Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis

Developmental Biology 265 (2004) 399 – 415 www.elsevier.com/locate/ydbio

Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis C.N. Tomes, a,b,* C.M. Roggero, a G. De Blas, a P.M. Saling, b and L.S. Mayorga a a

Laboratorio de Biologı´a Celular y Molecular, Instituto de Histologı´a y Embriologı´a (IHEM-CONICET), Facultad de Ciencias Me´dicas, CC56, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina b Department of Obstetrics and Gynecology, Duke University Medical Center, PO Box 3648, Durham, NC 27710, USA Received for publication 1 April 2003, revised 29 September 2003, accepted 30 September 2003

Abstract The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47—a tyrosine kinase inhibitor—abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intraacrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,Ndimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis. D 2003 Elsevier Inc. All rights reserved. Keywords: Acrosome reaction; Calcium; Capacitation; Exocytosis; Fusion; Kinase; Phosphatase; Sperm; Tyrosine phosphorylation

Introduction Exocytosis is a highly regulated, multistage process including targeting, tethering, docking, and fusion of secretory vesicles with the plasma membrane. During the last few years, several molecules involved in this complex cascade of events have been identified (Jahn and Sudhof, 1999). Protein tyrosine phosphorylation is an essential element in the control of fundamental cellular signaling events involved in growth, proliferation, and differentiation, including regulated exocytosis. In some cases, secretion depends on the phosphorylation of proteins on one or more tyrosine

* Corresponding author. Laboratorio de Biologı´a Celular y Molecular, Instituto de Histologı´a y Embriologı´a, Facultad de Ciencias Me´dicas, Universidad Nacional de Cuyo, Casilla de Correo 56, IHEM, 5500 Mendoza, Argentina. Fax: +54-261-449-4117. E-mail address: [email protected] (C.N. Tomes). 0012-1606/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2003.09.032

residues (Inoue et al., 2002; Sada et al., 2002) whereas, in others, it is dephosphorylation that governs exocytosis (Sterling et al., 2003). The initiation, extent, and termination of tyrosine phosphorylation is determined by the concerted activities of the phosphorylating (protein tyrosine kinases, PTK) and dephosphorylating (protein tyrosine phosphatases, PTP) enzymes. Both PTK (see, for instance, Cox et al., 1996; Kim et al., 1999; Parravicini et al., 2002; Zhang et al., 2002a) and PTP (Chen and Geng, 2001; Ostenson et al., 2002) have been implicated in secretion. Nevertheless, the specific protein substrates, as well as their regulation, remain largely unidentified. Also unknown is the exact stage of the exocytotic reaction where these proteins play their part. Exocytosis of the acrosome (the acrosome reaction, AR) is a terminal morphological alteration that sperm must undergo before penetration of the extracellular coat of the egg (zona pellucida). Although exocytosis of the acrosomal vesicle has some unique properties, the emerging body of

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data suggests that sperm use the same conserved machinery and regulatory components as those characterized for other secretory events (Tomes et al., 2002 and references therein). Tyrosine phosphorylation appears to display a key role in several aspects of sperm function. For instance, an increase in protein tyrosine phosphorylation has been shown to accompany capacitation, the acquired ability of sperm to interact productively with eggs (reviewed in Visconti and Kopf, 1998; Visconti et al., 1998). Accordingly, decapacitation by seminal plasma decreases both protein tyrosine phosphorylation and the subpopulation of cells exhibiting hyperactivated motility in ejaculated human sperm (Tomes et al., 1998). Protein tyrosine phosphorylation has been implicated in mammalian sperm AR since treatment with PTK inhibitors can block zona pellucida- (Leyton et al., 1992; Pukazhenthi et al., 1998) and progesterone- (Kirkman-Brown et al., 2002; Luconi et al., 1995; Meizel and Turner, 1996; Tesarik et al., 1993) induced exocytosis. Whether regulation of sperm protein phosphotyrosine content occurs through the activation of PTK, the inhibition of PTP, or both remains to be elucidated. Data supporting the involvement of PTKs has been derived from the identification of the src-related PTK c-yes in human sperm on one hand and the use of specific inhibitors on the other (see Leclerc and Goupil, 2002 and references therein). In contrast, information regarding the presence and role of PTPs in sperm function is lacking. Here, we use permeabilized human sperm to assess the role of protein tyrosine phosphorylation in acrosomal release. We find that challenging with calcium induces protein tyrosine phosphorylation on the sperm head in two waves, one preceding and one following acrosomal calcium efflux. We also show that both PTK and PTP activities are required in the signaling cascade leading to sperm exocytosis.

