**3. Sources of ROS in semen**

radicals (FR) [2, 3], which, under physiological conditions, are necessary for a normal cell function [4]. On the other hand, if FR concentrations become too high, either because of their overgeneration or due to low levels of antioxidant defense mechanisms, oxidative stress (OS)

Oxidative stress has been implicated in the pathogenesis of a variety of human diseases such as atherosclerosis, cancer, diabetes, liver damage, AIDS, Parkinson's disease and health complications associated with premature birth [6]. In the meantime, seminal OS is believed to be one of the main factors in the pathogenesis of sperm dysfunction in male sub- or infertility [7–9]. Several intrinsic and extrinsic factors have the ability to promote reactive oxygen species (ROS) generation in the testicular as well as post-testicular (e.g. epididymal) environment, resulting in defective spermatogenesis and altered sperm function [9]. As expected, approximately 25% of infertile patients exhibit higher ROS levels in semen as opposed to

Although the origin of ROS generation in semen and their roles in male reproduction have only recently been uncovered, numerous questions still remain unanswered, thus offering multiple strategies for future research. As such, the role of free radicals and oxidative stress in

A free radical (FR) is defined as any atom, molecule or a fragment of atoms and molecules with one or more unpaired electrons, capable of short independent existence. The abstraction or gain of one electron by a nonradical molecule may (or may not) convert it to a radical spe-

It is precisely the presence of an unpaired electron that results in certain common properties shared by most radicals. Free radicals are generally unstable and highly reactive. They can either donate an electron to or accept an electron from other molecules, thus behaving as

In cells, one-electron modification of molecules can yield sulfur-, oxygen-, carbon- and nitrogen-derived free radicals [14]. Furthermore, ions of transition metals have a radical nature [13]. The most common and important free radicals related to biological systems are oxygenderived radicals called reactive oxygen species (ROS) and nitrogen-derived molecules, defined as reactive nitrogen species (RNS) [15]. ROS represent a broad category of molecules including radical and non-radical oxygen derivatives [16]. Reactive nitrogen species are nitrogen-free radicals and commonly accepted as a subclass of ROS [13, 15]. A summary of the most common oxygen- and nitrogen-derived free radicals is provided in

emerges with unpredictable consequences on the cell behavior and survival [5].

fertility and subfertility is an area requiring continuous scientific attention.

cies [13]. Free radicals may have a positive, negative or a neutral charge [14]:

**2. Free radicals: general characteristics**

A → minus one electron → A+●.

B → plus one electron → B−●.

oxidants or reductants [13].

**Table 1**.

fertile men [7, 10–12].

118 Spermatozoa - Facts and Perspectives

Virtually every ejaculate may contain potential sources of ROS. Leukocytes activated by multiple factors, especially inflammation and infection, are among significant ROS producers in semen [17]. Subpopulations of leukocytes, which may be found in semen, mainly consist of polymorphonuclear (PMN) leukocytes (50–60%) and macrophages (20–30%) [18]. PMN leukocytes represent an important source of ROS due to their abundant presence in semen. Furthermore, external stimuli induce the activation of macrophages, leading to an oxidative burst and ROS overgeneration. Under normal circumstances, these monocytes are of paramount importance in defending male reproductive structures against nearby cells and pathogens [19].

The Endz test based on myeloperoxidase staining is an efficient technique to quantify seminal leukocytes during semen quality assessment [20]. According to the World Health Organization (WHO), if the leukocyte concentration in the ejaculate exceeds 1 × 106 /mL, leukocytospermia is present [21].

Numerous reports have studied possible relationships between seminal leukocytes and male reproductive dysfunction, resulting in two different directions. On the one hand, some studies failed to reveal any correlation between leukocytospermia and sperm damage [22], whereas inversely, other studies emphasized on a strong link between the presence of seminal leukocytes and abnormal sperm quality [23]. In particular, Sharma et al. [24] observed that even small numbers of white blood cells may be responsible for seminal OS, and hence subthreshold levels of leukocytes, as seen in ejaculates collected from otherwise healthy subjects, may not be considered safe as previously believed. Moreover, activated leukocytes may be responsible for a 100-fold increase in ROS production in comparison to non-activated white blood cells [25].

**3.1. Endogenous ROS production by sperm**

gen and the addition of a single electron [33].

