Abstract
In brief
This review focuses on the enzymatic antioxidant mechanisms to fight oxidative stress by spermatozoa, highlighting the differences among mammalian species. We discuss recent evidence about players that promote and fight oxidative stress and the need for novel strategies to diagnose and treat cases of male infertility associated with oxidative damage of the spermatozoon.
Abstract
The spermatozoon is very sensitive to high reactive oxygen species (ROS) levels due to its limited antioxidant system. A consortium of antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidases (GPXs), peroxiredoxins (PRDXs), thioredoxins, and glutathione-S-transferases, is necessary to produce healthy spermatozoa and to maintain sperm quality to ensure motility, capacitation, and DNA integrity. A delicate balance between ROS production and antioxidant enzymes is needed to ensure ROS-dependent sperm capacitation. GPX4 is an essential component of the mitochondrial sheath in mammalian spermatozoa, and GPX5 is a crucial antioxidant defence in the mouse epididymis to protect the sperm genome during the maturation of the spermatozoon. The mitochondrial superoxide (O2·–) production is controlled by SOD2, and the hydrogen peroxide (H2O2) generated by SOD2 activity and peroxynitrite (ONOO–) are scavenged mainly by PRDXs in human spermatozoa. PRDXs regulate the redox signalling necessary for sperm motility and capacitation, particularly by PRDX6. This enzyme is the first line of defence against oxidative stress to prevent lipid peroxidation and DNA oxidation by scavenging H2O2 and ONOO– through its peroxidase activity and repairing oxidized membranes by its calcium-independent phospholipase A2 activity. The success of antioxidant therapy in treating infertility resides in the proper diagnosis of the presence of oxidative stress and which type of ROS are produced. Thus, more research on the molecular mechanisms affected by oxidative stress, the development of novel diagnostic tools to identify infertile patients with oxidative stress, and randomized controlled trials are of paramount importance to generate personalized antioxidant therapy to restore male fertility.
Introduction
The spermatozoon is a terminal cell, with the only purpose of delivering the paternal genome inside the oocyte during fertilization. It also delivers to the oocyte a centriole to orchestrate cell division in the embryo and non-coding RNAs to modulate the pattern of embryonic growth. Because spermatozoa are translational silent, they must be equipped with all the necessary elements obtained during spermatogenesis and through the journey through the epididymis to accomplish this essential task for species survival. As an aerobic cell, spermatozoa are loaded with different antioxidant enzymes to protect the paternal genome’s precious cargo from the toxic effects of reactive oxygen species (ROS) that are typically produced during aerobic metabolism (Fig. 1). ROS have a dual role in spermatozoa; at high concentrations, they promote oxidative stress, damaging lipids, proteins, and DNA of the cell (Aitken et al. 2022), but at low and controlled amounts, they trigger the redox signalling necessary for physiological processes to ensure fertilization (O’Flaherty et al. 2006b , O’Flaherty 2015).
Antioxidant enzymes are fighting oxidative stress in mammalian spermatozoa. The paternal genome is protected against oxidative stress (generated by hydrogen peroxide (H2O2) and peroxynitrite (ONOO–) by 2-Cys PRDXs and PRDX6 and the nuclear form of GPX4). Different antioxidant enzymes scavenge reactive oxygen species (ROS); superoxide dismutase 2 (SOD2), present in mitochondria, prevents the increase of superoxide anion (O2·–). Due to this action, H2O2 is formed and removed by peroxiredoxins (PRDX2, 3, 5, and 6). SOD1 removes O2·– in the cytosol (not shown). ROS formation in the mitochondria (and other sites) promotes lipid peroxidation, altering the plasma membrane fluidity and inactivating the enzymes involved in motility, capacitation, and so on. PRDXs (namely 2-Cys PRDXs and PRDX6) fight against oxidative stress in different spermatozoon parts and prevent excessive lipid peroxidation formation. Sperm-specific thioredoxin (SpTRX2/3) is essential to reactivate 2-Cys PRDXs.
Citation: Reproduction 164, 6; 10.1530/REP-22-0200
Antioxidant enzymes are fighting oxidative stress in mammalian spermatozoa. The paternal genome is protected against oxidative stress (generated by hydrogen peroxide (H2O2) and peroxynitrite (ONOO–) by 2-Cys PRDXs and PRDX6 and the nuclear form of GPX4). Different antioxidant enzymes scavenge reactive oxygen species (ROS); superoxide dismutase 2 (SOD2), present in mitochondria, prevents the increase of superoxide anion (O2·–). Due to this action, H2O2 is formed and removed by peroxiredoxins (PRDX2, 3, 5, and 6). SOD1 removes O2·– in the cytosol (not shown). ROS formation in the mitochondria (and other sites) promotes lipid peroxidation, altering the plasma membrane fluidity and inactivating the enzymes involved in motility, capacitation, and so on. PRDXs (namely 2-Cys PRDXs and PRDX6) fight against oxidative stress in different spermatozoon parts and prevent excessive lipid peroxidation formation. Sperm-specific thioredoxin (SpTRX2/3) is essential to reactivate 2-Cys PRDXs.
Citation: Reproduction 164, 6; 10.1530/REP-22-0200
Antioxidant enzymes are fighting oxidative stress in mammalian spermatozoa. The paternal genome is protected against oxidative stress (generated by hydrogen peroxide (H2O2) and peroxynitrite (ONOO–) by 2-Cys PRDXs and PRDX6 and the nuclear form of GPX4). Different antioxidant enzymes scavenge reactive oxygen species (ROS); superoxide dismutase 2 (SOD2), present in mitochondria, prevents the increase of superoxide anion (O2·–). Due to this action, H2O2 is formed and removed by peroxiredoxins (PRDX2, 3, 5, and 6). SOD1 removes O2·– in the cytosol (not shown). ROS formation in the mitochondria (and other sites) promotes lipid peroxidation, altering the plasma membrane fluidity and inactivating the enzymes involved in motility, capacitation, and so on. PRDXs (namely 2-Cys PRDXs and PRDX6) fight against oxidative stress in different spermatozoon parts and prevent excessive lipid peroxidation formation. Sperm-specific thioredoxin (SpTRX2/3) is essential to reactivate 2-Cys PRDXs.
