Molecular Mechanisms Involved in the Impairment of Boar Sperm Motility by Peroxynitrite-Induced Nitrosative Stress

Abstract

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment. physiologically by spermatozoa, generating peroxynitrite (ONOO − ) by a very fast and irreversible reaction [13] due to its exothermic nature [15]. In the same way, peroxynitrite can diffuse into the cells and even cross cell membranes and at low concentrations contribute to modulate sperm functions, mainly capacitation, as demonstrated in human spermatozoa [13] or in cryopreserved bovine spermatozoa, where ONOO − might also act as an inducer of sperm capacitation [16]. However, peroxynitrite molecule has been involved in some diseases (reviewed in [17]), because it is a strong oxidizing and/or nitrating agent that covalently binds to a variety of biomolecules causing lipid peroxidation, protein oxidation, nitration of the aromatic-side chains of amino acids as tyrosine and tryptophan, DNA oxidation, and inactivation of different enzymes. Therefore, ONOO − causes oxidative stress that promotes protein damage by direct oxidation of proteins side-chain, but also by adduction of secondary products resulting from the oxidation of polyunsaturated fatty acids, lipid peroxidation [18].The molecular and functional effects of nitrosative stress induced by high concentrations of ONOO − in spermatozoa have been mainly investigated in the human species [5,7,8,10,13,19], where ROS and RNS promote a dose dependent increase of sperm tyrosine-nitrated proteins and S-glutathionylation and impair sperm motility and capacitation [5,13]. Among negative eff...

The Interaction of NO and H2S in Boar Spermatozoa under Oxidative Stress

“…The difference in the results could be attributed to the fact that, in our study, the positive effect might be masked by the presence of additionally induced oxidative stress. The reaction of NO with ROS results in the formation of peroxynitrite, which impairs sperm quality traits and the state of lipid peroxidation in boar spermatozoa at micromolar concentrations [ 24 ]. An interesting observation was made concerning the kinetic parameters of the CASA analysis.…”

“…Current RS classification includes reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS), whereof the last two named groups also involve both gasotransmitters, NO and H 2 S. As an example, in a recent study by Zhang et al [ 21 ], the negative impact of elevated concentrations of H 2 S on boar sperm motility was observed. Moreover, the presence of oxidative stress can potentiate the negative impact of high gasotransmitter levels, such as in the case of NO [ 6 ], which forms peroxynitrite that inhibits mitochondrial activity [ 22 ], reduces sperm motility [ 23 ], and increases lipid peroxidation of boar sperm membrane [ 24 ]. Thus, the effect of the gasotransmitter on sperm functionality and structural integrity depends not only on its concentration, but also on the specific cellular microenvironment and the presence of other RS.…”

The Roles of NO and H2S in Sperm Biology: Recent Advances and New Perspectives

“…In addition, Uribe et al [90] observed mitochondrial permeability transition (MPT) under nitrosative stress with biochemical traits of MPT-driven necrosis. On the contrary, Serrano et al [91] found that, although peroxinitrite induces oxidative stress in boar sperm, leading to lipid peroxidation and motility loss, it does not affect mitochondrial membrane potential.…”