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Claude Gagnon - One of the best experts on this subject based on the ideXlab platform.
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reactive oxygen species modulate independent protein phosphorylation pathways during human Sperm Capacitation
Free Radical Biology and Medicine, 2006Co-Authors: Cristian Oflaherty, Eve De Lamirande, Claude GagnonAbstract:Abstract Spermatozoa must undergo Capacitation to acquire fertilizing ability. Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide (H 2 O 2 ), and nitric oxide (NO ), are involved in this process. We investigated the roles and interactions of ROS, the ERK cascade, and the phosphoinositide 3-kinase (PI3K)/Akt axis during human Sperm Capacitation. Two different agents, fetal cord serum ultrafiltrate and bovine serum albumin, similarly promoted Capacitation and the associated phosphorylation of protein tyrosine residues (P-Tyr), threonine–glutamine–tyrosine (P-Thr-Glu-Tyr-P) motif, and MEK-like proteins (P-MEK-like proteins). Components of the ERK pathway modulated these phosphorylation events. ROS increased P-MEK-like proteins and NO induced P-Thr-Glu-Tyr-P, possibly by acting on or downstream of Ras. The PI3K/Akt axis participated in Capacitation and phosphorylation of Tyr and Thr-Glu-Tyr but not MEK-like proteins. H 2 O 2 and NO induced P-Tyr even in the presence of ERK pathway inhibitors, indicating that ROS also act downstream of this pathway. These new results indicate that ROS act on different transduction elements during Sperm Capacitation and regulate phosphorylation events that occur in parallel pathways that eventually lead to late phosphorylation of Tyr. These new data reinforce the concept that a complex network of differentially modulated pathways is needed for Spermatozoa to become capacitated.
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Activation of protein kinase A during human Sperm Capacitation and acrosome reaction.
Journal of andrology, 2002Co-Authors: Linda Lefièvre, Eve De Lamirande, Pablo E Visconti, Kula N. Jha, Claude GagnonAbstract:Spermatozoa undergo a variety of changes during their life that are prerequisites to their maturation and ability to fertilize eggs. Mammalian Sperm Capacitation and acrosome reaction are regulated by signal transduction systems involving cyclic adenosine monophosphate (cAMP) as a second messenger. This second messenger acts through the activation of protein kinase A (PKA) and indirectly regulates protein tyrosine phosphorylation. cAMP levels are controlled by a balance of phosphodiesterases (PDEs) and adenylyl cyclase (AC) enzymatic activities, which are responsible for its degradation and production, respectively. The aim of this study was to evaluate the possible relationship between the intracellular levels of cAMP and PDE and PKA activities during human Sperm Capacitation induced by fetal cord serum ultrafiltrate (FCSu) and acrosome reaction induced by calcium ionophore A23187. We report that PKA activity was higher in capacitating than in noncapacitating Spermatozoa and that intracellular levels of cAMP decreased but that PDE activity remained constant during Capacitation. The acrosome reaction induced by A23187 was associated with increases in cAMP and PKA activity but not in PDE activity. These results strongly suggest that net cAMP concentration is under the control of AC, since PDE activity is constant during Sperm Capacitation and the acrosome reaction. Moreover, the results suggest that low levels of cAMP are sufficient for Capacitation and PKA activation and/or that the cAMP concentration measured in whole Spermatozoa does not reflect the effective intracellular cAMP levels present in specific compartments of these cells.
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Nitric Oxide Regulates Human Sperm Capacitation and Protein-Tyrosine Phosphorylation In Vitro
Biology of reproduction, 1999Co-Authors: Maria Belén Herrero, Eve De Lamirande, Claude GagnonAbstract:The aim of the present study was to investigate whether the generation of nitric oxide by human Spermatozoa is associated with human Sperm Capacitation and with the tyrosine phosphorylation of Sperm proteins. Human Spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed Spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of Sperm proteins. A tight correlation between Capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human Sperm Capacitation and emphasize the importance of oxidoreduction reactions in the fine control of Sperm physiology.
