2.1.1. Spermatozoa

At least two distinct pathways have been implicated in ROS generation in mature spermatozoa. One such pathway is localized within the sperm plasma membrane and is linked to the activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, whilst the second is associated with electron leakage from the mitochondrial electron transport chain (ETC) [23]. The latter of these pathways represents the main source of ROS production in spermatozoa and occurs as a result of the premature exit of electrons from the respiratory chain. This leakage prevents the reduction of oxygen to water at cytochrome c oxidase, with the escaped electrons instead reacting with molecular oxygen (O2) to form the superoxide radical (O2•<sup>−</sup>). Basal levels of electron leakage can potentially occur from multiple sites within the ETC (reviewed in [24]), and, unlike the O2•<sup>−</sup> generated at the level of the plasma membrane that supports sperm capacitation [25], mitochondrial derived O2•<sup>−</sup> is generally associated with pathological damage. Indeed, when superoxide anion generation exceeds the limited antioxidant capacity of the sperm cell, it has the ability to propagate the formation of non-radicals, including hydrogen peroxide (H2O2), and in the presence of Fe3+, the formation of alternative radicals such the hydroxyl anion (OH−) via Haber-Weiss and Fenton reactions [26]. Additional ROS containing nitrogen atoms (such as NO, NO3<sup>−</sup>, NO<sup>−</sup>, N2O, and HNO3) are also able to be formed [27,28] and collectively, these powerful oxidants have the potential to trigger the peroxidation of membrane lipids and a concomitant loss of sperm function [29]. In studies conducted in rats it has been shown that the presence of dead spermatozoa can also promote higher than normal levels of H2O2 [30]. This response appears to be related to the apoptosis cascade during which the disruption of mitochondrial membranes leads to the release of cytochrome c and elevated ROS production [31].