Citation: Reproduction 164, 6; 10.1530/REP-22-0200
Infertility is a significant worldwide health issue that has increased in prevalence over the past two decades, currently touching approximately 17% of couples (Comhaire et al. 1987, Bushnik et al. 2012). Male factors cause half of these infertility cases, and oxidative stress, the net increase of ROS, has been identified as a common culprit in many conditions and diseases that lead to male infertility (Aitken 1994, Gharagozloo & Aitken 2011). These conditions include pollutants, drugs, and clinical disorders of various kinds (Anderson & Williamson 1988, Brennemann et al. 1997, Hasegawa et al. 1997, Turner 2001, Smith et al. 2006).
In this review, we will describe the sources of ROS, consequences of oxidative stress on sperm function, and possibilities of treating male infertility due to oxidative damage of mammalian spermatozoa.
Production of reactive oxygen species in mammalian spermatozoa
Pioneer works of McLeod in 1943 and Tosic and Walton in 1946, using human and bovine spermatozoa, respectively, demonstrated that mammalian spermatozoa produce ROS. The addition of catalase was able to prevent ROS damage to human (MacLeod 1943) and bovine spermatozoa by scavenging the hydrogen peroxide (H2O2) that can inhibit respiration and motility (Tosic & Walton 1946). Interestingly, spermatozoa lack endogenous catalase because this enzyme is present in peroxisomes that leave the transforming spermatids during spermiogenesis (Luers et al. 2006, Nenicu et al. 2007). Altogether, these findings could explain, in part, the high sensitivity of spermatozoa to oxidative stress.
The mitochondrion is an essential source of ROS in spermatozoa; superoxide anion (O2·–) is mainly produced at complexes I and II of the electron transfer chain during the oxidative phosphorylation process needed to generate energy (Ramalho-Santos et al. 2009, Aitken et al. 2012).
ROS can be produced at the plasma membrane level. It was demonstrated that a sperm oxidase is present in the plasma membrane of human, bovine, and equine spermatozoa that generates O2·– needed for capacitation since the addition of SOD added to the incubation medium prevented tyrosine phosphorylation, hyperactivation, and induction of acrosome reaction in these species (de Lamirande & Gagnon 1995, Leclerc et al. 1997, de Lamirande et al. 1998, O’Flaherty et al. 2003, 2006a , Musset et al. 2012). The exogenous addition of catalase to the capacitating medium also prevented capacitation, reinforcing the need for ROS produced at the plasma membrane level necessary for sperm capacitation (de Lamirande & Gagnon 1995, O’Flaherty et al. 2006b).
In human and stallion spermatozoa, the existence of NOX5 (NADPH oxidase isoform 5) was discovered, which produces ROS (e.g. O2·– that can then be converted into H2O2 spontaneously). The generation of O2·– and ONOO– (product of the combination of O2·– and NO·) is necessary for the change of motility in capacitated spermatozoa called hyperactivation (de Lamirande & Gagnon 1993, Herrero et al. 2001), while the presence of NOX5 in the sperm flagellum suggests a role for this oxidase in stimulating this form of movement (Musset et al. 2012). Interestingly, the efficiency of this redox stimulus has limits because overexpression of NOX5 has been associated with male infertility due to a severe decrease in sperm motility (Vatannejad et al. 2019).
Nitric oxide is also produced in mammalian spermatozoa. It has been implicated in sperm capacitation in human and bovine species (Herrero et al. 2000, Rodriguez et al. 2005, 2011, O’Flaherty et al. 2006b,c). The nitric oxide synthase is located in the cytosol and plasma membrane of the spermatozoon in humans, rodents, and bulls (Burnett et al. 1995, Herrero et al. 1996, O’Bryan et al. 1998, Meiser & Schulz 2003). Peroxynitrite is also associated with sperm capacitation (de Lamirande & Lamothe 2009), but, at high concentrations, it promotes damage, leading to a decrease in sperm motility, an increase of lipid peroxidation, and impairment of capacitation, as was observed in human and mouse spermatozoa (Fernandez & O’Flaherty 2018, Scarlata et al. 2020). Another source of ROS is l-amino acid oxidases, which produce H2O2 and were reported in human, equine, and ram spermatozoa (Shannon & Curson 1982a,b, Upreti et al. 1998, Aitken et al. 2015, Houston et al. 2015).
Enzymatic antioxidant system in mammalian spermatozoa
Because spermatozoa can produce ROS, these cells need an antioxidant system to maintain the ROS levels at low concentrations and avoid toxicity. As a terminal cell without the possibility to synthesize new proteins and with the limited amount of reduced glutathione (GSH), these cells are particularly vulnerable to ROS attack. In human spermatozoa, GSH is present at ~0.3 mM compared to the 10 mM concentration observed in somatic cells (Li 1975). A sufficient concentration of GSH is essential because it participates in the reactivation of certain antioxidant enzymes (e.g. PRDXs and glutathione peroxidases (GPXs)). Thus, mammalian spermatozoa need a set of antioxidant enzymes to control the significant ROS: O2·–, NO·, and ONOO–.
