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Pablo E Visconti - One of the best experts on this subject based on the ideXlab platform.
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Metabolic changes in mouse sperm during Capacitation.
Biology of Reproduction, 2020Co-Authors: Melanie Balbach, Lonny R Levin, Pablo E Visconti, María Gracia Gervasi, David Martin Hidalgo, Jochen BuckAbstract:Mammalian sperm are stored in the epididymis in a dormant state. Upon ejaculation, they must immediately start producing sufficient energy to maintain motility and support Capacitation. While this increased energy demand during Capacitation is well-established, it remains unclear how mouse sperm modify their metabolism to meet this need. We now show that capacitating mouse sperm enhance glucose uptake, identifying glucose uptake as a functional marker of Capacitation. Using an extracellular flux analyzer, we show that glycolysis and oxidative phosphorylation increase during Capacitation. Furthermore, this increase in oxidative phosphorylation is dependent on glycolysis, providing experimental evidence for a link between glycolysis and oxidative phosphorylation in mouse sperm.
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sperm Capacitation and acrosome reaction in mammalian sperm
Advances in Anatomy Embryology and Cell Biology, 2016Co-Authors: Cintia Stival, Pablo E Visconti, Lis Puga C Molina, Bidur Paudel, Mariano G Buffone, Dario KrapfAbstract:Physiological changes that endow mammalian sperm with fertilizing capacity are known as sperm Capacitation. As part of Capacitation, sperm develop an asymmetrical flagellar beating known as hyperactivation and acquire the ability to undergo the acrosome reaction. Together, these processes promote fertilizing competence in sperm. At the molecular level, Capacitation involves a series of signal transduction events which include activation of cAMP-dependent phosphorylation pathways, removal of cholesterol, hyperpolarization of the sperm plasma membrane, and changes in ion permeability. In recent years, new technologies have aided in the study of sperm signaling molecules with better resolution, at both spatial and temporal levels, unraveling how different cascades integrate and cooperate to render a fertilizing sperm. Despite this new information, the molecular mechanisms connecting Capacitation with acrosomal exocytosis and hyperactivation are not well understood. This review brings together results obtained in mammalian species in the field of sperm Capacitation with special focus on those pathways involved in the preparation to undergo the acrosomal reaction.
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flow cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during Capacitation
Biology of Reproduction, 2015Co-Authors: Jessica Escoffier, Alberto Darszon, Felipe A Navarrete, Doug Haddad, Celia M Santi, Pablo E ViscontiAbstract:ABSTRACT To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as Capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during Capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the Capacitation-associated hyperpolarization is blocked by high extracellular K+, by PKA inhibitors, and by SLO3 inhibitors in CD1 ...
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participation of the cl hco3 exchangers slc26a3 and slc26a6 the cl channel cftr and the regulatory factor slc9a3r1 in mouse sperm Capacitation
Biology of Reproduction, 2012Co-Authors: Julio C Chavez, Pablo E Visconti, Alberto Darszon, Enrique O Hernandezgonzalez, Eva Wertheimer, Claudia L TrevinoAbstract:ABSTRACT Sperm Capacitation is required for fertilization and involves several ion permeability changes. Although Cl− and HCO3− are essential for Capacitation, the molecular entities responsible for their transport are not fully known. During mouse sperm Capacitation, the intracellular concentration of Cl− ([Cl−]i) increases and membrane potential (Em) hyperpolarizes. As in noncapacitated sperm, the Cl− equilibrium potential appears to be close to the cell resting Em, opening of Cl− channels could not support the [Cl−]i increase observed during Capacitation. Alternatively, the [Cl−]i increase might be mediated by anion exchangers. Among them, SLC26A3 and SLC26A6 are good candidates, since, in several cell types, they increase [Cl−]i and interact with cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− channel present in mouse and human sperm. This interaction is known to be mediated and probably regulated by the Na+/H+ regulatory factor-1 (official symbol, SLC9A3R1). Our RT-PCR, immunocytoch...