●−) is considered to be the primary ROS produced by respiring cells, including

●− is predominantly generated through two reduced forms of ß-nicotin-

Physiological and Pathological Roles of Free Radicals in Male Reproduction

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

●− production could be inhibited by superoxide dismutase (SOD), which

●− generation through the NADPH oxidase [35, 36]. Lastly,

●− in spermatozoa is electron leakage from the mitochondrial

can undergo a series of cellular transformations to generate

), a highly toxic-free radical [13]. Interestingly, both high and low con-

●− important in

121

) it undergoes either

)—a membrane

●−, followed

●− to create

O2

can be either scavenged by glutathione

●− has the ability to interact with nitric oxide

), subsequent reactions of which may lead to either

spermatozoa [32]. It is a regular by-product of oxidative phosphorylation, created between complex I and III of the electron transport chain as a result of a monovalent reduction of oxy-

amide adenine dinucleotide phosphate (NADPH) oxidases that are similar to those found in phagocytic leukocytes: the NADH-dependent oxidoreductase located in the inner mitochondrial membrane and the NAD(P)H-oxidase found in the plasma membrane [34]. The hypoth-

cell signaling events in spermatozoa is based essentially on two observations. Firstly, adding pharmacological doses of NADPH to purified sperm suspensions has led to an increase in

●− production, subsequently leading to a decline in the sperm function [34, 35]. Secondly,

protects male reproductive cells against the toxic effects of NADPH [34]. Additionally, the cytoplasmic enzyme G6PD controls the rate of glucose flux and intracellular availability of NADPH through the hexose monophosphate shunt. This in turn serves as a source of elec-

permeable molecule [15], which is considered to be the major initiator of peroxidative damage

the highly reactive hydroxyl radical (OH●) through the Fenton and Haber-Weiss reactions,

The primary RNS species produced by male gametes is nitric oxide (NO●). Its production is catalyzed by nitric oxide synthase (NOS) in a redox reaction between L-arginine and oxygen,

centrations of NO● may result in significant alterations of the sperm function as a result of the

Inversely, physiological NO● levels are reported to have beneficial effects, acting in signal transduction pathways involved in spermatozoa motility, capacitation and acrosome reaction [37].

O2

●− is relatively unreactive, in the presence of hydrogen (H+

a spontaneous or SOD-catalyzed dismutation into hydrogen peroxide (H2

peroxidase (GPx) or catalase, catalyzing its dismutation into water and oxygen.

comprising a reduction of ferric (Fe3+) to ferrous ion (Fe2+) in the presence of O2

initiated by NADPH, and with L-citrulline as a byproduct. NO● interacts with O2

esis that these enzymes are primarily responsible for low-level generation of O2

Superoxide (O2

O2

In the male gamete, O2

such increased O2

trons by spermatozoa to fuel O2

in the plasma membrane of spermatozoa [27]. H2

O2

conversion to OH●. Furthermore, O2

●− as well as H2

(NO●) to generate peroxynitrite (ONOO<sup>−</sup>

**3.2. Endogenous RNS production by sperm**

[30].

another relevant source of O2

electron transport [34].

Although O2

Moreover, O2

O2

peroxynitrite (ONOO<sup>−</sup>

production of ONOO<sup>−</sup>

apoptosis or necrosis [30].

by the H2

Leukocytospermia has been furthermore associated with increased ROS production by spermatozoa, most likely triggered by a direct cell-to-cell contact of the leukocyte with the sperm cell or by the release of soluble products acting on the spermatozoon [23, 24].

Spermatozoa have also been reported to generate ROS independently of leukocytes, and this ability primarily depends on the maturation level of the sperm cell. During the epididymal transit, the main morphological change that takes place in the spermatozoon is the migration of the cytoplasmic droplet, a remnant of the cytoplasm associated with testicular sperm. The droplet migrates from the proximal to the distal position during maturation and is normally shed from spermatozoa during or shortly after ejaculation [26]. Failure to extrude excess cytoplasm during sperm differentiation and maturation traps a number of enzymes, including glucose-6-phosphate dehydrogenase (G6PD) and ß-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which have been associated with ROS generation through the formation of the NADPH intermediate [27]. As such, immature and functionally defective spermatozoa with abnormal head morphology and cytoplasmic retention are another important source of ROS in semen [12]. According to Gil-Guzman et al. [28] there is a strong positive correlation between immature spermatozoa and ROS production, which in turn is negatively correlated with semen quality. The study revealed that after a density gradient separation of human ejaculates, the layer of immature spermatozoa produced the highest levels of ROS. Furthermore, elevated concentrations of immature spermatozoa were accompanied by increased amounts of mature spermatozoa with damaged DNA [28].

