Redox signaling regulation in human spermatozoa: a primary role of peroxiredoxins

Abstract

Reactive oxygen species (ROS) play a dual role in mammalian spermatozoa. At high levels, they are detrimental to sperm function since they can promote oxidative stress that produces oxidation of protein, lipids, and sperm DNA. This oxidative damage is associated with male infertility. On the other hand, when ROS are produced at low levels, they participate in the redox signaling necessary for sperm capacitation. Capacitation-associated ROS are produced by the sperm oxidase, whose identity is still elusive, located in the plasma membrane of the spermatozoon. ROS, such as superoxide anion, hydrogen peroxide, nitric oxide, and peroxynitrite, activate protein kinases and inactivate protein phosphatases with the net increase of specific phosphorylation events. Peroxiredoxins (PRDXs), antioxidant enzymes that fight against oxidative stress, regulate redox signaling during capacitation. Among them, PRDX6, which possesses peroxidase and calcium-independent phospholipase A₂ (iPLA₂) activities, is the primary regulator of redox signaling and the antioxidant response in human spermatozoa. The lysophosphatidic acid signaling is essential to maintain sperm viability by activating the phosphatidylinositol 3-kinase/protein kinase (PI3K/AKT) pathway, and it is regulated by PRDX6 iPLA₂, protein kinase C (PKC), and receptor-type protein tyrosine kinase. The understanding of redox signaling is crucial to pave the way for novel diagnostic tools and treatments of male infertility.

INTRODUCTION

Infertility is on the rise; couples that struggle with infertility increased from 8% in the 1980s to 17.5% today worldwide.1,2,3 The underlying cause in half of these cases can be traced to men,1,2 and treatment efficacy remains poor because the causes are unknown in 30% of cases where men present normal semen analyses.4

The ejaculated spermatozoon cannot directly fertilize the egg. It must undergo a series of morphological and biochemical changes, collectively called sperm capacitation, occurring in the fallopian tubes or oviducts in the female genital tract.5,6 Sperm capacitation is an essential process for mammalian species and a critical selection process for preparing the best spermatozoon to reach and fertilize the egg.

During sperm capacitation, events occur in a timely fashion. One of the first events is the activation of the adenylyl cyclase/protein kinase A (PKA) signaling pathway that has been described in most of the species studied, including humans.7,8,9,10 Calcium uptake, increase of intracellular pH, cholesterol efflux, production of reactive oxygen species (ROS), and activation of phosphorylation pathways are other essential events necessary for capacitation.11,12,13,14,15,16,17

ROS ARE NECESSARY THROUGHOUT THE CAPACITATION PROCESS

In 1991, Bize et al.18 reported that the exogenous addition of hydrogen peroxide promoted sperm capacitation in hamster spermatozoa. This finding is the first indication that ROS can promote physiological processes in spermatozoa. Before, ROS were considered malicious molecules that affect sperm function and male fertility by inducing lipid peroxidation.19 Indeed, the toxic effect of ROS on spermatozoa was first reported by MacLeod20 in humans and Tosic and Walton21 in bulls. These toxic effects of ROS were then confirmed by others and in other species, suggesting a universal phenomenon of ROS-dependent damage of mammalian spermatozoa.22,23

The pioneering work of Claude Gagnon and Eve de Lamirande16 demonstrates the need for superoxide anion (O₂^•−), hydrogen peroxide (H₂O₂), nitric oxide (NO^•), and peroxynitrite (ONOO⁻) as signaling molecules necessary for human sperm capacitation.16,17 They demonstrated that O₂^•− and H₂O₂ are produced by a sperm oxidase at the level of the plasma membrane since the exogenous addition of superoxide dismutase (SOD) or catalase (CAT) prevented sperm capacitation induced by different biological fluids.24,25,26,27 In bull spermatozoa, heparin-induced capacitation was prevented by exogenous SOD but not by CAT, indicating that in the bovine species, O₂^•− is the primary ROS involved in capacitation.28,29 NO^• is also important to promote and sustain sperm capacitation as this ROS is produced during the entire duration of capacitation.16,17

