Biology of Reproduction, 2017, 97(4), 577–585 doi:10.1093/biolre/iox104 Review Advance Access Publication Date: 13 September 2017

Review Reactive oxygen species and modifications in spermatozoa†

∗ Cristian O’Flaherty1,2,3, and David Matsushita-Fournier2,3 Downloaded from https://academic.oup.com/biolreprod/article/97/4/577/4157784 by guest on 01 October 2021

1Department of Surgery (Urology Division), McGill University, Montreal,´ Quebec,´ Canada; 2Pharmacology and Therapeutics, Faculty of Medicine, McGill University, Montreal,´ Quebec,´ Canada and 3The Research Institute, McGill University Health Centre, Montreal,´ Quebec,´ Canada

∗Correspondence: The Research Insitute, McGill University Health Centre, room EM03212, 1001 Decarie Boulevard, Montreal,´ QC H4A 3J1, Canada. Fax: +514-933-4149; E-mail: cristian.ofl[email protected]

†Grant support: This work was supported by a grant from the Canadian Institutes of Health Research (MOP 133661 to C.O.). CO is the recipient of the Chercher Boursier Junior 2 salary award from the Fonds de la Recherche en SanteduQu´ ebec´ (33158).

Received 2 June 2017; Revised 1 August 2017; Accepted 11 September 2017

Abstract Cellular response to reactive oxygen species (ROS) includes both reversible redox signaling and irreversible nonenzymatic reactions which depend on the nature and concentration of the ROS involved. Changes in thiol/disulfide pairs affect protein conformation, enzymatic activity, ligand binding, and protein–protein interactions. During spermatogenesis and epididymal maturation, there are ROS-dependent modifications of the sperm chromatin and flagellar .The sperma- tozoon is regulated by redox mechanisms to acquire fertilizing ability. For this purpose, controlled amounts of ROS are necessary to assure sperm activation (motility and capacitation). Modifica- tions of the thiol groups redox status of sperm proteins are needed for spermatozoon to achieve fertilizing ability. However, when ROS are produced at high concentrations, the established ox- idative stress promotes pathological changes affecting sperm function and leading to infertility. Sperm proteins are sensitive to high levels of ROS and suffer modifications that impact on motility, capacitation, and the ability of the spermatozoon to recognize and bind to the zona pellucida and damage of sperm DNA. Thiol oxidation, tyrosine nitration, and S-glutathionylation are highlighted in this review as significant redox-dependent protein modifications associated with impairment of sperm function and alteration of paternal genome leading to infertility. , the primary antioxidant protection in spermatozoa, are affected by most of the protein modifications described in this review. They play a significant role in both physiological and pathological processes in mammalian spermatozoa. Summary Sentence Reactive oxygen species promote redox-dependent protein modifications that lead to impairment of sperm function.

Key words: reactive oxygen species, oxidative stress, redox signaling, spermatozoa, sperm activation, sperm motility, sperm capacitation.

Introduction that includes such as superoxide dismutase (SOD), cata- lase (CAT), glutathione (GPXs), thioredoxins (TRXs), Reactive oxygen species (ROS) are obligatory metabolic products of and peroxiredoxins (PRDXs), and other molecules with scavenging aerobic cells. They are kept at low levels by an antioxidant system properties such as glutathione (GSH), ubiquinol, vitamins C and E,

C The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. 577 For permissions, please e-mail: [email protected] 578 C. O’Flaherty and D. Matsushita-Fournier, 2017, Vol. 97, No. 4

Ta b l e 1 . Redox-dependent protein modifications associated with physiological processes in spermatozoa.

Modification Physiological processes Species Reference

Thiol oxidation Binding of spermatozoon with oviductal epithelium Bovine [142,143] Sperm motility Hamster, human, rat [144–147] Sperm capacitation Human, bovine [120,123,143,148] Sperm chromatin remodeling Human, mouse, equine, rabbit, rat [36,37,39,149–151] S-Nitrosylation Sperm motility Human [76,152] Tyrosine nitration Sperm capacitation Human [83,153]

Ta b l e 2 . Redox-dependent protein modifications associated with deleterious effects in spermatozoa.