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Paradoxical effect of reagents for sulfhydryl and disulfide groups on human Sperm Capacitation and superoxide production
Free radical biology & medicine, 1998Co-Authors: Eve De Lamirande, Claude GagnonAbstract:Spermatozoa must undergo Capacitation prior to fertilization. In humans, this process appears regulated by oxidoreduction reactions. We investigated the possibility that these reactions involved the sulfhydryl-disulfide pair, which offers a reversible regulation of cellular processes. The effects of reagents targeted for sulfhydryl and disulfide groups on human Sperm Capacitation, superoxide (O2-.) generation and protein tyrosine phosphorylation were evaluated. The sulfhydryl targeted agents, phenylarsine oxide (PAO), diamide, dithiopyridine (DTP), N-ethylmaleimide (NEM), maleimidylpropionyl biocytin (MPB), p-chloromercuribenzoic acid (PCMB), and bromobimane analogs (mBBr and qBBr) triggered Sperm Capacitation to levels comparable to those observed with a biological inducer, fetal cord serum ultrafiltrate (FCSu). Capacitation induced by NEM, MPB, PCMB, and PAO was prevented by superoxide dismutase (SOD) and associated with an increased Sperm production of O2-.. However, SOD did not affect the increase in protein tyrosine phosphorylation of Spermatozoa treated with NEM, PAO, or MPB. Disulfide reductants, dithiothreitol (DTT), thioredoxin (TRX), glutathione (GSH), tris-(2-carboxyethyl) phosphine (TCEP), and tris-(2-cyanoethyl) phosphine (TCP) partially to totally inhibited FCSu-induced Sperm Capacitation and O2-. production. TCEP, DTT, and TRX decreased the Capacitation-associated tyrosine phosphorylation of Sperm proteins. The strong time-dependent increase of Sperm membrane sulfhydryl groups exposed to the extracellular space occurring during the first hour of Capacitation could indicate an important rearrangement of sulfhydryl carrying proteins during the initiation of Capacitation. Therefore, protein sulfhydryl-disulfide status may be important for the regulation of human Sperm Capacitation and the mechanisms involved may be complex and multifactorial.
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Human Sperm Capacitation Induced by Biological Fluids and Progesterone, but Not by NADH or NADPH, Is Associated With the Production of Superoxide Anion
Journal of andrology, 1998Co-Authors: E. De Lamirande, A. Harakat, Claude GagnonAbstract:: Recent evidence indicated that human Sperm Capacitation is associated with an increased production of superoxide anion (O2.−). To further study the role and importance of (O2.−). in Capacitation, we investigated whether the (O2.−). generation is a general feature of capacitating Spermatozoa, irrespective of the inducer used, and is correlated with Capacitation levels and increased tyrosine phosphorylation of two Sperm proteins (p105/p81). We also studied the time courses of (O2.−). production and action. Percoll-washed human Spermatozoa were incubated in Ham's F-10 medium, supplemented or not supplemented with various Capacitation inducers and in the presence or absence of superoxide dismutase (SOD). Sperm Capacitation was measured by induction of the acrosome reaction with lysophosphatidylcholine, (O2.−). production was measured by chemiluminescence, and tyrosine phosphorylation was measured by immunodetection after electrophoresis and western blotting of Sperm proteins. Progesterone and ultrafiltrates of human fetal cord serum, follicular fluid, and seminal plasma individually promoted Sperm generation of (O2.−)., tyrosine phosphorylation of p105/p81, and Capacitation. Fetal cord serum ultrafiltrate triggered a five-fold higher (O2.−). production than the other inducers (1,700 ± 300 and 300 to 400 mV/10s/8×10 cells, respectively), a phenomenon possibly associated with the higher potency of this fluid to promote Sperm hyperactivation. The production of (O2.−). by Spermatozoa was rapid and transient. SOD prevented Sperm Capacitation triggered by the inducers mentioned above, but only when SOD was added at the beginning of incubation, and not after 30 minutes, indicating that the (O2.−). initiates a chain of early events leading to Sperm Capacitation. NADH and NADPH (5 mM) triggered Sperm Capacitation and phosphorylation of p105/p81, but these processes were not prevented by SOD or catalase, nor were they associated with an increased (O2.−). production. Therefore, these cofactors appeared to act by mechanisms different from those used by the other inducers studied. The Sperm enzyme responsible for the (O2.−). generation may be very different from the NADPH oxidase of neutrophils.