Superoxide dismutase
In ejaculated spermatozoa, O2·– levels are controlled by SOD, which is present in the cytosol (SOD1) and mitochondria (SOD2). Levels of SOD activity differ among species; compared with the activity found in human spermatozoa, donkeys and rats have 9 and 3 times more activity, respectively, and mice, bulls, and rabbits have ~4 times less activity (Cassani et al. 2005). Interestingly, SOD1 is the sole protection against O2·– in human spermatozoa, since SOD2 and SOD3 are negligible in these cells (Peeker et al. 1997).
The absence of SOD1 in mouse spermatozoa does not impact in vivo fertility (35); however, in vitro fertilization (IVF) rates are greatly diminished when Sod1 –/– spermatozoa are used compared to WT controls (98). The mitochondrial SOD2 plays an essential role in scavenging the O2·– inevitably formed during aerobic metabolism; thus, it is a critical antioxidant enzyme to protect mammalian spermatozoa. However, we cannot confirm this statement because Sod2 –/– mice die a few weeks after birth, showing neurodegeneration and myocardial injury and the absence of conditional knockout mouse models.
Glutathione peroxidases
Due to SOD activity, H2O2 is produced, which can be harmful if not scavenged immediately. Somatic cells have several enzymes to remove excess H2O2, catalase, restricted to peroxisomes, GPXs, and PRDXs, widely distributed in organelles. In the case of mammalian spermatozoa, the defence against H2O2 is limited to a few antioxidant enzymes. Indeed, catalase, present in peroxisomes, is removed from spermatids in the residual body during spermiogenesis (Luers et al. 2006, Nenicu et al. 2007). Mitochondrial GPX4 (mGPX4) is incorporated during spermatogenesis into the mitochondrial sheath and becomes inactive enzymatically (Ursini et al. 1999). The GPX4 isoform is essential for spermatozoa since its deficiency or absence has been associated with male infertility (Foresta et al. 2002). The nuclear form of GPX4 participates in the protection of sperm DNA in mice. Still, it is not essential as nGPX4 conditional knockout male mice are fertile (Conrad et al. 2005), suggesting other mechanisms to protect the sperm DNA.
Others GPX isoforms have been identified in male mice that are critical for maintaining the health of spermatozoa within the epididymis. GPX3 and GPX5 are highly expressed in mouse epididymis (Schwaab et al. 1998, Chabory et al. 2009), but they are absent in the testes, epididymes, and seminal plasma in humans (Hall et al. 1998). These findings highlight the species differences in fighting against oxidative stress in the male reproductive system.
Peroxiredoxins
The PRDX family comprises six isoforms involved in protecting cells against oxidative stress and participating in redox signalling of physiological processes. These are non-selenium enzymes capable of scavenging H2O2, organic hydroperoxides, and ONOO– that are widely distributed in all compartments of human spermatozoa (O’Flaherty 2014) and found in mouse and bovine spermatozoa (O’Flaherty 2018, Mostek-Majewska et al. 2021). The pharmacological inhibition of PRDX6 increases the mitochondrial derived oxidative stress with a reduction of sperm viability and increases sperm DNA oxidation and lipid peroxidation (Fernandez & O’Flaherty 2018). Male mice lacking the PRDX6 gene are subfertile, displaying low sperm quality (Ozkosem et al. 2016). PRDX2 is another antioxidant enzyme found in the mitochondrial sheath of mouse and boar spermatozoa (Manandhar et al. 2009). Still, it seems not to be essential since the PRDX2 mouse knockout model is fertile (Lee et al. 2003).
For the normal functioning of PRDXs, which protect against high levels of ROS and regulate redox signalling during sperm capacitation (O’Flaherty 2015), these enzymes need to be reactivated by different systems. PRDX1–5, also called 2-Cys PRDXs because they contain two cysteine residues in their active site, are reactivated by the thioredoxin–thioredoxin reductase–NADPH system. The importance of this reactivation system has been demonstrated in human spermatozoa by pharmacological inhibition of thioredoxin reductase and enzymes that provide NADPH, such as glucose-6-phosphate dehydrogenase, NADP-dependent isocitrate dehydrogenase, and malic enzyme (Fig. 2). This inhibition promoted increased mitochondrial production levels, reduced mitochondrial membrane potential, and increased lipid peroxidation and sperm DNA oxidation (Fernandez & O’Flaherty 2018). The need for an active molecular mechanism to reactivate 2-Cys PRDX activity in spermatozoa has been confirmed using a mouse model lacking sperm-specific Txndc2 and Txndc3. In these knockout males, decreased sperm motility and increased oxidative damage in lipids and DNA were observed as the males aged (Smith et al. 2013).
2-Cys peroxiredoxins are reactivated by the thioredoxin (TRX)/TRD/NAPDH system to protect spermatozoa against oxidative stress. PRDXs scavenge H2O2 (1) and become inactive because the thiol groups in its active site are oxidized (2). This thiol oxidation is reversed by the TRX–TRX reductase (TRD) system that uses NADPH as reducing equivalents. The sources of NADPH in human spermatozoa are glucose-6-phosphate dehydrogenase (G6PDH), NAD+-dependent isocitrate dehydrogenase (NADP-ICDH), and the malic enzyme (ME) (3). Higher amounts of H2O2 promote further oxidation inactivating the enzyme by converting it into its hyperoxidized form. The hyperoxidized form of PRDXs is reduced by sulfiredoxin (SRX) or sestrins, using ATP, and reduced GSH converts the PRDX into the reduced and active form (4).