ROS are capable of inducing cyclic adenosine monophosphate (cAMP) production by activating adenylyl cyclase, present in spermatozoa,30,31 and promoting the early phosphorylation of PKA substrates.10,32 The PKA pathway is necessary to activate later on tyrosine kinases and promote the protein tyrosine phosphorylation, a marker for sperm capacitation.7,8 Other phosphorylation events, such as the activation of the extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) pathways, are also promoted and regulated by ROS. NO^• is produced by nitric oxide synthase (NOS) that is activated by the PI3K/AKT pathway during capacitation. Then, NO^• regulates the ERK pathway by interacting with Ras,33,34 and H₂O₂, produced by the sperm oxidase, activates PKC.35,36,37,38,39 Interestingly, these pathways and the PKA and protein tyrosine kinase (PTK) pathways are not involved in regulating ROS production during capacitation.24,35,40

The regulation of phosphorylations during sperm capacitation is complex, and crosstalk among major phosphorylation events that are timely activated has been described in the human spermatozoon (Figure 1).32,36,39 It is plausible to consider that these phosphorylation events occur because of the inactivation of protein phosphatases. Indeed, calyculin A, an inhibitor of protein phosphatases, promotes human sperm capacitation.41 It was reported that serine/threonine protein phosphatase inactivation is necessary for human sperm capacitation to sustain the increased levels of phosphorylation observed during this process.42 However, how phosphatases are inactivated and thus allow the promotion of capacitation is not well understood.43

Figure 1.

Reactive oxygen species activate and regulate phosphorylation events during human sperm capacitation. ROS, such as O₂^•−, H₂O_2, and NO^•, generated by the sperm oxidase at the level of the plasma membrane, activate PKA, PKC, ERK, and PI3K pathways involved in human sperm capacitation. ROS also inhibit protein phosphatases (e.g., PP1 and PP2A), ERK protein phosphatases (e.g., MKP-3), and PTP to ensure high levels of phosphorylation in the capacitating spermatozoa. There is a crosstalk among these phosphorylation pathways through phosphorylated substrates but also by the generation of NO^•, as it is in the case of the PI3K/AKT pathway that activates NOS, a substrate of AKT. Nitric oxide then reacts with Ras, activating the ERK pathway known to be essential for human sperm capacitation. ROS: reactive oxygen specie; PKA: protein kinase A; p-PKA substrates: phosphorylated PKA substrates; PP1: protein phosphatase 1; PP2A: protein phosphatase A2; H₂O₂: hydrogen peroxide; NO^•: nitric oxide; O₂^•−: superoxide anion; Shc: Src homology and collagen protein; Grb2: growth factor receptor-bound protein 2; Sos: guanine nucleotide exchange factor son of sevenless; Ras: rat sarcoma; Raf: rapidly accelerated fibrosarcoma; MEK: mitogen-activated kinase kinase; ERK: extracellular-regulated kinase; p-ERK substrates: phosphorylated ERK substrates; MKP-3: mitogen-activated protein phosphatase 3; PDK1: 3-phosphoinositide-dependent protein kinase 1; p-PI3K: phosphorylated phosphatidylinositol 3-kinase; p-AKT: phosphorylated serine/threonine kinase 1; NOS: nitric oxide synthase; NO^•: nitric oxide; PKC: protein kinase C; p-MEK-like proteins: phosphorylated mitogen-activated protein kinase-like proteins; PTK: protein tyrosine kinase; PTP: protein tyrosine phosphatase; Tyr: tyrosine; p-Tyr: phosphorylated tyrosine. Created with Biorender.com.