Eve De Lamirande - One of the best experts on this subject based on the ideXlab platform.
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Control of superoxide and nitric oxide formation during human Sperm Capacitation
Free radical biology & medicine, 2009Co-Authors: Eve De Lamirande, Geneviève Lamothe, Michèle VillemureAbstract:We studied the modulation of superoxide anion (O(2).(-)) and nitric oxide (NO.) generation during human Sperm Capacitation (changes needed for the acquisition of fertility). The production of NO. (diaminofluorescein-2 fluorescence assay), but not that of O(2).(-) (luminescence assay), related to Sperm Capacitation was blocked by inhibitors of protein kinase C, Akt, protein tyrosine kinase, etc., but not by those of protein kinase A. Extracellular calcium (Ca(2+)) controlled O(2).(-) synthesis but extra- and intracellular Ca(2+) regulated NO. formation. Zinc inhibited Capacitation and formation of O(2).(-) and NO.. Zinc chelators (TPEN and EDTA) and sulfhydryl-targeted compounds (diamide and N-ethylmaleimide) stimulated Capacitation and formation of O(2).(-) and NO.; superoxide dismutase (SOD) and nitric oxide synthase inhibitor (L-NMMA) prevented these events. Diphenyliodonium (flavoenzyme inhibitor) blocked Capacitation and related O(2).(-) synthesis but promoted NO. formation, an effect canceled by SOD and L-NMMA. NADPH induced Capacitation and NO. (but not O(2).(-)) synthesis and these events were blocked by L-NMMA and not by SOD. Integration of these data on O(2).(-) and NO. production during Capacitation reinforces the concept that a complex, but flexible, network of factors is involved and probably is associated with rescue mechanisms, so that Spermatozoa can achieve successful fertilization.
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reactive oxygen species modulate independent protein phosphorylation pathways during human Sperm Capacitation
Free Radical Biology and Medicine, 2006Co-Authors: Cristian Oflaherty, Eve De Lamirande, Claude GagnonAbstract:Abstract Spermatozoa must undergo Capacitation to acquire fertilizing ability. Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide (H 2 O 2 ), and nitric oxide (NO ), are involved in this process. We investigated the roles and interactions of ROS, the ERK cascade, and the phosphoinositide 3-kinase (PI3K)/Akt axis during human Sperm Capacitation. Two different agents, fetal cord serum ultrafiltrate and bovine serum albumin, similarly promoted Capacitation and the associated phosphorylation of protein tyrosine residues (P-Tyr), threonine–glutamine–tyrosine (P-Thr-Glu-Tyr-P) motif, and MEK-like proteins (P-MEK-like proteins). Components of the ERK pathway modulated these phosphorylation events. ROS increased P-MEK-like proteins and NO induced P-Thr-Glu-Tyr-P, possibly by acting on or downstream of Ras. The PI3K/Akt axis participated in Capacitation and phosphorylation of Tyr and Thr-Glu-Tyr but not MEK-like proteins. H 2 O 2 and NO induced P-Tyr even in the presence of ERK pathway inhibitors, indicating that ROS also act downstream of this pathway. These new results indicate that ROS act on different transduction elements during Sperm Capacitation and regulate phosphorylation events that occur in parallel pathways that eventually lead to late phosphorylation of Tyr. These new data reinforce the concept that a complex network of differentially modulated pathways is needed for Spermatozoa to become capacitated.
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Activation of protein kinase A during human Sperm Capacitation and acrosome reaction.
