**4. Physiological roles of ROS**

Aerobic metabolism utilizing oxygen is essential for energy requirements of reproductive cells, and free radicals do play a significant role in physiological processes occurring within the male reproductive tract. Spermatozoa themselves produce small amounts of ROS that are essential for a variety of physiological processes such as capacitation, hyperactivation, acrosome reaction and sperm-oocyte fusion [30].

#### **4.1. Sperm maturation**

**3.3. External sources of ROS**

122 Spermatozoa - Facts and Perspectives

production of reactive species such as O2

malnourished individuals [44].

related etiologies.

ROS generation can be exacerbated by a multitude of environmental, infectious and lifestyle-

A wide range of industrial by-products and waste chemicals (e.g. polychlorinated biphenyls, nonylphenol or dioxins) have been associated with several adverse health effects, many of which are related to male infertility. These chemicals have been shown to increase the

●− and H2

by increased oxidative damage to the sperm lipids, proteins and DNA [39].

motility and vitality depending on the duration of exposure to radiation [40].

impair spermatogenesis [38]. Persistent environmental contaminants, such as heavy metals and pesticides, may also lead to OS, particularly among workers exposed to such pollutants. These individuals often present with a decreased semen volume and density, accompanied

Radiation is a natural source of energy with significant effects on living organisms. Mobile devices are becoming more accessible to the general population, particularly to adolescent males and men of reproductive age. Cell phones release radiofrequency electromagnetic radiation, exposure to which has shown to increase the risk of oligo-, astheno- or teratozoospermia. Furthermore, *in vitro* studies have demonstrated that EMR induces ROS generation and DNA fragmentation in human spermatozoa, alongside a decreased sperm concentration,

Various components of cigarette smoke have been associated with OS exacerbation. Cigarettes contain a broad array of free radical-inducing agents such as nicotine, cotinine, hydroxycotinine, alkaloids and nitrosamines [41, 42]. The prime component of tobacco is nicotine, which is a well-known ROS producer in spermatozoa with detrimental effects on the sperm count, motility and morphology. Moreover, smokers exhibited a lower hypo-osmotic swelling test percentage, indicating a weaker plasma membrane integrity when compared to non-smokers [41]. Smoking increases ROS production by causing leukocytospermia as shown by Saleh et al. [42], who also demonstrated that in smokers, the seminal ROS and total antioxidant capacity score was increased—a direct indication of oxidative imbalance in affected ejaculates. A different study showed that levels of seminal plasma antioxidants were diminished in smokers. This was furthermore confirmed by the presence of increased levels of 8-hydroxy-2′-deoxyguanosine [43]. By directly affecting the liver, alcohol intake increases ROS production while simultaneously decreasing the antioxidant capacity of the body. Although alcohol consumption has been repeatedly associated with systemic OS, its effect on semen parameters has not been explored to a larger extent. In a study comprising 8344 subjects, moderate alcohol consumption did not negatively affect semen parameters [44]. Nevertheless, it was revealed that chronic drinkers had reduced levels of testosterone, possibly due to an impaired hypothalamic-pituitary axis and damage to the Leydig cells [45]. Increased alcohol levels block gonadotropin-releasing hormone, leading to reduced luteinizing hormone and testosterone levels. Furthermore, alcohol has been shown to increase ROS generation when consumed by

Lastly, diet may affect semen parameters. In a Danish study, men with the highest saturated fat intake presented with a significantly lower total sperm count and concentration in

O2

in the testes, damage sperm DNA and

During transit and storage in the epididymis, spermatozoa undergo membrane, nuclear and enzymatic remodeling, involving the release, attachment and rearrangement of surface proteins [6, 30, 51]. Such changes are based on the assembly of several signal transduction pathways necessary for the subsequent ability of spermatozoa to undergo hyperactivation and capacitation.

ROS are essential for a proper chromatin packing during the maturation of mammalian spermatozoa, leading to a characteristic chromatin stability. This unique chromatin architecture results from an extensive inter- and intra-molecular disulfide bond stabilization between the cysteine residues of protamines—small nuclear proteins that replace histones during spermatogenesis. Oxidation of the thiol groups in protamines takes place during the transport of spermatozoa from the caput to the cauda epididymis [52]. As demonstrated by Aitken et al. [53], a spontaneous luminol peroxidase signal indicating the presence of ROS was exclusive to mature spermatozoa collected from the cauda region. ROS may act as oxidizing agents in this process, hence facilitating the formation of disulfide bonds, increasing chromatin stability and protecting DNA from possible damage [30, 52]. As spermatozoa possess minimal to none repair mechanisms [9], chromatin condensation is a crucial protective mechanism, in which ROS actually protect male gametes against future oxidative insults.