ROS are also proposed to inactivate phosphatases to ensure that the human spermatozoon carries on the phosphorylation events to achieve capacitation.35 The regulation of the well-known tyrosine phosphorylation events associated with sperm capacitation is the activation of protein tyrosine kinases and/or the inactivation of protein tyrosine phosphatases (PTP). Hydrogen peroxide reacts with the Cys118 and Cys215 residues present in the active site of PTP1B, forming a disulfide bridge and promoting the inactivation of the enzyme.44,45 ROS also oxidize serine/threonine protein phosphatases, and therefore, they become inactivated.46,47 Other phosphatases need to be inactivated to allow the completion of sperm capacitation; for instance, ERK phosphatases such as the mitogen-activated protein phosphatase 3 (MKP-3) can be oxidized and inactivated by ROS, as seen in neurons.48 Although ROS are known regulators of phosphatase activity in other cells, their role in sperm capacitation is still to be revealed.

SOURCES OF ROS TO SUSTAIN SPERM CAPACITATION

During sperm capacitation, there is a rise in ROS levels and lipid peroxidation in healthy spermatozoa that will be capable of fertilizing oocytes.29,40,49,50,51 The production of ROS starts at the early stages of the capacitation process and lasts for hours as measured in in vitro conditions. The sources of ROS that participate in the capacitation of mammalian spermatozoa are not elucidated. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) isoforms are a group of enzymes that produce O₂^•− and H₂O₂ in different cell types and are recognized as pivotal players in redox signaling.52 In human spermatozoa, NOX1, NOX2, and NOX4 are not present,53 and apparently, the only member present is NOX5, which it is also present in equine but not mouse spermatozoa.53 NOX5 is located in the sperm midpiece and flagellum,53,54 activated by calcium, and has been associated with motility by activating H_v1 voltage-regulated proton channel.53 In infertile men, it was reported that an overexpression of NOX5 was associated with oxidative stress.55 The role of NOX5 as the source of O₂^•− and H₂O₂ in sperm capacitation is questionable as the effects described to stimulate hyperactivation in human spermatozoa were done with short incubation times (approximately 10 min)53 which are insufficient to promote the physiological processes involved in sperm capacitation.

The identity of the O₂^•− and H₂O₂ source during sperm capacitation is still elusive. It has been demonstrated that the O₂^•− is an early event during capacitation that occurs at the level of the sperm plasma membrane, as was shown in humans and bulls.25,28,29 One of the targets of O₂^•− is the adenylyl cyclase31,56 that produces cAMP to activate the PKA pathway, an early event during sperm capacitation.32 Another important source of ROS in spermatozoa is NOS, located in different spermatozoa compartments.57,58 This differential location suggests an important role of NO^• in these parts of the cell. NO^• can also activate the adenylyl cyclase.30

The incubation of bull or human spermatozoa with NADPH promotes capacitation,29,59 and, this treatment is not associated with O₂^•− but NO^•.40 This phenomenon can be explained by the fact that NADPH is a substrate for the sperm NOS. These findings and the absence of NOX isoforms in many species (e.g., NOX5 is absent in mouse spermatozoa, and NOX1, NOX2, and NOX4 are not present in human spermatozoa) reinforce the fact that the sperm oxidase is not a member of the NOX family.

Moreover, NO^• production is complex and regulated at least by PKC, PTK, ERK, PI3K, AKT,51 and citrate metabolism,60 whereas that of O₂^•− seems to occur upstream of most of the cascades.51 It is important to highlight the novel role of citrate as a substrate needed to provide NO^• for human sperm capacitation. Citrate is abundant in seminal plasma61 and present at low levels in the seminal plasma of infertile men with obstructive azoospermia, oligoasthenozoospermia, and oligoasthenoteratozoospermia.62,63,64 Citrate is a nonglycolytic energy source capable of supporting sperm capacitation in mouse and human spermatozoa.65 Moreover, it was recently demonstrated that citrate participates in generating NO^• through a series of biochemical reactions involving the ATP-citrate lyase (ACLY) and malic enzyme60 (Figure 2). Human spermatozoa contain mitochondrial citrate carrier,66 and its inhibition prevented capacitation (measured by levels of tyrosine phosphorylation and acrosome reaction), thus suggesting that human spermatozoa utilize extracellular citrate or the one generated by the Krebs cycle in the mitochondrion for NO^• production during sperm capacitation.60

Figure 2.