Journal of andrology, 2002Co-Authors: Linda Lefièvre, Eve De Lamirande, Pablo E Visconti, Kula N. Jha, Claude GagnonAbstract:Spermatozoa undergo a variety of changes during their life that are prerequisites to their maturation and ability to fertilize eggs. Mammalian Sperm Capacitation and acrosome reaction are regulated by signal transduction systems involving cyclic adenosine monophosphate (cAMP) as a second messenger. This second messenger acts through the activation of protein kinase A (PKA) and indirectly regulates protein tyrosine phosphorylation. cAMP levels are controlled by a balance of phosphodiesterases (PDEs) and adenylyl cyclase (AC) enzymatic activities, which are responsible for its degradation and production, respectively. The aim of this study was to evaluate the possible relationship between the intracellular levels of cAMP and PDE and PKA activities during human Sperm Capacitation induced by fetal cord serum ultrafiltrate (FCSu) and acrosome reaction induced by calcium ionophore A23187. We report that PKA activity was higher in capacitating than in noncapacitating Spermatozoa and that intracellular levels of cAMP decreased but that PDE activity remained constant during Capacitation. The acrosome reaction induced by A23187 was associated with increases in cAMP and PKA activity but not in PDE activity. These results strongly suggest that net cAMP concentration is under the control of AC, since PDE activity is constant during Sperm Capacitation and the acrosome reaction. Moreover, the results suggest that low levels of cAMP are sufficient for Capacitation and PKA activation and/or that the cAMP concentration measured in whole Spermatozoa does not reflect the effective intracellular cAMP levels present in specific compartments of these cells.
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Nitric Oxide Regulates Human Sperm Capacitation and Protein-Tyrosine Phosphorylation In Vitro
Biology of reproduction, 1999Co-Authors: Maria Belén Herrero, Eve De Lamirande, Claude GagnonAbstract:The aim of the present study was to investigate whether the generation of nitric oxide by human Spermatozoa is associated with human Sperm Capacitation and with the tyrosine phosphorylation of Sperm proteins. Human Spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed Spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of Sperm proteins. A tight correlation between Capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human Sperm Capacitation and emphasize the importance of oxidoreduction reactions in the fine control of Sperm physiology.
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Paradoxical effect of reagents for sulfhydryl and disulfide groups on human Sperm Capacitation and superoxide production
Free radical biology & medicine, 1998Co-Authors: Eve De Lamirande, Claude GagnonAbstract:Spermatozoa must undergo Capacitation prior to fertilization. In humans, this process appears regulated by oxidoreduction reactions. We investigated the possibility that these reactions involved the sulfhydryl-disulfide pair, which offers a reversible regulation of cellular processes. The effects of reagents targeted for sulfhydryl and disulfide groups on human Sperm Capacitation, superoxide (O2-.) generation and protein tyrosine phosphorylation were evaluated. The sulfhydryl targeted agents, phenylarsine oxide (PAO), diamide, dithiopyridine (DTP), N-ethylmaleimide (NEM), maleimidylpropionyl biocytin (MPB), p-chloromercuribenzoic acid (PCMB), and bromobimane analogs (mBBr and qBBr) triggered Sperm Capacitation to levels comparable to those observed with a biological inducer, fetal cord serum ultrafiltrate (FCSu). Capacitation induced by NEM, MPB, PCMB, and PAO was prevented by superoxide dismutase (SOD) and associated with an increased Sperm production of O2-.. However, SOD did not affect the increase in protein tyrosine phosphorylation of Spermatozoa treated with NEM, PAO, or MPB. Disulfide reductants, dithiothreitol (DTT), thioredoxin (TRX), glutathione (GSH), tris-(2-carboxyethyl) phosphine (TCEP), and tris-(2-cyanoethyl) phosphine (TCP) partially to totally inhibited FCSu-induced Sperm Capacitation and O2-. production. TCEP, DTT, and TRX decreased the Capacitation-associated tyrosine phosphorylation of Sperm proteins. The strong time-dependent increase of Sperm membrane sulfhydryl groups exposed to the extracellular space occurring during the first hour of Capacitation could indicate an important rearrangement of sulfhydryl carrying proteins during the initiation of Capacitation. Therefore, protein sulfhydryl-disulfide status may be important for the regulation of human Sperm Capacitation and the mechanisms involved may be complex and multifactorial.