Sertoli cells have also been revealed to have the ability to generate ROS, which may be inhibited by the addition of scavestrogens (J811 and J861). Scavestrogens are derivates of 17alphaestradiol and serve as effective FR-quenching molecules that able to inhibit iron-catalyzed cell damage *in vitro*. As such, Sertoli cells may play a vital role in ROS-mediated spermatogenesis. Due to currently limited evidence, there is a need to further understand the function of Sertoli cells in the process of ROS generation [29, 30].

Varicocele is defined as the excessive dilation of the *pampiniform venous plexus* around the spermatic cord and this endogenous condition is highly linked to testicular and seminal OS. While its role in male infertility is well researched, recent studies have linked higher grades of varicocele with higher ROS levels [29]. In addition, research has shown that spermatozoa from varicocele patients tend to have high levels of oxidative DNA damage [31]. The most common management option is varicocelectomy, which has been effective in the reduction of ROS levels in affected patients [29, 31].

#### **3.1. Endogenous ROS production by sperm**

Numerous reports have studied possible relationships between seminal leukocytes and male reproductive dysfunction, resulting in two different directions. On the one hand, some studies failed to reveal any correlation between leukocytospermia and sperm damage [22], whereas inversely, other studies emphasized on a strong link between the presence of seminal leukocytes and abnormal sperm quality [23]. In particular, Sharma et al. [24] observed that even small numbers of white blood cells may be responsible for seminal OS, and hence subthreshold levels of leukocytes, as seen in ejaculates collected from otherwise healthy subjects, may not be considered safe as previously believed. Moreover, activated leukocytes may be responsible for a 100-fold increase in ROS production in comparison to non-activated white

Leukocytospermia has been furthermore associated with increased ROS production by spermatozoa, most likely triggered by a direct cell-to-cell contact of the leukocyte with the sperm

Spermatozoa have also been reported to generate ROS independently of leukocytes, and this ability primarily depends on the maturation level of the sperm cell. During the epididymal transit, the main morphological change that takes place in the spermatozoon is the migration of the cytoplasmic droplet, a remnant of the cytoplasm associated with testicular sperm. The droplet migrates from the proximal to the distal position during maturation and is normally shed from spermatozoa during or shortly after ejaculation [26]. Failure to extrude excess cytoplasm during sperm differentiation and maturation traps a number of enzymes, including glucose-6-phosphate dehydrogenase (G6PD) and ß-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which have been associated with ROS generation through the formation of the NADPH intermediate [27]. As such, immature and functionally defective spermatozoa with abnormal head morphology and cytoplasmic retention are another important source of ROS in semen [12]. According to Gil-Guzman et al. [28] there is a strong positive correlation between immature spermatozoa and ROS production, which in turn is negatively correlated with semen quality. The study revealed that after a density gradient separation of human ejaculates, the layer of immature spermatozoa produced the highest levels of ROS. Furthermore, elevated concentrations of immature spermatozoa were accom-

cell or by the release of soluble products acting on the spermatozoon [23, 24].

panied by increased amounts of mature spermatozoa with damaged DNA [28].

cells in the process of ROS generation [29, 30].

els in affected patients [29, 31].

Sertoli cells have also been revealed to have the ability to generate ROS, which may be inhibited by the addition of scavestrogens (J811 and J861). Scavestrogens are derivates of 17alphaestradiol and serve as effective FR-quenching molecules that able to inhibit iron-catalyzed cell damage *in vitro*. As such, Sertoli cells may play a vital role in ROS-mediated spermatogenesis. Due to currently limited evidence, there is a need to further understand the function of Sertoli

Varicocele is defined as the excessive dilation of the *pampiniform venous plexus* around the spermatic cord and this endogenous condition is highly linked to testicular and seminal OS. While its role in male infertility is well researched, recent studies have linked higher grades of varicocele with higher ROS levels [29]. In addition, research has shown that spermatozoa from varicocele patients tend to have high levels of oxidative DNA damage [31]. The most common management option is varicocelectomy, which has been effective in the reduction of ROS lev-

blood cells [25].

120 Spermatozoa - Facts and Perspectives

Superoxide (O2 ●−) is considered to be the primary ROS produced by respiring cells, including spermatozoa [32]. It is a regular by-product of oxidative phosphorylation, created between complex I and III of the electron transport chain as a result of a monovalent reduction of oxygen and the addition of a single electron [33].

