Sperm redox biology challenges the role of antioxidants as a treatment for male factor infertility

Abstract

In spite of inconclusive or negative outcomes from clinical studies, oral antioxidants are still largely prescribed to infertile men to improve sperm motility and/or reduce sperm DNA damage, on the basis of the assumption that it is an oxidative damage and it will be corrected by antioxidants. We aimed to challenge this view by examining the available experimental evidence. The regulation of sperm motility may suffer several pathologic derangements, including alterations of the flagellum, impaired function of the activating phosphatases and kinases, impaired function of the extracellular vesicles of either epididymal or prostatic origin, deranged Ca2+ trafficking, and infection/inflammation of the male accessory glands. None of the aforementioned issues seem to be directly dependent on the redox balance and to indicate a direct role for oral antioxidants treatment. Indeed, antioxidants may generate reductive imbalances resulting in an increase in the nicotinamide adenine dinucleotide reduced/nicotinamide adenine dinucleotide oxidized ratio, which sustains reactive oxygen species generation in mitochondria, potentially leading to increased sperm DNA damage, whereas a shortage of nicotinamide adenine dinucleotide oxidized may jeopardize the pol(ADP-ribose) polymerase-based DNA repair mechanisms at the time of histone to protamine transition, resulting in unresolved double-strand breaks and defective protamination, which further increases DNA vulnerability. The occurrence of reactive oxygen species and oxidative damages does not necessarily imply a shortage of antioxidant defenses, and the possibility that a different problem is in place should be considered. On this base, the current attitude to prescribe oral antioxidants to infertile men without demonstration of antioxidant shortage or true oxidative imbalance should be reconsidered. In spite of inconclusive or negative outcomes from clinical studies, oral antioxidants are still largely prescribed to infertile men to improve sperm motility and/or reduce sperm DNA damage, on the basis of the assumption that it is an oxidative damage and it will be corrected by antioxidants. We aimed to challenge this view by examining the available experimental evidence. The regulation of sperm motility may suffer several pathologic derangements, including alterations of the flagellum, impaired function of the activating phosphatases and kinases, impaired function of the extracellular vesicles of either epididymal or prostatic origin, deranged Ca2+ trafficking, and infection/inflammation of the male accessory glands. None of the aforementioned issues seem to be directly dependent on the redox balance and to indicate a direct role for oral antioxidants treatment. Indeed, antioxidants may generate reductive imbalances resulting in an increase in the nicotinamide adenine dinucleotide reduced/nicotinamide adenine dinucleotide oxidized ratio, which sustains reactive oxygen species generation in mitochondria, potentially leading to increased sperm DNA damage, whereas a shortage of nicotinamide adenine dinucleotide oxidized may jeopardize the pol(ADP-ribose) polymerase-based DNA repair mechanisms at the time of histone to protamine transition, resulting in unresolved double-strand breaks and defective protamination, which further increases DNA vulnerability. The occurrence of reactive oxygen species and oxidative damages does not necessarily imply a shortage of antioxidant defenses, and the possibility that a different problem is in place should be considered. On this base, the current attitude to prescribe oral antioxidants to infertile men without demonstration of antioxidant shortage or true oxidative imbalance should be reconsidered. DIALOG: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/xfnr-d-21-00045Essential points•Mechanisms regulating sperm motility do not involve the redox balance, and the role of mitochondria in sperm bioenergetics is controversial.•Reactive oxygen species are generated in mitochondria in conditions of reductive rather than of oxidative stress.