Haim Breitbart - One of the best experts on this subject based on the ideXlab platform.
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Remodeling of the Actin Cytoskeleton During Mammalian Sperm Capacitation
2016Co-Authors: Acrosome Reaction, Gili Cohen, Ephraim Brener, Keren Shternall, Joel Rivlin, Sara Rubinstein, Haim BreitbartAbstract:The Sperm acrosome reaction and penetration of the egg fol-low zona pellucida binding only if the Sperm has previously un-dergone 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 meth-yl-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 sug-gest that actin polymerization may represent an important reg-ulatory pathway in with Sperm Capacitation, whereas F-actin breakdown occurs before the acrosome reaction. acrosome reaction, gamete biology, in vitro fertilization, Sperm, Sperm capacitatio
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AKAP3 degradation in Sperm Capacitation is regulated by its tyrosine phosphorylation
Biochimica et biophysica acta, 2015Co-Authors: Ruth Vizel, Pnina Hillman, Debby Ickowicz, Haim BreitbartAbstract:Abstract Background The A-kinase anchoring protein (AKAP) family is essential for Sperm motility, Capacitation and the acrosome reaction. PKA-dependent protein tyrosine phosphorylation occurs in mammalian Sperm Capacitation including AKAP3. In a recent study, we showed that AKAP3 undergoes degradation under Capacitation conditions. Thus, we tested here whether AKAP3 degradation might be regulated by its tyrosine phosphorylation. Methods The intracellular levels of AKAP3 were determined by western blot (WB) analysis using specific anti-AKAP3 antibodies. Tyrosine phosphorylation of AKAP3 was tested by immunoprecipitation and WB analysis. Acrosome reaction was examined using FITC-pisum sativum agglutinin. Results AKAP3 is degraded and undergoes tyrosine-dephosphorylation during Sperm Capacitation and the degradation was reduced by inhibition of tyrosine phosphatase and enhanced by inhibition of tyrosine kinase. Sperm starvation or inhibition of mitochondrial respiration, which reduce cellular ATP levels, significantly accelerated AKAP3 degradation. Treatment with vanadate, or Na+ or bicarbonate depletion, reduced AKAP3-degradation and the AR rate, while antimycin A or NH4Cl elevated both AKAP3-degradation and the AR degree. Treatment of Sperm with NH4Cl enhanced PKA-dependent phosphorylation of four proteins, further supporting the involvement of AKAP3-degradation in Capacitation. To demonstrate more specifically that Sperm Capacitation requires AKAP3-degradation, we inhibited AKAP3-degradation using anti-AKAP3 antibody in permeabilized cells. The anti-AKAP3-antibody induced significant inhibition of AKAP3-degradation and of the AR rate. Conclusion Sperm Capacitation process requires AKAP3-degradation, and the degradation degree is regulated by the level of AKAP3 tyrosine phosphorylation. General significance Better understanding of the molecular mechanisms that mediate Sperm Capacitation can be used for infertility diagnosis, treatment and the developing of male contraceptives.
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Zn2+-stimulation of Sperm Capacitation and of the acrosome reaction is mediated by EGFR activation.