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ion channels phosphorylation and mammalian sperm Capacitation
Asian Journal of Andrology, 2011Co-Authors: Pablo E Visconti, Dario Krapf, Jose L De La Vegabeltran, Juan Jose Acevedo, Alberto DarszonAbstract:Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to Capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to Capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve Capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.
Gregory S. Kopf - One of the best experts on this subject based on the ideXlab platform.
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Bovine sperm Capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on Capacitation-associated protein tyrosine phosphorylation.
Molecular Reproduction and Development, 2004Co-Authors: Hannah Galantino-homer, Harvey M. Florman, Bayard T. Storey, Ina Dobrinski, Gregory S. KopfAbstract:Mammalian sperm Capacitation is the obligatory maturational process leading to the development of the fertilization-competent state. Heparin is known to be a unique species-specific inducer of bovine sperm Capacitation in vitro and glucose a unique inhibitor of this induction. Heparin-induced Capacitation of bovine sperm has been shown to correlate with protein kinase A (PKA)-dependent protein tyrosine phosphorylation driven by an increase in intracellular cAMP. This study examines the possible roles of cyclic nucleotide phosphodiesterase (PDE) activity and intracellular alkalinization on bovine sperm Capacitation and the protein tyrosine phosphorylation associated with it. Measurement of whole cell PDE kinetics during Capacitation reveals neither a substantial change with heparin nor one with glucose: PDE activity is effectively constitutive in maintaining intracellular cAMP levels during Capacitation. In contrast to a transient increase in intracellular pH, a sustained increase in medium pH by switching from 5% CO2/95% air incubation to 1% CO2/99% air incubation over 4 hr in the absence of heparin resulted in an increase in protein tyrosine phosphorylation and in the extent of induced acrosome reaction comparable to that observed following heparin-induced Capacitation in 5% CO2. These results suggest that increased bicarbonate-dependent adenylyl cyclase activity, driven by alkalinization, increases intracellular cAMP and so increases PKA activity mediating protein tyrosine phosphorylation. Quantitative analysis of the lactic acid production rate by bovine sperm glycolysis accounts fully for intracellular acidification sufficient to offset heparin-induced alkalinization, thus inhibiting Capacitation. The mechanism by which heparin uniquely induces intracellular alkalinization in bovine sperm leading to Capacitation remains obscure, inviting future investigation. Mol. Reprod. Dev. 67: 487–500, 2004. © 2004 Wiley-Liss, Inc.
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involvement of a na hco 3 cotransporter in mouse sperm Capacitation
Journal of Biological Chemistry, 2003Co-Authors: Ignacio A Demarco, Gregory S. Kopf, Jose L De La Vegabeltran, Alberto Darszon, Felipe Espinosa, Jennifer Edwards, Julian Sosnik, Joel W Hockensmith, Pablo E ViscontiAbstract:Abstract Mammalian sperm are incapable of fertilizing eggs immediately after ejaculation; they acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes sperm undergo in the female reproductive tract that render sperm able to fertilize constitute the phenomenon of “sperm Capacitation.” We have demonstrated that Capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins and that these events are regulated by an HCO /cAMP-dependent pathway involving protein kinase A. Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. Here we present evidence that, in addition to its role in the regulation of adenylyl cyclase, HCO has a role in the regulation of plasma membrane potential in mouse sperm. Addition of HCO but not Cl− induces a hyperpolarizing current in mouse sperm plasma membranes. This HCO -dependent hyperpolarization was not observed when Na+ was replaced by the non-permeant cation choline+. Replacement of Na+ by choline+ also inhibited the Capacitation-associated increase in protein tyrosine phosphorylation as well as the zona pellucida-induced acrosome reaction. The lack of an increase in protein tyrosine phosphorylation was overcome by the presence of cAMP agonists in the incubation medium. The lack of a hyperpolarizing HCO current and the inhibition of the Capacitation-dependent increase in protein tyrosine phosphorylation in the absence of Na+ suggest that a Na+/HCO cotransporter is present in mouse sperm and is coupled to events regulating Capacitation.