Modification Associated outcome Species Reference

Thiol oxidation Male infertility Human [38,39,42] Downloaded from https://academic.oup.com/biolreprod/article/97/4/577/4157784 by guest on 01 October 2021 Impairment of sperm motility Hamster, human, rat [42,154,155] Blockage of sperm-egg fusion Mouse [156,157] 4-HNE protein adducts Impairment of sperm motility Human, equine [62,158,159] S-Glutathionylation Impairment of sperm motility Human [34] Impairment of sperm capacitation Human [34] Tyrosine nitration Impairment of sperm motility Human [34,80] Impairment of sperm capacitation Human [34] Sulfonation Impairment of sperm motility Human, rat [42,60,154] Impairment of epididymal maturation Rat [154]

andsoon[1]. However, when the antioxidant system is dysregu- (Tables 1 and 2). Some of these modifications are reversible, thus lated, and the production of ROS is exacerbated, then these active allowing a tight regulation of cellular processes involved in redox molecules become harmful by-products of cellular metabolism [1]. signaling [29,30]. A schematic representation of the major redox- Mammalian spermatozoar are sensitive to ROS, such as the super-r protein modifications is shown in Figure 1. − oxide anion (O2r ), hydrogen peroxide (H2O2), nitric oxide (NO ), − hydroxyl (HO ), and peroxynitrite anion (ONOO ). When ROS Thiol oxidation levels are increased, sperm function is affected leading to infertil- Cysteine, a sulfur-containing amino acid, is a potent nucleophile un- ity [2–6]. This increase in ROS levels is denominated oxidative stress der physiological conditions. This remarkable reactivity is due to and is the result of an excessive production of ROS and/or a decrease the thiol (-SH) group. The formation of disulfide bridges (-SS-) by in the antioxidant defense system [7,8]. The oxidative damage tar- thiol oxidation is a common strategy to fold a protein generating gets all cell components, reducing sperm motility and mitochondrial a structure to assure, for instance, enzymatic activity or interaction activity [5,9,10]. The first evidence of a relationship between oxida- with receptors, plasma membrane components, etc. A specific ratio tive damage and male infertility was demonstrated by the pioneering -SS-/–SH within a protein molecule is essential to assure its function, work of Thaddeus Mann and Bayard Storey [11,12]. and this rate can be affected by ROS. An oxidative stress will oxidize The infertile population has been increasing over the past few free -SH, thus preventing the formation of -SS- where and when it decades [13]. However, treatment efficiency is poor because the is needed during a physiological process and will translate in the cause is unknown in 40%–50% of cases [14]. Several factors are impairment of protein function. A good example of this situation is related to infertility such as exposure to environmental pollutants, the effects of high levels of ROS on the ATP production by human chemicals, drugs, smoke, toxins, radiation, and even diseases [15– spermatozoa. It was observed that elevated levels of ROS, gener- 18]. A common feature of the above is the production of oxidative ated by direct addition of H2O2r or by adding xanthine-xanthine stress. In such conditions, vital cell components (proteins, lipids, and − oxidase system (generator of O2 and of H2O2) to the incubation DNA) are oxidized compromising cell function and survival [8,19]. medium, reduce human sperm motility in a dose- and time-dependent In at least 25% of cases, elevated levels of ROS are detected in both manner [31]. This reduction was correlated with a decrease in the semen and spermatozoa from infertile patients [2–5]. In some cases ATP production by the spermatozoon [32], thus suggesting that the of male infertility, the antioxidant system present in semen [20,21]is impairment of sperm motility was a depletion of energy. Human not sufficient to protect spermatozoa from ROS-dependent damages. spermatozoa depend on aerobic oxidation of glucose accomplish by Spermatozoa from infertile patients have peroxidation of membrane the conjunction of glycolysis, Krebs cycle, and the oxidative phos- lipids [22], DNA fragmentation and oxidation of bases [23,24], low phorylation by the electron transport chain. In 1992, de Lamirande mitochondrial membrane potential [25,26], and inactivation of en- and Gagnon [32] suggested that one possible target of ROS could zymes associated with motility [27,28]. be the glyceraldehyde 3-phosphate dehydrogenase, which is linked to the fiber sheath explaining the drop in ATP production observed Protein modifications in spermatozoa due to reactive in ROS-treated spermatozoa. Recently, it was demonstrated that the oxygen species glyceraldehyde 3-phosphate dehydrogenase, a key of the gly- Sperm proteins are the target of redox-dependent modifications that colytic pathway, can be inactivated by oxidation of the –SH in its will lead, depending on the levels of ROS, to either the activa- by exogenous H2O2 [33]. tion/inactivation of signaling pathways important for sperm phys- An appropriate level of thiol oxidation in proteins is necessary for iology or to oxidative damage and impairment of vital functions sperm motility [144–147]. However, the machinery that makes the ROS-dependent protein modifications in spermatozoa, 2017, Vol. 97, No. 4 579 Downloaded from https://academic.oup.com/biolreprod/article/97/4/577/4157784 by guest on 01 October 2021