Citrate metabolism during human sperm capacitation. The citrate provided by seminal plasma (prostate origin) can enter the human spermatozoon through the SLC13A5 sodium-citrate cotransporter. In the cytosol, it is converted into acetyl-CoA and oxaloacetate by the ACLY. The mitochondrial citrate can also be exported to the cytosol through the CIC located in the inner mitochondrial membrane, which has the same fate as the citrate from the seminal plasma. Then, the oxaloacetate is converted into malate by the MDH and to pyruvate and NADPH by the ME and can enter into the mitochondria through the MPC. The NAPDH is then utilized by the NOS to generate the NO^• necessary for human sperm capacitation. SLC13A5: solute carrier family 13 member 5; acetyl-CoA: acetyl coenzyme A; ACLY: adenosine triphosphate citrate lyase; CIC: mitochondrial citrate carrier; MDH: malate dehydrogenase; ME: malic enzyme: NADP⁺: oxidized nicotinamide adenine dinucleotide phosphate; NADPH: reduced nicotinamide adenine dinucleotide phosphate; MPC: mitochondrial pyruvate carrier; NOS: nitric oxide synthase; NO^•: nitric oxide. Created with Biorender.com.

Human spermatozoa contain citrate provided by the seminal plasma (provided by the prostate) or by exiting the mitochondria through the mitochondrial citrate carrier (CIC) to the cytosol. The ACLY converts the cytosolic citrate into acetyl-coenzyme A (acetyl-CoA) and oxaloacetate. Then, oxaloacetate is converted by the malate dehydrogenase into malate, which is further converted into pyruvate by the malic enzyme generating NADPH. The NAPDH is used by NOS to generate NO^• for sperm capacitation. The pyruvate is then recycled by entering into mitochondria through the mitochondrial pyruvate carrier (MPC) to provide energy for spermatozoa.

Based on in vitro studies, the participation of ROS during sperm capacitation varies according to the species in question. O₂^•− is needed during the first 30 min of capacitation.25,28,29 H₂O₂ and NO^• are also produced at the beginning of incubation under capacitating conditions, with high levels lasting for hours, then decrease slowly but steadily until the end of capacitation.16,17 Tyrosine nitration, a posttranslational protein modification induced by peroxynitrite (ONOO⁻), increases steadily during the first 2 h, reaches a maximum of 3 h and slowly decreases.51 This phosphorylation event supports the need for O₂^•− and NO^•, which is necessary to form ONOO⁻ at later stages of capacitation.

The premature ROS production by spermatozoa is prevented by semenogelin and zinc, two decapacitation factors that prevent O₂^•− production and therefore, capacitation.67,68 Once the sperm oxidase is active, this enzyme is no longer inhibited from producing ROS. Thus, the only way to avoid toxic effects due to oxidative stress is to control the intracellular ROS levels to ensure capacitation. Thankfully, the spermatozoon is equipped with all members of the peroxiredoxin family responsible for controlling ROS levels and avoiding oxidative damage.69

PEROXIREDOXINS (PRDXS) PLAY A DUAL ROLE OF PROTECTING SPERMATOZOA AGAINST OXIDATIVE STRESS AND REGULATING SPERM CAPACITATION

The spermatozoon is highly sensitive to high levels of ROS or oxidative stress. Its inability to fight oxidative stress effectively is due to the limited antioxidant system and the particularities of some of its components. The sperm cytoplasm is very scarce; thus, cytoplasmic antioxidant enzymes such as SOD1 are present in low amounts.70 There are limitations to fighting against high levels of H₂O₂ in spermatozoa because catalase is absent in the ejaculated spermatozoa. During spermiogenesis, catalase is removed when peroxisomes and other organelles leave the spermatids in the residual body.71 Another candidate to remove H₂O₂ is glutathione peroxidases (GPXs); however, the participation of GPX isoforms in the antioxidant response is limited to the nucleus through the nuclear GPX4 because the mitochondrial GPX4 is part of the mitochondrial sheath and inactive as an antioxidant.72,73,74

