**5.2. DNA damage**

The unique sperm chromatin packing alongside antioxidant molecules present in the seminal plasma provide notable protection to sperm DNA against oxidative damage. Nevertheless, spermatozoa lack any specific DNA repair mechanisms and hence depend on the oocyte for eventual DNA repair following fertilization. ROS-associated catalysis and apoptosis are considered to be the primary mechanisms that induce DNA fragmentation in spermatozoa [72].

DNA bases and phosphodiester backbones are believed to be most susceptible to ROSassociated peroxidative damage. At the same time, sperm mitochondrial DNA is more vulnerable to oxidative insults when compared to the nuclear genome [73]. Furthermore, because of the structure of the Y chromosome as well as its inability to repair double strand breaks, Y-bearing spermatozoa are more susceptible to DNA damage than X-carrying counterparts [74]. Y-bearing spermatogonia can be a target of mutations in the euchromatic Y region (Yq11), known as the azoospermia factor, resulting in infertility [75].

Various types of DNA abnormalities may occur in sperm that have been exposed to ROS artificially. These include base modifications, production of base-free sites, deletions, frame shifts, DNA crosslinks and chromosomal rearrangements. OS has also been associated with high frequencies of single- and double-strand DNA breaks. ROS can also cause gene mutations, such 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 involved [74].

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 is currently available [77].

### **5.3. Protein oxidation**

leukocytes as an extracellular source. O2

motility and sperm-oocyte fusion [68–71].

●) or it can be converted into H2

mark the beginning of the initiation stage, as neither O2

oxyl radical (HO2

128 Spermatozoa - Facts and Perspectives

to generate lipid HO2

alkoxyl radical and HO2

finally ceases [70].

death [68].

**5.2. DNA damage**

spermatozoa [72].

●− can be directly protonated to create the hydroper-

O2

may be subsequently con-

is not energetically rich

●

O2

●− nor H2

O2 *via* SOD. H2

●, which may be transformed into lipid peroxides through available

● through the Fenton and Haber-Weiss reaction, subsequently act-

verted into OH● *via* the Fenton reaction involving ferrous iron. Generation of OH● and HO2

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

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

ing 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

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

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

The unique sperm chromatin packing alongside antioxidant molecules present in the seminal plasma provide notable protection to sperm DNA against oxidative damage. Nevertheless, spermatozoa lack any specific DNA repair mechanisms and hence depend on the oocyte for eventual DNA repair following fertilization. ROS-associated catalysis and apoptosis are considered to be the primary mechanisms that induce DNA fragmentation in

DNA bases and phosphodiester backbones are believed to be most susceptible to ROSassociated peroxidative damage. At the same time, sperm mitochondrial DNA is more vulnerable to oxidative insults when compared to the nuclear genome [73]. Furthermore, because of the structure of the Y chromosome as well as its inability to repair double strand breaks, Y-bearing spermatozoa are more susceptible to DNA damage than X-carrying counterparts [74]. Y-bearing spermatogonia can be a target of mutations in the euchromatic Y region (Yq11),

Various types of DNA abnormalities may occur in sperm that have been exposed to ROS artificially. These include base modifications, production of base-free sites, deletions, frame shifts, DNA crosslinks and chromosomal rearrangements. OS has also been associated with high frequencies of single- and double-strand DNA breaks. ROS can also cause gene mutations, such

known as the azoospermia factor, resulting in infertility [75].

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 nonspecific [78].

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

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 mammalian pathologies [78, 79].

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

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 responsible for the suppressive effects of ROS on sperm functions.

#### **5.4. Apoptosis**

Usually, when cellular components undergo serious damage, apoptosis or programmed cell death is initiated. During spermatogenesis, abnormal spermatozoa are eliminated primarily through apoptosis. The exact mechanism of action is not fully understood yet; however, previous studies have speculated that ROS serve as an activator of the mitochondria to release the signaling cytochrome c [82, 83]. This molecule initiates a cascade of events involving caspases 3 and 9, eventually leading to sperm apoptosis. The Fas-protein may be also an integral component in the apoptotic pathway. When Fas-ligand or anti-Fas antibody binds to Fas, apoptosis is initiated [83]. An additional mechanism involves the inflammatory production of ROS, primarily hypochlorous acid (HOCl), which is a product of H2 O2 and chloride ion. This molecule oxidizes a variety of cellular components, thus causing apoptosis [84]. Said et al. [85] emphasized that HOCl is associated with elevated levels of apoptotic markers in spermatozoa.