Figure 1. Redox-dependent protein modifications. Depending on the type of ROS produced, different protein modifications are produced to either control cellular processes or, when these ROS are at high levels, promote damage by inactivation protein function (i.e. enzymatic activity, alteration of conformational structure, etc.). Hydrogen peroxide (H2O2) and other peroxides promote different protein modifications depending on the levels that they are present in the cell; mild oxidative stress will promote mostly thiol oxidation and S-glutathionylation. These redox-dependent modifications are easily reverted by antioxidant (non- and enzymatic compounds). A strong oxidative stress lead to sulfonation, a modification that is more difficult to revert (it requires an enzymatic reaction withr − energy consumption). 4-HNE forms protein adducts that irreversibly inactivates enzymes such as succinate dehydrogenaser in spermatozoa. Superoxide (O2 ) − − spontaneously or by superoxide dismutase activity dismutates to H2O2 or it could be combined with nitric oxide (NO ) to give ONOO . Nitric oxide and ONOO promote S-nitrosylation and tyrosine nitration that could participates in physiological and pathological processes. spermatozoon to move is very sensitive to ROS [42,154,155]; levels Peroxiredoxins are selenium-free enzymes with one (PRDX6) or that decrease motility significantly do not impair the viability of these two cysteines (PRDX1 to 5) in their active site that are highly reactive spermatozoa [34]. But most importantly, ROS alter motility differ- with H2O2 and other peroxides. They compose the primary antiox- ently; for instance, 100 μMH2O2 promotes a significant decrease idant system in ejaculated human spermatozoa since the amount of in motility and inhibits capacitation of human spermatozoa com- reduced GSH is insufficient (∼0.1 mM), the absence of CAT, and in- paredr to nontreated controls. However, 100 μM DA-NONOate, activation of mitochondrIal GPX4 as ROS scavenger [40,41]. They aNO donor, do not affect sperm motility and even stimulate ca- are considered essential elements of the antioxidant defense of sper- pacitation [34]. It is also important to highlight that 200–500 μM matozoa since infertile men have low amounts of PRDXs with high DA-NONOate inhibits sperm capacitation without impairing motil- levels of thiol oxidation. This modification promotes the inactiva- ity or viability [34]. Altogether, these findings indicate that there is tion of their enzymatic activity and thus generating an increase of a need to identify which specifics ROS are at high levels in infertile oxidative stress and DNA damage [42]. Mouse spermatozoa lacking men to better find treatment strategies to avoid their toxic effects. PRDX6 display low motility and high levels of lipid peroxidation (a Another possible target of ROS is tubulin, a structural protein marker of oxidative stress) and poor sperm quality. This poor sperm of the sperm flagellum. We found that increasing concentrations of quality is associated with subfertility which is exacerbated with age

