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• W1968238097 abstract "To acquire fertilization competence, spermatozoa should undergo several biochemical changes in the female reproductive tract, known as capacitation. The capacitated spermatozoon can interact with the egg zona pellucida resulting in the occurrence of the acrosome reaction, a process that allowed its penetration into the egg and fertilization. Sperm capacitation requires actin polymerization, whereas F-actin must disperse prior to the acrosome reaction. Here, we suggest that the actin-severing protein, gelsolin, is inactive during capacitation and is activated prior to the acrosome reaction. The release of bound gelsolin from phosphatidylinositol 4,5-bisphosphate (PIP2) by PBP10, a peptide containing the PIP2-binding domain of gelsolin, or by activation of phospholipase C, which hydrolyzes PIP2, caused rapid Ca2+-dependent F-actin depolymerization as well as enhanced acrosome reaction. Using immunoprecipitation assays, we showed that the tyrosine kinase SRC and gelsolin coimmunoprecipitate, and activating SRC by adding 8-bromo-cAMP (8-Br-cAMP) enhanced the amount of gelsolin in this precipitate. Moreover, 8-Br-cAMP enhanced tyrosine phosphorylation of gelsolin and its binding to PIP2(4,5), both of which inactivated gelsolin, allowing actin polymerization during capacitation. This actin polymerization was blocked by inhibiting the Src family kinases, suggesting that gelsolin is activated under these conditions. These results are further supported by our finding that PBP10 was unable to cause complete F-actin breakdown in the presence of 8-Br-cAMP or vanadate. In conclusion, inactivation of gelsolin during capacitation occurs by its binding to PIP2 and tyrosine phosphorylation by SRC. The release of gelsolin from PIP2 together with its dephosphorylation enables gelsolin activation, resulting in the acrosome reaction. To acquire fertilization competence, spermatozoa should undergo several biochemical changes in the female reproductive tract, known as capacitation. The capacitated spermatozoon can interact with the egg zona pellucida resulting in the occurrence of the acrosome reaction, a process that allowed its penetration into the egg and fertilization. Sperm capacitation requires actin polymerization, whereas F-actin must disperse prior to the acrosome reaction. Here, we suggest that the actin-severing protein, gelsolin, is inactive during capacitation and is activated prior to the acrosome reaction. The release of bound gelsolin from phosphatidylinositol 4,5-bisphosphate (PIP2) by PBP10, a peptide containing the PIP2-binding domain of gelsolin, or by activation of phospholipase C, which hydrolyzes PIP2, caused rapid Ca2+-dependent F-actin depolymerization as well as enhanced acrosome reaction. Using immunoprecipitation assays, we showed that the tyrosine kinase SRC and gelsolin coimmunoprecipitate, and activating SRC by adding 8-bromo-cAMP (8-Br-cAMP) enhanced the amount of gelsolin in this precipitate. Moreover, 8-Br-cAMP enhanced tyrosine phosphorylation of gelsolin and its binding to PIP2(4,5), both of which inactivated gelsolin, allowing actin polymerization during capacitation. This actin polymerization was blocked by inhibiting the Src family kinases, suggesting that gelsolin is activated under these conditions. These results are further supported by our finding that PBP10 was unable to cause complete F-actin breakdown in the presence of 8-Br-cAMP or vanadate. In conclusion, inactivation of gelsolin during capacitation occurs by its binding to PIP2 and tyrosine phosphorylation by SRC. The release of gelsolin from PIP2 together with its dephosphorylation enables gelsolin activation, resulting in the acrosome reaction. Mammalian sperm must undergo a series of biochemical modifications in the female reproductive tract prior to productive sperm-egg interaction and the occurrence of the acrosome reaction (AR). 