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Michaela Luconi - One of the best experts on this subject based on the ideXlab platform.
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src activation triggers capacitation and Acrosome Reaction but not motility in human spermatozoa
Human Reproduction, 2008Co-Authors: Gabriele Varano, Elisabetta Baldi, Gianni Forti, Adriana Lombardi, Giulia Cantini, Michaela LuconiAbstract:BACKGROUND: Protein tyrosine phosphorylation is one of the main processes associated with sperm activation. Although this process and its targets have been well characterized, only few tyrosine kinases have been identified so far and their roles in spermatozoa are still largely unknown. In this study, we report the presence and localization of Src kinase in ejaculated human spermatozoa and investigate its role in regulating the processes underlying sperm activation. METHODS AND RESULTS: Specific anti-Src antibodies, against different epitopes of the protein, identified a single band of approximately 70 kDa relating to a protein which is mainly localized in the post-acrosomal region of the head, neck and midpiece. By immunoprecipitation and immunofluorescence techniques performed with antibodies against Src phosphorylated at Tyr416, which identifies the active kinase, we showed an increased phosphorylation during sperm capacitation. Blocking Src activity with SU6656 resulted in a significant reduction in the protein tyrosine phosphorylation. Moreover, this inhibitor also blocked the progesterone-induced Acrosome Reaction and interfered with the calcium response to progesterone evaluated in fura-2-loaded spermatozoa. No effect on sperm motility and hyperactivation resulted from incubation with SU6656. CONCLUSIONS: We identified a novel Src isoform in human spermatozoa, which appears to be involved in regulating sperm capacitation, calcium fluxes, tyrosine phosphorylation and Acrosome Reaction.
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src activation triggers capacitation and Acrosome Reaction but not motility in human spermatozoa
Human Reproduction, 2008Co-Authors: Gabriele Varano, Elisabetta Baldi, Gianni Forti, Adriana Lombardi, Giulia Cantini, Michaela LuconiAbstract:BACKGROUND: Protein tyrosine phosphorylation is one of the main processes associated with sperm activation.Although this process and its targets have been well characterized, only few tyrosine kinases have been identifiedso far and their roles in spermatozoa are still largely unknown. In this study, we report the presence and localizationof Src kinase in ejaculated human spermatozoa and investigate its role in regulating the processes underlying spermactivation. METHODS AND RESULTS: Specific anti-Src antibodies, against different epitopes of the protein, ident-ifiedasinglebandof 70 kDarelatingtoaproteinwhichismainlylocalizedin thepost-acrosomalregionofthehead,neck and midpiece. By immunoprecipitation and immunofluorescence techniques performed with antibodies againstSrc phosphorylated at Tyr416, which identifies the active kinase, we showed an increased phosphorylation duringsperm capacitation. Blocking Src activity with SU6656 resulted in a significant reduction in the protein tyrosine phos-phorylation. Moreover, this inhibitor also blocked the progesterone-induced Acrosome Reaction and interferedwith the calcium response to progesterone evaluated in fura-2-loaded spermatozoa. No effect on sperm motilityand hyperactivation resulted from incubation with SU6656. CONCLUSIONS: We identified a novel Src isoformin human spermatozoa, which appears to be involved in regulating sperm capacitation, calcium fluxes, tyrosinephosphorylation and Acrosome Reaction.