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regulation of human sperm Capacitation by a cholesterol efflux stimulated signal transduction pathway leading to protein kinase a mediated up regulation of protein tyrosine phosphorylation
Molecular Human Reproduction, 1999Co-Authors: Joseph E Osheroff, Pablo E Visconti, Juan Pablo Valenzuela, Alexander J Travis, Juan G Alvarez, Gregory S. KopfAbstract:: Protein tyrosine phosphorylation is an important intracellular event accompanying the in-vitro Capacitation of mouse, bovine and human spermatozoa. Here, we demonstrate that bovine serum albumin (BSA) and NaHCO(3) are required for protein tyrosine phosphorylation in ejaculated human spermatozoa. The absence of protein tyrosine phosphorylation in media minus these two constituents could be recovered by addition to the media of cAMP analogues and/or phosphodiesterase inhibitors. Since BSA is postulated to modulate Capacitation by removal of cholesterol from the sperm plasma membrane, we determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Incubation of spermatozoa in media containing BSA resulted in the release of significant amounts of cholesterol when compared with media devoid of BSA. Preloading BSA with cholesterol-SO(4) inhibited protein tyrosine phosphorylation, as well as Capacitation, and this inhibitory effect was overcome by the addition of dibutyryl cAMP plus isobutylmethylxanthine (IBMX). The functional significance of BSA-mediated cholesterol release, protein tyrosine phosphorylation and Capacitation was confirmed by examining the effects of the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin or OH-propyl-beta-cyclodextrin. Both cyclodextrins caused cholesterol efflux from the spermatozoa, increased protein tyrosine phosphorylation, and stimulated Capacitation. Therefore, cholesterol release is associated with the activation of a signal transduction pathway involving protein kinase A and tyrosine kinase second messenger systems, and resulting in protein tyrosine phosphorylation and Capacitation.
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cholesterol efflux mediated signal transduction in mammalian sperm cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm Capacitation
Developmental Biology, 1999Co-Authors: Pablo E Visconti, Juan G Alvarez, Stephanie A Connors, Xiaoping Ning, Miguel W Fornes, Paula Stein, Gregory S. KopfAbstract:Abstract We previously demonstrated that mouse sperm Capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca 2+ , and NaHCO 3 , all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this Capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate Capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO − 4 to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as Capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO − 4 and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the Capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm Capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO − 3 is necessary for the Capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO − 3 or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO − 3 effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm.
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regulation of protein phosphorylation during sperm Capacitation
Biology of Reproduction, 1998Co-Authors: Pablo E Visconti, Gregory S. KopfAbstract:After leaving the testis, mammalian spermatozoa from many species are morphologically differentiated but have acquired neither progressive motility nor the ability to fertilize a metaphase II-arrested egg. During epididymal transit, sperm acquire the ability to move progressively; however, they are still fertilization incompetent. Fertilization capacity is gained after residence in the female tract for a finite period of time. The physiological changes that confer on the sperm the ability to fertilize are collectively called ‘‘Capacitation.’’ Capacitation was first described and defined independently by Chang [1, 2] and Austin [3, 4]. The definition of this poorly understood phenomenon has been modified and narrowed over the years. Although fertilization still represents the benchmark endpoint of a capacitated sperm, the ability of the sperm to undergo a regulated acrosome reaction (e.g., in response to the zona pellucida) can be taken as an earlier, upstream endpoint of this extratesticular maturational event. It must be stressed at this point that Capacitation is also correlated with changes in sperm motility patterns, designated as sperm hyperactivation, in a number of species [5, 6]. There are examples of cases in which Capacitation and hyperactivation can be dissociated experimentally [7], but one cannot yet argue that hyperactivation of motility represents an event completely independent of the Capacitation process [6]. Therefore, when one attempts to understand the process of Capacitation at the molecular level, it is necessary to consider events occurring both in the head (i.e., acrosome reaction) and in the tail (i.e., motility changes). The physiological site of Capacitation in vivo is the oviduct or the uterus, depending on the species [5]. However, Capacitation in vitro has been accomplished using cauda and/or ejaculated sperm incubated under a variety of conditions in defined media that mimic the electrolyte composition of the oviduct fluid. In most cases, these media contain energy substrates such as pyruvate, lactate, and glucose (depending on the species); a protein source that usually is serum albumin; NaHCO3; and Ca21. The action of these media components to promote Capacitation at the molecular level is poorly understood and will be discussed in this review. This review is not intended to provide an ex-
Haim Breitbart - One of the best experts on this subject based on the ideXlab platform.
