**5. Oxidative stress (OS)**

The term oxidative stress refers to a critical imbalance between ROS production and antioxidant defense mechanisms available to the biological system [15]. According to Sies [5], it is a disturbance in the prooxidant-antioxidant balance in favor of the former, leading to potential cellular damage.

Essentially, OS may result from:


by excessive activation of 'natural' FR-generating systems (e.g. phagocytic oxidative outburst during chronic inflammatory diseases) [5, 15]. This mechanism is normally thought to be more relevant to mammalian diseases and is frequently the target of attempted therapeutic intervention.

OS can result in:

on the AR [6, 32, 64]. ●NO has also been reported to increase the percentage of sperm undergoing the AR [37]. At the same time, results regarding the specific ROS are conflicting. The majority of

Moreover, ROS act as signal transducers in the AR. Elevated ROS production may occur upon interaction with the *cumulus oophorus*, thereby enhancing the signal for exocytosis initiated by either progesterone or the zona pellucida. *In vivo*, binding of the zona pellucida and a certain stimulus *via* progesterone on capacitated spermatozoa initiates this process and is associated with an influx of extracellular Ca2+ into the cytosol [6]. *In vitro* studies indicate that ROS can induce the Ca2+ influx and initiates the biochemical cascade associated with the AR [53, 64].

A link exists between enhanced ROS levels and increased sperm-oocyte fusion. High rates of sperm-oocyte fusion are correlated with increased expression of phosphorylated tyrosine proteins [6], suggesting that sperm-oocyte fusion is related to the events of capacitation and

that the addition of catalase or SOD significantly decreased the fusogenicity, whereas the

●− significantly increased the fusogenicity [53, 64]. Ultimately, ROS are thought to increase membrane fluidity using two mechanisms: (1) deesterification of membrane phospholipids and (2) activation of phospholipase A2 (PLA2) [65]. Once the zona pellucida and corona radiata are penetrated by the sperm cell, the oocyte prevents eventual polyspermy by turning the vitelline layer into a hard envelope. o,o-Dityrosine crosslinks catalyzed by ovoperoxidase lead to the formation of a single macromolecular struc-

the envelope formation. With our understanding of ROS and their spermicidal effect, H<sup>2</sup>

The term oxidative stress refers to a critical imbalance between ROS production and antioxidant defense mechanisms available to the biological system [15]. According to Sies [5], it is a disturbance in the prooxidant-antioxidant balance in favor of the former, leading to potential

**1.** Diminished antioxidants, e.g. mutations affecting antioxidant defense enzymes or toxic

**2.** Increased ROS or RNS generation either by exposure to increased levels of toxins that act as reactive species themselves or are metabolized to induce further biological oxidation or

O2

proves to be an effective spermicide agent against polyspermy [66, 67].

and negative effects of catalase, thus suggesting that H<sup>2</sup>

●− contribute to the increase in fertilization rates as revealed by the fact

serves as the substrate to ovoperoxidase to provide for

O2 is

O2

O2

the major species responsible for a proper AR [58, 64].

studies note positive effects of H<sup>2</sup>

126 Spermatozoa - Facts and Perspectives

**4.5. Sperm-oocyte fusion**

O2

O2 or O2

ture acting as the envelope [66]. H2

**5. Oxidative stress (OS)**

Essentially, OS may result from:

agents that deplete such mechanisms [5].

cellular damage.

and O2

AR. Both H2

addition of H2


An intricate cellular architecture of spermatozoa renders them to be particularly sensitive to OS. Sperm plasma membranes contain large quantities of polyunsaturated fatty acids (PUFAs). On the other hand, their cytoplasm contains low concentrations of scavenging enzymes [68]. OS usually results in a decreased sperm motion and viability, accompanied by a rapid loss of ATP, axonemal damage, increased midpiece morphology defects, followed by alterations in the sperm capacitation and acrosome reaction [32]. Lipid peroxidation has been repeatedly postulated to be the key mechanism of ROS-induced sperm damage, possibly leading to male reproductive dysfunction [68].

#### **5.1. Lipid peroxidation (LPO)**

Sperm plasma membranes are largely composed of PUFAs, which are exceptionally susceptible to oxidative damage due to the presence of more than two carbon–carbon double bonds [68]. These fatty acids maintain the fluidity of membranes [69]. ROS attack PUFAs, leading to a cascade of chemical reactions called lipid peroxidation (LPO). As the LPO proceeds, more than 60% of PUFAs may be lost. LPO affects most prominent structural and functional characteristics of the membrane, including fluidity, ion gradients, receptor transduction, transport processes as well as enzymatic activities. As a result, properties that are crucial for a normal fertilization are impaired [68, 69].