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intracellular events and signaling pathways involved in sperm acquisition of fertilizing capacity and Acrosome Reaction
Frontiers in Bioscience, 2000Co-Authors: Elisabetta Baldi, Michaela Luconi, Lorella Bonaccorsi, Monica Muratori, Gianni FortiAbstract:Two processes, namely capacitation and Acrosome Reaction, are of fundamental importance in the fertilization of oocyte by spermatozoon. Physiologically occurring in the female genital tract, capacitation is a complex process, which renders the sperm cell capable for specific interaction with the oocyte. During capacitation, modification of membrane characteristics, enzyme activity and motility properties of spermatozoa render these cells able to penetrate oocyte investments and responsive to stimuli that induce Acrosome Reaction prior to fertilization. Physiological Acrosome Reaction occurs upon interaction of the spermatozoon with the zona pellucida protein ZP3. This is followed by liberation of several acrosomal enzymes and other constituents that facilitate penetration of the zona and expose molecules on the sperm equatorial segment that allows fusion of sperm membrane with the oolemma. The molecular mechanisms and the signal transduction pathways mediating the processes of capacitation and Acrosome Reaction have been partially defined, and appear to involve modifications of intracellular calcium and other ions, lipid transfer and phospholipid remodeling in sperm plasma membrane as well as changes in protein phosphorylation. Some of the kinases and phosphorylated proteins that are involved in the processes of capacitation and Acrosome Reaction have been now characterized. Characterization of sperm receptors to physiological inducers of Acrosome Reaction is in progress. This review summarizes the main signal transduction pathways involved in capacitation and Acrosome Reaction.Furthermore, the mechanisms underlying sperm DNA fragmentation are also briefly reviewed.
Masaru Okabe - One of the best experts on this subject based on the ideXlab platform.
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The Acrosome Reaction: A Historical Perspective
Advances in anatomy embryology and cell biology, 2016Co-Authors: Masaru OkabeAbstract:Acrosome Reaction is often referred to as acrosomal exocytosis, but it differs significantly from normal exocytosis. While the vesicle membrane initially holding excreting molecules remains on the cell surface during exocytosis, the outer acrosomal membrane and plasma membrane are lost by forming vesicles during Acrosome Reaction. In this context, the latter process resembles a release of exosome. However, recent experimental data indicate that the most important roles of Acrosome Reaction lie not in the release of acrosomal contents (or “vesiculated” plasma and outer acrosomal membrane complexes) but rather in changes in sperm membrane. This review describes the mechanism of fertilization vis-a-vis sperm membrane change, with a brief historical overview of the half-century study of Acrosome Reaction.
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most fertilizing mouse spermatozoa begin their Acrosome Reaction before contact with the zona pellucida during in vitro fertilization
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Eiji Fujiwara, Yasutaka Kakiuchi, Yuhkoh Satouh, Kazuyoshi Chiba, Shoji A. Baba, Masaru Okabe, Noritaka HirohashiAbstract:To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the Acrosome Reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm–oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm–zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the Acrosome Reaction using fluorescence microscopy. We found that the spermatozoa that began the Acrosome Reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the Acrosome Reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization.
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Disruption of Mouse CD46 Causes an Accelerated Spontaneous Acrosome Reaction in Sperm
Molecular and cellular biology, 2003Co-Authors: Naokazu Inoue, Tomoko Nakanishi, Masahito Ikawa, Misako Matsumoto, Midori Nomura, Tsukasa Seya, Masaru OkabeAbstract:Human membrane cofactor protein (MCP, CD46) is a ubiquitously expressed protein known to protect cells from complement attack. Interestingly, when we examined the expression of mouse CD46, which we recently cloned, the message was found only in testis and the protein was found on the inner acrosomal membrane of sperm. In order to elucidate the function of CD46, we produced mice carrying a null mutation in the CD46 gene by using homologous recombination. Despite the absence of CD46, the mice were healthy and both sexes were fertile. However, to our surprise, the fertilizing ability of males appeared to be facilitated by disruption of the CD46 gene, as the average number of pups born from CD46(-/-) males was significantly greater than that of wild-type males. It was also revealed that the incidence of the spontaneous Acrosome Reaction doubled in CD46(-/-) sperm compared to that in wild-type sperm. It was assumed that this increase caused the heightened fertilizing ability found in CD46(-/-) sperm. These data suggest that CD46 may have some role in regulating sperm Acrosome Reaction.