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mechanism of sperm Capacitation and the acrosome reaction role of protein kinases
Asian Journal of Andrology, 2012Co-Authors: Debby Ickowicz, Maya Finkelstein, Haim BreitbartAbstract:: Mammalian sperm must undergo a series of biochemical and physiological modifications, collectively called Capacitation, in the female reproductive tract prior to the acrosome reaction (AR). The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca(2+) during both Capacitation and the AR. In the present review, we summarize some of the signaling events that are involved in Capacitation. During the Capacitation process, phosphatidyl-inositol-3-kinase (PI3K) is phosphorylated/activated via a protein kinase A (PKA)-dependent cascade, and downregulated by protein kinase C α (PKCα). PKCα is active at the beginning of Capacitation, resulting in PI3K inactivation. During Capacitation, PKCα as well as PP1γ2 is degraded by a PKA-dependent mechanism, allowing the activation of PI3K. The activation of PKA during Capacitation depends mainly on cyclic adenosine monophosphate (cAMP) produced by the bicarbonate-dependent soluble adenylyl cyclase. This activation of PKA leads to an increase in actin polymerization, an essential process for the development of hyperactivated motility, which is necessary for successful fertilization. Actin polymerization is mediated by PIP(2) in two ways: first, PIP(2) acts as a cofactor for phospholipase D (PLD) activation, and second, as a molecule that binds and inhibits actin-severing proteins such as gelsolin. Tyrosine phosphorylation of gelsolin during Capacitation by Src family kinase (SFK) is also important for its inactivation. Prior to the AR, gelsolin is released from PIP(2) and undergoes dephosphorylation/activation, resulting in fast F-actin depolymerization, leading to the AR.
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hyper activated motility in sperm Capacitation is mediated by phospholipase d dependent actin polymerization
Developmental Biology, 2012Co-Authors: Sarit Barsheshet Itach, Nir Etkovitz, Maya Finklestein, Haim BreitbartAbstract:In order to fertilize the oocyte, sperm must undergo a series of biochemical changes in the female reproductive tract, known as Capacitation. Once capacitated, spermatozoon can bind to the zona pellucida of the egg and undergo the acrosome reaction (AR), a process that enables its penetration and fertilization of the oocyte. Important processes that characterize sperm Capacitation are actin polymerization and the development of hyper-activated motility (HAM). Previously, we showed that Phospholipase D (PLD)-dependent actin polymerization occurs during sperm Capacitation, however the role of this process in sperm Capacitation is not yet known. In the present study, we showed for the first time the involvement of PLD-dependent actin polymerization in sperm motility during mouse and human Capacitation. Sperm incubated under Capacitation conditions revealed a time dependent increase in actin polymerization and HAM. Inhibition of Phosphatidic Acid (PA) formation by PLD using butan-1-ol, inhibited actin polymerization and motility, as well as in vitro fertilization (IVF) and the ability of the sperm to undergo the AR. The inhibition of sperm HAM by low concentration of butan-1-ol is completely restored by adding PA, further indicating the involvement of PLD in these processes. Furthermore, exogenous PA enhanced rapid actin polymerization that was followed by a rise in the HAM, as well as an increased in IVF rate. In conclusion, our results demonstrate that PLD-dependent actin polymerization is a critical step needed for the development of HAM during mouse and human sperm Capacitation.
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Role and regulation of PI3K in sperm Capacitation and the acrosome reaction.