The defense against H₂O₂, organic hydroperoxides, and ONOO⁻ is accomplished by the PRDXs family.75 All six isoforms are present and differentially located in subcellular compartments of human spermatozoa, and all are active as antioxidants.76,77 They are also expressed in the testis and epididymis of human and mouse.78,79,80 All PRDXs can remove ROS, but PRDX6 stands alone within the family since it is a moonlight enzyme with three enzymatic activities (peroxidase, calcium-independent phospholipase A₂ [iPLA₂], and acyltransferase activities) necessary to fight against high levels of ROS and lipid peroxidation. Although the 2-Cys PRDXs (PRDX1–5) can scavenge ROS as PRDX6,81 the latter is more efficient and essential for male fertility since its absence promotes infertility in the knockout males,82 and this abnormal reproductive phenotype worsens with aging.83,84

PRDX4 is another member of the family that is important to control oxidative stress in mouse testis to allow spermatogenesis to proceed. Indeed, Prdx4^−/− male mice showed abnormal spermatogenesis with elevated germ cell death and high levels of oxidative stress as the male age.85 Noteworthy, Prdx4^−/− males have normal in vivo and in vitro fertility, suggesting that PRDX4 is not essential in supporting male fertility.85 In boars, the levels of PRDX4 have been correlated with litter size, suggesting the use of this isoform as a biomarker of fertility.86

The scavenging properties of PRDX6 are based on its peroxidase activity (Figure 3); using a knock-in mouse model lacking PRDX6 peroxidase activity generated using an amino acid substitution to replace the active cysteine in 47 position to a serine (C47S), we found that C47S males were infertile having spermatozoa displaying low motility and high levels of protein tyrosine nitration and DNA damage.87 The oxidative damage observed in C47S knock-in males is due to an inability to remove ONOO⁻.88,89

Figure 3.

The impairment of PRDX6 peroxidase activity leads to increased levels of oxidative stress in human spermatozoa. (1) PRDX6 reacts with ROS (e.g., H₂O₂, ONOO^-, and organic hydroperoxide) and become inactive. (2) The oxidized PRDX6 can then be reactivated to its reduced state by GSTPi utilizing reduced glutathione in this reaction (GSH). (3) Further oxidation promotes PRDX6-SO₂, which irreversibly inactivates the enzyme and leads to the formation of high molecular mass complexes. (4) The inactivation of GSTIPi with ezatiostat or the removal of GSH by ethacrynic acid or the irreversible inactivation of the PRDX6 activity promotes the increase of mito O₂^•−, ONOO⁻ and the reduction of mitochondrial membrane potential potential. PRDX6: peroxiredoxin 6; ROS: reactive oxygen species; H₂O₂: hydrogen peroxide; ONOO⁻: peroxynitrite; PRDX6-SH: reduced peroxiredoxin; PRDX6-SOH: oxidized peroxiredoxin; PRDX6-SO₂: sulfonated peroxiredoxin 6; GSTPi: glutathione-S-transferase isoform Pi; GSH: reduced glutathione; mito O₂^•−: mitochondrial superoxide anion; MMP: mitochondrial membrane potential. Created with Biorender.com.

We demonstrated that the PRDX6 is essential to remove ONOO⁻ in human spermatozoa.82 The pharmacological inhibition of glutathione-S-transferase Pi (GSTpi) and the elimination of reduced glutathione (GSH) promotes the inactivation of PRDX6 peroxidase activity, leading to an increase in mitochondrial superoxide anion (mito O₂^•−) and ONOO⁻ and the reduction of mitochondrial membrane potential82 (Figure 3). The further oxidation of PRDX6, due to strong oxidative stress, promotes the hyperoxidation of the enzyme, which leads to its irreversible inactivation (Figure 3).