2The abbreviations used are: ARacrosome reactionPIP2phosphatidylinositol 4,5-bisphosphatePLCphospholipase C8-Br-cAMP8-bromo-cAMPEGFREGF receptorBAPTA/AM1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)PSAP. sativum agglutinin. The mechanisms of these modifications, collectively called capacitation, are not well characterized. There is no clear recognizable marker of successful capacitation, although several cellular changes are known to have taken place, including cholesterol efflux from the plasma membrane, an increase in membrane permeability to bicarbonate and calcium ions, and an increase in cAMP and protein kinase A (PKA)-dependent protein tyrosine phosphorylation (reviewed in Refs. 1Breitbart H. Cell. Mol. Biol. 2003; 49: 321-327PubMed Google Scholar, 2Visconti P.E. Moore G.D. Bailey J.L. Leclerc P. Connors S.A. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1139-1150Crossref PubMed Google Scholar). Another important process that occurs during sperm capacitation is actin polymerization; however, at the end of the capacitation, the polymers must disperse to achieve the AR (3Brener E. Rubinstein S. Cohen G. Shternall K. Rivlin J. Breitbart H. Biol. Reprod. 2003; 68: 837-845Crossref PubMed Scopus (202) Google Scholar). It has been suggested that an increase in F-actin creates a network between the plasma and the outer acrosomal membranes, and the dispersion of F-actin between the two membranes is needed to enable the AR (3Brener E. Rubinstein S. Cohen G. Shternall K. Rivlin J. Breitbart H. Biol. Reprod. 2003; 68: 837-845Crossref PubMed Scopus (202) Google Scholar, 4Breitbart H. Cohen G. Rubinstein S. Reproduction. 2005; 129: 263-268Crossref PubMed Scopus (161) Google Scholar, 5Spungin B. Margalit I. Breitbart H. J. Cell. Sci. 1995; 108: 2525-2535Crossref PubMed Google Scholar, 6Arnoult C. Kazam I.G. Visconti P.E. Kopf G.S. Villaz M. Florman H.M. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 6757-6762Crossref PubMed Scopus (187) Google Scholar). acrosome reaction phosphatidylinositol 4,5-bisphosphate phospholipase C 8-bromo-cAMP EGF receptor 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) P. sativum agglutinin. A better understanding of the mechanisms responsible for actin remodeling requires a closer look at the actin-binding proteins. The presence of actin-binding proteins such as calicin (7von Bülow M. Heid H. Hess H. Franke W.W. Exp. Cell Res. 1995; 219: 407-413Crossref PubMed Scopus (78) Google Scholar), the capping protein, CPβ3 (8von Bülow M. Rackwitz H.R. Zimbelmann R. Franke W.W. Exp. Cell Res. 1997; 233: 216-224Crossref PubMed Scopus (53) Google Scholar), CPα3 (9Tanaka H. Yoshimura Y. Nishina Y. Nozaki M. Nojima H. Nishimune Y. FEBS Lett. 1994; 355: 4-10Crossref PubMed Scopus (95) Google Scholar), destrin, thymosin β10, testis-specific actin-capping protein (10Howes E.A. Hurst S.M. Jones R. J. Androl. 2001; 22: 62-72PubMed Google Scholar), acinderin (11Pelletier R. Trifaro J.M. Carbajal M.E. Okawara Y. Vitale M.L. Biol. Reprod. 1999; 60: 1128-1136Crossref PubMed Scopus (42) Google Scholar), Arp-T1 and -T2 (12Heid H. Figge U. Winter S. Kuhn C. Zimbelmann R. Franke W. Exp. Cell Res. 2002; 279: 177-187Crossref PubMed Scopus (55) Google Scholar), and gelsolin (13de las Heras M.A. Valcarcel A. Perez L.J. Moses D.F. Tissue & Cell. 1997; 29: 47-53Crossref PubMed Scopus (25) Google Scholar) in mammalian sperm suggests that the assembly, of G-actin to form F-actin, as well as the disassembly of F-actin are well controlled events. In this study, we focused on the actin-binding protein, gelsolin. Gelsolin severs assembled actin filaments and caps the fast growing plus end of free or newly severed filaments in response to Ca2+ ions. Phosphoinositides release gelsolin from actin filament ends, providing sites for actin assembly (14Gremm D. Wegner A. Eur. J. Biochem. 