observed is highly correlated with sperm LPO. Furthermore, the ability of antioxidants to revive sperm motility is evidence that LPO is a major cause for motility loss in spermato-

Physiological and Pathological Roles of Free Radicals in Male Reproduction

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131

Because ROS have both physiological and pathological functions, biological systems have developed defense systems to maintain ROS levels within a certain range. Whenever ROS levels become pathologically elevated, antioxidants scavenge them to minimize any potential

Antioxidants are defined as molecules that dispose, scavenge and inhibit the formation of ROS or oppose their actions. According to Ďuračková [13], antioxidants can protect cells

• Non-enzymatic (e.g. vitamin C, vitamin E, vitamin A, carotenoids, albumin, glutathione,

Due to the size and small volume of cytoplasm, as well as the low concentrations of scavenging enzymes, spermatozoa have limited antioxidant defense possibilities. Mammalian spermatozoa predominantly contain enzymatic antioxidants, including SOD and glutathione peroxidases (GPx), which are mainly located in the midpiece. A few non-enzymatic antioxidants, such as vitamins C and E, transferrin and ceruloplasmin, are present in the plasma

Under normal circumstances, the seminal plasma is an important protectant of spermatozoa against any possible ROS formation and distribution. Seminal plasma contains both enzymatic antioxidants, as well as an array of non-enzymatic antioxidants (e.g. ascorbate, urate,

Studies have shown that antioxidants protect spermatozoa from ROS generating abnormal spermatozoa, scavenge ROS produced by leukocytes, prevent DNA fragmentation, improve semen quality, reduce cryodamage to spermatozoa, block premature sperm maturation and

Superoxide dismutases are metal-containing enzymes that catalyze the conversion of two superoxides into oxygen and hydrogen peroxide, which is less toxic than superoxide [1, 13]:

●− → Mn+ − SOD + O2 (1)

**6. The role of antioxidants in male reproduction**

against OS *via* three mechanisms: prevention, interception and repair.

membrane of spermatozoa and act as preventive antioxidants [16].

vitamin E, pyruvate, glutathione, albumin, taurine and hypotaurine) [9].

• Enzymatic (e.g. superoxide dismutases, catalase and glutathione peroxidases).

Antioxidants may be divided into two dominant categories:

zoa [68, 69].

oxidative damage [1].

uric acid, pyruvate, etc.) [13].

generally stimulate sperm vitality [91, 92].

M(n+1)+ − SOD + O2

**6.1. Superoxide dismutases (SOD)**

Numerous studies have focused to study apoptosis in spermatozoa. Various authors [35, 86] have reported increased ROS levels and apoptotic markers measured by fluorescence in samples of infertile subjects. In deer spermatozoa, it was demonstrated that H2 O2 addition stimulates apoptosis, whereas O2 ●− and OH● do not have this ability [86]. Meanwhile studies in primate, murine and boar spermatozoa indicated that NO● was correlated with apoptosis possibly through caspase activation [87, 88].

On the other hand, in certain males, abortive apoptosis appears to fail in the clearance of spermatozoa that are marked for elimination by apoptosis. As such, the subsequent population of ejaculated spermatozoa may exhibit an array of anomalies consistent with characteristics typical for cells that are in the process of apoptosis. Apoptotic failures may lead to a decreased sperm count resulting in subfertility [82, 83].

#### **5.5. Effects on sperm motility**

Spermatozoa motility is an important prerequisite to secure their distribution in the female sexual system, followed by an effective passage through the cervical mucus and penetration into the egg [89]. Increased ROS levels have been repeatedly correlated with a decreased sperm motility [10–12, 90], although the exact mechanism involved is still not completely understood. One hypothesis suggests that H2 O2 diffuses across the membranes into the cells and inhibits the activity of vital enzymes such as NADPH oxidase [6]. At the same time, a decreased G6PDH leads to a reduced availability of NADPH accompanied by a build-up of oxidized glutathione. Such changes may lead to a decline in the intracellular antioxidant levels and a subsequent peroxidation of membrane phospholipids [65].

Another hypothesis presents a series of interrelated events leading to a decreased phosphorylation of axonemal proteins, followed by sperm immobilization, both of which are linked to a reduced membrane fluidity crucial for sperm-oocyte fusion [10, 32]. When spermatozoa are incubated with selected ROS overnight, loss of motion characteristics observed is highly correlated with sperm LPO. Furthermore, the ability of antioxidants to revive sperm motility is evidence that LPO is a major cause for motility loss in spermatozoa [68, 69].