Materials and methods Reagents Streptolysin O (SLO) was obtained from Corgenix (Peterborough, UK). Tyrphostin A47 was from LC Laboratories (Woburn, MA). bis(N,N-dimethylhydroxoamido)hydroxovanadate (DMHV) and ethyl 3,4-dephostatin were from Calbiochem (San Diego, CA). Mouse monoclonal antiphosphotyrosine antibody (clone 4G10, subclass IgG2b) (Druker et al., 1989) was from Upstate Biotechnology Incorporated (UBI, Lake Placid, NY). Another mouse monoclonal to phosphotyrosine (clone PY20, subclass IgG2b) (Glenney et al., 1988) was purchased from ICN (Aurora, OH). Monoclonal anti-PTP1B antibody, clone 15 (purified IgG2a) was from Transduction Laboratories (Lexington, KY). Mouse monoclonal anti-PTP1B, clone FG61G (purified IgG2a) (Schievella et al., 1993) was a kind gift from Dr. Nicholas Tonks (Cold Spring Harbor Laboratory). Horseradish peroxidase and TRITC-conjugated goat anti-

mouse-IgG were from Kirkegaard and Perry Laboratories Inc (KPL, Gaithersburg, MD). Reduced, carboxyamidomethylated, and maleylated lysozyme (RCML) phosphorylated on tyrosyl residues prepared as in Tonks et al. (1988a) was a gift from Dr. Nicholas Tonks. 32P-labeled poly(glutamic acid-tyrosine) (random copolymer), 4:1 ratio, ([32P]poly (Glu-Tyr)) prepared as in Burridge and Nelson (1995) was a gift from Dr. Keith Burridge (University of North Carolina, Chapel Hill). Nickel – nitrilotriacetic acid agarose was from Qiagen (Hilden, Germany) and glutathione –sepharose was from Amersham Biosciences Argentina (Buenos Aires, Argentina). Prestained molecular weight standards were from BRL-Life Technologies, Inc. (Gaithersburg, MD). Onitrophenyl EGTA-AM (EGTA-NP) was from Molecular Probes (Eugene, OR). Methanol was purchased from Merck Quı´mica (Buenos Aires, Argentina). All other chemicals were reagent or analytical grade and were purchased from Sigma Chemical Co. (St Louis, MO). The expression plasmid pGEX2T containing the cDNAencoding human Rab3A was generously provided by Drs. M.I. Colombo and P.D. Stahl (Washington University, St. Louis, MO). GST-Rab3A was expressed in Escherichia coli strain XL1-blue (Stratagene, La Jolla, CA), purified, prenylated, and activated as previously described (Yunes et al., 2000). A plasmid encoding the light chain of BoNT/E (BoNT/E-LC) was a kind gift from Dr. T. Binz (Medizinische Hochschule Hannover, Hannover, Germany). His6-tagged BoNT/E-LC was expressed and purified as in Vaidyanathan et al. (1999). Human sperm preparation procedure After at least 2 days of abstinence, semen samples were provided by masturbation from healthy volunteer donors who were free from sexually transmitted diseases. Semen was allowed to liquefy for 30– 60 min at 37jC. For Western blot and PTP experiments, sperm were isolated on a discontinuous two-step PercollR (Pharmacia, Uppsala, Sweden) gradient. Sperm washed with phosphate buffered saline (PBS) were incubated F1 mM pervanadate (made fresh by mixing stocks of H2O2 and Na3VO4) for 15 min at 37jC, and lysed to extract proteins immediately (noncapacitated samples) or capacitated in Human Tubal Fluid media (Irvine Scientific, Santa Ana, CA) supplemented with 7.5% human serum (HTF media) at a concentration of 5 –10
106/ml at 37jC in a humidified atmosphere of 5% CO2 in air for 5 h. Where indicated, an additional 3 h incubation F50 AM tyrphostin A47 and/or various concentrations of sodium pervanadate in HTF were performed. After removing HTF by washing twice with PBS (200
g for 10 min), sperm pellets were processed for SDS-PAGE or for phosphatase assays. Total motility was evaluated by diluting 2 Al of sperm suspension with 8 Al of HTF on a glass slide, covering the suspension with a 22
22 mm2 coverslip (thickness #1), and examining with phase contrast optics at a magnification of 400
. Multiple fields were viewed and