LPO is a self-propagating process that may be divided into three phases: the initiation phase, the propagation phase and the termination phase. Before any of these processes takes place, O2 ●− is generated either intracellularly through the NADPH system or through leukocytes as an extracellular source. O2 ●− can be directly protonated to create the hydroperoxyl radical (HO2 ●) or it can be converted into H2 O2 *via* SOD. H2 O2 may be subsequently converted into OH● *via* the Fenton reaction involving ferrous iron. Generation of OH● and HO2 ● mark the beginning of the initiation stage, as neither O2 ●− nor H2 O2 is not energetically rich enough to initiate LPO directly [70]. During the initiation phase, one hydrogen is taken from unsaturated lipids to form lipid radicals. These radicals subsequently interact with oxygen to generate lipid HO2 ●, which may be transformed into lipid peroxides through available antioxidants, stabilizing the sperm plasma membrane. Nevertheless, during the propagation stage, in the presence of a transition metal ion, lipid peroxides will be transformed into alkoxyl radical and HO2 ● through the Fenton and Haber-Weiss reaction, subsequently acting upon additional lipids until the damage is widespread and irreversible [68–70]. During the termination phase, two radicals react with each other to form a stable product and LPO finally ceases [70].

as point mutation and polymorphism, resulting in decreased semen quality. These changes may be observed especially during the prolonged meiotic prophase, when the spermatocytes are particularly sensitive to damage and widespread degeneration can occur [72–74]. Also, mutations in the mitochondrial DNA (mtDNA) may cause a defect of mitochondrial energy metabolism and therefore lower levels of mutant mtDNA may compromise sperm motility *in vivo* [76]. Other mechanisms such as denaturation and DNA base-pair oxidation may also be

Physiological and Pathological Roles of Free Radicals in Male Reproduction

http://dx.doi.org/10.5772/intechopen.70793

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Increased DNA damage has become a serious issue during artificial reproduction techniques (ARTs), as it has been correlated with decreased fertilization rates *in vitro* and increased early embryo death. Unfortunately, no successful method to prevent or treat sperm DNA damage

Proteins are a critical target for oxidation because of their abundance and high rate constants for interactions with diverse ROS. As such, protein damage is a major consequence of both intracellular and extracellular oxidative insults. ROS may attack both the side chains and backbone, and the extent of the insult depends on multiple factors. In some cases, the damage is limited to specific residues, whereas in case of other ROS, the damage is widespread and

Oxidative attacks on proteins generally result in site-specific amino acid modifications, fragmentation of the peptide chain, aggregation of cross-linked reaction products, altered electric

The resulting products of protein oxidation include reactive hydroperoxides, which may be employed as biomarkers for protein oxidation *in vitro* and *in vivo*. As protein damage is usually non-repairable, oxidation may have deleterious consequences, including the loss (or sometimes gain) of enzymatic, structural or signaling function, fragmentation, unfolding, altered interactions with other proteins and modified turnovers. Generally, oxidized proteins are degraded by proteasomal and lysosomal pathways; however, in some cases, such altered material is poorly degraded and may accumulate within cells contributing to multiple mam-

The amino acids in a peptide differ in their susceptibility to oxidative insults, while various ROS differ in their potential reactivity. Primary, secondary and tertiary protein structures alter the relative susceptibility of certain amino acids. Sulfur-containing amino acids and par-

According to Mammoto et al. [81], protein oxidation in spermatozoa leads to a blocked spermegg fusion, the capacity to penetrate the zona pellucida, as well as sperm-egg binding. Sinha et al. [80] showed that oligospermia is linked to a quantitative reduction in the SH-groups in spermatozoa. Thus, oxidation of the sperm SH-proteins may be a notable mechanism respon-

ticularly thiol (−SH) groups are very susceptible to ROS-associated damage [79, 80].

sible for the suppressive effects of ROS on sperm functions.

charge and increased susceptibility or extreme tolerance to proteolysis [79].

involved [74].

is currently available [77].

**5.3. Protein oxidation**

nonspecific [78].

malian pathologies [78, 79].

Numerous pathological effects of LPO on the sperm function are currently known. Overall, LPO causes DNA and protein damage through oxidation of lipid peroxyl or alkoxyl radicals. DNA fragmentation by LPO can occur *via* base modifications, strand breaks or crosslinks [71]. LPO generally results in loss of membrane fluidity and subsequently a decreased sperm motility and sperm-oocyte fusion [68–71].

Furthermore, during LPO, ROS initiate a cascade of events involving the xanthine and xanthine oxidase system and deplete the ATP production which may ultimately lead to sperm death [68].