2000; 267: 4339-4345Crossref PubMed Scopus (53) Google Scholar, 15Yin H.L. Zaner K.S. Stossel T.P. J. Biol. Chem. 1980; 255: 9494-9500Abstract Full Text PDF PubMed Google Scholar, 16Yin H.L. BioEssays. 1987; 7: 176-179Crossref PubMed Scopus (173) Google Scholar, 17Janmey P.A. Stossel T.P. Nature. 1987; 325: 362-364Crossref PubMed Scopus (492) Google Scholar). In vitro studies suggested that gelsolin is phosphorylated by pp60(c-SRC) and that this phosphorylation is enhanced by phosphoinositide 4,5-bisphosphate (PIP2(4,5)) (18De Corte V. Gettemans J. Vandekerckhove J. FEBS Lett. 1997; 401: 191-196Crossref PubMed Scopus (74) Google Scholar) and can interfere with the binding of gelsolin to the actin filaments (19De Corte V. Demol H. Goethals M. Van Damme J. Gettemans J. Vandekerckhove J. Protein Sci. 1999; 8: 234-241Crossref PubMed Scopus (54) Google Scholar). Previous work identified SRC in human spermatozoa, and it appears to be involved in regulating sperm capacitation, calcium fluxes, tyrosine phosphorylation, and the acrosome reaction (20Varano G. Lombardi A. Cantini G. Forti G. Baldi E. Luconi M. Hum. Reprod. 2008; 23: 2652-2662Crossref PubMed Scopus (55) Google Scholar). In a recent study, we reported that the epidermal growth factor receptor (EGFR) is partially activated during sperm capacitation by the cAMP/PKA/SRC mechanisms. It was suggested that EGFR is phosphorylated at the SRC-specific site, suggesting that SRC is active during capacitation (21Etkovitz N. Tirosh Y. Chazan R. Jaldety Y. Daniel L. Rubinstein S. Breitbart H. Dev. Biol. 2009; 334: 447-457Crossref PubMed Scopus (61) Google Scholar, 22Lax Y. Rubinstein S. Breitbart H. FEBS Lett. 1994; 339: 234-238Crossref PubMed Scopus (61) Google Scholar). Additionally, EGFR-mediated signaling is known to activate phospholipase Cγ (PLCγ), to hydrolyze PIP2(4,5), and to cause the subsequent release of PIP2(4,5)-bound proteins. In vitro studies have shown that gelsolin modulates the activity of several signaling enzymes, including PLC and phospholipase D through interactions with PIP2(4,5) (23Banno Y. Nakashima T. Kumada T. Ebisawa K. Nonomura Y. Nozawa Y. J. Biol. Chem. 1992; 267: 6488-6494Abstract Full Text PDF PubMed Google Scholar, 24Cheng J. Weber J.D. Baldassare J.J. Raben D.M. J. Biol. Chem. 1997; 272: 17312-17319Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 25Sun H. Lin K. Yin H.L. J. Cell Biol. 1997; 138: 811-820Crossref PubMed Scopus (83) Google Scholar, 26Liscovitch M. Chalifa V. Pertile P. Chen C.S. Cantley L.C. J. Biol. Chem. 1994; 269: 21403-21406Abstract Full Text PDF PubMed Google Scholar, 27Steed P.M. Nagar S. Wennogle L.P. Biochemistry. 1996; 35: 5229-5237Crossref PubMed Scopus (62) Google Scholar). It appears that actin polymerization during sperm capacitation depends on phospholipase D activity, which is regulated by the cross-talk between PKA and protein kinase C (PKC) (1Breitbart H. Cell. Mol. Biol. 2003; 49: 321-327PubMed Google Scholar, 28Cohen G. Rubinstein S. Gur Y. Breitbart H. Dev. Biol. 2004; 267: 230-241Crossref PubMed Scopus (98) Google Scholar). Moreover, we showed elsewhere that PIP2(4,5), the cofactor for phospholipase D activation and F-actin production during sperm capacitation, is mediated by phosphatidylinositol 4-kinase (PI4K) but not by phosphatidylinositol 3-kinase (PI3K) activity (29Etkovitz N. Rubinstein S. Daniel L. Breitbart H. Biol. Reprod. 