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>100 spermatozoa were scored as either immotile or showing flagellar movement. Viability was measured with 1 Ag/ ml propidium iodide (Molecular Probes); the percentage of live sperm was estimated by counting >100 sperm with epifluorescence optics at a magnification of 400
. SLO permeabilization and AR assay For AR assays, indirect immunofluorescence, and confocal microscopy, highly motile sperm were recovered following a swim-up separation for 1 h in Gamete Preparation Medium (GPM, Aubonne, Switzerland) at 37jC in an atmosphere of 5% CO2 – 95% air. Concentration was then adjusted to 5– 10
106/ml, and incubation proceeded for at least 2 h under conditions that support capacitation (GPM medium, 37jC, 5% CO2 –95% air). Permeabilization was accomplished as described (Yunes et al., 2000). Briefly, washed spermatozoa were resuspended in cold PBS containing 0.4 unit/ml SLO for 15 min at 4jC. Cells were washed once with PBS, resuspended in ice-cold sucrose buffer (250 mM sucrose, 20 mM HEPES-K, 0.5 mM EGTA, pH 7) containing 2 mM DTT, inhibitors were added when indicated, and further incubated for 5 –15 min at 37jC. After addition of stimulants to the sperm suspensions, incubation proceeded at 37jC for 15 min. Ten microliters of each reaction mixture were spotted on eight-well slides, air dried, and fixed and permeabilized in ice-cold methanol for 30 s. Acrosomal status was evaluated by staining with fluorescein isothiocyanate-coupled Pisum sativum according to Mendoza et al. (1992). At least 200 cells were scored using a Nikon Optiphot II microscope (Nikon Corporation, Tokyo, Japan) equipped with epifluorescence optics. Negative (no stimulation) and positive (10 AM calcium) controls were included in all experiments. For each experiment, the data were normalized by subtracting the number of reacted spermatozoa in the negative control from all values and expressing the result as a percentage of the AR observed in the positive control. Mouse sperm preparation procedure Mouse sperm were obtained by rupturing the excised caudae epididymides of mature Swiss outbred mice (Charles River Breeding Co., Wilmington, MA or Harlan Sprague Dawley, Indianapolis, IN) in 20 mM Tris –HCl pH 7.4, 130 mM NaCl, 2 mM CaCl2 (TNC buffer), washed once by centrifugation at 100
g for 10 min, and lysed immediately (noncapacitated samples) or resuspended in CM media (a Krebs Ringer– bicarbonate medium supplemented with pyruvate, lactate, glucose, and 20 mg/ml BSA; Saling and Storey, 1979). Capacitation was achieved by incubating sperm (107 cells/ml) in CM for 60– 120 min at 37jC in 5% CO2 – 95% air. Proteins from washed, noncapacitated and capacitated sperm were extracted in 1% TX-100 lysis buffer (see below) and processed for SDS-PAGE or PTP assays. Soluble and particulate fractions were recovered by