H2O2 promoted an increase in thiol oxidation levels of α-tubulin [2, 3]. The absence of PRDX4 also leads to loss of spermatogenic cells in human spermatozoa [1]. Thiol oxidation of α-tubulin impaired and increase of apoptosis in the testis without a significant reduction microtubule polymerization and thus affecting the appropriate func- of fertility of the knockout males [43]. Although not measured, the tioning of the flagellum [35]. authors concluded that the spermatogenic cell loss and the increase Sperm chromatin remodeling is completed during epididymal in apoptosis are due to an oxidative stress generated by the absence maturation [36,37]. During this process, an appropriate balance of of PRDX4. thiol oxidation in protamines assures a healthy sperm chromatin Peroxiredoxins are highly sensitive to ROS, and an active reduc- structure. Both reduction and over oxidation of protamine thiols are tant system must be taken in place to maintain active their associated with male infertility [38,39]. activity [44]. In the case of 2-Cys PRDXs, this re-activation is done 580 C. O’Flaherty and D. Matsushita-Fournier, 2017, Vol. 97, No. 4 by the TRX/TRX reductase/NADPH system. The functional deletion with proteins and DNA and thus promoting its deleterious effects of thioredoxin domain-containing proteins Txndc2 and Txndc3 in observed in spermatozoa [62,63] that may include enzyme inacti- mouse spermatozoa correlated with an increase of age-related oxida- vation and mutagenesis [66,67]. It has been reported that acrolein tive stress [45]. The thiol oxidized PRDX6 cannot be reduced by the impairs the activity of the TRX/TRD system and PRDX [68]. TRXs and requires the presence of reduced GSH and glutathione- S- pi (GSTpi) [46]. Ascorbate can also reduce PRDX6 S-Glutathionylation as it was reported in yeast [47]. However, later studies using yeast Glutathione is a water-soluble tripeptide ubiquitously distributed in and mammalian somatic cells revealed that the GSH/GSTpi system tissues. It is the predominant nonprotein source of intracellular SH is the physiological mechanism to reduce PRDX6 [48–50]. Since the groups (∼1–10 mM in most of the mammalian cells) present in the GSH content is very low in spermatozoa [51], it is possible that cytosol (∼90% of the total GSH), mitochondria, endoplasmic reticu- ascorbate may be necessary to maintain the peroxidase activity of lum, and possibly in the nucleus [69,70]. Protein S-glutathionylation PRDX6. In mouse, we demonstrated that PRDX6 is important to occurs when GSH reacts with protein SH groups, often resulting in protect the paternal genome from oxidative damage [52,53]. The