2007; 77: 263-273Crossref PubMed Scopus (58) Google Scholar). Even though the role of gelsolin in somatic cells is well established, the presence and the possible role of this protein in the male gamete are not fully understood. In guinea pig sperm, gelsolin and actin were detected in a mixture of plasma and outer acrosomal membranes, and both proteins were absent from the membranes of capacitated spermatozoa (30Cabello-Agüeros J.F. Hernández-González E.O. Mújica A. Cell Motil. Cytoskeleton. 2003; 56: 94-108Crossref PubMed Scopus (45) Google Scholar). We propose that during sperm capacitation, gelsolin is inactive to maintain actin polymerization; at some point prior to the AR, gelsolin is activated, leading to the occurrence of the AR. We further suggest that gelsolin inactivation occurs via two mechanisms as follows: first, the binding of gelsolin to PIP2(4,5), and the second, the phosphorylation of tyrosine residues on gelsolin by SRC, which was shown to release gelsolin from the actin filaments (19De Corte V. Demol H. Goethals M. Van Damme J. Gettemans J. Vandekerckhove J. Protein Sci. 1999; 8: 234-241Crossref PubMed Scopus (54) Google Scholar). The data described here confirm this hypothesis and provide a better understanding of the regulation of gelsolin during sperm capacitation and in the acrosome reaction. PBP10 (polyphosphoinositide-binding peptide, rhodamine B-conjugated), PP1, U73122, and A23187 were obtained from Calbiochem. Capacitation medium, F-10 (Ham's) nutrient mixture with l-glutamine, was purchased from Biological Industries (Kibbutz Beit Haemek, IL). Goat polyclonal anti-gelsolin (C-20), mouse monoclonal anti-SRC, (H-12), anti-β-actin HRP-conjugated antibodies, secondary donkey anti-goat IgG, protein G PLUS-agarose (sc-2002), and anti-PIP2(4,5) (PIP2 2C11, sc-53412) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Alexa Fluor 568 donkey anti-goat IgG was purchased from Invitrogen. All other chemicals were purchased from Sigma, unless otherwise stated. Human semen was initially liquefied. The semen was then loaded on a Percoll gradient (80, 40, and 20%) and centrifuged for 30 min at 6750 rpm at room temperature. The lower layer containing the sperm was collected and washed twice in Ham's F-10 and then recentrifuged and allowed to “swim up” after the last wash at 37 °C. The motile cells were collected and resuspended in capacitation medium, and the pellet was discarded. Only sperm preparations that contained at least 70% motile sperm were used in the experiments. Human sperm (1 × 107 cells/ml) were capacitated by incubation in capacitation media (Ham's F-10) supplemented with 3 mg/ml BSA. The cells were incubated in this capacitation medium for 3 h at 37 °C in 5% CO2. The capacitation state of the sperm was confirmed after the 3-h incubation by examining the ability of the sperm to undergo the acrosome reaction. Washed cells (1 × 107 cells/ml) were capacitated for 3 h at 37 °C in capacitation medium. The inhibitors indicated for the various experiments were added after 3 h of incubation for 10–20 min, and the inducers were then added for another 60 min of incubation. The percentage of acrosome-reacted sperm was determined microscopically on air-dried sperm smears using FITC-conjugated Pisum sativum agglutinin (PSA). An aliquot of spermatozoa was smeared on a glass slide and allowed to air-dry. The sperm were then permeabilized by adding methanol for 15 min at room temperature. The cells were washed three times at 5-min intervals with TBS, air-dried, and then incubated with FITC-PSA (60 mg/ml) at room temperature in the dark for 60 min, washed twice with H2O at 5-min intervals, and mounted with FluoroGuard Antifade (Bio-Rad). For each experiment, at least 150 cells per slide on duplicate slides were evaluated (a total of 300 cells per experiment). Cells with green staining over the acrosomal cap were considered acrosome-intact; those with equatorial green staining