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the subcellular fractionation procedure detailed in Tomes et al. (1996). PTP assay Sperm incubated and washed as described were lysed in cold 50 mM Tris –HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, 5 Ag/ml trypsin inhibitor, 5 Ag/ml pepstatin, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine, 1% TX-100. After sonication for 2
15 s and extraction for 30 – 60 min at 4jC, the sperm extracts were clarified by centrifugation at 12,000
g for 5 min and used immediately or stored at 20jC. PTP activity assays measure the release of 32Pi from labeled substrate. Briefly, sperm extracts were incubated with [32P]poly(Glu-Tyr) in a reaction mixture (final volume 100 Al) containing 50 mM HEPES, pH 7.4, 1 mM EDTA, 2 mM DTT, and 2 mg/ml BSA for 10 min at 30jC. Reactions were stopped by precipitating the excess of substrate with 20% ice-cold TCA. After incubating on ice for 15 min, pellets were recovered by centrifuging 10 min
12,000 g and measuring the radioactivity released in 90 Al of the supernatant. The protocol using RCML as PTP substrate has been detailed elsewhere (Tonks et al., 1988a). SDS-PAGE and Western blots Proteins were extracted in PTP lysis buffer (Figs. 7G and 8B) or in sample buffer (Fig. 6; Tomes et al., 1998), separated on polyacrylamide slab gels according to Laemmli (1970) and transferred to 0.45 Am Immobilon (Millipore Corp., Bedford, MA) membranes (Towbin et al., 1979). Nonspecific reactivity was blocked by incubation for 1 h at room temperature with 5% gelatin (PY20), 5% skim milk (FG6), or 1% BSA (clone 15) dissolved in washing buffer (Tomes et al., 1996). Blots were incubated with PY20 (0.2 Ag/ml), FG6 (1 Ag/ml), or clone 15 (1 Ag/ml) in blocking solution for 60 –120 min at room temperature. Horseradish peroxidase-conjugated goat antimouse-IgG was used as secondary antibody with 45-min incubations. Excess first and second antibodies were removed by washing 5
10 min in washing buffer. Detection was accomplished with a chemiluminescence system (ECL, Amersham Biosciences, Piscataway, NJ) and subsequent exposure to Kodak XAR film (Eastman Kodak, Rochester, NY) for 5 –30 s. Indirect immunofluorescence in SLO-permeabilized sperm Sperm were permeabilized with SLO as described and treated with 350 nM BoNT/E-LC (preactivated by incubation for 15 min at 37jC with 5 mM DTT) to prevent exocytosis (Tomes et al., 2002). Following addition of inhibitors, 10 AM free calcium and incubation at 37jC for 15 min, sperm pellets were washed in PBS and fixed and permeabilized in 2% paraformaldehyde – 0.1% TX-100 in PBS for 10 min at room temperature. After centrifugation (5000
g for 5 min), sperm were incubated in PBS

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containing 50 mM glycine for 30 min at room temperature, pelleted, and washed twice with PBS containing 0.4% polyvinylpyrrolidone (PVP, average M.W. = 40,000; ICN, PBS/PVP). Sperm pellets were suspended in blocking solution (5% BSA in PBS/PVP) and incubated for 1 h at room temperature. Antiphosphotyrosine antibodies 4G10 or PY20 were diluted 1:100 (10 Ag/ml) in blocking solution, added to the sperm pellets, and incubated overnight at 4jC. After washing with PBS/PVP (3
), TRITC-goat antimouse IgG (10 Ag/ml in 1% BSA in PBS/PVP) was added and incubated for 1 h at RT. After washing as before, samples were spotted on glass slides, mounted in Gelvatol, and examined with an Eclipse TE300 Nikon microscope equipped with a Hamamatsu Orca 100 camera operated with the MetaMorph software (Universal Imaging Corp., USA). Alternatively, smears were allowed to air dry, and stained with FITC – PSA as done for AR assays. Samples were mounted in a mixture of propylgalate – DABCO (3:1) or Gelvatol, and examined in a Nikon C1 confocal microscope. Images were recorded with the program EZ-C1 (Nikon) and processed with MetaMorph. Indirect immunofluorescence in non-SLO-permeabilized sperm Cells maintained in GPM for 3 h under capacitating conditions were washed with PBS (2
) and allowed to air dry on 9-mm round coverslips before fixing in 2% paraformaldehyde and permeabilizing with 0.1% TX-100. All following procedures were as described above, except that an anti-PTP1B (FG6, 20 Ag/ml) IgG was used instead of the antiphosphotyrosine antibodies. Statistical analysis Differences between experimental and control conditions were tested by a two-way ANOVA and Fisher’s protected least-significant difference tests. Percentages (not normalized) were transformed to the arc sine before analysis. Only significant differences ( P < 0.05) between experimental groups are discussed in the text.

Results Calcium stimulates protein tyrosine phosphorylation in human sperm during the AR Protein phosphorylation is a dynamic process in which the phosphorylation state of a protein reflects the competing action of kinases and phosphatases. Extensive investigation in a variety of systems indicates that exocytosis is one process that heavily depends on phosphorylation. A widespread methodology in the study of exocytosis is the use of SLO permeabilization, which we have recently applied to the human sperm AR (Yunes et al., 2000). Permeabilization