enzyme inactivation [71–74]. This mechanism may appear detrimen- Downloaded from https://academic.oup.com/biolreprod/article/97/4/577/4157784 by guest on 01 October 2021 fact ascorbic acid protected human sperm DNA against oxidative tal for the cell, but it is protective because it prevents further protein damage [54] supports the hypothesis yet to be proven that PRDX6 oxidation and it is reversible [8,75]. may be reduced by ascorbate rather than by the GSH/GST system in Mammalian spermatozoa have a little amount of GSH (1–13 spermatozoa. nmoles GSH/109 spermatozoa) and particularly in humans is ap- Rat epididymal spermatozoa, collected after 24 h of the end of proximately 0.3 mM [51] compared to the 10 mM found in most treatment for 2 weeks with tert-BHP, a compound that generates an somatic cells [5]. Thus, the contribution of this antioxidant com- oxidative stress in vivo, showed increasing levels of thiol oxidation pound in the defense against oxidative stress is limited. This restric- of PRDX1 and PRDX6, the most abundant PRDXs in the rat sper- tion will impact on antioxidant enzymes, for instance, PRDXs that matozoa [4]. This treatment was meant to generate the oxidative require GSH for reduction of their thiol groups after ROS oxidized stress during the epididymal maturation, and this PRDXs oxidation them. This particular topic will be discussed later in this review. reflects the scavenging activity of these enzymes in an attempt to fight Human and mouse sperm proteins are subjected to S- against the oxidative stress caused. Moreover, the total amount of glutathionylation; we observed that mouse spermatozoa, lacking PRDX1, PRDX4, and PRDX6 increased in the treated spermatozoa, PRDX6, show higher levels of this modification compared to wild- suggesting an active transfer of these enzymes from the epididymal type controls [34]. Human spermatozoa treated with H O or tet- epithelium to the maturing spermatozoa [4]. This active transfer of 2 2 butyl hydroperoxide (tert-BHP) showed higher levels of glutathiony- antioxidant enzymes has also been reported for other proteins in- lated proteins than nontreated controls [34]. Although the increase cluding GPX5 and TRX [55–59]. of S-glutathionylation was an antioxidant response, it was not suf- Infertile men have a lower quantity of PRDXs in seminal plasma ficient as the oxidative stress generated either by lacking PRDX6 or and spermatozoa than healthy donors [42]. Sperm PRDX6 was low by exogenously increasing the levels of ROS severely affected sperm in 67% and 39% varicocele and idiopathic infertile patients, respec- motility. tively. Sperm PRDX1 was only low in 42% of varicocele patients The response of human spermatozoa to high levels of H O or [42]. In most of the cases of infertility, higher levels of thiol oxida- 2 2 tert-BHP depended on the concentration of each ROS; H O gener- tion of PRDX1 and PRDX6 were observed in sperm from these in- 2 2 ates S-glutathionylation at lower concentrations than tert-BHP, indi- fertile men. The thiol oxidation ratio (thiol oxidized PRDX/reduced cating a more reactive molecule capable of damaging the spermato- PRDX) negatively and positively correlated with sperm motility and zoon. Interestingly, this significant reduction in total and progressive DNA damage and lipid peroxidation, respectively [42]. Interestingly, motility was observed in viable spermatozoa [34]. This observation sperm levels of high molecular mass complexes of hyperoxidized highlights the differential effects of ROS on sperm functions. PRDX6 were greater in both infertile men groups than in donors and S-Glutathionylation was observed in the cytosolic and Triton the PRDX6 thiol oxidation ratio correlated positively with lipid per- X-100-insoluble fractions in human spermatozoa treated with high oxidation in spermatozoa [42]. These higher molecular mass com- concentration of H O [34]. Members of the human sperm antiox- plexes contain the sulfonated form of PRDXs (PRDX-SO ) a kind of 2 2 2 idant system may be inactivated by S-glutathionylation and there- redox-dependent modification that occurs when a strong oxidative fore trigger a strong oxidative stress associated with the impairment stress is established [60]. of sperm motility and capacitation. PRDX1, PRDX5, and PRDX6 Another significant modification of thiol groups by ROS is the are found in the Triton X-100-soluble fraction, and PRDX6 is also formation of protein adducts with electrophiles such as aldehyde found in the cytosolic and Triton X-100 soluble fractions [60]. We 4-hydroxynonenal (4-HNE) and acrolein, products of lipid peroxi- then can hypothesize that S-glutathionylation also contributes with dation [61]. Both electrophiles promoted an increase in mitochon- the inactivation of these PRDXs, explaining the high oxidative stress drial ROS production and reduction, DNA damage and reduction associated with poor sperm quality observed in infertile men [42]. of motility in spermatozoa [62,63]. The 4-HNE-dependent protein modification promotes the inactivation of succinate dehydrogenase and dynein heavy chain, both important proteins of the motility S-Nitrosylation andr tyrosine nitration − machinery of human spermatozoa. The modification of heat shock Nitric oxide (NO ) or its derivatives (e.g.r ONOO ) generate S- protein A2 by 4-HNE promoted its degradation by the proteasome nitrosylation in proteins. High levels of NO and ONOO− can mod- system in male germ cells [64]. The recent evidence that the inhibi- ify structural proteins and enzymes, thus altering cellular function. tion of arachidonate 15-lipoxygenase prevented the 4-HNE-induced However, low levels of these ROS are involved in redox signaling protein damage in male germ cells [65] supports the potential advan- [6, 7]. About 240 s-nitrosylated proteinsr were identified in human tage of pharmacological inhibition of this enzyme to protect germ spermatozoa treated with the NO donor nitrosocysteine [76]. En- cells from oxidative damage. Acrolein is capable of forming adducts zymes involved in energy production, motility, ion channels, and in ROS-dependent protein modifications in spermatozoa, 2017, Vol. 97, No. 4 581

