main isoform of NOX in spermatozoa, has been recognized as the main active generators of reactive oxygen species (ROS), including superoxide anion (O 2 -. ) and hydrogen peroxide (H2O2). ROS have been shown to play important roles in many physiological and pathological conditions in spermatozoa. The present study aims to investigate the alterations of NOX5 protein expression and oxidative stress (OS) status in asthenozoospermic men compared to normozoospermic men. METHODS: Semen samples were collected from 25 asthenozoospermic men and 28 normozoospermic men. In this study, NOX5 protein expression was evaluated by Western blotting. An OS status was evaluated by measuring of ROS (O 2 -. and H2O2), DNA damage and plasma membrane integrity in spermatozoa. RESULTS: The protein expression of NOX5 (p < 0.0001) was remarkably higher in asthenozoospermic men in comparison to normozoospermic men. In addition, the percentages of intracellular O 2 -. (p < 0.0001), H2O2 (p < 0.0001) in viable spermatozoa, apoptotic sperm cells with altered plasma membrane (p < 0.001) and DNA damage (p = 0.001) were significantly increased in asthenozoospermic men compared to normozoospermic men. CONCLUSIONS: The present study provides evidence that the overexpression of NOX5 protein may induce excessive ROS production and oxidative stress damages to DNA and plasma membrane integrity in asthenozoospermic men.
Subject(s)
Asthenozoospermia/genetics , Asthenozoospermia/metabolism , NADPH Oxidase 5/genetics , Oxidative Stress/physiology , Spermatozoa/metabolism , Adult , Case-Control Studies , Cell Membrane/metabolism , DNA Damage/genetics , DNA Fragmentation , Gene Expression Regulation, Enzymologic , Humans , Infertility, Male/genetics , Infertility, Male/metabolism , Male , Reactive Oxygen Species/metabolism , Semen/metabolism , Semen AnalysisABSTRACT
Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 µm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and motility parameters. These findings, although preliminary, suggest that resveratrol-induced improvement of cryopreserved sperm functions may be mediated through activation of AMP-activated protein kinase, indicating the importance of AMP-activated protein kinase activity for human spermatozoa functions. Further investigations are required to elucidate the mechanism by which resveratrol ameliorates oxidative stress-mediated damages in an AMP-activated protein kinase-dependent mechanism.