or no staining were considered acrosome-reacted. Sperm lysates were prepared by the addition of lysis buffer that contained 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 6% SDS, protease inhibitor mixture 1:100 (Calbiochem), 50 μm NaF, 50 μm pyrophosphate, 1 mm phenylmethylsulfonyl fluoride (PMSF), and 0.2 mm Na3VO4, to the pellet. The mixture was vortexed for 10 min at room temperature. Lysates were then centrifuged at 14,000 × g for 5 min at 4 °C; the supernatant was removed, and the protein concentration was determined by the Bradford method (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214351) Google Scholar). Sample buffer (2×) was added to the supernatant and boiled for 5 min. The extracts were separated on 10% SDS-polyacrylamide gels and electrophoretically transferred (200 mA for 1 h) to nitrocellulose membranes. Western blotting was performed using a buffer composed of 25 mm Tris, pH 8.2, 192 mm glycine, and 20% methanol. The nitrocellulose membranes were blocked with 5% milk (for anti-gelsolin and anti-actin) or 5% BSA (for anti-p-SRC and anti-SRC) in Tris-buffered saline, pH 7.6, containing 0.1% Tween 20 (TBST), for 30 min at room temperature. Gelsolin and actin were immunodetected using a polyclonal anti-gelsolin antibody (diluted 1:3000), horseradish peroxidase (HRP)-conjugated anti-actin (1:3000) and a polyclonal anti-SRC antibody (diluted 1:3000). The membranes were incubated overnight at 4 °C with the primary antibodies. Next, the membranes were washed three times with TBST and incubated for 1 h at room temperature with the appropriate secondary antibody diluted at 1:10,000. The membranes were washed three times with TBST and visualized by enhanced chemiluminescence (Amersham Biosciences). Sperm cells were spread on microscope slides. After air-drying, the sperm cells were fixed in 2% formaldehyde in TBS for 10 min, placed in 0.2% Triton X-100 in TBS for 30 min, washed three times at 5-min intervals in distilled water, air-dried, and then incubated with phalloidin-FITC (4 μm in TBS) for 60 min, washed four times with distilled water at 10-min intervals, and mounted with FluoroGuard Antifade (Bio-Rad). Sperm suspensions were incubated in Ham's F-10 (3 × 107/ml) for 3 h, and the cells were lysed as described previously (29Etkovitz N. Rubinstein S. Daniel L. Breitbart H. Biol. Reprod. 2007; 77: 263-273Crossref PubMed Scopus (58) Google Scholar). Briefly, the sperm cells were washed once with TBS. An equal volume of lysis solution that contained 1.5% Triton X-100 was added, and the suspension was vortexed vigorously (10 min at 4 °C). At the end of this incubation, the mixture was centrifuged at 12,000 × g for 5 min. The supernatant (Triton-soluble G-actin) was collected. The Triton-insoluble content was determined by the addition of 50 μl of lysis buffer containing 6% SDS. The mixture was vortexed vigorously for 10 min. The sample was centrifuged at 12,000 × g for 5 min, and the supernatant (Triton-insoluble F-actin) was collected. Sample buffer was added, and the extracted proteins were separated by SDS-PAGE and immunoblotted, as indicated. For immunocytochemistry, sperm cells were spread on glass slides, air-dried, fixed in formaldehyde (4%) for 10 min, dipped in 0.5% Triton X-100 in TBS for 30 min, and washed three times at 5-min intervals with TBS. Nonspecific reactive sites were blocked for 30 min at room temperature with TBS containing 10% donkey serum. The cells were incubated for 24 h at 4 °C with goat polyclonal anti-gelsolin (C-20) antibody diluted 1:50 in TBS containing 1% donkey serum. Next, the slides were washed once in TBS-T and twice (for 5 min) in TBS. The bound antibody was detected using Alexa Fluor 568 donkey anti-goat IgG (a 1:200 dilution), incubated for 1 h at 37 °C, and