Lipid peroxidation is one of the most common oxidative damages with a high negative impact on sperm motility.90,91 The PRDX6 iPLA₂ activity is essential to protect membranes from lipid peroxidation through this and the acyltransferase activities. PRDX6 repairs oxidized membranes by removing the oxidized phospholipid and replacing it with a reduced one.92 Using the D140A mouse strain, lacking only the PRDX6 iPLA₂ activity due to an amino acid substitution of aspartate in position 140 to alanine (D140A), we discovered that this enzymatic activity is also essential for male fertility to prevent lipid peroxidation and DNA damage in spermatozoa.87 It is known that lipid peroxidation products such as 4-hydroxynonenal (4-HNE), measured in our study, can form DNA adducts generating mutations,93 that can be present in the paternal genome and contribute to infertility.

When human spermatozoa were treated with MJ33, a lipid analog that inhibits the PRDX6 iPLA₂ activity, we observed an increase in mitochondrial O₂^•−, hydroxyl radical and 4-HNE, and a reduction of mitochondrial membrane potential.82 These findings highlight the primary role of PRDX6 in repairing the oxidized sperm plasma membrane (Figure 4). Therefore, the PRDX6 activities (peroxidase and iPLA₂) are essential to protecting human and mouse spermatozoa and probably other mammal species.

Figure 4.

Peroxiredoxin 6 calcium-independent phospholipase A₂ activity is essential to fight against oxidative stress to protect sperm DNA. (1) High levels of H₂O₂ (or O₂^•−) and Fe²⁺ oxidize the sn-2 unsaturated fatty acids in phospholipids (e.g., phosphatidylcholine), promoting lipid peroxidation of sperm membranes. (2) PRDX6 calcium-independent phospholipase A₂ activity hydrolyzes the phospholipid hydroperoxide, generating lysophospholipid and FAOOH. (3) The lysophospholipid is deacylated with a reduced acyl-CoA by the PRDX6 LPCAT to regenerate the reduced phospholipid. The inhibition of PRDX6 iPLA₂ activity by MJ33 generates oxidative stress by increasing mito O₂^•−, of HO^•, 4-HNE (a subproduct of lipid peroxidation) and sperm DNA oxidation, and a decrease of mitochondrial membrane potential. H₂O₂: hydrogen peroxide; Fe²⁺: iron ion; MJ33: 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol lithium; PRDX6-iPLA₂: peroxiredoxin 6 calcium-independent phospholipase A₂ activity; FAOOH: fatty acid hydroperoxide; PRDX6 LPCAT: PRDX6 lysophospholipid acyltransferase activity; FA:CoA: acyl-coenzyme A; mito O₂^•−: mitochondrial superoxide anion; MMP: mitochondrial membrane potential; HO^•: hydroxyl radical; 4-HNE: 4-hydroxynonenal; sperm DNA oxidation: sperm deoxyribonucleic acid oxidation. Created with Biorender.com.

PRDXs are the major protectors of spermatozoa against oxidative damage and regulate the redox signaling necessary for sperm capacitation. ROS can trigger and regulate the molecular mechanisms the spermatozoon needs to acquire fertilizing ability. Thus, a regulatory mechanism to prevent oxidative damage during capacitation was sought. Semenogelin and zinc are components of the seminal plasma recognized as decapacitation factors.67,68,94 Although removing these molecules from ejaculated spermatozoa allows the cell to produce ROS upon stimulation with capacitation inducers,67,68,95 the regulation of ROS levels during the entire duration of capacitation was unknown until recently.

The seminal plasma contains significant amounts of antioxidant enzymes, including glutathione peroxidases, catalase, SOD, and PRDXs, to protect spermatozoa against oxidative stress.96 Low GPX-like and catalase-like activity was found in infertile men, whereas no changes in SOD-like activity were detected.97,98 PRDX2 and PRDX5 were found in large amounts in the seminal plasma of asthenozoospermic men,99 indicating increased levels of antioxidant enzymes to fight against oxidative stress. These could be due to the epididymal epithelium’s active production of epididymosomes containing these enzymes.100 It is known that human and bovine epididymosomes contain PRDXs and other antioxidant enzymes such as thioredoxin (TRX) and GPX5.100,101,102