antioxidant defense were identified as modified by s-nitrosylation, Growing evidence highlights the importance of H2O2 signaling indicating that this modification may be involved in redox regula- in cell physiology [98–100]. tion of sperm physiological processes. Of interest, PRDX1, GST, and The redox regulation is essential for sperm capacitation thioredoxin domain-containing protein 3 and 11 carried this modi- [101–104]. Low levels of ROS trigger early, intermediate, and late fication (for a complete list of s-nitrosylated proteins, see Lefievre` et phosphorylation events [105–109] that culminate with the increased al. [76]). r capacitation-associated Tyr phosphorylation [101,102]. , − Peroxiredoxins are susceptible to NO or ONOO attack; for GPXs, and PRDXs are recognized scavengers of H2O2, but PRDXs instance, S-nitrosylation impairs the ability of PRDX2 to reduce are more versatile with a dual action as antioxidant and as modula- peroxide, thus promoting neuronal cell oxidative stress [77] and, as tors of H2O2-dependent signaling [44,98,100,110–114]. This dual mentioned above, PRDX1 was identified as one of the S-nitrosylated action of PRDXs is necessary for mammalian spermatozoa because proteins due to nitroso-cysteine treatment in human spermatozoa these cells lack CAT (peroxisomes, containing the enzyme, are elimi- [76]. nated from germ cells during spermatogenesis [115,116], and GPX4 Tyrosiner (Tyr) nitration is also a protein modification promoted is a structural protein involved in the mitochondrial sheath with- by NO and ONOO−. A Tyr residue reacts with these ROS pro- out antioxidant capacity in the ejaculated spermatozoa [117]. Other Downloaded from https://academic.oup.com/biolreprod/article/97/4/577/4157784 by guest on 01 October 2021 ducing a nitro (-NO2) group. This modification will alter protein GPXs such as GPX2, 3, and 5 are not present in human spermato- function leading to either a physiological or pathological effect, de- zoa [118,119], and inhibition of either CAT or the GPX system did pending on the protein target and the level of ROS generated [78,79]. not increase the levels of lipid peroxidation in human spermatozoa It has been reported that spermatozoa from asthenozoospermic pa- [40]. Thus, the highly abundant and reactive PRDXs with different tients had high levels of Tyr nitration determined by immunocy- ROS become major players not only in the protection against ox- tochemistry [80]. Human spermatozoa treated with DA-NONOate idative damage but the regulation of the redox signaling in human show increased levels of Tyr nitration in a dose-dependent man- spermatozoa [40–42,60,101–103]. ner, a modification associated with the impairment of sperm motil- We observed differential subcellular localization of the six PRDX ity [34]. Tyr-nitrated proteins were located in the Triton X-100- isoforms in human spermatozoa and that PRDX1, PRDX4, PRDX5, insoluble fraction of human spermatozoa. Immunocytochemistry and PRDX6 react with different concentrations of H2O2 [60]. In- studies showed that these modified proteins were mainly found in terestingly, PRDX6 is highly abundant and the only member of the the flagellum in nonpermeabilized spermatozoa but also in the head family present in all the subcellular compartments of human sper- μ when the sperm cells were permeabilizedr with methanol [34,81]. matozoa and to react with H2O2 at levels (50 M) that promote Protein targets for NO and ONOO− that display Tyr nitra- CAP, as well as with other ROS [60]. tion and may account for impairment of sperm motility are enzymes The thiol oxidation is also associated with physiological func- belong to glycolysis (glyceraldehyde 3-P- dehydrogenase and eno- tions; in human spermatozoa, a temporal