followed by one wash with TBS-T and two washes with H2O at 5-min intervals. The slides were then mounted with FluoroGuard Antifade. Nonspecific staining was determined by incubating the sperm without the primary antibody. No staining was detected. All images were captured on an Olympus AX70 microscope at 400× magnification. This microscope was equipped with an Olympus DP50 digital camera and with the Viewfinder Lite version 1 software (Pixera Corp., Los Gatos, CA). All fluorescence determinations were performed under nonsaturated conditions. Both the experiments and staining were performed on the same day, and the sperm were photographed within 24 h to reduce the loss of fluorescence. All cell preparations from a single experiment were photographed in the same session and for the same exposure period. The fluorescence intensity was quantified using the MetaMorph ImageJ software (National Institutes of Health), and the background intensity was subtracted. For F-actin, all experiments were carried out in duplicate, and at least 100 cells (5–7 pictures) per slide were quantified for fluorescence intensity. For gelsolin, at least 50 cells were quantified for fluorescence intensity. Human spermatozoa (5 × 107 cells/ml) were isolated and capacitated as described previously. After incubation, the sperm samples were transferred into glass tubes and sonicated on ice for 1 min using the “Vibra Cell” (Sonics & Materials Inc., Danbury, CT) with an intensity setting of 40. The cells were then sonicated for 15 s to facilitate the separation of sperm heads and tails (32Nixon B. MacIntyre D.A. Mitchell L.A. Gibbs G.M. O'Bryan M. Aitken R.J. Biol. Reprod. 2006; 74: 275-287Crossref PubMed Scopus (119) Google Scholar). Following sonication, the samples were layered over a 75% Percoll cushion in 2-ml tubes and centrifuged at 700 rpm for 15 min to isolate the heads and tails in separate fractions. The pellet that formed contained the sperm heads, although the top layer contained the sperm membranes. The tails resided at the interface between the two liquid layers. The heads and tails were removed and diluted with F-10 media. The purity of each fraction was assessed by microscopy prior to analysis. The head and tail samples were then centrifuged at 1400 rpm for 5 min, and the supernatant was removed, and the remaining pellet was SDS-extracted as described previously. Sperm cells (5 × 107 cells/ml) were centrifuged at 4000 rpm for 10 min and washed with cold TBS. Equal amounts of protein (100 mg for spermatozoa) were sonicated three times in homogenization buffer containing the following: 20 mm Tris-HCl, pH 7.5; 0.25 m sucrose; 2 mm EGTA; 2 mm EDTA; 1 mm benzamidine; 1 mm Na3VO4; 10% (w/v) glycerin; 5 mm NaF; 30 mm NaH2PO4; 25 μg/ml leupeptin; 4 μg/ml aprotinin; 1.5 μg/ml pepstatin; 2 μg/ml antipain; and 1 mm PMSF, and incubated for 30 min at 4 °C. Next, the mixture was centrifuged at 14,000 rpm for 5 min. For immunoprecipitation, 50 μl of protein A/G plus-agarose was added for preclearing the suspension, and the samples were incubated for 2 h at 4 °C. The mixture was centrifuged at 7500 rpm for 5 min, and the supernatants were then incubated with 2 μg of anti-SRC/p-Thy/PIP2(4,5) overnight at 4 °C. The next day, 50 μl of protein A/G-agarose beads was added and further incubated for 2 h with gentle agitation at 4 °C. The beads were then subjected to three washes with 0.1% Triton in TBS buffer and boiled for 5 min with SDS sample buffer. Data are expressed as means ± S.D. of at least three experiments for all determinations. Statistical significance was calculated by Student's t test or by analysis of variance with Bonferroni's post hoc comparison test using SPSS software (Chicago). In our previous studies, we showed that actin polymerization occurs during sperm capacitation (3Brener E. Rubinstein S. Cohen G. Shternall K. Rivlin J. Breitbart H. Biol. Reprod. 