The pharmacological inhibition of 2-Cys PRDXs by directly inhibiting with thiostrepton A, and the inhibition of the PRDX6 iPLA₂ with MJ33, prevented capacitation in human spermatozoa without altering their viability.50 This inhibition was associated with an increased lipid peroxidation, an indication of the ongoing oxidative stress due to the inhibition of PRDX activities. Interestingly, capacitation was prevented even when the PRDX inhibitors were added 15 min, 30 min, 60 min, or 120 min after the beginning of the incubation with the capacitation inducer, indicating the need for PRDXs to control the levels of ROS during the entire process of sperm capacitation.50 Recently, the role of PRDXs in regulating the redox signaling during bovine sperm capacitation has been suggested since the inhibition of PRDXs increased S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and S-nitrosylation of glutathione-S-transferase omega 2 (GSTO2) and SOD2.103 This posttranslational modification promotes a reduction of energy supply and antioxidant protection. SOD2 is important to control the levels of O₂^•− in the sperm mitochondria, and polymorphisms of SOD2 were found in infertile men.104 GSTO2, found in the perinuclear theca of eutherian spermatozoa,105 plays an oxidative/reductive role in regulating sperm function during sperm capacitation.106 The inhibition of GSTO2 in mouse spermatozoa promoted increased lipid peroxidation in these cells and significant reduction of their in vitro fertilization potential.106

In bull spermatozoa, the pharmacological inhibition of 2-Cys PRDXs promoted oxidation of glycolytic enzymes, including GAPDH, pyruvate kinase complex, alpha-enolase, and the lactate dehydrogenase A. These enzymes are needed for re-oxidation of NADH to ensure the continuation of the glycolysis metabolic pathway.107

PRDX6 IS ESSENTIAL FOR THE MAINTENANCE OF SPERM VIABILITY AND DNA INTEGRITY

Maintaining cell viability is crucial for spermatozoa to face the different environments they are exposed to since ejaculation.108 Changes in pH and metabolites present in the male and female reproductive fluids impact the spermatozoon’s performance as they reach and traverse the female reproductive tract. Ultimately, spermatozoa need to colonize the crypts of the epithelial oviduct to be capacitated. Finally, the capacitated spermatozoon will fertilize the oocyte to deliver the paternal genome.109,110

The lysophosphatidic acid (LPA) signaling is essential in maintaining germ cell viability during mouse spermatogenesis. However, the role of this lipid in human spermatozoa was largely unknown. We discovered that the exogenous addition of LPA prevented the impairment of sperm viability observed when human spermatozoa were treated with MJ33,111 the specific inhibitor of PRDX6 iPLA₂ activity.82 The inhibition of PRDX6 iPLA₂ activity prevented the phosphorylation of the PI3K/AKT pathway, known to maintain sperm viability and DNA integrity in human spermatozoa.112 The LPA is a product of PRDX6 iPLA₂ activity that prevented apoptosis in fibroblasts and increased phospho-PI3K levels, leading to an increase in the proliferation of lung cancer cells. We observed that LPA prevented the increase in lipid peroxidation and Annexin V positive cells, and restored sperm viability due to inhibition of PRDX6 iPLA₂ activity.111 Based on these findings, we can conclude that LPA, produced by PRDX6 iPLA₂ activity, is essential in maintaining sperm viability and DNA integrity in humans.82,111 Notably, immunocytochemistry studies revealed that PRDX6 colocalizes in areas of the sperm nucleus with DNA oxidation.113

We recently characterized the LPA signaling in human spermatozoa (Figure 5).114 LPA receptors (LPARs) 1, 3, 5, and 6 are G protein-coupled receptors that are located on the sperm plasma membrane and involved in the maintenance of sperm viability since spermatozoa treated with Ki16425, an inhibitor of LPAR1, LPAR2 and LPAR3, promoted impairment of cell viability in human spermatozoa. It was observed that male mice lacking LPAR1, LPAR2 and LPAR3, have impaired spermatogenesis with germ cell loss.115 The LPARs exert a compensatory mechanism since the knockout animals for the individuals LPAR show a minimal reduction in sperm production.115 The LPA signaling regulates the PI3K/AKT pathway and depends on PLC, PKC, and receptor-type tyrosine kinase.