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acrosin accelerates the dispersal of sperm acrosomal proteins during Acrosome Reaction
Journal of Biological Chemistry, 1998Co-Authors: Kazuo Yamagata, Masaru Okabe, Keitaro Murayama, Kiyotaka Toshimori, Tomoko Nakanishi, Shinichi Kashiwabara, Tadashi BabaAbstract:Abstract Using homologous recombination, we have previously produced male mice carrying a disruptive mutation (Acr −/−) in the acrosin gene. AlthoughAcr −/− mouse sperm lacking the acrosin protease activity still penetrated the zona pellucida and fertilized the egg, the mutant sperm exhibited a delay in penetration of the zona pellucida solely at the early stages after insemination. To further elucidate the role of acrosin in fertilization, we have examined the involvement of acrosin in the Acrosome Reaction of sperm using theAcr −/− mutant mice. When the ability of sperm to adhere (attach) and bind to the zona pellucida of cumulus-free eggs was assessed in vitro, no significant difference was observed among Acr +/+,Acr +/−, and Acr −/−mouse sperm. Immunocytochemical analysis demonstrated that the release of several acrosomal proteins from the Acrosome ofAcr −/− mouse sperm was significantly delayed during the calcium ionophore- and solubilized zona pellucida-induced Acrosome Reaction, despite normal membrane vesiculation. These data indicate that the delayed sperm penetration of the zona pellucida in the Acr −/− mouse results from the altered rate of protein dispersal from the Acrosome and provide the first evidence that the major role of acrosin is to accelerate the dispersal of acrosomal components during Acrosome Reaction.
Pablo E Visconti - One of the best experts on this subject based on the ideXlab platform.
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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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mouse sperm membrane potential hyperpolarization is necessary and sufficient to prepare sperm for the Acrosome Reaction
Journal of Biological Chemistry, 2012Co-Authors: Jose L De La Vegabeltran, Pablo E Visconti, Dario Krapf, Claudia L Trevino, Claudia Sanchezcardenas, Enrique O Hernandezgonzalez, Eva Wertheimer, Alberto DarszonAbstract:Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pHi) and Ca2+ ([Ca2+]i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K+ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the Acrosome Reaction induced either by depolarization with high K+ or by addition of solubilized zona pellucida (sZP). Moreover, K+ and sZP were also able to increase [Ca2+]i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K+ concentrations inhibited the agonist-induced Acrosome Reaction as well as the increase in [Ca2+]i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the Acrosome Reaction.
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snare complex assembly is required for human sperm Acrosome Reaction
Developmental Biology, 2002Co-Authors: Claudia N Tomes, Pablo E Visconti, Marcela Alejandra Michaut, Gerardo A De Blas, Ulf Matti, Luis S MayorgaAbstract:Abstract Exocytosis of the Acrosome (the Acrosome Reaction) is a terminal morphological alteration that sperm must undergo prior to penetration of the extracellular coat of the egg. Ca 2+ is an essential mediator of this regulated secretory event. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, NSF, and synaptotagmin VI in the human sperm Acrosome Reaction. Interestingly, Rab3A elicits an exocytotic response of comparable magnitude to that of Ca 2+ . Here, we report a direct role for the SNARE complex in the Acrosome Reaction. First, the presence of SNARE proteins is demonstrated by Western blot. Second, the Ca 2+ -triggered Acrosome Reaction is inhibited by botulinum neurotoxins BoNT/A, -E, -C, and -F. Third, antibody inhibition studies show a requirement for SNAP-25, SNAP-23, syntaxins 1A, 1B, 4, and 6, and VAMP 2. Fourth, addition of bacterially expressed SNAP-25 and SNAP-23 abolishes exocytosis. Acrosome Reaction elicited by Rab3-GTP is also inhibited by BoNT/A, -C, and -F. Taken together, these results demonstrate a requirement for members of all SNARE protein families in the Ca 2+ - and Rab3A-triggered Acrosome Reaction. Furthermore, they indicate that the onset of sperm exocytosis relies on the functional assembly of SNARE complexes.