Molecular and Cellular Endocrinology, 2009Co-Authors: Haim Breitbart, Sara Rubinstein, Tali Rotman, Nir EtkovitzAbstract:Abstract Mammalian spermatozoa undergo several signaling and biochemical transformations in the female genital tract, collectively called Capacitation. The capacitated spermatozoon binds to the egg zona pellucida, where it undergoes the acrosome reaction (AR), a process enabling it to penetrate and fertilize the egg. Actin polymerization occurs in sperm Capacitation and depolymerization prior to the AR. In this review we describe the possible role and regulation of PI3K in sperm Capacitation and the acrosome reaction. We claim that PI3K is activated by protein kinase A and suppressed by protein kinase C. Only partial activation of PI3K is seen during the Capacitation time, however towards the end of incubation, full activation is observed. Actin polymerization during Capacitation is independent on PI3K activity, suggesting that the enzyme is not involved in sperm Capacitation. However, the full activation of PI3K towards the end of the Capacitation suggests that it might mediate the AR, as indeed was found.
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crosstalk between protein kinase a and c regulates phospholipase d and f actin formation during sperm Capacitation
Developmental Biology, 2004Co-Authors: Gili Cohen, Sara Rubinstein, Haim BreitbartAbstract:Mammalian spermatozoa should reside in the female reproductive tract for a certain time before gaining the ability to fertilize. During this time, the spermatozoa undergo a series of biochemical processes collectively called Capacitation. We recently demonstrated that actin polymerization is a necessary step in the cascade leading to Capacitation. We demonstrate here for the first time a role for phospholipase D (PLD) in the induction of actin polymerization and Capacitation in spermatozoa. The involvement of PLD is supported by specific inhibition of F-actin formation during sperm Capacitation by PLD inhibitors and the stimulation of fast F-actin formation by exogenous PLD or phosphatidic acid (PA). Moreover, PLD activity is enhanced during Capacitation before actin polymerization. Protein kinase A (PKA), known to be active in sperm Capacitation, and protein kinase C (PKC), involved in the acrosome reaction, can both activate PLD and actin polymerization. We suggest that PKA- and PKC-dependent signal transduction pathways can potentially lead to PLD activation; however, under physiological conditions, actin polymerization depends primarily on PKA activity. Activation of PKA during Capacitation causes inactivation of phospholipase C, and as a result, PKC activation is prevented. It appears that PKA activation promotes sperm Capacitation whereas early activation of PKC during Capacitation would jeopardize this process.
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remodeling of the actin cytoskeleton during mammalian sperm Capacitation and acrosome reaction
Biology of Reproduction, 2003Co-Authors: Ephraim Brener, Gili Cohen, Sara Rubinstein, Keren Shternall, Joel Rivlin, Haim BreitbartAbstract:The sperm acrosome reaction and penetration of the egg follow zona pellucida binding only if the sperm has previously undergone the poorly understood maturation process known as Capacitation. We demonstrate here that in vitro Capacitation of bull, ram, mouse, and human sperm was accompanied by a time-dependent increase in actin polymerization. Induction of the acrosome reaction in capacitated cells initiated fast F-actin breakdown. Incubation of sperm in media lacking BSA or methyl-b-cyclodextrin, Ca21, or NaHCO3, components that are all required for Capacitation, prevented actin polymerization as well as Capacitation, as assessed by the ability of the cells to undergo the acrosome reaction. Inhibition of F-actin formation by cytochalasin D blocked sperm Capacitation and reduced the in vitro fertilization rate of metaphase II-arrested mouse eggs. It has been suggested that protein tyrosine phosphorylation may represent an important regulatory pathway that is associated with sperm Capacitation. We show here that factors known to stimulate sperm protein tyrosine phosphorylation (i.e., NaHCO3, cAMP, epidermal growth factor, H2O2, and sodium vanadate) were able to enhance actin polymerization, whereas inhibition of tyrosine kinases prevented F-actin formation. These data suggest that actin polymerization may represent an important regulatory pathway in with sperm Capacitation, whereas F-actin breakdown occurs before the acrosome reaction. acrosome reaction, gamete biology, in vitro fertilization, sperm, sperm Capacitation
Claude Gagnon - One of the best experts on this subject based on the ideXlab platform.