inhibition of PRDXs by lase) and the Krebs cycle (aconitase, α-ketoglutarate dehydrogenase, thiol oxidation allows the increase of ROS at levels that will not malate dehydrogenase, and dihydro lipoamide dehydrogenase) (see promote damage but will trigger the capacitation-associated phos- references in Morielli and O’Flaherty [34]). By inactivating these phorylation events such as phosphorylation of PKA substrates and enzymes by Tyr nitration, the production of ATP is severely dimin- Tyr residues [120]. The inhibition of 2-Cys PRDXs with thiostrep- ished leading to an impairment of sperm motility. α-tubulin also ton promoted the prevention of sperm capacitation and the estab- can be modified by Tyr nitration [82], interfering with appropriate lishment of oxidative stress. Thus, PRDX1–5 are important to as- microtubule polymerization in the sperm flagellum. sure the acquisition of fertilizing ability by the human spermatozoon Tyrosine nitration was found mostly in the Triton X-100- [120]. insoluble fraction [34], where PRDX1, PRDX5, and PRDX6 werer PRDX6 is the only member of the family with phospholipase found [60]. It is possible then that PRDXs are the target of NO or A2 activity which is important to remove lipid peroxides from ONOO− and be inactivated by Tyr nitration, leading to an increase the membranes [121,122]. When 1-Hexadecyl-3-(trifluoroethyl)-sn- in ROS levels that will impair sperm motility and capacitation. glycero-2-phospho-methanol lithium (MJ33) was present in the Tyrosine nitration is also associated with sperm capacitation. capacitation medium, spermatozoa not only were unable to un- Levels of Tyr nitration increased in spermatozoa exposed to dergo capacitation but also they displayed higher levels of lipid ONOO− in a dose-dependent manner but at concentrations that peroxidation compared to those capacitated without the inhibitor. do not affect sperm motility [34,83]. Ther prevention of the time- These results indicate that PRDX6 is dominant in the protection − dependent Tyr nitration by SOD (O2 scavenger) or L-NMMA of human spermatozoa against lipid peroxidation to assure nor- (inhibitor of nitric oxide synthase (NOS)) that prevent capacitation mal function as the other 2-Cys PRDXs were not inhibited by in spermatozoa under capacitating conditions strongly suggests that MJ33 [120]. ONOO− is endogenously produced during human sperm capacita- Human sperm capacitation is associated with rapid and reversible tion [84]. changes in protein -SH groups that appear to be redox regulated [123,124]. This shift in the redox status of thiol groups is a very dynamic mechanism of switching on or off protein activity. The Redox signaling during sperm capacitation increases or decreases in -SH content were prevented by exogenous Phosphorylation of proteins (e.g. enzymes, receptors and transcrip- addition of SOD or CAT to the capacitating medium [124]. Some tion factors) [85–92] and the redox regulation (which involves the sperm proteins, with molecular mass and isoelectric point similar to oxidation of a signaling molecule) [93–96] represent two primary those of PRDX1, PRDX4, and PRDX6 [125,126], are oxidized by mechanisms regulating cell function. In essence, they resemble on– H2O2 during capacitation. This evidence supports the findings that off switches for proteins [93,96]. Under physiological conditions, the thiol oxidation of PRDXs allows the redox signaling necessary to reversibility of these reactions is spontaneous or assured by reductive trigger phosphorylations events occurring during sperm capacitation pathways or are catalyzed by enzymes [93,97]. [105,107–109,127]. 582 C. O’Flaherty and D. Matsushita-Fournier, 2017, Vol. 97, No. 4

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