Figure 5.

Lysophosphatidic acid signaling is regulated by PRDX6 and kinases to maintain human sperm viability. Lysophosphatidic acid, produced by the PRDX6-iPLA₂, binds to the G protein-coupled receptors LPAR1, 3, 5, or 6, present in the human sperm plasma membrane. This interaction activates phospholipase C and produces diacylglycerol, a second messenger and activator of PKC, to activate the PI3K/AKT pathway. As a result, AKT substrates are phosphorylated, which prevents apoptotic-like changes (e.g., oxidative stress and DNA damage) to maintain sperm viability. The LPA signaling is also regulated by receptor-type protein tyrosine kinase, leading to the phosphorylation of PI3K. LPA: lysophosphatidic acid; LPAR: lysophosphatidic acid receptor; PRDX6-iPLA₂: peroxiredoxin 6 calcium-independent phospholipase A₂ activity; PLC: phospholipase C; DAG: diacylglycerol; PKC: protein kinase C: PI3K: phosphatidylinositol 3-kinase; AKT: serine/threonine kinase 1; p-AKT substrates: phosphorylated serine/threonine kinase 1 substrates; RT-PTK: receptor-type protein tyrosine kinase. Created with Biorender.com.

EFFECTS OF EXPOSURE TO ENVIRONMENTAL FACTORS AND PEROXIREDOXIN EXPRESSION AND CONSEQUENCES FOR MALE FERTILITY

It is known the relationships between exposure to environmental factors (e.g., smoking, plasticizers, pesticides, pollution, chemotherapy, etc.) and oxidative stress and their negative impact on male fertility.116 The maternal exposure to di-n-butyl phthalate promoted a differential expression of PRDX6 in rat fetus’s testis,117,118 that could explain the testicular dysgenesis observed triggered by phthalates.119 PRDXs play a protective role against oxidative stress in gonocytes,120 the fetal precursors of spermatogonia in the fetal testis.121 Exposure of GC-2 cells (a germ cell line) to mono-(2-ethyl)phthalate increased mitochondrial PRDX3 expression.122

Radiation is often used to treat cancer patients. However, they impact reproductive organs and may lead to infertility. These treatments promote significant oxidative stress as part of their mechanism of action to kill cancer cells. The exposure to ionizing radiation promoted overexpression of PRDX1 and PRDX2 in mouse testes,123 which explains in part the radiation resistance of Sertoli and Leydig cells.124,125

FUTURE PERSPECTIVES FOR TREATMENTS OF MALE INFERTILITY

Currently, diagnosis of the presence of oxidative stress in the semen of infertile men is not performed at fertility clinics. The lack of such a test is due to the complexity of the development of oxidative stress (levels, sources of ROS acting, etc.) and the difficulties in translating current methods to be accessible for the clinic. Efforts in diagnosis and treatment have been made. Treatment of spermatozoa from asthenozoospermic patients with transactivating transduction PRDX2 (TAT-PRDX2) fusion protein increases their motility and reduces DNA damage after 12 h of incubation in vitro.126 Knowing that PRDXs are in low quantities in idiopathic infertile patients,127,128 the supplementation with recombinant PRDXs may increase sperm quality and functions to improve assisted reproductive technology outcomes to treat male infertility.

CONCLUSIONS

The redox signaling is essential to ensure sperm function. Reactive oxygen species are produced at low levels in human spermatozoa to promote and regulate sperm capacitation. PRDXs, particularly PRDX6, are antioxidant enzymes that protect mammalian spermatozoa against oxidative stress and regulate ROS, maintaining them at physiological levels to ensure sperm capacitation. Dysregulation of the PRDX system promotes the establishment of oxidative stress that is associated with male infertility.127 A deep knowledge of redox signaling during sperm capacitation, motility, and maintenance of viability will help develop novel diagnostic tools and treatments for male infertility.

COMPETING INTERESTS

The author declares no competing interests.