Origin of ROS in Semen and Sperm

Reduced forms of oxygen, collectively termed ROS, interact with various types of molecules and include free radicals that are charged molecules with unpaired electrons. Examples of free radicals are superoxide anion (O- •) and hydroxyl radical (•OH) . Not all ROS are free radicals, however, and molecules such as hydrogen peroxide (H2O2), ozone, and singlet oxygen are also ROS and contribute to oxidative physiology and pathology [60, 61].

Within an ejaculate, there are two potential sources of ROS: spermatozoa [62, 63] and leukocytes [64]. It is believed that spermatozoa and leukocytes possess similar mechanisms for ROS generation: an NADPH oxidase (NOX) located in or near the cell membrane [60, 65-68].

In mammalian semen, leukocytes are not normally part of an ejaculate and are not present in significant numbers in normal ejaculates in most species. Human semen, however, commonly contains leukocytes and, hence, sperm have a consistent production of ROS and exposure to the subsequent oxidative effects. Leukocytes utilize the generation of ROS in the oxidative burst of microbial-killing phagocytosis and are capable of generating a significant amount of ROS in semen [69].

All NOX family members are transmembrane proteins with an NAD(P)H binding site at the carboxylic end, an FAD-binding region, six conserved transmembrane domains, and four highly conserved heme-binding histidines. It has been reported that human spermatozoa contain a NOX similar to that reported in phagocytic leukocytes [66]. However, NAD(P)H failed to stimulate extracellular (O- •) production as detected by MCLA (2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one), whereas addition of progesterone or ultrafiltrates of fetal cord serum, follicular fluid, or seminal plasma (stimulators of sperm) to human spermatozoa did stimulate (O- •) generation [70]. Another group using a highly sensitive spin trapping technique has also reported the absence of NOX activity in human spermatozoa [71]. Richer and Ford [72] proposed that the majority of the chemiluminescence promoted by NAD(P)H is derived from redox cycling independent of O- • generation by spermatozoa [72]. Recent research has determined that lucigenin chemiluminescence in response to NAD(P)H in rat epididymal sperm preparations is due to the presence of an NAD(P)H-dependent cytochrome P450 reductase that originates largely from contaminating epididymal cells [73] . This enzyme directly reduces lucigenin and tetrazolium salts, thus initiating their redox cycling with molecular oxygen and production of (O- •). Consequently, not only is the existence of an NAD(P)H oxidase in spermatozoa subject to scrutiny, but the

Fig. 3.1 Conversion of diatomic oxygen to ROS in sperm. NAD(P)H oxidase shown as NOX; superoxide dismutase shown as SOD

mechanism of (O- •) detection is susceptible to interference. In addition to driving ROS production in sperm by exogenous NAD(P)H, the reaction can be inhibited using the classic inhibitor diphenylene iodonium (DPI). Although DPI has been shown to be a less specific inhibitor than earlier proposed, it has been shown to inhibit ROS production in sperm from a variety of species (reviewed by Aitken and Curry) [36]. As these authors point out, the NOX family influence in sperm ROS has not been clearly elucidated. Mouse sperm [74] and stallion [68] sperm have been shown to express NOX2 and NOX5, respectively. Further, dual oxidase (DUOX) has been recently identified in human sperm using a proteomic method [75]. These studies suggest that NOX systems are capable of generating ROS in sperm, but it is not known whether this mechanism predominates during normal sperm function or during cryopreservation as a stress response.

The major ROS that are generated by sperm and play significant biological roles are the superoxide anion (O- •) which is the most common ROS generated by spermatozoa, and rapidly dismutates either spontaneously or catalyzed by superoxide dismutase (SOD) to H2O2 , which is the second major ROS in sperm (Fig. 3.1). Hydrogen peroxide is not a free radical, and in contrast to (O- •) it is more stable and can readily cross the plasma membrane [76]. An increase in ROS generation has been attributed to abnormal or damaged spermatozoa [77-79]. Generation of ROS by sperm has been the subject of a number of studies in recent years to elucidate the subcellular origin of reduced oxygen forms. In different cell types, ROS can be produced variably by intracellular oxidases and peroxidases but also by cytochrome p450, nitric oxide synthase, and leakage of electrons from the electron transport chain (reviewed in [61, 67]). ROS generation continues as a cascade of reactions as discussed for Fig. 3.1 culminating in H. O2 production. In many cell types, reactive nitrogen species (RNS) are concurrently produced that are also agents of oxidative stress to cells [60]. As ROS are processed by enzyme systems, they may also react with RNS products, such as nitric oxide (NO), to generate per-oxinitrite (superoxide + NO), hypochlorous acid (H2O2 + NO), and the iron-catalyzed Fenton reaction to result in hydroxyl radical (•HO). RNS have been shown to affect sperm function and fertility [80, 81].

Sperm mitochondria also contribute to ROS production through active leakage of electrons and have been shown to be a significant source of ROS leading to cell damage [82,83]. Mitochondrial ROS production has also been associated with cell regulation and initiation of apoptosis, or programmed cell death [ 84] . As apoptotic cell processes have been associated with sperm cryoinjury and death, it seems reasonable that mitochondria, the source of energy for sperm motility, could be specifically damaged during cryopreservation leading to a major source of oxidative change in sperm.

When generated by sperm or leukocytes in semen, ROS interact with numerous molecules, including lipids, proteins, carbohydrates, and nucleic acids, and cause often irreversible effects. Sperm lipid membranes contain an abundance of highly unsatu-rated fatty acids and are, therefore, very susceptible to oxidative damage [52, 67],

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