Developmental biology, 2014Co-Authors: Yulia Michailov, Debbi Ickowicz, Haim BreitbartAbstract:Extracellular zinc regulates cell proliferation via the MAP1 kinase pathway in several cell types, and has been shown to act as a signaling molecule. The testis contains a relatively high concentration of Zn(2+), required in both the early and late stages of Spermatogenesis. Despite the clinical significance of this ion, its role in mature Sperm cells is poorly understood. In this study, we characterized the role of Zn(2+) in Sperm Capacitation and in the acrosome reaction. Western blot analysis revealed the presence of ZnR of the GPR39 type in Sperm cells. We previously demonstrated the presence of active epidermal growth factor receptor (EGFR) in Sperm, its possible transactivation by direct activation of G-protein coupled receptor (GPCR), and its involvement in Sperm Capacitation and in the acrosome reaction (AR). We show here that Zn(2+) activates the EGFR during Sperm Capacitation, which is mediated by activation of trans-membrane adenylyl cyclase (tmAC), protein kinase A (PKA), and the tyrosine kinase, Src. Moreover, the addition of Zn(2+) to capacitated Sperm caused further stimulation of EGFR and phosphatydil-inositol-3-kinase (PI3K) phosphorylation, leading to the AR. The stimulation of the AR by Zn(2+) also occurred in the absence of Ca(2+) in the incubation medium, and required the tmAC, indicating that Zn(2+) activates a GPCR. The AR stimulated by Zn(2+) is mediated by GPR39 receptor, PKA, Src and the EGFR, as well as the EGFR down-stream effectors PI3K, phospholipase C (PLC) and protein kinase C (PKC). These data support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways in Sperm Capacitation and the acrosome reaction.
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Dissociation between AKAP3 and PKARII Promotes AKAP3 Degradation in Sperm Capacitation
PloS one, 2013Co-Authors: Pnina Hillman, Ruth Vizel, Debby Ickowicz, Haim BreitbartAbstract:Ejaculated Spermatozoa must undergo a series of biochemical modifications called Capacitation, prior to fertilization. Protein-kinase A (PKA) mediates Sperm Capacitation, although its regulation is not fully understood. Sperm contain several A-kinase anchoring proteins (AKAPs), which are scaffold proteins that anchor PKA. In this study, we show that AKAP3 is degraded in bovine Sperm incubated under Capacitation conditions. The degradation rate is variable in Sperm from different bulls and is correlated with the Capacitation ability. The degradation of AKAP3 was significantly inhibited by MG-132, a proteasome inhibitor, indicating that AKAP3 degradation occurs via the proteasomal machinery. Treatment with Ca2+-ionophore induced further degradation of AKAP3; however, this effect was found to be enhanced in the absence of Ca2+ in the medium or when intracellular Ca2+ was chelated the degradation rate of AKAP3 was significantly enhanced when intracellular space was alkalized using NH4Cl, or when Sperm were treated with Ht31, a peptide that contains the PKA-binding domain of AKAPs. Moreover, inhibition of PKA activity by H89, or its activation using 8Br-cAMP, increased AKAP3 degradation rate. This apparent contradiction could be explained by assuming that binding of PKA to AKAP3 protects AKAP3 from degradation. We conclude that AKAP3 degradation is regulated by intracellular alkalization and PKARII anchoring during Sperm Capacitation.
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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.
Pablo E Visconti - One of the best experts on this subject based on the ideXlab platform.
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Manipulation of bicarbonate concentration in Sperm Capacitation media improves in vitro fertilisation output in porcine species
Journal of animal science and biotechnology, 2019Co-Authors: Cristina Soriano-Úbeda, Pablo E Visconti, Carmen Matás, Jon Romero-aguirregomezcorta, Francisco A. García-vázquezAbstract:The in vivo concentration of bicarbonate (HCO3−), one of the essential Sperm capacitating effectors, varies greatly in the different environments Sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro Sperm Capacitation and fertilisation media usually contains a standard concentration of 25 mmol/L, and one of the main problems presented is the unacceptable high incidence of polySpermy. This work hypothesised that by modifying the HCO3− concentration of the medium, the output of in vitro Sperm Capacitation and fertilisation could be increased. Once exposed to the Capacitation medium, the intracellular pH (pHi) of Spermatozoa increased immediately even at low concentrations of HCO3−, but only extracellular concentrations of and above 15 mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15 mmol/L of HCO3− stimulated Sperm linear motility and increased other late events in Capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25 mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3− concentration to 15 mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%). Optimising HCO3− concentrations allows for establishing an IVF method that significantly reduced porcine polySpermy and increased the production of viable zygotes. A concentration of 15 mmol/L of HCO3− in the medium is sufficient to trigger the in vitro Sperm Capacitation and increase the fertilisation efficiency in porcine.