•Double-strand breaks in sperm DNA are not correlated with oxidative damages but are associated with defective histone to protamine transition.•Reductive stress may impair DNA repair from poly-ADP-ribose polymerase, resulting in both double-strand breaks and impaired protamine transition.•Metabolic perturbances from inappropriate antioxidant treatments may jeopardize men’s reproductive competence as well as embryo development. DIALOG: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/xfnr-d-21-00045 •Mechanisms regulating sperm motility do not involve the redox balance, and the role of mitochondria in sperm bioenergetics is controversial.•Reactive oxygen species are generated in mitochondria in conditions of reductive rather than of oxidative stress.•Double-strand breaks in sperm DNA are not correlated with oxidative damages but are associated with defective histone to protamine transition.•Reductive stress may impair DNA repair from poly-ADP-ribose polymerase, resulting in both double-strand breaks and impaired protamine transition.•Metabolic perturbances from inappropriate antioxidant treatments may jeopardize men’s reproductive competence as well as embryo development. Male factor infertility accounts for 40%–50% of cases of couple infertility (1Brugh III, V.M. Lipshultz L.I. Male factor infertility: evaluation and management.Med Clin North Am. 2004; 88: 367-385Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Effective treatments for male factor infertility are well established only for few medical conditions (i.e., hypogonadotropic hypogonadism and male accessory gland infection), and therapeutic strategies used to improve sperm parameters are most often empirical. Antioxidants belong to the category of empirical medical treatments of male factor infertility because, despite their widespread clinical use, the supporting evidence is insufficient or conflicting. It has been postulated that antioxidants could protect the sperm from oxidative stress that may arise from an imbalance between the production of reactive oxygen species (ROS) and the natural antioxidant defenses, thus avoiding damages to the sperm cell membrane and sperm DNA, with the consequent impairment of sperm motility and fertilizing ability. Unfortunately, such a strategy seems to be ineffective. The most recently released version of the Cochrane review on this subject (2Smits R.M. Mackenzie-Proctor R. Yazdani A. Stankiewicz M.T. Jordan V. Showell M.G. Antioxidants for male subfertility.Cochrane Database Syst Rev. 2019; 3CD007411PubMed Google Scholar) included 61 studies with a total population of 6,264 subfertile men, but only 12 studies out of the 44 included in the meta-analysis reported on live birth or clinical pregnancy. The initial analysis, on the basis of 124 live births from 750 couples in 7 relatively small studies, brought the conclusion that antioxidants improved live birth rates compared with no treatment or placebo (odds ratio, 1.79; 95% confidence interval, 1.20–2.67; P=.005; I2 = 40%; low-quality evidence). However, when studies at high risk of bias were removed from the analysis, there was no evidence of increased live birth in the treated group compared with the control group (Peto odds ratio, 1.38; 95% confidence interval, 0.89–2.16; participants, 540 men; 5 randomized controlled trials; P=.15; I2 = 0%). A more recent multicenter, double-blind, randomized, placebo-controlled trial evaluating 174 infertile men demonstrated that antioxidants were unable to affect sperm motility as well as sperm DNA fragmentation and confirmed the lack of any beneficial effect on cumulative live birth rate compared with placebo (3Steiner A.Z. Hansen K.R. Barnhart K.T. Cedars M.I. Legro R.S. Diamond M.P. et al.The effect of antioxidants on male factor infertility: the Males, Antioxidants, and Infertility (MOXI) randomized clinical trial.Fertil Steril. 2020; 113: 552-560.e3Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Later on, the same cohort of patients was investigated for a possible correlation between the serum levels of administered antioxidants (α-tocopherol, zinc, and selenium) and semen parameters and couple outcomes: their baseline serum levels were within the normal limits and were not correlated with semen parameters or clinical outcomes, and in the treated group, the increased level of vitamins failed as well to benefit the same parameters (4Knudtson J.F. Sun F. Coward R.M. Hansen K.R. Barnhart K.T. Smith J. et al.The relationship of plasma antioxidant levels to semen parameters: the Males, Antioxidants, and Infertility (MOXI) randomized clinical trial.J Assist Reprod Genet. 