Citation: Reproduction 164, 6; 10.1530/REP-22-0200
2-Cys peroxiredoxins are reactivated by the thioredoxin (TRX)/TRD/NAPDH system to protect spermatozoa against oxidative stress. PRDXs scavenge H2O2 (1) and become inactive because the thiol groups in its active site are oxidized (2). This thiol oxidation is reversed by the TRX–TRX reductase (TRD) system that uses NADPH as reducing equivalents. The sources of NADPH in human spermatozoa are glucose-6-phosphate dehydrogenase (G6PDH), NAD+-dependent isocitrate dehydrogenase (NADP-ICDH), and the malic enzyme (ME) (3). Higher amounts of H2O2 promote further oxidation inactivating the enzyme by converting it into its hyperoxidized form. The hyperoxidized form of PRDXs is reduced by sulfiredoxin (SRX) or sestrins, using ATP, and reduced GSH converts the PRDX into the reduced and active form (4).
Citation: Reproduction 164, 6; 10.1530/REP-22-0200
2-Cys peroxiredoxins are reactivated by the thioredoxin (TRX)/TRD/NAPDH system to protect spermatozoa against oxidative stress. PRDXs scavenge H2O2 (1) and become inactive because the thiol groups in its active site are oxidized (2). This thiol oxidation is reversed by the TRX–TRX reductase (TRD) system that uses NADPH as reducing equivalents. The sources of NADPH in human spermatozoa are glucose-6-phosphate dehydrogenase (G6PDH), NAD+-dependent isocitrate dehydrogenase (NADP-ICDH), and the malic enzyme (ME) (3). Higher amounts of H2O2 promote further oxidation inactivating the enzyme by converting it into its hyperoxidized form. The hyperoxidized form of PRDXs is reduced by sulfiredoxin (SRX) or sestrins, using ATP, and reduced GSH converts the PRDX into the reduced and active form (4).
Citation: Reproduction 164, 6; 10.1530/REP-22-0200
Among PRDXs, PRDX6 is the only one with peroxidase and calcium-independent phospholipase A2 (iPLA2) activities. Whereas PRDX6 peroxidase activity is vital to scavenge H2O2, organic hydroperoxides, and ONOO–, the PRDX6 iPLA2 activity is important to repair oxidized membranes (Fisher 2018). In human and mouse spermatozoa, these two PRDX6 activities are essential to maintain sperm quality to ensure fertility. Indeed, males lacking either the peroxidase or iPLA2 activities (C47S and D140A knock-in mouse strains) are subfertile, showing low motility, failure to undergo capacitation and acrosome reaction, and the presence of high levels of lipid peroxidation, DNA oxidation, and tyrosine nitration in their spermatozoa. Such changes indicate the presence of significant oxidative damage to lipids and proteins in the spermatozoa, thereby explaining the low sperm quality and infertility in these mice (Bumanlag et al. 2022).
Glutathione-S-transferases
Different glutathione-S-transferases (GSTs) isoforms are present in the testes, epididymides, and spermatozoa from different mammalian species, including humans, goats, boars, buffalos, and rodents (Veri et al. 1993, Gopalakrishnan et al. 1998, Mukherjee et al. 1999, Raijmakers et al. 2003, Kumar et al. 2014, Llavanera et al. 2020). GSTs are being discovered as crucial elements in the antioxidant response to sustain male fertility. The specific inhibition of GSTO2 promotes oxidative stress in spermatozoa impairing capacitation and fertilization in mice (Hamilton et al. 2019). Polymorphisms of GSTM1, GSTM3, GSTP1, and GSTT1 have been associated with infertility in men (reviewed in Llavanera et al. 2019).
GSTP1 is important to reactivate PRDX6 peroxidase activity in spermatozoa (Fernandez & O’Flaherty 2018) (Fig. 3). The inhibition of GST-P1 and the depletion of GSH in spermatozoa promote an increase of oxidative stress, leading to lipid peroxidation and DNA oxidation and resulting in the impairment of sperm viability (Fernandez & O’Flaherty 2018).
PRDX6 scavenges hydrogen peroxide and becomes inactive by thiol oxidation of the thiol residue in the active site (1). Further oxidation by H2O2 promotes the formation of the hyperoxidized form, which is irreversible inactive (2). The thiol oxidation of PRDX6 is reduced by the glutathione-S-transferase pi (GST-P1) using reduced glutathione (GSH) (3).
Citation: Reproduction 164, 6; 10.1530/REP-22-0200
PRDX6 scavenges hydrogen peroxide and becomes inactive by thiol oxidation of the thiol residue in the active site (1). Further oxidation by H2O2 promotes the formation of the hyperoxidized form, which is irreversible inactive (2). The thiol oxidation of PRDX6 is reduced by the glutathione-S-transferase pi (GST-P1) using reduced glutathione (GSH) (3).
Citation: Reproduction 164, 6; 10.1530/REP-22-0200
PRDX6 scavenges hydrogen peroxide and becomes inactive by thiol oxidation of the thiol residue in the active site (1). Further oxidation by H2O2 promotes the formation of the hyperoxidized form, which is irreversible inactive (2). The thiol oxidation of PRDX6 is reduced by the glutathione-S-transferase pi (GST-P1) using reduced glutathione (GSH) (3).
Citation: Reproduction 164, 6; 10.1530/REP-22-0200
Recently, it was demonstrated that GST-P1 forms a heterocomplex with c-Jun N-terminal kinase (JNK) and thus prevents oxidative stress since the pharmacological disturbance of this complex was associated with impairment of sperm motility and increased production of mitochondrial ROS in boar spermatozoa (Llavanera et al. 2021).
GPX5 and PRDXs protect the maturing mammalian spermatozoa against oxidative stress in the epididymis
During epididymal maturation, many biochemical processes that can promote ROS are produced to prepare the spermatozoa with all the necessary elements to acquire motility and to be able to recognize the oocyte at the time of fertilization. In mice, GPX5 plays a vital role in protecting the maturing spermatozoa and particularly the paternal genome. Certainly, male mice lacking GPX5, highly expressed in the epididymis, showed increased sperm DNA oxidation and lipid peroxidation with reduced sperm motility (Chabory et al. 2009). However, GPX5 is absent in the human reproductive system or spermatozoa (Hall et al. 1998). This finding strengthens the different strategies that mammalian species have to fight against oxidative stress in the epididymis.