allows entrance of exogenous proteins that usually are not able to penetrate the plasma membrane and can be exploited to block the function of target proteins. In the present report, we use this strategy to analyze the role of protein tyrosine phosphorylation in human sperm ARs. First, we investigated the localization of tyrosine-phosphorylated proteins in SLO-permeabilized human sperm by indirect immunofluorescence. The loss of tyrosine phosphorylation in the sperm head during the AR would introduce an underestimation of the level of phosphorylation achieved in response to a calcium stimulus. To circumvent this problem, the final events of the AR—fusion between the plasma and outer acrosomal membranes but not the preceding signaling cascade—was blocked by cleavage of the Q-SNARE protein SNAP-25 with His6-tagged BoNT/E-LC. The vast majority of cells exhibited phosphotyrosine staining on three different compartments of spermatozoa: the principal piece (Fig. 1B, asterisk), the principal piece plus the whole acrosome (Figs. 1B,F), and the principal piece plus the equatorial segment (Fig. 1D). Double staining with FITC – PSA and examination with a confocal microscope revealed correspondence between the phosphotyrosine and the PSA signals on the acrosomal region. Only acrosome-intact cells (i.e. labeled with FITC – PSA; Figs. 1A, C, and E) displayed tyrosine phosphorylation on the acrosomal region (Figs. 1B and F) or in the equatorial segment (Fig. 1D) whereas acrosome-reacted cells (i.e. not stained with FITC –PSA; Fig. 1A, asterisk) failed to exhibit these patterns of staining (Fig. 1B, asterisk). Identical results were observed when another antiphosphotyrosine monoclonal antibody (4G10) was used instead of PY20 (data not shown). The phosphotyrosine immunolocalization described here shows good correspondence with that recently reported in nonpermeabilized human sperm (Ficarro et al., 2003; Sakkas et al., 2003), suggesting that permeabilization of sperm plasma membrane with SLO does not alter the detection of tyrosinephosphorylated proteins. We reasoned that if this posttranslational modification plays a role during the AR, then we should be able to detect changes in tyrosine phosphorylation when we stimulate the cells to undergo exocytosis. Following incubation under capacitating conditions and SLO permeabilization, cells were exposed to 350 nM of His6-tagged BoNT/E-LC to minimize the onset of exocytosis before detection of phosphotyrosine residues with the PY20 antibody. Lack of AR in these cells was corroborated with FITC – PSA (Fig. 2B). Under resting conditions, 19% of the cells exhibited phosphotyrosine staining on the head (Fig. 2A, ‘‘control’’). Challenging with calcium for 15 min doubled the percentage (37%) of cells depicting phosphotyrosine staining in this region (Fig. 2A, ‘‘Ca’’). This increase was precluded by preincubation (15 min) with the tyrosine kinase inhibitor tyrphostin A47 before stimulating the cells (22%; Fig. 2A, ‘‘tyrpA47 + Ca’’). Despite these alterations in PY levels in sperm heads, the percentage of the population undergoing ARs remained largely unchanged (10 –13%, panel B) as a

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Fig. 1. Localization of tyrosine-phosphorylated proteins in SLO-permeabilized human sperm. After permeabilization with SLO, human sperm samples were fixed, permeabilized, and exposed to the antiphosphotyrosine antibody PY20. Cells were dual labeled with a TRITC-coupled antimouse antibody (red staining, B,D,F) and FITC – PSA (green staining, A,C,E). Two labeling patterns were detected on the sperm head, acrosomal (F and arrowheads in B) and equatorial (arrowheads in D). All sperm present strong labeling in the principal piece of the flagellum. Acrosome-reacted sperm (asterisk in A) did not present head staining (asterisk in B). The yellow acrosomal signal in panel G indicates overlap between the green and red signals seen in panels E and F. Shown are fluorescence micrographs recorded with a confocal microscope. Scale bars are depicted in panels A, C, and E.

function of BoNT/E-LC. These data suggest that tyrosine kinase activity is significantly induced when the signaling cascades leading to the AR are triggered by stimulating sperm with calcium. The observed localization of phosphotyrosine residues on the sperm head is consistent with the notion that this posttranslational modification plays a role in sperm exocytosis.