Likewise, peroxides have been associated with formation of the mitochondrial capsule a coat surrounding sperm mitochondria providing protection against possible proteolytic degradation [54]. It is suggested that during spermatogenesis peroxides may oxidize the active form of phospholipid hydroperoxide glutathione peroxidase (PHGPx), creating an intermediate that subsequently interacts with thiol groups to form a seleno-disulfide bond. The resulting mitochondrial capsule is made out of a complex protein network rich in disulfide bonds. Mitochondria require such protection as their proper function is crucial for metabolism, cell cycle control and oxidative balance [51, 53, 54].

The results of the above studies show that ROS can positively enhance sperm capacitation,

ecules in the biochemical pathways, and depending on the *in vitro* method used to induce capacitation, specific ROS involved may therefore differ. Several studies have confirmed the lack of molecular specificity in the activation of capacitation and tyrosine phosphorylation, as both SOD and catalase have been shown to negate the positive effect exogenously induced

Although physiological ROS levels are necessary for capacitation, their overgeneration may trigger apoptosis. When the levels of oxysterols and lipid aldehydes increase, cell-mediated

Hyperactivation is an incompletely understood process to be observed in the final maturation stage of spermatozoa and is considered a subcategory of capacitation. Normally spermatozoa exhibit a low amplitude flagellar movement accompanied by low, linear velocity. In the hyperactivated state, spermatozoa movement is of high amplitude, asymmetric flagellar movement, pronounced lateral head displacement and non-linear trajectory, allowing the sperm to penetrate the *cumulus oophorus* and zona pellucida surrounding the oocyte. Furthermore, hyperactive motility may enable the progressive movement through the oviduct by preventing stagnation, adding yet another benefit to the sperm function [62]. The biochemistry of hyperactivation is poorly understood, but it is known to involve a rise in cAMP activity and pH [58], increased generation of ROS, an initial influx of bicarbonate ions and an

zoa, as the presence of SOD, but not catalase, reduced the percentage of spermatozoa exhibiting hyperactivity in a variety of culture media [58]. *In vitro* experiments have also revealed

with this process as well; however, its activity may be dependent upon NO● regulation. This hypothesis was confirmed by *in vitro* experiments, according to which catalase vital against

Acrosome reaction (AR) is related to the release of proteolytic enzymes, primarily acrosin and hyaluronidase, in order to degrade the zona pellucida of the oocyte. Once degraded, hyperactive motility propels the spermatozoa into the perivitelline space, at which point the spermatozoa may eventually fuse with the oocyte [63]. Compared to the slow, reversible process of capacitation, this

is a permanent, fast-acting step associated with a respiratory burst (rapid extracellular O2

duction) increasing the tyrosine phosphorylation of specific proteins [57, 64]. O2

O2

important roles in regulating hyperactivation in mammalian epididymis. H2

toxicity prevents NO●-induced capacitation and hyperactivation [60].

●− is a vital trigger of sperm motility hyperactivation [30]. At the same time, NO● plays

●− is considered nearly essential for hyperactivation in mammalian spermato-

●− and H2

O2

Physiological and Pathological Roles of Free Radicals in Male Reproduction

may stimulate different mol-

125

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

●− production, lipid peroxi-

O2

may interact

●− pro-

●− produced *via*

, and these two molecules may have a positive effect

but diverge over the specific ROS involved. Both O<sup>2</sup>

suicide may occur accompanied by an enhanced mitochondrial O2

dation (LPO), cytochrome c release and subsequent caspase activation [53, 61].

capacitation and hyperactivation [59].

**4.3. Motility and hyperactivation**

increase in intracellular Ca2+ concentrations [62].

Extracellular O2

**4.4. Acrosome reaction**

NADPH oxidase may dismutate into H2

that O2

H2 O2

Although several studies have reported improved sperm DNA integrity and reduced ROS production as a result of daily antioxidant consumption [55], an unusual decondensation of sperm DNA has been revealed as well [56]. Hence it may be hypothesized that high antioxidant levels may alter the oxidative conditions necessary for a proper formation of the interand intra-molecular disulfide bonds, leading to a lower DNA compaction.

### **4.2. Capacitation**

Capacitation is a prominent process of final maturation that spermatozoa undergo in the female reproductive tract, during which sperm motility changes from a progressive state to a highly energetic one. It is hypothesized that capacitation occurs exclusively in mature spermatozoa in order to reach the oocyte taking advantage of hyperactive motility and an increased responsiveness to chemotactic agents. Numerous receptors on the sperm head become activated, providing energy to the sperm to penetrate the zona pellucida. As such, capacitation sets up the path necessary for subsequent hyperactivation and acrosome reaction [57]. Most prominent molecular processes associated with capacitation include Ca2+ and HCO3 − influx, cholesterol efflux, increased cAMP activity, ROS generation, pH, protein phosphorylation and membrane hyperpolarization [32, 58].