2021; 38: 3005-3013Crossref PubMed Scopus (0) Google Scholar). Thus, in the absence of a defined shortage, extra amounts of antioxidants do not seem to be of benefit. Despite that the evidence in support of antioxidants as treatment of male factor infertility is scanty, their use is still growing, and they are commonly prescribed to infertile men even in the lack of any evidence of vitamin shortage or of confirmed oxidative imbalance and/or increased oxidative aggression, that is, only on the basis of the backing rationale. The present narrative review aimed to provide a summary of the mechanisms underlying the regulation of sperm motility, which is the main intended target of antioxidant administration, and new insights into the sperm redox metabolism and its relationship with sperm DNA integrity to verify whether there would be room for the use of antioxidant treatment to restore such conditions. The sperm flagellum consists of 9 doublet microtubules surrounding 2 central singlet microtubules, thus forming a 9 + 2 structure termed axoneme, associated with radial spokes and dynein arms. The axoneme is approximately 50 μm long and composed by the midpiece, formed by a sheath of mitochondria helically assembled around 9 outer dense fibers, each connected to an axonemal doublet microtubule, the principal piece, where 2 outer dense fibers are replaced by the longitudinal columns of the fibrous sheath, which are connected to each other by transverse ribs, and the endpiece, which contains only the axoneme surrounded by the plasma membrane (5Lehti M.S. Sironen A. Formation and function of sperm tail structures in association with sperm motility defects.Biol Reprod. 2017; 97: 522-536Crossref PubMed Scopus (87) Google Scholar, 6Linck R.W. Chemes H. Albertini D.F. The axoneme: the propulsive engine of spermatozoa and cilia and associated ciliopathies leading to infertility.J Assist Reprod Genet. 2016; 33: 141-156Crossref PubMed Scopus (47) Google Scholar). Each doublet microtubule is composed of a complete, 13-protofilament, A-microtubule and partial, 10-protofilament, B-tubule and contains a high level of acetylated tubulin. Conformational changes in tubulin and its associated proteins allow dynamic bending and twisting of doublet microtubules. The sliding movement of microtubules is due to the activation of the adenosine triphosphatases of the dynein arms, which are permanently anchored to each A-tubule and directed to the B-tubules of their next doublet microtubules: dynein arms transduce the chemical energy of adenosine triphosphate (ATP) into mechanical energy (6Linck R.W. Chemes H. Albertini D.F. The axoneme: the propulsive engine of spermatozoa and cilia and associated ciliopathies leading to infertility.J Assist Reprod Genet. 2016; 33: 141-156Crossref PubMed Scopus (47) Google Scholar, 7Serohijos A.W. Chen Y. Ding F. Elston T.C. Dokholyan N.V. A structural model reveals energy transduction in dynein.Proc Natl Acad Sci U S A. 2006; 103: 18540-18545Crossref PubMed Scopus (25) Google Scholar). The outer and inner dynein arms are composed of heavy chains, which possesses the sites of both ATP hydrolysis and ATP-sensitive microtubule binding, and are able to transduce the energy produced by hydrolysis into mechanical force applied to the microtubule surface, intermediate chains that participates to the structural attachment of dynein arms to microtubules, and light chains that bind to the microtubule-binding domain and seem to act as regulatory switch that senses the curvature of the axoneme and tunes the flagellar beating (8King S.M. The dynein microtubule motor.Biochim Biophys Acta. 2000; 1496: 60-75Crossref PubMed Scopus (285) Google Scholar, 9Toda A. Nishikawa Y. Tanaka H. Yagi T. Kurisu G. The complex of outer-arm dynein light chain-1 and the microtubule-binding domain of the γ heavy chain shows how axonemal dynein tunes ciliary beating.J Biol Chem. 2020; 295: 3982-3989Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). In some cases, male factor infertility due to loss of sperm motility is caused by mutations in the genes for dynein and dynein-associated polypeptides leading to primary ciliary dyskinesia (6Linck R.W. Chemes H. Albertini D.F. The axoneme: the propulsive engine of spermatozoa and cilia and associated ciliopathies leading to infertility.J Assist Reprod Genet. 