When male rats were exposed to an in vivo oxidative stress induced by tert-butyl hydroperoxide (tert-BHP) treatment during the epididymal transit, an increase in PRDX1 and PRDX6, but not of SOD1, was observed in spermatozoa from the cauda epididymis, suggesting the activation of mechanisms to protect the maturing spermatozoa when facing high levels of ROS (Liu & O’Flaherty 2017). This increase of PRDXs in epididymal spermatozoa was accompanied by a rise in PRDX1 and PRDX6 in cauda epididymis, suggesting that the epididymal epithelium is not only fighting against the established oxidative stress but also supplying spermatozoa with antioxidant enzymes through epididymosomes. It is known that epididymosomes (at least from bovines and humans) are loaded with antioxidant enzymes and interact with epididymal spermatozoa during their maturation (Sullivan et al. 2007, Thimon et al. 2008, Girouard et al. 2011).
Role of antioxidant enzymes in the maintenance of sperm viability, motility, and capacitation
Spermatozoa must reside for a specific time in the oviduct, which varies among species, to acquire the fertilizing ability and respond to the signals sent by the oocyte to accomplish fertilization. Thus, biochemical mechanisms are put in place to ensure sperm viability during the spermatozoon’s entire lifespan to guarantee the delivery of the paternal genome at the time of fertilization. Oxidative stress promotes apoptotic like changes in spermatozoa, including externalizing phosphatidylserine, activating caspases, and releasing cytochrome C from mitochondria. These changes are associated with loss of motility, DNA oxidation, and cell death (Aitken & Koppers 2011).
The PRDX6 iPLA2 activity regulates the PI3K/AKT pathway to maintain sperm viability
The phosphoinositide-3-phosphate kinase (PI3K) pathway is essential for maintaining sperm viability since it prevents these apoptotic like changes from occurring (Koppers et al. 2011). It is regulated by PRDX6 iPLA2 activity since the inhibition of this activity with the lipid analogue MJ33 prevented the phosphorylation of PI3K and AKT, which was associated with increased levels of mitochondrial O2·– and lipid peroxidation-dependent impairment of sperm viability (Fernandez & O’Flaherty 2018, Fernandez et al. 2019). Moreover, lysophosphatidic acid (LPA), a product of PRDX6 iPLA2 activity, added to MJ33-treated spermatozoa prevented the loss of viability by maintaining phosphorylation of PI3K and AKT substrates in human spermatozoa (Fernandez et al. 2019). These findings confirm the need for PRDX6 and an active LPA-dependent lipid signalling to maintain sperm viability.
S-glutathionylation and tyrosine nitration of sperm proteins and lipid peroxidation impair sperm motility and capacitation
The toxic effect of ROS on spermatozoa is dose dependent. High ROS levels that do not impair viability heavily promote the inhibition of motility and capacitation (Morielli & O’Flaherty 2015).
The oxidative stress impairs sperm motility and capacitation because lipids of the plasma membrane and flagellum proteins are oxidized and, therefore, unable to perform their function. For instance, tubulin, the major protein of the sperm flagellum, is susceptible to S-glutathionylation and tyrosine nitration. These redox-dependent protein modifications were found in the flagellum of human spermatozoa challenged with oxidative stress (Morielli & O’Flaherty 2015). The energy machinery is also susceptible to oxidative stress; glyceraldehyde-3-phosphate dehydrogenase, a key enzyme in glycolysis, is inactivated by oxidation of thiol groups of the enzyme’s active site by ROS (Elkina et al. 2011). Other enzymes of the Krebs cycle, such as a-ketoglutarate dehydrogenase, and malate dehydrogenase, involved in the generation of NADH for the oxidative phosphorylation to produce ATP in spermatozoa, are susceptible to S-glutathionylation and tyrosine nitration (Morielli & O’Flaherty 2015).
It is well recognized in andrology that high lipid peroxidation levels have detrimental effects on sperm function. However, it is important to highlight that low lipid peroxidation levels occur in spermatozoa undergoing capacitation to ensure fertilization in humans and mice (Kodama et al. 1996, Lee et al. 2017). Lipid peroxidation plays its toxic role on spermatozoa at different levels, affecting sperm structures, plasma membrane fluidity, and inactivation of key enzymes involved in the molecular mechanisms for motility and capacitation (Alvarez & Aitken 2012, Walters et al. 2020b ). A major culprit of this toxic effect is 4-hydroxynonenal (4-HNE), a subproduct obtained during the oxidation of lipids. It was demonstrated that 4-HNE inactivates the succinate dehydrogenase in complex I of the respiratory chain, promoting the increase of mitochondrial O2·– (Aitken et al. 2012). Due to this enzymatic inactivation, more 4-HNE is produced due to the established oxidative stress, creating a vicious circle and leading to an impairment of sperm motility and increase in sperm DNA oxidation (Aitken et al. 2012). As mentioned above, a similar situation was observed when PRDX6 iPLA2 activity was pharmacologically inactivated: high levels of mitochondrial O2·–, increase of 4-HNE, and a significant reduction of sperm viability (Fernandez & O’Flaherty 2018).
Enzymes involved in glycolysis (phosphofructokinase, aldolase A, fructose-bisphosphate, phosphoglycerate kinase, and pyruvate kinase), in the re-oxidation of NADH, like lactate dehydrogenase C, to maintain active glycolysis, enzymes belonging to Krebs cycle (malate dehydrogenase 2), to electron transport chain (succinate dehydrogenase complex, subunit A, and ubiquinol-cytochrome c reductase), are inactivated by forming a complex with 4-HNE (Aitken et al. 2012, Baker et al. 2015).