Protein tyrosine phosphorylation is required for human sperm AR triggered by calcium and Rab3A Based on the observation that calcium induces protein tyrosine phosphorylation in sperm, we went on to analyze the role of PTK in the human sperm AR. Our SLO permeabilization approach allows us to separate the process

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Fig. 2. Calcium elicits tyrosine phosphorylation in human sperm. (A) Human sperm were capacitated, SLO-permeabilized, and incubated for 15 min at 37jC in the presence of 350 nM His6-BoNT/E-LC to block exocytosis. When indicated, aliquots were incubated F 80 AM tyrphostin A47 for 15 min at 37jC, followed by 0.5 mM CaCl2 (10 AM free ion) and additional 15-min incubation. Afterward, cells were processed for indirect immunofluorescence using PY20 (10 Ag/ml) as primary antibody. The percentage of labeled heads was calculated scoring the acrosomal (Fig. 1B, arrowheads) plus equatorial (Fig. 1D, arrowheads) fluorescent patterns over the entire population of stained cells. At least 150 cells were scored per condition. The data represent the mean F SEM of three independent experiments. (B) Acrosomal status of sperm treated as in A assessed with FITC – PSA. At least 400 cells were scored per condition.

of capacitation from the distinct process of exocytosis since, during and after permeabilization, cells are maintained in conditions that do not support capacitation (low temperature, unsupplemented simple saline solutions, brief incubation). The separation of these two processes is mandatory to reveal the role of tyrosine phosphorylation in acrosomal exocytosis, a task that has proven difficult to investigate in a heterogeneous population of intact sperm cells that are engaged in capacitation as well as acrosomal exocytosis. First, a specific antibody against phosphotyrosine (4G10) was introduced into SLO-permeabilized human sperm before challenging with Ca2+ or GTP-loaded Rab3A. Addition of GTP-loaded recombinant Rab3A to SLO-permeabilized human sperm induced an exocytotic response of magnitude comparable to that of Ca2+ (Fig. 3A, ‘‘Rab’’). Pretreatment with the antiphosphotyrosine antibody inhibited the AR by 90% (Fig. 3A, ‘‘anti-PY + Ca’’, ‘‘anti-PY + Rab’’). Inhibition was not observed when the antibody was preincubated with ortho-phospho-D,L-tyrosine (Fig. 3A, ‘‘antiPY* + Ca’’), suggesting its inhibitory effect was specific due to binding to endogenous phosphotyrosyl groups. These data indicate that tyrosine-phosphorylated proteins are involved in calcium- and Rab3A-triggered acrosomal exocytosis in human sperm. These observations do not allow us to distinguish whether the modification of tyrosine residues by PTK(s) occurs before or after permeabilization and/or as a function of sperm challenge with inducers. To discern between these possibilities, permeabilized sperm cells were incubated with the PTK inhibitors genistein and tyrphostins A47 and A51, or with tyrphostin A1 as negative control, before Ca2+

stimulation. Several different concentrations of each inhibitor were tested to determine the optimal inhibitory dose and to minimize nonspecific effects. Thus, the calculated IC50 under our assay conditions were 85 AM for genistein and 29 AM for tyrphostin A47 (data not shown). The results in Fig. 3B show that calcium-triggered exocytosis was inhibited by 80% by 185 AM genistein, and by over 90% by 80 AM tyrphostins A47 and A51. The inactive analog tyrphostin A1 had no effect. Our data suggest that active PTKs—and therefore de novo phosphorylations—are required for calcium-triggered exocytosis in permeabilized human sperm. Moreover, it is suitable to speculate that the calcium stimulus is responsible—directly or indirectly—for the activation of those PTKs. Calcium triggers sperm protein tyrosine phosphorylation in two waves, one before and one after the efflux of intra-acrosomal calcium In an attempt to refine our understanding of the sequence of signaling events leading to the AR, we used a reversible blocker of exocytosis that prevents the AR by sequestering intra-acrosomal calcium (De Blas et al., 2002). The reversible blocker of choice was the photolabile calcium chelator Onitrophenyl-EGTA (NP-EGTA). UV photolysis of NP-EGTA releases the caged calcium rapidly and in high photochemical yield (Ellis-Davies and Kaplan, 1994). In our SLO-permeabilized human sperm model, the membrane-permeable compound NP – EGTA – AM crosses the plasma and outer acrosomal membranes, accumulating inside the acrosome and thus precluding the availability of intra-acrosomal calci-