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cyclic adenosine 3 5 monophosphate dependent regulation of protein tyrosine phosphorylation in relation to human sperm Capacitation and motility
Biology of Reproduction, 1996Co-Authors: P Leclerc, E De Lamirande, Claude GagnonAbstract:The involvement of cAMP in the process of sperm Capacitation has been the subject of several studies. In addition, the importance of protein-tyrosine phosphorylation in this process has been investigated, although only a few studies have been reported in the human. Since agents regulating the intracellular concentrations of cAMP affect sperm Capacitation rates, the role of cAMP on the expression of phosphotyrosine-containing proteins was investigated during human sperm Capacitation. Fetal cord serum ultrafiltrate, a known Capacitation inducer in human spermatozoa, caused an increase in the phosphotyrosine content of 105- and 81-kDa proteins (p105 and p81), the two major phosphotyrosine-containing proteins of human spermatozoa. Similar effects were observed when spermatozoa were incubated with phosphodiesterase inhibitors or cell-permeant cAMP analogs, suggesting that cAMP is involved in these two processes. Forskolin, an adenylyl cyclase activator, also caused an increase in both sperm Capacitation rates and tyrosine phosphorylation of p105 and p81, while 12-O-tetradecanoyl phorbol 13-acetate stimulated both Capacitation and tyrosine phosphorylation of p105 and p81 only when spermatozoa were incubated in the presence of bicarbonate, in agreement with its reported effects on cAMP production and hamster sperm Capacitation. The inhibition of these phenomena by cAMP-dependent protein kinase inhibitors, and the stimulation by protein phosphatase inhibitors, suggest that Ser/Thr protein phosphorylation plays an important role in the regulation of both sperm Capacitation and protein-tyrosine phosphorylation pathways. However, observations that both calyculin A and okadaic acid stimulated sperm Capacitation, whereas only calyculin A increased p105 and p81 phosphotyrosine content and sperm velocity, suggest that protein phosphatase PP1 is involved in the two latter phenomena while PP2A mediates sperm Capacitation. These results suggest that divergent pathways might regulate tyrosine phosphorylation of p105 and p81 and sperm Capacitation after cAMP-dependent phosphorylation of an intermediate protein.
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low levels of nitric oxide promote human sperm Capacitation in vitro
Journal of Andrology, 1995Co-Authors: Armand Zini, E De Lamirande, Claude GagnonAbstract:: The influence of nitric oxide on human sperm hyperactivation and Capacitation, as well as its mechanism of action and its possible origin from spermatozoa were studied. Percoll-washed spermatozoa from healthy volunteers were incubated in Ham's F-10 medium supplemented or not with the nitric oxide-releasing agents, diethylamine-NONOate or spermine-NONOate, in combination or not with superoxide dismutase or catalase (scavengers for the superoxide anion and for hydrogen peroxide, respectively), or with sodium nitrate, sodium nitrite, or preincubated NONOates. Sperm hyperactivation, Capacitation, and nitric oxide synthase activity were determined. High concentrations (0.3 to 1 mM) of NONOates reduced sperm motility. However, a lower concentration (0.1 mM) of the two NONOates had no effect on the percentage of sperm motility or of hyperactivation but resulted in a significant increase in sperm Capacitation (24% +/- 4%) when compared to that of control spermatozoa (Ham's F-10 alone, 12% +/- 2%). Nitric oxide released by the NONOates appeared responsible for this effect because sodium nitrate or nitrite or preincubated NONOates (to exhaust the formation of nitric oxide) had no influence on sperm Capacitation. Catalase, but not superoxide dismutase, abolished the capacitating action of the NONOates. No nitric oxide synthase activity was detected in spermatozoa, whether they were in their basal state or already capacitated. Furthermore, the nitric oxide synthetase inhibitor L-NG nitroarginine methyl ester did not block sperm Capacitation induced by fetal cord serum ultrafiltrate. It is therefore concluded that, although spermatozoa do not possess detectable nitric oxide synthase activity, low levels of nitric oxide induce human sperm Capacitation, and this action likely involves hydrogen peroxide.