Arachidonate 15-lipoxygenase (ALOX15), which is responsible for catalyzing lipid peroxidation via the oxygenation of polyunsaturated fatty acid (PUFA) substrates, is also implicated in 4-HNE production in germ cells (Walters et al. 2018). ALOX15 metabolizes arachidonic acid, linoleic acid, and docosahexaenoic acid, thus producing increased lipid peroxidation levels in human spermatozoa. Recently, it was demonstrated that infertile men have high levels of ALOX15 in their spermatozoa (Walters et al. 2020a ).
The role of antioxidant enzymes in the protection of the paternal genome
Protecting the sperm DNA is essential to ensure the quality of producing a healthy embryo. Many factors can affect sperm DNA quality, including drugs, diseases, and even lifestyles (Anderson & Williamson 1988, Brennemann et al. 1997, Hasegawa et al. 1997, Turner 2001, Smith et al. 2006). DNA fragmentation, oxidation, and changes in sperm chromatin are observed in infertile patients (Lewis et al. 2013, Dorostghoal et al. 2017, Esteves et al. 2017). Approximately 9000 genomic regions of 150–1000 bp are highly susceptible to oxidative damage in human spermatozoa, particularly those located in chromosome 15 (Xavier et al. 2019). The oxidative damage of the paternal genome can produce various outcomes, from the most severe (e.g. miscarriages) to the reduction of fertilization rates or even health problems of the child.
GPX5 and PRDX6, guardians of the sperm DNA integrity
The sperm DNA is protected by antioxidant enzymes during the entire journey of the cell until fertilization is accomplished. GPX5, highly expressed in the epididymis, is vital to protect sperm DNA against oxidative stress in the aging male mouse, whereas PRDXs, particularly PRDX6, are essential to maintain sperm DNA quality in human and mouse spermatozoa (Ozkosem et al. 2016, Fernandez & O’Flaherty 2018). It was observed that the inhibition of PRDX6 peroxidase activity leads to an increase in lipid peroxidation and 4-HNE formation, which in turn will promote the impairment of the mitochondrial membrane potential in human spermatozoa (Fernandez & O’Flaherty 2018).
The transit through the epididymis is essential to complete the maturation of the spermatozoon. During this journey, spermatozoa acquire the ability to move and are further transformed by the removal of excess cytoplasm and remodelling of the sperm chromatin. Due to this chromatin remodelling, the sperm DNA is exposed to ROS normally or pathologically produced in the epididymis. Thus, the epididymis has a battery of antioxidant enzymes to prevent oxidative stress and associated damage to vital sperm structures. The antioxidant enzymatic protection varies among species; as mentioned above, GPX5 is an important protector of spermatozoa in the aging male mouse; however, PRDXs and, in particular, PRDX6 protect the maturing spermatozoa in young and old mice and rats. Indeed, male mice or rats treated with an in vivo oxidative stress induced by t-BHP during the sperm epididymal transit show decreased motility and increased DNA oxidation and lipid peroxidation in their spermatozoa collected 24 h after the end of treatment (Ozkosem et al. 2016, Liu & O’Flaherty 2017). These oxidative damages were accompanied by a selective increase of PRDX1 and 6, but not SOD1, in spermatozoa and a segment-specific increase in PRDX expression (Liu & O’Flaherty 2017).
The oxidative damage generated by t-BHP treatment during only 15 days in male rats promotes chronic oxidative damage that took 3–9 weeks to be resolved in spermatozoa and the epididymal epithelium (Wu et al. 2020). Lipid peroxidation was higher in caput and cauda epididymis of treated rats compared to untreated controls, observing higher levels in the cauda than in caput epididymis. Although lipid peroxidation decreased in the epididymis to a similar level to untreated controls, the inhibition of sperm motility and sperm DNA oxidation persisted during the entire 9-week period, indicating that the balance between ROS levels and the antioxidant system has been compromised. The origin of oxidative damage in spermatozoa could be due to problems during epididymal maturation or spermatogenesis. The increased expression of PRDX6 in caput epididymis only at 3 weeks and in cauda epididymis during 3–6 weeks post-treatment supports the active response of the epididymal epithelium to control the oxidative stress and minimize the damage to spermatozoa.
Moreover, the absence of changes in lipid peroxidation or expression of PRDXs in testes during the same period of 9 weeks demonstrates that the epididymis is a more sensitive organ, compared to testes, to oxidative stress (Wu et al. 2020). However, we cannot rule out the possibility that spermatozoa are being damaged during spermatogenesis. For instance, we did not evaluate changes in the epigenome or miRNA content in that study.
Similarly, Prdx6 –/– males are also subjected to chronic oxidative stress with severe damage to sperm chromatin, including low DNA compaction, DNA fragmentation and oxidation, and low protamination (Ozkosem et al. 2015, 2016). The deletion of PRDX6 peroxidase or iPLA2 activities produced increased levels of sperm DNA oxidation in male mice, which were exacerbated by the t-BHP treatment (Bumanlag et al. 2022). These findings indicate that both enzymatic activities of PRDX6 are essential to maintaining sperm DNA integrity.
The sperm DNA damage observed in these mouse models challenged with in vivo oxidative stress (produced by t-BHP treatment) is similar to that found in cancer survivors after chemotherapy, a therapy that generates a significant amount of ROS (Look & Musch 1994). Sperm chromatin in cancer survivors is extensively damaged and takes up to 2 years to return to normality, although sperm DNA damage is still observed (O’Flaherty et al. 2012). These data suggest that long-term damage is produced by oxidative stress; thus, it is imperative to treat males suffering from oxidative damage to avoid infertility.