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Manipulation of bicarbonate concentration in Sperm Capacitation media improves in vitro fertilisation output in porcine species
BMC, 2019Co-Authors: Cristina Soriano-Úbeda, Pablo E Visconti, Carmen Matás, Jon Romero-aguirregomezcorta, Francisco A. García-vázquezAbstract:Abstract Background The in vivo concentration of bicarbonate (HCO3 −), one of the essential Sperm capacitating effectors, varies greatly in the different environments Sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro Sperm Capacitation and fertilisation media usually contains a standard concentration of 25 mmol/L, and one of the main problems presented is the unacceptable high incidence of polySpermy. This work hypothesised that by modifying the HCO3 − concentration of the medium, the output of in vitro Sperm Capacitation and fertilisation could be increased. Results Once exposed to the Capacitation medium, the intracellular pH (pHi) of Spermatozoa increased immediately even at low concentrations of HCO3 −, but only extracellular concentrations of and above 15 mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15 mmol/L of HCO3 − stimulated Sperm linear motility and increased other late events in Capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25 mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3 − concentration to 15 mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%). Conclusions Optimising HCO3 − concentrations allows for establishing an IVF method that significantly reduced porcine polySpermy and increased the production of viable zygotes. A concentration of 15 mmol/L of HCO3 − in the medium is sufficient to trigger the in vitro Sperm Capacitation and increase the fertilisation efficiency in porcine
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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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understanding the molecular basis of Sperm Capacitation through kinase design
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Pablo E ViscontiAbstract:Thirty years ago, on July 25, Steptoe and Edwards reported the birth of Louise Joy Brown, the first successful “Test-Tube” baby (1). This achievement followed a lack of success of in vitro fertilization experiments for almost 80 years since the first attempts in 1878. These early failures were due mainly to a lack of comprehension of Sperm physiology. In the early 1950s, Chang (2) and Austin (3) demonstrated independently that Sperm had to be in the female reproductive tract for a finite period before acquiring fertilizing capacity. This phenomenon is known as Sperm Capacitation. What made this finding a necessary step for the consequent development of in vitro fertilization was the understanding that certain factors in the female were needed for the Sperm to become fertile. A logical follow-up of the discovery of Sperm Capacitation occurred some years later when Chang demonstrated mammalian in vitro fertilization conclusively by showing that eggs from a black rabbit fertilized in vitro by capacitated Sperm from a black male, and transferred to a white female, resulted in the birth of a litter of black offspring (4).
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Activation of protein kinase A during human Sperm Capacitation and acrosome reaction.
Journal of andrology, 2002Co-Authors: Linda Lefièvre, Eve De Lamirande, Pablo E Visconti, Kula N. Jha, Claude GagnonAbstract:Spermatozoa undergo a variety of changes during their life that are prerequisites to their maturation and ability to fertilize eggs. Mammalian Sperm Capacitation and acrosome reaction are regulated by signal transduction systems involving cyclic adenosine monophosphate (cAMP) as a second messenger. This second messenger acts through the activation of protein kinase A (PKA) and indirectly regulates protein tyrosine phosphorylation. cAMP levels are controlled by a balance of phosphodiesterases (PDEs) and adenylyl cyclase (AC) enzymatic activities, which are responsible for its degradation and production, respectively. The aim of this study was to evaluate the possible relationship between the intracellular levels of cAMP and PDE and PKA activities during human Sperm Capacitation induced by fetal cord serum ultrafiltrate (FCSu) and acrosome reaction induced by calcium ionophore A23187. We report that PKA activity was higher in capacitating than in noncapacitating Spermatozoa and that intracellular levels of cAMP decreased but that PDE activity remained constant during Capacitation. The acrosome reaction induced by A23187 was associated with increases in cAMP and PKA activity but not in PDE activity. These results strongly suggest that net cAMP concentration is under the control of AC, since PDE activity is constant during Sperm Capacitation and the acrosome reaction. Moreover, the results suggest that low levels of cAMP are sufficient for Capacitation and PKA activation and/or that the cAMP concentration measured in whole Spermatozoa does not reflect the effective intracellular cAMP levels present in specific compartments of these cells.