In the male gamete, O2 ●− is predominantly generated through two reduced forms of ß-nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that are similar to those found in phagocytic leukocytes: the NADH-dependent oxidoreductase located in the inner mitochondrial membrane and the NAD(P)H-oxidase found in the plasma membrane [34]. The hypothesis that these enzymes are primarily responsible for low-level generation of O2 ●− important in cell signaling events in spermatozoa is based essentially on two observations. Firstly, adding pharmacological doses of NADPH to purified sperm suspensions has led to an increase in O2 ●− production, subsequently leading to a decline in the sperm function [34, 35]. Secondly, such increased O2 ●− production could be inhibited by superoxide dismutase (SOD), which protects male reproductive cells against the toxic effects of NADPH [34]. Additionally, the cytoplasmic enzyme G6PD controls the rate of glucose flux and intracellular availability of NADPH through the hexose monophosphate shunt. This in turn serves as a source of electrons by spermatozoa to fuel O2 ●− generation through the NADPH oxidase [35, 36]. Lastly, another relevant source of O2 ●− in spermatozoa is electron leakage from the mitochondrial electron transport [34].

Although O2 ●− is relatively unreactive, in the presence of hydrogen (H+ ) it undergoes either a spontaneous or SOD-catalyzed dismutation into hydrogen peroxide (H2 O2 )—a membrane permeable molecule [15], which is considered to be the major initiator of peroxidative damage in the plasma membrane of spermatozoa [27]. H2 O2 can be either scavenged by glutathione peroxidase (GPx) or catalase, catalyzing its dismutation into water and oxygen.

Moreover, O2 ●− as well as H2 O2 can undergo a series of cellular transformations to generate the highly reactive hydroxyl radical (OH●) through the Fenton and Haber-Weiss reactions, comprising a reduction of ferric (Fe3+) to ferrous ion (Fe2+) in the presence of O2 ●−, followed by the H2 O2 conversion to OH●. Furthermore, O2 ●− has the ability to interact with nitric oxide (NO●) to generate peroxynitrite (ONOO<sup>−</sup> ), subsequent reactions of which may lead to either apoptosis or necrosis [30].

#### **3.2. Endogenous RNS production by sperm**

The primary RNS species produced by male gametes is nitric oxide (NO●). Its production is catalyzed by nitric oxide synthase (NOS) in a redox reaction between L-arginine and oxygen, initiated by NADPH, and with L-citrulline as a byproduct. NO● interacts with O2 ●− to create peroxynitrite (ONOO<sup>−</sup> ), a highly toxic-free radical [13]. Interestingly, both high and low concentrations of NO● may result in significant alterations of the sperm function as a result of the production of ONOO<sup>−</sup> [30].

Inversely, physiological NO● levels are reported to have beneficial effects, acting in signal transduction pathways involved in spermatozoa motility, capacitation and acrosome reaction [37].

#### **3.3. External sources of ROS**

ROS generation can be exacerbated by a multitude of environmental, infectious and lifestylerelated etiologies.

comparison to those with the lowest saturated fat intake [46]. These observations were supported by a later report focused on studying the link between dairy food intake and male fertility and revealing that a low-fat dairy diet may lead to a higher spermatogenesis [47]. On the other hand, omega-3 fatty acids and omega-6 fatty acids were shown to improve sperm count, motility and morphology [48]. With regard to obesity and its relation to semen parameters, currently available data are conflicting. In a study on Iranian men, it was found that overweight men tend to have lower sperm counts [49]. Inversely, a different study reported that underweight subjects had lower sperm counts than normal and overweight men [48]. Moreover, a study comprising Tunisian men revealed that sperm concentration, motility and

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123

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, acro-

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

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

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

ROS actually protect male gametes against future oxidative insults.

morphology did not vary across different BMI values [50].

**4. Physiological roles of ROS**

some reaction and sperm-oocyte fusion [30].

**4.1. Sperm maturation**

capacitation.

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 production of reactive species such as O2 ●− and H2 O2 in the testes, damage sperm DNA and 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 by increased oxidative damage to the sperm lipids, proteins and DNA [39].

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

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 malnourished individuals [44].

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 comparison to those with the lowest saturated fat intake [46]. These observations were supported by a later report focused on studying the link between dairy food intake and male fertility and revealing that a low-fat dairy diet may lead to a higher spermatogenesis [47]. On the other hand, omega-3 fatty acids and omega-6 fatty acids were shown to improve sperm count, motility and morphology [48]. With regard to obesity and its relation to semen parameters, currently available data are conflicting. In a study on Iranian men, it was found that overweight men tend to have lower sperm counts [49]. Inversely, a different study reported that underweight subjects had lower sperm counts than normal and overweight men [48]. Moreover, a study comprising Tunisian men revealed that sperm concentration, motility and morphology did not vary across different BMI values [50].