Oxidative stress and male infertility
Human infertility is rising, with approximately 17 couples touched by this disease (Comhaire & World Health Organization 1997, Bushnik et al. 2012). Although some treatments, such as IVF and intracytoplasmic sperm injection (ICSI), were designed to help infertile couples, these artificial reproductive technologies have low efficiency. Indeed, the pregnancy rate per cycle started is 33% and 265% for IVF and ICSI, respectively. Moreover, these techniques can transmit genetic defects to the child since the genomic integrity of the spermatozoa is unknown (Corabian & Hailey 1999, Agarwal et al. 2005, Hansen et al. 2005, Moawad et al. 2017). Emerging evidence suggests that ART treatment may predispose individuals to an increased risk of chronic aging-related diseases such as obesity, type 2 diabetes, and cardiovascular disease (Chen & Heilbronn 2017).
The male factor is an important culprit of human idiopathic infertility since up to 35% of infertility cases are exclusively due to problems in the man (Aitken 2020). The causes of dysfunctional spermatozoa are many and are still unknown. Moreover, the disruption of molecular mechanisms that can account for the infertility phenotype is not currently evaluated in the clinic.
As explained in the above sections, oxidative stress is a major culprit of male infertility. Indeed, high levels of ROS can impair motility, capacitation, acrosome reaction, and sperm quality, particularly leading to severe damage to the sperm DNA (Lavranos et al. 2012, Morielli & O’Flaherty 2015, Xavier et al. 2019).
With a limited antioxidant system, the inactivation of key antioxidant enzymes in the spermatozoon due to oxidative stress will further increase ROS production and negatively impact sperm quality and function. Low amounts of PRDXs were found in men with clinical varicocele or idiopathic infertility (Gong et al. 2012). In particular, PRDX1 was found in low quantities in spermatozoa from varicocele infertile patients, whereas PRDX6 amounts in infertile men (with both varicocele or idiopathic infertility) were present in low abundance in spermatozoa. Moreover, higher thiol-oxidation levels of PRDXs, which promote enzymatic inactivation, were observed in spermatozoa from infertile compared to fertile men. Thiol-oxidized PRDX1 and PRDX6 levels were increased in varicocele and idiopathic infertile male cohorts, respectively. This inactivation of PRDX1 and PRDX6 was associated with asthenozoospermia, high lipid peroxidation levels, and sperm DNA fragmentation (Gong et al. 2012). Altogether, these findings support the concept that low levels and inactivation of PRDXs are associated with male infertility.
The genetic deletion of PRDX6 promotes male infertility in mice (Ozkosem et al. 2016). The number of pups sired by Prdx6 –/– males was significantly lower than those generated by WT males. Moreover, a delay in the weaning time of pups from the knockout males was observed, suggesting problems in the offspring’s development. Interestingly, Prdx6 –/– spermatozoa produced lower IVF rates and were incapable of producing blastocysts (Moawad et al. 2017) compared to C57Bl6/J (WT) or CD1 male controls. Noteworthy, when spermatozoa from WT or CD1 controls were treated with the inhibitor of PRDX6 iPLA2 activity, they also produced lower fertilization rates and a reduced number of blastocysts compared to the untreated controls. These findings are exciting and can be translated to humans when IVF failure observed in some infertile couples could be related to dysfunctional PRDX6 in spermatozoa.
The pharmacological inactivation of PRDXs, and particularly of PRDX6, or the genetic ablation of PRDX6, leads to increased levels of mitochondrial O2·– , leading to impairment of motility, capacitation, and poor sperm quality, evidenced by increased levels of DNA oxidation, lipid peroxidation and tyrosine nitration, S-glutathionylation, and carbonylation in mammalian spermatozoa that are associated with subfertility (Ozkosem et al. 2016, Lee et al. 2017, Moawad et al. 2017, Fernandez & O’Flaherty 2018, Fernandez et al. 2019). This abnormal phenotype is worsened in aging mouse males (Ozkosem et al. 2015). Moreover, the specific inactivation of PRDX6 iPLA2 (pharmacologically or by knock-in gene strategy) is sufficient to promote infertility and poor sperm quality in mice and impair sperm capacitation and viability. It promotes DNA oxidation in human spermatozoa (Lee et al. 2017, Fernandez & O’Flaherty 2018, Bumanlag et al. 2022).
Although Sod1 –/– are fertile under in vivo conditions of fertilization (Ho et al. 1998), they show problems in fertilizing oocytes in vitro, producing lower levels of two-cell embryos compared to WT controls (Tsunoda et al. 2012). Interestingly, SOD1 knockout male mice show decreased fertility upon age (Selvaratnam & Robaire 2016).
Based on the activity of the antioxidant enzymes described above, we can recognize different lines of defence against oxidative stress, depending on which ROS is produced in the spermatozoon. First, SOD (mitochondrial and cytoplasmic isoforms) scavenges the O2·– generated in mitochondria and sperm membranes. Then, the H2O2 produced by this action or spontaneously is removed by GPXs and PRDXs, with a significant variation among mammalian species. However, if the O2·– and H2O2 levels are excessively high, GPXs and PRDXs become inactive, and strong oxidative stress occurs. An example is the NOX5 overexpression observed in asthenozoospermic men (Vatannejad et al. 2019).
An important line of defence is PRDX6, which can react with H2O2 at low concentrations in spermatozoa (O’Flaherty & de Souza 2011). This activity is important for both regulation of redox signalling during sperm capacitation (Lee et al. 2017, Moawad et al. 2017) and protecting mammalian spermatozoa against oxidative stress and maintaining fertility (Ozkosem et al. 2016, Moawad et al. 2017, Fernandez & O’Flaherty 2018).
