Hydrogen Peroxide Modifies Human Sperm Peroxiredoxins in a Dose-Dependent Manner

Abstract

Low levels of reactive oxygen species (ROS) modulate signaling pathways required for human sperm activation, but high levels impair sperm function, leading to infertility. Peroxiredoxins (PRDXs) are enzymes with a dual role as ROS scavengers and modulators of ROS-dependent signaling. The present study aimed to characterize PRDXs in human spermatozoa and possible modifications resulting from hydrogen peroxide (H(2)O(2)). We found PRDX1, PRDX4, PRDX5, and PRDX6 in both seminal plasma and spermatozoa. Using immunocytochemistry, we demonstrated that these PRDXs are differentially localized in the head, acrosome, mitochondrial sheath, and flagellum. These observations were confirmed by immunoblotting using cytosolic, Triton-soluble and -insoluble, and head and flagella sperm fractions. PRDXs are dose-dependently modified by H(2)O(2), as seen by the formation of disulfide bridges and high-molecular-mass complexes. This first study, to our knowledge, on PRDXs in human spermatozoa indicates that PRDX1, PRDX4, PRDX5, and PRDX6 are modified when spermatozoa are challenged with H(2)O(2). This suggests that PRDXs may protect these cells at high levels of H(2)O(2) but could also control H(2)O(2) levels within different cell compartments so that normal sperm activation can occur.

Oxidative stress impairs function and increases redox protein modifications in human spermatozoa

“…It is noteworthy that the strong signal of tyrosine nitration and S-glutathionylation on the sperm head (Fig. 8) corresponds with the localization of PRDXs (O'Flaherty & de Souza 2011, O'Flaherty 2014. It is then plausible that the redox-dependent modifications of PRDXs are causing the increase in levels of sperm DNA damage observed in infertile men (Gong et al 2012).…”

“…PRDXs are highly abundant and differentially distributed in all sub-compartments of the human spermatozoon and are considered the first line of defense against oxidative stress in human spermatozoa (O'Flaherty & de Souza 2011). Our laboratory reported that PRDX1 and PRDX6 form w170 kDa protein complexes detected in spermatozoa treated with high concentrations of H 2 O 2 (0.25-5 mM) and also in spermatozoa from infertile men (O'Flaherty & de Souza 2011, Gong et al 2012. There is increasing evidence supporting the involvement of PRDXs in the regulation of redox signaling, especially H 2 O 2 -dependent signaling (Rhee et al 2005, Fourquet et al 2008.…”

“…ROS-treated spermatozoa were fractionated into cytosolic, triton-soluble, and triton-insoluble fractions as described previously (O'Flaherty & de Souza 2011). Briefly, cells were frozen at K80 8C for 15 min and then thawed at 37 8C in order to disrupt sperm membranes and allow the release of the cytosolic content.…”

“…Aliquots containing 10!10 6 cells were then smeared onto superfrost plus slides (Fisher Scientific, Montreal, QC, Canada), and allowed to dry at room temperature. Dried cells were permeabilized with methanol as described previously (O'Flaherty & de Souza 2011). Cells were rehydrated with PBS supplemented with triton!K100 (PBS-T) and blocked with 5% goat serum in PBS-T for 30 min at 20 8C.…”

Unravelling the bull fertility proteome

“…However, high levels impair sperm function, leading to infertility. 45 High levels of showed an overexpression of this protein in the ERCR+ group vs. the ERCRÀ. Acrosomal biogenesis is divided into four different steps, Golgi, cap, elongation (acrosome), and maturation phases.…”