2003; 68: 837-845Crossref PubMed Scopus (202) Google Scholar, 28Cohen G. Rubinstein S. Gur Y. Breitbart H. Dev. Biol. 2004; 267: 230-241Crossref PubMed Scopus (98) Google Scholar). Gelsolin, an actin-severing protein, is expected to be localized with the F-actin fraction in the cell when activated. The data in Fig. 1 reveal that gelsolin levels during capacitation remained constant (Fig. 1A). The Triton X-100 sperm fractions revealed that before capacitation most of the actin was in the form of monomers, i.e. the G-actin form (soluble fraction), although at the end of capacitation most of the actin was in the form of polymers, i.e. the F-actin form (insoluble fraction) (Fig. 1B). Thus, gelsolin is localized in the G-actin fraction before capacitation, although at the end of capacitation, gelsolin is localized in the F-actin fraction (Fig. 1, B and C). We have shown elsewhere in bovine sperm that before capacitation there is almost no F-actin in the sperm head, as most of it is localized to the tail midpiece, whereas after capacitation, there is a significant increase in F-actin in the head (28Cohen G. Rubinstein S. Gur Y. Breitbart H. Dev. Biol. 2004; 267: 230-241Crossref PubMed Scopus (98) Google Scholar). Here, we demonstrated that the amount of F-actin in human sperm heads was low before capacitation, increased in capacitated cells, and was reduced again in acrosome-reacted cells (Fig. 2A). Moreover, immunocytochemical staining revealed an increase of gelsolin staining in the sperm head during capacitation (Fig. 2B). Interestingly, the increase of gelsolin in the head could not be seen when intracellular calcium was chelated by BAPTA/AM (Fig. 2B), indicating the importance of calcium ions for this translocation. Further support for the increase of gelsolin in the head is seen by Western blot analysis of separated tail and heads, showing that most of the gelsolin was localized in the tail before capacitation, whereas after capacitation there was a significant decrease in its amount in the tail and a significant increase in the head (Fig. 2C). These data suggest that gelsolin translocates from the sperm tail to the head during capacitation. Gelsolin is an actin-severing protein that causes F-actin depolymerization, and its activation is regulated by calcium ions and phosphoinositides (14Gremm D. Wegner A. Eur. J. Biochem. 2000; 267: 4339-4345Crossref PubMed Scopus (53) Google Scholar, 15Yin H.L. Zaner K.S. Stossel T.P. J. Biol. Chem. 1980; 255: 9494-9500Abstract Full Text PDF PubMed Google Scholar, 16Yin H.L. BioEssays. 1987; 7: 176-179Crossref PubMed Scopus (173) Google Scholar). Relatively low calcium concentrations cause conformational changes in the C terminus of gelsolin, which expose its binding site to F-actin, whereas higher calcium concentrations cause a second conformational change exposing the catalytic site (33Kinosian H.J. Newman J. Lincoln B. Selden L.A. Gershman L.C. Estes J.E. Biophys. J. 1998; 75: 3101-3109Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To test gelsolin activity, the cells were incubated under capacitation conditions to increase the intracellular levels of F-actin. At this point, gelsolin was activated by increasing the intracellular calcium using the calcium ionophore A23187 or by activating the EGFR by EGF. The data in Fig. 3 reveal a rapid decrease in F-actin levels when intracellular calcium concentrations increased. This effect was blocked when intracellular calcium was chelated using BAPTA/AM. Moreover, a rapid decrease in F-actin was also induced by adding the peptide PBP10 (Fig. 3), which is a peptide derived from the PIP2(4,5)-binding domain of gelsolin. This peptide competes