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Fig. 3. Protein tyrosine phosphorylation is required for human sperm AR. (A) SLO-permeabilized human sperm were treated for 15 min at 37jC in the presence of 10 Ag/ml (67 nM) of the antiphosphotyrosine antibody 4G10 pretreated (anti-PY* + Ca) or not (anti-PY + Ca) with 60 AM orthophospho-D,L-tyrosine. Acrosomal exocytosis was evaluated by FITC – PSA binding after an additional 15-min incubation at 37jC in the absence or presence of 0.5 mM CaCl2 (10 AM free ion, grey bars) or 300 nM GTP-g-Sbound Rab3A (black bars). (B) SLO-permeabilized human sperm were treated for 15 min at 37jC in the presence of 185 AM genistein (gen), 80 AM tyrphostin A47 (tyrpA47), 80 AM tyrphostin A51 (tyrpA51), or 80 AM tyrphostin A1 (tyrp A1). Acrosomal exocytosis was evaluated by FITC – PSA binding after an additional 15-min incubation at 37jC in the presence of 0.5 mM CaCl2 (10 AM free ion). The values were normalized as described in Materials and methods. Actual percentages of reacted sperm for negative (control, 0 mM CaCl2) and positive (Ca, 0.5 mM CaCl2, no further additions) controls ranged between 17 – 25% and 27 – 36%, respectively. The data represent the mean F SEM of at least three independent experiments.

um. By using flash photolysis of the NP-EGTA, we have previously shown that the occurrence of the calcium- and Rab3A-triggered AR relies on the efflux of intra-acrosomal calcium through IP3-sensitive calcium channels (De Blas et

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al., 2002). By combining the NP – EGTA – AM strategy with the experimental approach described in Fig. 2, we assessed whether a Ca2+-induced increase in sperm head tyrosine phosphorylation takes place before or following intra-acrosomal calcium efflux. When permeabilized cells were preloaded with 10 AM NP – EGTA –AM, challenging with 10 AM calcium induced a moderate but significant (13 – 23%) augmentation in tyrosine phosphorylation when the cells remained in the dark (Fig. 4, ‘‘NP’’ vs. ‘‘NP!Ca’’). When NP-EGTA was converted by UV flash to products with negligible calcium affinity, the percentage of cells displaying head staining increased even further (38%; Fig. 4, ‘‘NP!Ca!hr’’), reaching a level of staining comparable to the maximum obtained with calcium in the absence of NPEGTA (42%; Fig. 4‘‘Ca’’). We interpret these two discrete increases as a consequence of accumulative and sequential phosphorylation events triggered by external and intra-acrosomal calcium, respectively. Addition of tyrphostin A47 (80 AM) to cells treated previously with calcium had no effect on the percentage of cells exhibiting tyrosine phosphorylation on the head when the cells were kept in the dark (27%; Fig. 4, ‘‘NP!Ca!tyrpA47’’). In other words, tyrphostin—when added after the stimulus—was unable to suppress the first round of tyrosine phosphorylation elicited by external calcium. In contrast, tyrphostin abolished the additional increase in tyrosine phosphorylation accomplished by UV illumination (28%; Fig. 4, ‘‘NP!Ca!tyrpA47!hr’’), maintaining the percentage of stained heads identical to that of cells challenged with calcium but not illuminated (23%; Fig. 4, ‘‘NP!Ca’’). In other words, tyrphostin suppressed phosphorylations that occurred because of PTKs activated by the efflux of intra-acrosomal calcium. In summary, our data support the notion that sperm head tyrosine phosphorylation takes place in two waves, the first shortly after cytoplasmic calcium increase and the second in a later step, achieved after intra-acrosomal calcium efflux. The increase in tyrosine phosphorylation appears to be due to calcium-triggered PTK activation, which also takes place in two waves, one before and one following intravesicular calcium release. Remarkably, 10 AM cytosolic calcium is not sufficient to elicit maximum tyrosine phosphorylation. Sperm must contain PTKs that presumably require a specific microenvironment for optimal activity, with higher and/or more localized calcium concentrations achieved upon efflux of intra-acrosomal calcium. Alternatively, a component activated by intraacrosomal calcium release, instead of calcium itself, could be responsible for the activation of these late-acting PTKs. Protein tyrosine phosphorylation is required in the AR after the efflux of intra-acrosomal calcium To investigate the correlation between the calcium-induced increase on sperm head tyrosine phosphorylation and acrosomal exocytosis, we applied the NP-EGTA approach to AR assays. Once again, the protocol allows for an AR inducer to prepare the fusion machinery up to the point