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human sperm hyperactivation and Capacitation as parts of an oxidative process
Free Radical Biology and Medicine, 1993Co-Authors: De Lamirande E, Claude GagnonAbstract:Capacitation of spermatozoa is essential for fertilization ans is visually characterized by hyperactivated motility. Previous reports have shown that foetal cord serum (FCS) and superoxide anion, O2⋅, can trigger human sperm hyperactivation (HA) and Capacitation and that superoxide dismutase (SOD) could prevent these processes. We investigated further the role of O2⋅ and FCS components in human spern HA and Capacitation. Percoll-washed spermatozoa were incubated, at 37°C, in Ham's F-10 medium with 75% of FCS, dialyzed FCS (> 12kdD), ultrafiltrate from FCS (FCSu;<3kD), or xanthine + xanthine oxidase + catalase (X + XO + cat). Spermatozoa incubated with FCSu were also supplemented with catalse to prevent the loss of motility often after 2–3 h of incubation. FCS and dialyzed FCS induced significant levels of HA (10 ±1% and 7.7 ±0.7%, respectively) that were, however, lower than those observed with FCSu (19 ± 1%) or X + XO + cat (16 ± 2%). Similar results were obtained when the lysophorphatidylcholine-induced acrosome raction (LPC-AR, a measure of sperm Capacitation) was evaluated. The presence of SOD in the incubation medium blocked the induction of HA and capaciatation by FCS. FCSu, X + XO + cat, as well as the spontaenous HA and Capacitation. The enzymatic activity of SOD was needed for the prevention of these processes. Desferioxamine, up to 100 μM, had no effect on HA and LPC-AR iniduced by FCSu and X + XO + cat. Addition of SOD to already hyperactivated spermatozoa reversed the HA. These data suggest that spermatozoa need a sustaned O2⋅ generation to maintain HA and proceed to Capacitation. We hypothesize that FDSu or the O2⋅ generated by X + XO + cat activate enzymeds, possible a reduced nicotinamide adeninie dinucleotide dinucleotide phosphate [NAD(P)H] oxidase at the level of sperm membrane.
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a positive role for the superoxide anion in triggering hyperactivation and Capacitation of human spermatozoa
International Journal of Andrology, 1993Co-Authors: E De Lamirande, Claude GagnonAbstract:Summary Capacitation of human spermatozoa is essential for fertilization, and is characterized visually by hyperactivated motility. We have investigated whether reactive oxygen species could induce these two events in human spermatozoa. The addition of xanthine + xanthine oxidase + catalase (X+XO+CAT: generation of superoxide anion and removal of hydrogen peroxide) and foetal cord serum (FCS), a known biological inducer of Capacitation and hyperactivation, to spermatozoa, induced levels of hyperactivation (15.4 ± 1.6% and 8.0 ± 1.0%, respectively) which were significantly higher than that of controls (5.4 ± 0.6%). The hyperactivation measured was part of the Capacitation process. Furthermore, the addition of superoxide dismutase prevented the Capacitation and hyperactivation induced by X+XO+CAT or by FCS. These results suggest that the superoxide anion may be involved in Capacitation and hyperactivation of human spermatozoa.
Janice L Bailey - One of the best experts on this subject based on the ideXlab platform.