P. Leclerc - One of the best experts on this subject based on the ideXlab platform.
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Regulation of protein-tyrosine phosphorylation and human Sperm Capacitation by reactive oxygen derivatives.
Free radical biology & medicine, 1997Co-Authors: P. Leclerc, Eve De Lamirande, Claude GagnonAbstract:Spermatozoa undergoing Capacitation, a necessary prerequisite event to successful fertilization that can be induced in vitro by reactive oxygen species (ROS), generate superoxide anion (O2.-). Because, in neutrophils, the generation of O2.- is associated with tyrosine phosphorylation of several proteins, the aim of the present study was to investigate the association between protein-tyrosine phosphorylation and ROS-induced human Sperm Capacitation. Human Spermatozoa express two major phosphotyrosine-containing proteins of 105 and 81 kDa, the phosphotyrosine content of which is increased when Spermatozoa are incubated under capacitating conditions. Superoxide dismutase and catalase abolish both Sperm Capacitation and tyrosine phosphorylation of p105 and p81, suggesting the involvement of O2.- and hydrogen peroxide in these two processes. Inhibitors of NADPH oxidase, the enzyme responsible for the neutrophil's respiratory burst, decrease both p105 and p81 tyrosine phosphorylation and Sperm Capacitation while hydrogen peroxide stimulates these two processes. Tyrosine phosphorylation of p105 and p81 occurs through a herbimycin A-sensitive tyrosine kinase, and Sperm incubation with phosphotyrosine-protein phosphatase inhibitors results in an increase in phosphotyrosine content of these two proteins. Indirect immunocytochemical studies reveal phosphotyrosine-containing proteins mostly in the principal piece of the flagellum, in agreement with the localization of p105 and p81 in the human Sperm fibrous sheath. Although tyrosine phosphorylation of p105 and p81 and Sperm Capacitation are related in a time-dependent fashion, some discrepancies are observed in the regulation of these two processes according to the redox status of the Spermatozoa.
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regulation of protein tyrosine phosphorylation and human Sperm Capacitation by reactive oxygen derivatives
Free Radical Biology and Medicine, 1997Co-Authors: P. Leclerc, Eve De Lamirande, Claude GagnonAbstract:Abstract Spermatozoa undergoing Capacitation, a necessary prerequisite event to successful fertilization that can be induced in vitro by reactive oxygen species (ROS), generate superoxide anion (O 2 − ). Because, in neutrophils, the generation of O 2 − is associated with tyrosine phosphorylation of several proteins, the aim of the present study was to investigate the association between protein-tyrosine phosphorylation and ROS-induced human Sperm Capacitation. Human Spermatozoa express two major phosphotyrosine-containing proteins of 105 and 81 kDa, the phosphotyrosine content of which is increased when Spermatozoa are incubated under capacitating conditions. Superoxide dismutase and catalase abolish both Sperm Capacitation and tyrosine phosphorylation of p105 and p81, suggesting the involvement of O 2 − and hydrogen peroxide in these two processes. Inhibitors of NADPH oxidase, the enzyme responsible for the neutrophil's respiratory burst, decrease both p105 and p81 tyrosine phosphorylation and Sperm Capacitation while hydrogen peroxide stimulates these two processes. Tyrosine phosphorylation of p105 and p81 occurs through a herbimycin A-sensitive tyrosine kinase, and Sperm incubation with phosphotyrosine-protein phosphatase inhibitors results in an increase in phosphotyrosine content of these two proteins. Indirect immunocytochemical studies reveal phosphotyrosine-containing proteins mostly in the principal piece of the flagellum, in agreement with the localization of p105 and p81 in the human Sperm fibrous sheath. Although tyrosine phosphorylation of p105 and p81 and Sperm Capacitation are related in a time-dependent fashion, some discrepancies are observed in the regulation of these two processes according to the redox status of the Spermatozoa. Copyright © 1997 Elsevier Science Inc.