2016; 33: 141-156Crossref PubMed Scopus (47) Google Scholar). Large amounts of ATP are required to promote sperm motility. The 2 available metabolic pathways of ATP production in sperm are mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis. Oxidative phosphorylation generates 30 molecules of ATP per molecule of glucose, while the net yield of ATP in glycolysis is 2 molecules per molecule of oxidized glucose. The mature sperm cell contains 72–80 mitochondria, which are functionally and somewhat morphologically different compared with somatic mitochondria. Although several studies have provided indirect demonstrations in support of a key role played by sperm mitochondria in the modulation of sperm motility, for example, mitochondrial abnormalities in asthenozoospermic men, disrupted mitochondrial membrane potential, and/or abnormal mitochondrial DNA copy number in asthenozoospermic/low-quality semen samples (10Amaral A. Lourenço B. Marques M. Ramalho-Santos J. Mitochondria functionality and sperm quality.Reproduction. 2013; 146: R163-R174Crossref PubMed Scopus (270) Google Scholar), the results of other studies challenged that hypothesis. Since mitochondria are confided to the midpiece, if mitochondrial ATP is the only source of energy for the axoneme, it would have to diffuse some distance to supply the full length of the axoneme: this, however, has not been supported by mathematical models (11Turner R.M. Moving to the beat: a review of mammalian sperm motility regulation.Reprod Fertil Dev. 2006; 18: 25-38Crossref PubMed Scopus (200) Google Scholar). In addition, the need for glucose to maintain mammalian sperm functions cannot be satisfied by replacing glucose with other OXPHOS substrates (10Amaral A. Lourenço B. Marques M. Ramalho-Santos J. Mitochondria functionality and sperm quality.Reproduction. 2013; 146: R163-R174Crossref PubMed Scopus (270) Google Scholar, 12du Plessis S.S. Agarwal A. Mohanty G. van der Linde M. Oxidative phosphorylation versus glycolysis: what fuel do spermatozoa use?.Asian J Androl. 2015; 17: 230-235Crossref PubMed Google Scholar). Furthermore, the inhibition of mitochondrial ATP production by carbonyl cyanide m-chlorophenylhydrazine had no effect on ATP content and motility parameters (13Mukai C. Okuno M. Glycolysis plays a major role for adenosine triphosphate supplementation in mouse sperm flagellar movement.Biol Reprod. 2004; 71: 540-547Crossref PubMed Scopus (284) Google Scholar). Thus, although they seem to be involved in the modulation of sperm capacitation, as described in the subsection “Hyperactivated Sperm Motility,” the role of mitochondria in fueling human sperm motility remains questionable (14Boguenet M. Bouet P.E. Spiers A. Reynier P. May-Panloup P. Mitochondria: their role in spermatozoa and in male infertility.Hum Reprod Update. 2021; 27: 697-719Crossref PubMed Scopus (9) Google Scholar). On the other hand, the evidence is suggestive of the key role played by sperm glycolysis in providing the required amount of ATP production for its metabolic needs (15Danshina P.V. Qu W. Temple B.R. Rojas R.J. Miley M.J. Machius M. et al.Structural analyses to identify selective inhibitors of glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme.Mol Hum Reprod. 2016; 22: 410-426Crossref PubMed Scopus (16) Google Scholar). Mammalian sperm have distinct glycolytic isoenzymes and express specific isoforms of proteins essential for glycolysis. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS), phosphoglycerate kinase-2, and lactate dehydrogenase-C4 are encoded by genes expressed only during spermatogenesis, but 5 other enzymes in the glycolytic pathway have unique structural or functional properties in spermatogenic cells (16Miki K. Qu W. Goulding E.H. Willis W.D. Bunch D.O. Strader L.F. et al.Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility.Proc Natl Acad Sci U S A. 2004; 101: 16501-16506Crossref PubMed Scopus (460) Google Scholar). Glyceraldehyde 3-phosphate dehydrogenase-S in sperm is tightly bound to the fibrous sheath; it is expressed only in the postmeiotic period of spermatogenesis, when it replaces the somatic isoenzyme glyceraldehyde 3-phosphate dehydrogenase (17Welch J.E. Brown P.L. O’Brien D.A. Magyar P.L. Bunch D.O. Mori C. et al.Human glyceraldehyde 3-phosphate dehydrogenase-2 gene is expressed specifically in spermatogenic cells.J Androl. 2000; 21: 328-338PubMed Google Scholar). Glyceraldehyde 3-phosphate dehydrogenase-S catalyzes the oxidation and phosphorylation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, which represents a crucial step in glycolysis. Its targeted deletion (GAPDS −/− mice) results in glycolysis blockade and in a fourfold accumulation of the substrate glyceraldehyde 3-phosphate compared with wild-type (WT) mice (16Miki K. Qu W. Goulding E.H. Willis W.D. Bunch D.O. Strader L.F. et al.Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility.Proc Natl Acad Sci U S A. 2004; 101: 16501-16506Crossref PubMed Scopus (460) Google Scholar). Glyceraldehyde 3-phosphate dehydrogenase-S −/− mice were found to be infertile (whereas heterozygous GAPDS +/− mice were fertile), despite their normal sperm count (compared with WT animals) and morphology, their intact testis weight and histology, and their normal mating behavior. Infertility was probably due to the profound motility defects: although approximately 50% of sperm displayed some movement immediately after isolation from the cauda epididymis, the slow-moving flagellar beat rarely resulted in forward movement (3.2 ± 1.5% of sperm). Sperm ATP content was significantly reduced (approximately 10.4% of WT mice) and further declined when sperm were incubated at 37°C in 5% CO2 and air for 4 hours, whereas sperm ATP content in WT mice did not change when incubated for the same time period under identical conditions. Interestingly, sperm mitochondrial oxygen consumption was not affected in GAPDS −/− mice (16Miki K. Qu W. Goulding E.H. Willis W.D. Bunch D.O. Strader L.F. et al.Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility.Proc Natl Acad Sci U S A. 2004; 101: 16501-16506Crossref PubMed Scopus (460) Google Scholar). In addition to GAPDS, other glycolytic enzymes are tightly bound to the fibrous sheath (hexokinase 1, aldolase, lactate dehydrogenase A), some of which are specific isoenzymes that contain modifications at their N-terminus capable of anchoring the proteins of the fibrous sheath (18Gu N.H. Zhao W.L. Wang G.S. Sun F. Comparative analysis of mammalian sperm ultrastructure reveals relationships between sperm morphology, mitochondrial functions and motility.Reprod Biol Endocrinol. 2019; 17: 66Crossref PubMed Scopus (33) Google Scholar), so that the latter has been proposed to play as a scaffold for anchoring these glycolytic enzymes along the length of the flagellum to provide a localized source of ATP (19Krisfalusi M. Miki K. Magyar P.L. O’Brien D.A. Multiple glycolytic enzymes are tightly bound to the fibrous sheath of mouse spermatozoa.Biol Reprod. 2006; 75: 270-278Crossref PubMed Scopus (148) Google Scholar). These findings support the hypothesis that glycolysis is the preferred metabolic pathway that fuels sperm motility in pre-ejaculatory sperm. On the other hand, the actual role of mitochondria in pre-ejaculatory and postejaculatory sperm bioenergetics remains an unsolved puzzle. At the end of spermatogenesis, sperm cells are immotile or display a finely twitching movement. They are also immature and unable to fertilize an egg, unless used for intracytoplasmic sperm injection. It is during the epididymal transit that sperm interact with locally synthesized proteins, undergo a series of biochemical and structural changes, and lose their cytoplasmic droplets; their plasma membrane surface proteins undergo changes in phospholipid composition and of surface proteins; and finally they acquire progressive motility (20Machtinger R. Laurent L.C. Baccarelli A.A. Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation.Hum Reprod Update. 2016; 22: 182-193PubMed Google Scholar, 21Freitas M.J. Vijayaraghavan S. Fardilha M. Signaling mechanisms in mammalian sperm motility.Biol Reprod. 2017; 96: 2-12PubMed Google Scholar). When motility is activated, the specific activities of enzymes in the glycolytic pathway increase, with a consequent increase in ATP production. The sperm glycolytic pathway is under the control of male germline-specific isozymes, arising from genes or alternatively spliced transcripts. An increase in the cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activation is supposed to initiate motility in epididymal sperm, probably following changes in protein phosphorylation. An important signaling event responsible for sperm motility acquisition seems to be the control of sperm phosphoprotein phosphatase 1 (PP1) activity. Phosphoprotein phosphatase 1 activity is high in caput epididymis, where sperm are immotile, whereas it is inactive in the cauda epididymis, where sperm are motile (21Freitas M.J. Vijayaraghavan S. Fardilha M. Signaling mechanisms in mammalian sperm motility.Biol Reprod. 2017; 96: 2-12PubMed Google Scholar). The PP1γ2 isoform, produced by alternative splicing of the phosphatase catalytic subunit gamma (PPP1cc) gene transcript, is the only PP1 isoform expressed in meiotic and postmeiotic germ cells in the testis and spermatozoa. In PPP1cc (−/−) knockout mice, replacement of PP1γ2 with PP1γ1 did not hamper spermatogenesis; however, its subcellular localization and distribution were altered, and motility was impaired with significantly attenuated flagellar beat amplitude. Mature caudal epididymal sperm from PPP1cc (−/−) mice had lower ATP levels compared with normal WT sperm, even if supplemented with energy substrates (22Dudiki T. Joudeh N. Sinha N. Goswami S. Eisa A. Kline D. et al.The protein phosphatase isoform PP1γ1 substitutes for PP1γ2 to support spermatogenesis but not normal sperm function and fertility.Biol Reprod. 2019; 100: 721-736Crossref PubMed Scopus (3) Google Scholar). Phosphoprotein phosphatase 1 activity is regulated by PP1 regulatory subunit 2 (PPP1R2): when PPP1R2 is phosphorylated by glycogen synthase kinase 3 (GSK3), PP1 is active, and sperm are immotile (caput epididymis) (21Freitas M.J. Vijayaraghavan S. Fardilha M. Signaling mechanisms in mammalian sperm motility.Biol Reprod. 2017; 96: 2-12PubMed Google Scholar). Indeed, GSK3 activity is negatively correlated with sperm motility regulation in both cauda and caput, and it is 6 times more active in caput than in caudal sperm (21Freitas M.J. Vijayaraghavan S. Fardilha M. Signaling mechanisms in mammalian sperm motility.Biol Reprod. 2017; 96: 2-12PubMed Google Scholar). However, in vitro disruption of PP1γ2/PPP1R2 interaction was not able to completely abolish sperm motility: after 2 hours of incubation with PPP1R2-BM bioportides, 19 ± 7.2% of bovine sperm still retained progressive motility (23Silva J.V. Freitas M.J. Santiago J. Jones S. Guimarães S. Vijayaraghavan S. et al.Disruption of protein phosphatase 1 complexes with the use of bioportides as a novel approach to target sperm motility.Fertil Steril. 2021; 115: 348-362Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). This is surprising because it has been suggested that PPP1R2 activity is central in controlling PP1 activity in mammalian sperm. Indeed, very recently, it has been demonstrated in the mouse that catalytic activity is regulated by 3 distinct protein phosphatase inhibitors, PPP1R2, PPP1R7, and PPP1R11 (24Goswami S. Korrodi-Gregório L. Sinha N. Bhutada S. Bhattacharjee R. Kline D. et al.Regulators of the protein phosphatase PP1γ2, PPP1R2, PPP1R7, and PPP1R11 are involved in epididymal sperm maturation.J Cell Physiol. 