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a differential mechanism is involved during heparin and cryopreservation induced Capacitation of bovine spermatozoa
Biology of Reproduction, 2003Co-Authors: Nathaly Cormier, Janice L BaileyAbstract:After ejaculation, mammalian spermatozoa must undergo Capacitation to fertilize. Capacitation of bovine spermatozoa occurs in vitro in medium supplemented with heparin. Semen cryopreservation is an important tool for assisted reproduction, although the fertility of frozen-thawed spermatozoa is reduced, possibly due to precocious Capacitation-like changes that are known to occur. Our purpose was to clarify the mechanisms involved in bull sperm cryoCapacitation induced by cryopreservation. Our general hypothesis is that the signaling pathways that lead to Capacitation are triggered by the cryopreservation procedure. Ejaculated bovine semen was divided into two aliquots and diluted in extender; one was then kept fresh, whereas the second was cryopreserved. Western blots of extracted sperm proteins with anti-phosphotyrosine antibody showed that Capacitation, induced by either heparin in fresh sperm or cryopreservation (cryoCapacitation), is associated with a differential profile of phosphotyrosine-containing proteins. Immunolocalization of phosphotyrosine-containing proteins in the fresh and cryopreserved spermatozoa showed that, after thawing, cryocapacitated sperm displayed labeling over the acrosomal region, whereas for fresh sperm, this labeling appeared after 5-h incubation with heparin. The chlortetracycline assay and the ability of the sperm to undergo the lysophosphatidylcholine-induced acrosome reaction were used to confirm that a subpopulation of cryopreserved sperm is capacitated at thawing, irrespective of heparin inclusion. Since glucose is known to inhibit heparin-induced Capacitation, the semen extender was modified to include glucose as a means of inhibiting cryoCapacitation; however, cryoCapacitation was not prevented according to the chlortetracycline assay and profile of phosphotyrosine-containing sperm proteins.
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Capacitation is associated with tyrosine phosphorylation and tyrosine kinase like activity of pig sperm proteins
Biology of Reproduction, 2001Co-Authors: Steve Tardif, Charlotte Dube, Simone Chevalier, Janice L BaileyAbstract:Capacitation represents the final maturational steps that render mammalian sperm competent to fertilize, either in vivo or in vitro. Capacitation is defined as a series of events that enables sperm to bind the oocyte and undergo the acrosome reaction in response to the zona pellucida. Although the molecular mechanisms involved are not fully understood, sperm protein phosphorylation is associated with Capacitation. The hypothesis of this study is that protein tyrosine phosphorylation and kinase activity mediate Capacitation of porcine sperm. Fresh sperm were incubated in noncapacitating or capacitating media for various times. Proteins were extracted with SDS, subjected to SDS-PAGE, and immunoblotted with an antiphosphotyrosine antibody. An Mr 32 000 tyrosine-phosphorylated protein (designated as p32) appeared only when the sperm were incubated in capacitating medium and concomitant with Capacitation as assessed by the ionophore-induced acrosome reaction. The p32 was soluble in Triton X-100. Fractionation of sperm proteins with Triton X-114 demonstrated that after Capacitation, this tyrosine phosphoprotein is located in both the cytosol and the membrane. Enzyme renaturation of sperm proteins was conducted in gels with or without either poly glu:tyr (a tyrosine kinase substrate) or kemptide (a protein kinase A substrate). An Mr 32 000 enzyme with kinase behavior was observed in all gels but was preferentially phosphorylated on tyrosine, as assessed by phosphorimagery and by thin layer chromotography to identify the phosphoamino acids. Indirect immunolocalization showed that the phosphotyrosine residues redistribute to the acrosome during Capacitation, which is an appropriate location for a protein involved in the acquisition of fertility. gamete biology, kinases, signal transduction, sperm, sperm Capacitation,
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Capacitation of mouse spermatozoa i correlation between the Capacitation state and protein tyrosine phosphorylation
Development, 1995Co-Authors: Pablo E Visconti, Janice L Bailey, Grace D Moore, Patricia Oldsclarke, Gregory S. KopfAbstract:The molecular basis of mammalian sperm Capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that Capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for Capacitation, prevent the sperm from undergoing Capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of Capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm Capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo Capacitation in vitro, do not display this Capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
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Capacitation of mouse spermatozoa ii protein tyrosine phosphorylation and Capacitation are regulated by a camp dependent pathway
Development, 1995Co-Authors: Pablo E Visconti, P Leclerc, Janice L Bailey, Grace D Moore, Patricia Oldsclarke, Stephanie A Connors, Gregory S. KopfAbstract:In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129–1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of M(r) 40,000-120,000 was correlated with the Capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the Capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support Capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote Capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the Capacitation-dependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting Capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentration-dependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and Capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.