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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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roles of bicarbonate camp and protein tyrosine phosphorylation on capacitation and the spontaneous Acrosome Reaction of hamster sperm
Biology of Reproduction, 1999Co-Authors: Pablo E Visconti, J Stewartsavage, Aida Blasco, Licia Battaglia, Patricia V Miranda, Gregory S Kopf, Jorge G TezonAbstract:Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO 3 -buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO 3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous Acrosome Reaction, did not take place. Addition of 25 mM Na-HCO 3 to NaHCO 3 -free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous Acrosome Reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2,2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO 3 -containing medium inhibited the Acrosome Reaction, with maximal inhibition at 600 μM, and with an EC 50 of 100 μM. Increasing either extracellular or intracellular pH did not induce the Acrosome Reaction in NaHCO 3 -free medium. In contrast, addition of 500 μM dibutyryl cAMP (dbcAMP), alone or together with 100 μM 1-methyl-3-isobutylxanthine (IBMX), induced the Acrosome Reaction in spermatozoa incubated in NaHCO 3 -free medium. These compounds also partially reversed the inhibition of the Acrosome Reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce Acrosome Reactions in cells incubated in Ca 2+ -free medium. When hamster sperm were incubated in the absence of NaHCO 3 or in the presence of NaHCO 3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO 3 , the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX. The induction of the Acrosome Reaction by calcium ionophore A23187 in NaHCO 3 -free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO 3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation.
Alberto Darszon - One of the best experts on this subject based on the ideXlab platform.
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acrosomal alkalization triggers ca2 release and Acrosome Reaction in mammalian spermatozoa
Journal of Cellular Physiology, 2018Co-Authors: Julio C Chavez, Jose L De La Vegabeltran, Omar Jose, Paulina Torres, Takuya Nishigaki, Claudia L Trevino, Alberto DarszonAbstract:The sperm Acrosome Reaction (AR), an essential event for mammalian fertilization, involves Ca2+ permeability changes leading to exocytosis of the acrosomal vesicle. The Acrosome, an intracellular Ca2+ store whose luminal pH is acidic, contains hydrolytic enzymes. It is known that acrosomal pH (pHacr ) increases during capacitation and this correlates with spontaneous AR. Some AR inducers increase intracellular Ca2+ concentration ([Ca2+ ]i ) through Ca2+ release from internal stores, mainly the Acrosome. Catsper, a sperm specific Ca2+ channel, has been suggested to participate in the AR. Curiously, Mibefradil and NNC55-0396, two CatSper blockers, themselves elevate [Ca2+ ]i by unknown mechanisms. Here we show that these compounds, as other weak bases, can elevate pHacr , trigger Ca2+ release from the Acrosome, and induce the AR in both mouse and human sperm. To our surprise, μM concentrations of NNC55-0396 induced AR even in nominally Ca2+ free media. Our findings suggest that alkalization of the Acrosome is critical step for Ca2+ release from the Acrosome that leads to the Acrosome Reaction.
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mouse sperm membrane potential hyperpolarization is necessary and sufficient to prepare sperm for the Acrosome Reaction
Journal of Biological Chemistry, 2012Co-Authors: Jose L De La Vegabeltran, Pablo E Visconti, Dario Krapf, Claudia L Trevino, Claudia Sanchezcardenas, Enrique O Hernandezgonzalez, Eva Wertheimer, Alberto DarszonAbstract:Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pHi) and Ca2+ ([Ca2+]i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K+ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the Acrosome Reaction induced either by depolarization with high K+ or by addition of solubilized zona pellucida (sZP). Moreover, K+ and sZP were also able to increase [Ca2+]i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K+ concentrations inhibited the agonist-induced Acrosome Reaction as well as the increase in [Ca2+]i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the Acrosome Reaction.