Numerous of studies on both human and animal spermatozoa indicate that H2 O2 is the primary ROS responsible for capacitation to occur. This process is associated with an increase in tyrosine phosphorylation, and it has been shown that the amount and banding pattern of tyrosine phosphorylation by adding exogenous H2 O2 was similar to that observed during endogenous ROS production, providing evidence that H2 O2 may be responsible for the enhancement of capacitation [32, 57, 58]. This hypothesis was further confirmed by Rivlin et al. [59] who showed that catalase decreased, while H2 O2 increased the tyrosine phosphorylation in a dose-dependent manner, thereby solidifying the involvement of H2 O2 in the process of capacitation.

At the same time, de Lamirande and Gagnon [58] indicated that O2 ●− may be also involved in this process. Of note is also the role of NO●, which is present in the female genital tract. NO● may initiate the acrosome reaction, the effects of which are likely achieved through a complex mechanism involving H2 O2 [59, 60].

Finally, a combination of O2 ●−and NO● forms ONOO<sup>−</sup> , which allows oxysterol to be produced. Oxysterol, which removes cholesterol from the lipid bilayer, inhibits tyrosine phosphate and promotes cyclic adenosine 3′,5′-monophosphate (cAMP) production [60]. This process is vital as cAMP must increase in concentration for capacitation to occur. cAMP and its subsequent pathways involve protein kinase A, which phosphorylates MEK (extracellular signal-regulated kinase)-like proteins as well as tyrosine present in fibrous sheath proteins [57, 58].

The results of the above studies show that ROS can positively enhance sperm capacitation, but diverge over the specific ROS involved. Both O<sup>2</sup> ●− and H2 O2 may stimulate different molecules in the biochemical pathways, and depending on the *in vitro* method used to induce capacitation, specific ROS involved may therefore differ. Several studies have confirmed the lack of molecular specificity in the activation of capacitation and tyrosine phosphorylation, as both SOD and catalase have been shown to negate the positive effect exogenously induced capacitation and hyperactivation [59].

Although physiological ROS levels are necessary for capacitation, their overgeneration may trigger apoptosis. When the levels of oxysterols and lipid aldehydes increase, cell-mediated suicide may occur accompanied by an enhanced mitochondrial O2 ●− production, lipid peroxidation (LPO), cytochrome c release and subsequent caspase activation [53, 61].

### **4.3. Motility and hyperactivation**

intermediate that subsequently interacts with thiol groups to form a seleno-disulfide bond. The resulting mitochondrial capsule is made out of a complex protein network rich in disulfide bonds. Mitochondria require such protection as their proper function is crucial for

Although several studies have reported improved sperm DNA integrity and reduced ROS production as a result of daily antioxidant consumption [55], an unusual decondensation of sperm DNA has been revealed as well [56]. Hence it may be hypothesized that high antioxidant levels may alter the oxidative conditions necessary for a proper formation of the inter-

Capacitation is a prominent process of final maturation that spermatozoa undergo in the female reproductive tract, during which sperm motility changes from a progressive state to a highly energetic one. It is hypothesized that capacitation occurs exclusively in mature spermatozoa in order to reach the oocyte taking advantage of hyperactive motility and an increased responsiveness to chemotactic agents. Numerous receptors on the sperm head become activated, providing energy to the sperm to penetrate the zona pellucida. As such, capacitation sets up the path necessary for subsequent hyperactivation and acrosome reaction [57]. Most

> − influx,

is the pri-

in the pro-

O2

may be responsible for the

O2

●− may be also involved in

was similar to that observed dur-

increased the tyrosine phosphory-

, which allows oxysterol to be pro-

prominent molecular processes associated with capacitation include Ca2+ and HCO3

Numerous of studies on both human and animal spermatozoa indicate that H2

lation in a dose-dependent manner, thereby solidifying the involvement of H2

cholesterol efflux, increased cAMP activity, ROS generation, pH, protein phosphorylation

mary ROS responsible for capacitation to occur. This process is associated with an increase in tyrosine phosphorylation, and it has been shown that the amount and banding pattern

enhancement of capacitation [32, 57, 58]. This hypothesis was further confirmed by Rivlin

this process. Of note is also the role of NO●, which is present in the female genital tract. NO● may initiate the acrosome reaction, the effects of which are likely achieved through a complex

duced. Oxysterol, which removes cholesterol from the lipid bilayer, inhibits tyrosine phosphate and promotes cyclic adenosine 3′,5′-monophosphate (cAMP) production [60]. This process is vital as cAMP must increase in concentration for capacitation to occur. cAMP and its subsequent pathways involve protein kinase A, which phosphorylates MEK (extracellular signal-regulated kinase)-like proteins as well as tyrosine present in fibrous sheath

●−and NO● forms ONOO<sup>−</sup>

O2

O2

O2

metabolism, cell cycle control and oxidative balance [51, 53, 54].