with gelsolin binding to PIP2(4,5) causing the release of gelsolin from PIP2(4,5) and enabling its activity on F-actin (34Cunningham C.C. Vegners R. Bucki R. Funaki M. Korde N. Hartwig J.H. Stossel T.P. Janmey P.A. J. Biol. Chem. 2001; 276: 43390-43399Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). F-actin depolymerization by PBP10 was also prevented by chelating intracellular calcium using BAPTA/AM (Fig. 3), suggesting the dependence of gelsolin activation on calcium ions. In our previous study, we showed that F-actin depolymerization must occur in capacitated sperm prior to the acrosome reaction (3Brener E. Rubinstein S. Cohen G. Shternall K. Rivlin J. Breitbart H. Biol. Reprod. 2003; 68: 837-845Crossref PubMed Scopus (202) Google Scholar). Thus, the acrosome reaction cannot occur if F-actin is polymerized. Similarly, the F-actin depolymerization shown in Fig. 3 occurs before the acrosome reaction (Fig. 4). Increasing the intracellular calcium concentration or adding PBP10 to capacitated sperm induced a significant increase in the acrosome reaction rate in sperm that was pretreated with the intracellular calcium chelator BAPTA/AM (Fig. 4). The data in FIGURE 3, FIGURE 4 suggest that gelsolin is activated by an increase in the intracellular calcium levels of capacitated cells, enabling the acrosome reaction. PIP2(4,5) can bind to gelsolin, and the hydrolysis of PIP2(4,5) by PLC releases the bound gelsolin, resulting in gelsolin activation in Sertoli cells (35Guttman J.A. Janmey P. Vogl A.W. J. Cell Sci. 2002; 115: 499-505Crossref PubMed Google Scholar). Here, we demonstrate that F-actin depolymerization and acrosome reaction induced by the calcium ionophore, A23187, or EGF in capacitated sperm were significantly reduced when PLC is blocked by U73122 (FIGURE 5, FIGURE 6). However, when actin depolymerization or the acrosome reaction was induced by the peptide PBP10, there was no effect on F-actin depolymerization by the PLC inhibitor U73122 (FIGURE 5, FIGURE 6). These data suggest that PLC mediates actin depolymerization prior to the acrosome reaction. In addition, sperm PLC requires micromolar levels of calcium for half-maximal activation similar to the calcium concentration found after the sperm-zona interaction (6Arnoult C. Kazam I.G. Visconti P.E. Kopf G.S. Villaz M. Florman H.M. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 6757-6762Crossref PubMed Scopus (187) Google Scholar), suggesting the possible activation of PLC at this time. Stimulation of capacitated spermatozoa with progesterone or with isolated zona pellucida leads to activation of calcium-dependent phospholipase C (36Baldi E. Luconi M. Bonaccorsi L. Forti G. J. Reprod. Immunol. 2002; 53: 121-131Crossref PubMed Scopus (80) Google Scholar). Our results show that PLCγ was phosphorylated on Tyr-782 at the end of the capacitation period, an indication of its activation. This phosphorylation could also be seen when AR was induced by the Ca2+ ionophore, A23187, and was reduced after 1 h (Fig. 5B). The induction of F-actin depolymerization by EGF, the physiological ligand of EGFR, a known activator of PLCγ, and the phosphorylation/activation of PLCγ indicate that the PLCγ isoform mediates F-actin depolymerization. The induction of F-actin breakdown by PBP10 suggests that this reaction is mediated by gelsolin.FIGURE 6PLCγ activity is requ" @default.
• W1968238097 created "2016-06-24" @default.
• W1968238097 creator A5029790971 @default.
• W1968238097 creator A5046615745 @default.
• W1968238097 creator A5063165730 @default.
• W1968238097 date "2010-12-01" @default.
• W1968238097 modified "2023-10-15" @default.
• W1968238097 title "Role and Regulation of Sperm Gelsolin Prior to Fertilization" @default.
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