The nuclear factor-erythroid 2-related factor 2 (Nrf2) is a crucial transcription factor in the cellular antioxidant defence since it promotes the expression of several antioxidant enzymes and allows the production of reduced GSH by increasing levels of enzymes involved in its production (Wild & Mulcahy 2000, Nguyen et al. 2003). Mutations in the Nrf2 promoter are associated with sperm DNA damage in infertile men (Aydos et al. 2021). Moreover, Nrf2 knockout males have reduced fertility with increased oxidative stress and sperm DNA damage (Nakamura et al. 2010).
As it occurs with other ROS, ONOO– has an impact on sperm quality and function. Low ONOO– levels promote sperm capacitation, but high levels are associated with low sperm motility, impairment of capacitation, and increased sperm DNA oxidation (Fernandez & O’Flaherty 2018). Tyrosine nitration is a consequence of the ONOO– reaction with proteins. This protein modification can either regulate sperm capacitation (de Lamirande & Lamothe 2009) or inactivate essential proteins altering sperm function (Vignini et al. 2006, Morielli & O’Flaherty 2015, Uribe et al. 2015, Fernandez & O’Flaherty 2018). Indeed, studies using the knock-in mouse strain C47S, lacking only the PRDX6 peroxidase activity, revealed higher tyrosine nitration levels in spermatozoa compared to WT controls and associated with subfertility in the mouse (Bumanlag et al. 2022). High levels of NOS and tyrosine nitration were observed in spermatozoa of infertile men (Salvolini et al. 2012). Peroxynitrite promotes lipid peroxidation (Radi et al. 1991), and the pharmacological inhibition of PRDX6 peroxidase activity leads to an increase of ONOO– and lipid peroxidation, which is triggered and enhanced by this free radical in human spermatozoa, suggesting the need of the PRDX6 peroxidase activity to regulate levels of ONOO– and prevent high levels of lipid peroxidation (Fernandez & O’Flaherty 2018). Altogether, these studies highlight the role of ONOO– in spermatozoa as a culprit of male infertility.
Antioxidant treatments for male infertility
Antioxidant therapy is a common clinical strategy to improve sperm quality and function in infertile men. The treatments include vitamins E (α-tocopherol) and C (ascorbic acid), carotenoids, carnitines, n -acetylcysteine, coenzyme Q, and micronutrients such as folate, selenium, zinc, and resveratrol. These compounds have been used alone or in combination to restore male fertility, and their properties and success or not to treat infertile men have been explained in many recent reviews (Henkel et al. 2019, Garolla et al. 2020, Agarwal et al. 2021, Amorini et al. 2021, de Ligny et al. 2022). Based on these review articles, the results obtained are controversial due to the small number of infertile patients, including the lack of proof for the existence of oxidative damage, different doses and combination used, and so on. Some small controlled trials suggested that such treatments are beneficial to achieving live births (de Ligny et al. 2022), whereas others did not benefit (Zini & Al-Hathal 2011). This discrepancy is likely based on the lack of tools in clinics to establish whether oxidative stress is responsible for infertility in a given patient. Currently, no assay can determine whether a semen sample contains fertile spermatozoa. The quest to find markers of male infertility is rendered difficult due to the multifactorial nature of this disease. Then, it becomes imperative to design novel diagnostic strategies to determine the presence of oxidative stress and what types of ROS are generated as a cause of infertility when evaluating a patient at the fertility clinic.
The pharmacological inhibition of PRDX6 peroxidase activity in human spermatozoa, the ablation of the PRDX6 gene, or its genetic mutation to promote the inactive form of the PRDX6 peroxidase (C47S knock-in mouse strain) demonstrated the production of high levels of ONOO–, lipid peroxidation, and tyrosine nitration and DNA oxidation associated with abnormal sperm quality and infertility (Ozkosem et al. 2016, Fernandez & O’Flaherty 2018, Bumanlag et al. 2022). We recently published a study using a special diet with a combination of tocopherol mixed with a high percentage of γ-tocopherol and ascorbic acid to treat infertility in Prdx6 –/– male mice (Scarlata et al. 2020). We found that males fed with this enriched γ-tocopherol/ascorbic acid diet restored fertility and normal sperm quality. Among tocopherol isoforms, γ-tocopherol is better than -tocopherol to scavenge ONOO– (Jiang et al. 2001, Galli et al. 2004). Our study highlights the need to identify the type of ROS associated with oxidative stress, a culprit of male infertility, before an attempt to restore fertility. Without this essential and logical diagnosis, we cannot prescribe an antioxidant treatment. Moreover, it can be useless to treat the infertile man if oxidative stress is not involved in developing the disease. Such a therapeutic approach may even cause detrimental effects since high levels of antioxidants can negatively alter sperm chromatin and disturb the delicate redox balance needed for the spermatozoon to achieve capacitation and be capable of fertilizing the oocyte (Ménézo et al. 2007, O’Flaherty 2015).
Conclusions
Male infertility is a multifactorial disease whose causes are often difficult to address. One of the major causes of unsuccessful oocyte fertilization is the oxidative damage of different structures or ROS-dependent inactivation of key enzymes of the spermatozoon. Although antioxidant therapy is part of the treatment of the infertile man, the proper diagnosis of the presence of oxidative stress as a cause of infertility is crucial for the success of this treatment. More research is necessary to understand better the molecular mechanisms that produce ROS and are affected by oxidative stress to set the basis of novel diagnostic tools and personalized treatment to help infertile men achieve fatherhood.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work was supported by a grant from the Canadian Institutes of Health Research (PJT165962) to C O.
Author contribution statement
Both authors contributed towards researching, writing, discussing, and editing this review.
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