**4.2. Capacitation**

124 Spermatozoa - Facts and Perspectives

cess of capacitation.

mechanism involving H2

proteins [57, 58].

Finally, a combination of O2

and membrane hyperpolarization [32, 58].

of tyrosine phosphorylation by adding exogenous H2

et al. [59] who showed that catalase decreased, while H2

O2

ing endogenous ROS production, providing evidence that H2

At the same time, de Lamirande and Gagnon [58] indicated that O2

[59, 60].

and intra-molecular disulfide bonds, leading to a lower DNA compaction.

Hyperactivation is an incompletely understood process to be observed in the final maturation stage of spermatozoa and is considered a subcategory of capacitation. Normally spermatozoa exhibit a low amplitude flagellar movement accompanied by low, linear velocity. In the hyperactivated state, spermatozoa movement is of high amplitude, asymmetric flagellar movement, pronounced lateral head displacement and non-linear trajectory, allowing the sperm to penetrate the *cumulus oophorus* and zona pellucida surrounding the oocyte. Furthermore, hyperactive motility may enable the progressive movement through the oviduct by preventing stagnation, adding yet another benefit to the sperm function [62]. The biochemistry of hyperactivation is poorly understood, but it is known to involve a rise in cAMP activity and pH [58], increased generation of ROS, an initial influx of bicarbonate ions and an increase in intracellular Ca2+ concentrations [62].

Extracellular O2 ●− is considered nearly essential for hyperactivation in mammalian spermatozoa, as the presence of SOD, but not catalase, reduced the percentage of spermatozoa exhibiting hyperactivity in a variety of culture media [58]. *In vitro* experiments have also revealed that O2 ●− is a vital trigger of sperm motility hyperactivation [30]. At the same time, NO● plays important roles in regulating hyperactivation in mammalian epididymis. H2 O2 may interact with this process as well; however, its activity may be dependent upon NO● regulation. This hypothesis was confirmed by *in vitro* experiments, according to which catalase vital against H2 O2 toxicity prevents NO●-induced capacitation and hyperactivation [60].

#### **4.4. Acrosome reaction**

Acrosome reaction (AR) is related to the release of proteolytic enzymes, primarily acrosin and hyaluronidase, in order to degrade the zona pellucida of the oocyte. Once degraded, hyperactive motility propels the spermatozoa into the perivitelline space, at which point the spermatozoa may eventually fuse with the oocyte [63]. Compared to the slow, reversible process of capacitation, this is a permanent, fast-acting step associated with a respiratory burst (rapid extracellular O2 ●− production) increasing the tyrosine phosphorylation of specific proteins [57, 64]. O2 ●− produced *via* NADPH oxidase may dismutate into H2 O2 , and these two molecules may have a positive effect 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 studies note positive effects of H<sup>2</sup> O2 and negative effects of catalase, thus suggesting that H<sup>2</sup> O2 is the major species responsible for a proper AR [58, 64].

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

Physiological and Pathological Roles of Free Radicals in Male Reproduction

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

127

**2.** Cell and tissue injury: OS can cause damage to all molecular targets: DNA, proteins and lipids. Often it is not clear which is the first point of attack, since injury mechanisms may

**3.** Cell death: This process may occur by two mechanisms, necrosis or apoptosis. During necrotic cell death, the cell swells and ruptures, releasing its contents into surrounding areas and affecting adjacent cells. The intracellular content can include antioxidants such as catalase or glutathione (GSH) as well as prooxidants such as copper and iron. As such, necrosis may lead to further oxidative insults in the internal milieu [3–5, 15]. During apoptosis, the cell's own "suicide mechanism" gets activated. As such, apoptotic cells do not release their content into surrounding environment and apoptosis does not cause damage

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

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

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

●− is generated either intracellularly through the NADPH system or through

**1.** Adaptation: Usually by upregulation of antioxidant defense systems.

therapeutic intervention.

to the neighboring cells [5].

**5.1. Lipid peroxidation (LPO)**

fertilization are impaired [68, 69].

takes place, O2

leading to male reproductive dysfunction [68].

OS can result in:

overlap [5].

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].