2019; 234: 3105-3118Crossref PubMed Scopus (8) Google Scholar). Binding of PPP1R2, PPP1R11, and PPP1R7 to PP1γ2 changes during the sperm transit through the epididymis: in caudal sperm, all 3 inhibitors are bound to PP1γ2, rendering it inactive, whereas in caput sperm phosphorylation of PPP1R11 prevents the binding of PPP1R7 to PP1γ2, which is, therefore, catalytically active. Whether such a complex regulation of PPP1 activity could be applicable to mammalian and human sperm motility regulation requires further studies. The study of extracellular vesicles in the male reproductive tract has brought interesting findings in this field. Epididymal secretory epithelial cells secrete also extracellular vesicles, termed epididymosomes, by apocrine secretion (25Sullivan R. Epididymosomes: a heterogeneous population of microvesicles with multiple functions in sperm maturation and storage.Asian J Androl. 2015; 17: 726-729PubMed Google Scholar). Two main classes of epididymosomes have been identified: CD9-positive epididymosomes, which preferentially bind live sperm, and epididymal sperm binding protein 1-enriched epididymosomes, which bind dead sperm in the caput and cauda epididymis. CD9-positive epididymosomes are small (10–100 nm) vesicles rich in P25b and GliPriL1, involved in the sperm-egg interaction, macrophage migration inhibitory factor and AKR1B1, proteins involved in sperm motility (25Sullivan R. Epididymosomes: a heterogeneous population of microvesicles with multiple functions in sperm maturation and storage.Asian J Androl. 2015; 17: 726-729PubMed Google Scholar). Epididymal sperm binding protein 1 is transferred to dead sperm by epididymosomes in the presence of zinc, this probably being a mechanism for the recognition of defective sperm in the epididymis (26D’Amours O. Frenette G. Bordeleau L.J. Allard N. Leclerc P. Blondin P. et al.Epididymosomes transfer epididymal sperm binding protein 1 (ELSPBP1) to dead spermatozoa during epididymal transit in bovine.Biol Reprod. 2012; 87: 94Crossref PubMed Google Scholar). A slightly acidic intraluminal pH (6.5) is required to allow the interaction between epididymosomes and sperm (21Freitas M.J. Vijayaraghavan S. Fardilha M. Signaling mechanisms in mammalian sperm motility.Biol Reprod. 2017; 96: 2-12PubMed Google Scholar). Epididymosomes transfer proteins as well as microRNAs to the sperm. Although the contribution of sperm to epididymosome interaction in the acquisition of sperm motility has not been fully demonstrated to date, bovine studies revealed that they carry proteins that participate in the acquisition of sperm motility and fertilization ability, including macrophage migration inhibitory factor and enzymes of the polyol pathways known to promote sperm motility (20Machtinger R. Laurent L.C. Baccarelli A.A. Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation.Hum Reprod Update. 2016; 22: 182-193PubMed Google Scholar). Moreover, an experimental study in the domestic cat model demonstrated that the exposure of immature sperm cells to the epididymosomes resulted in higher and sustained motility compared with that of immature sperm incubated in plain buffer medium (27Rowlison T. Ottinger M.A. Comizzoli P. Key factors enhancing sperm fertilizing ability are transferred from the epididymis to the spermatozoa via epididymosomes in the domestic cat model.J Assist Reprod Genet. 2018; 35: 221-228Crossref PubMed Scopus (22) Google Scholar). Extracellular vesicles have also been demonstrated in the human seminal plasma, termed prostasomes. They measure 50–500 nm and contain proteins, lipids, nucleic acids, and, interestingly, glycolytic enzymes able to produce ATP when supplied to substrate, as well as glucose transporters, such as GLUT3, GLUT5, and GLUT14, and adenosine triphosphatases (28Ronquist K.G. Ek B. Stavreus-Evers A. Larsson A. Ronquist G. Human prostasomes express glycolytic enzymes with capacity for ATP production.Am J Physiol Endocrinol Metab. 2013; 304: E576-E582Crossref PubMed Scopus (35) Google Scholar). Because approximately one-half of the protein kinase activity in human prostatic fluid has been found to be associated with prostasomes and coincubation of sperm with prostasomes was found to result in a tenfold increase in total protein phosphorylation compared with controls (29Stegmayr B. Brody I. Ronquist G. A biochemical and ultrastructural study on the endogenous protein kinase activity of secretory granule membranes of prostatic origin in human seminal plasma.J Ultrastruct Res. 1982; 78: 206-214Crossref PubMed Scopus (44) Google Scholar), it has been hypothesized that ATP formed by prostasomes could represent a subst