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particulate and soluble adenylyl cyclases participate in the sperm Acrosome Reaction
Biochemical and Biophysical Research Communications, 2007Co-Authors: Carmen Beltran, Victor D Vacquier, Gary W Moy, Yanqiu Chen, Jochen Buck, Lonny R Levin, Alberto DarszonAbstract:cAMP is important in sea urchin sperm signaling, yet the molecular nature of the adenylyl cyclases (ACs) involved remained unknown. These cells were recently shown to contain an ortholog of the mammalian soluble adenylyl cyclase (sAC). Here, we show that sAC is present in the sperm head and as in mammals is stimulated by bicarbonate. The Acrosome Reaction (AR), a process essential for fertilization, is influenced by the bicarbonate concentration in seawater. By using functional assays and immunofluorescence techniques we document that sea urchin sperm also express orthologs of multiple isoforms of transmembrane ACs (tmACs). Our findings employing selective inhibitors for each class of AC indicate that both sAC and tmACs participate in the sperm Acrosome Reaction.
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ca2 entry through store operated channels in mouse sperm is initiated by egg zp3 and drives the Acrosome Reaction
Molecular Biology of the Cell, 2000Co-Authors: Christine M B Otoole, Alberto Darszon, Christophe Arnoult, Richard A Steinhardt, Harvey M FlormanAbstract:Fertilization occurs after the completion of the sperm Acrosome Reaction, a secretory event that is triggered during gamete adhesion. ZP3, an egg zona pellucida glycoprotein, produces a sustained i...
Haim Breitbart - One of the best experts on this subject based on the ideXlab platform.
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bovine sperm Acrosome Reaction induced by g protein coupled receptor agonists is mediated by epidermal growth factor receptor transactivation
Developmental Biology, 2009Co-Authors: Nir Etkovitz, Sara Rubinstein, Yitshak Tirosh, Reut Chazan, Yael Jaldety, Limor Daniel, Haim BreitbartAbstract:We have previously demonstrated the presence of active epidermal growth factor receptor (EGFR) and its involvement in sperm capacitation and the Acrosome Reaction; however, the mechanism of EGFR activation was not clear. We show here that the sperm EGFR can be transactivated by angiotensin II or by lysophosphatydic acid, two ligands which activate specific G-protein-coupled receptors (GPCR), or by directly activating protein kinase A using 8Br-cAMP. This transactivation occurs in noncapacitated sperm and is mediated by PKA, SRC and a metalloproteinase. We also show that the EGFR is activated in sperm incubated under in vitro capacitation conditions, without any added ligand, but not in bicarbonate-deficient medium or when PKA is blocked. Despite the fact that EGFR is activated in capacitated sperm, this state is not sufficient to induce the Acrosome Reaction. We conclude that the EGFR is stimulated during capacitation via PKA activation, while further activation of the EGFR in capacitated sperm is required in order to induce the Acrosome Reaction. The Acrosome Reaction can be induced by GPCR via the transactivation of the EGFR by a signaling pathway involving PKA, SRC and metalloproteinase and the EGFR down-stream effectors PI3K, PLC and PKC.
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role of actin cytoskeleton in mammalian sperm capacitation and the Acrosome Reaction
Reproduction, 2005Co-Authors: Haim Breitbart, Gili Cohen, Sara RubinsteinAbstract:In order to fertilize, the mammalian spermatozoa should reside in the female reproductive tract for several hours, during which they undergo a series of biochemical modifications collectively called capacitation. Only capacitated sperm can undergo the Acrosome Reaction after binding to the egg zona pellucida, a process which enables sperm to penetrate into the egg and fertilize it. Polymerization of globular (G)-actin to filamentous (F)-actin occurs during capacitation, depending on protein kinase A activation, protein tyrosine phosphorylation, and phospholipase D activation. F-actin formation is important for the translocation of phospholipase C from the cytosol to the sperm plasma membrane during capacitation. Prior to the occurrence of the Acrosome Reaction, the F-actin should undergo depolymerization, a necessary process which enables the outer acrosomal membrane and the overlying plasma membrane to come into close proximity and fuse. The binding of the capacitated sperm to the zona pellucida induces a fast increase in sperm intracellular calcium, activation of actin severing proteins which break down the actin fibers, and allows the Acrosome Reaction to take place.
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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, Keren Shternall, Joel Rivlin, Sara Rubinstein, 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