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Fig. 4. Calcium elicits two sequential rounds of tyrosine phosphorylation in human sperm. Human sperm were capacitated, SLO-permeabilized, and incubated for 15 min at 37jC in the presence of 350 nM His6-BoNT/E-LC to block exocytosis. One aliquot was untreated (control), another was incubated 15 min at 37jC with 0.5 mM CaCl2 (10 AM free ion, Ca), and the rest was loaded with 10 AM NP – EGTA – AM for 15 min at 37jC in the dark. When indicated, 0.5 mM CaCl2 was added and incubations proceeded for additional 15 min in the dark, followed by another 15 min incubation in the presence (NP!Ca!tyrpA47!) or absence (NP!Ca!) of 80 AM tyrphostin A47. UV flash photolysis of the chelator was induced at the end of the incubation period (NP!hr, NP!Ca!hr, NP!Ca!tyrpA47!hr). The rest of the tubes were not illuminated (NP!, NP!Ca!, NP!Ca!tyrpA47!). After a final 5-min incubation, samples were processed for PY20 immunofluorescence as described. Percentage of head labeling was evaluated as explained in the legend to Fig. 2. The data represent the mean F SEM of three independent experiments.

when intra-acrosomal calcium release is required. This calcium is kept unavailable by the chelator NP-EGTA. The inhibitors to test are added, and finally release of the caged calcium with a flash of UV light restores the intra-acrosomal pool. Thus, if an inhibitor blocks exocytosis when added after an inducer has initiated the AR, it suggests that the target(s) of the inhibitor is required downstream of intra-

acrosomal calcium release. When permeabilized cells were preloaded with 10 AM NP – EGTA – AM, challenging with 10 AM calcium failed to elicit exocytosis as long as the cells were kept in the dark (Fig. 5, top, NP!Ca). When NPEGTA was converted by UV flash to products with negligible calcium affinity, exocytosis was restored (Fig. 5, top, NP!Ca!hr). These data demonstrate that cytosolic calci-

Fig. 5. A late protein tyrosine phosphorylation event is required for human sperm AR. Top bars: SLO-permeabilized human sperm were loaded with 10 AM NP – EGTA – AM, followed by addition of 0.5 mM CaCl2 and incubated for 15 min at 37jC in the dark. As control, an aliquot was kept in the dark (NP!Ca) and another illuminated at the end of the incubation (NP!Ca!hr). NP – EGTA – AM-loaded sperm were treated F antiphosphotyrosine antibody 4G10 (10 Ag/ml) followed by addition of 0.5 mM CaCl2 and further incubation for 15 min at 37jC in the dark (NP!anti-PY!Ca!hr). Alternatively, NP – EGTA – AMloaded sperm were treated first with 0.5 mM CaCl2 and then with 10 Ag/ml of 4G10 and incubated as before (NP!Ca!anti-PY!hr). In all cases, UV flash photolysis of the chelator was induced at the end of the incubation period. Bottom bars: Sperm were treated as described above, except that 4G10 was replaced with 185 AM genistein (gen), 80 AM tyrphostin A47 (tyrp A47), or 80 AM tyrphostin A51 (tyrp A51). Actual percentages of reacted sperm for negative (control, 0 mM CaCl2) and positive (Ca, 0.5 mM CaCl2) controls ranged between 15 – 34% and 24 – 54%, respectively. The data represent the mean F SEM of at least three independent experiments.

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um is not sufficient to trigger exocytosis but requires the availability of intra-acrosomal calcium. Having shown the requirement of tyrosine-phosphorylated proteins (Fig. 3) and of intra-acrosomal calcium (Fig. 5, top; De Blas et al., 2002) in the calcium-induced AR, we investigated the temporal relationship between the two. First, we loaded permeabilized sperm with the caged calcium buffer NP –EGTA –AM. In one case, the antiphosphotyrosine antibody 4G10 was added and the cells were preincubated for 15 min before addition of calcium. Following these treatments, uncaging of intraacrosomal calcium was achieved by flash photolysis of the NP – EGTA –AM (Fig. 5, top, ‘‘NP!anti-PY!Ca!hr’’). As expected, AR was blocked since the inhibitor was present throughout the experiment. In a second experimental setting, we added calcium to NP – EGTA – AM-loaded sperm and incubated for 15 min before addition of 4G10 in the dark. UV flash photolysis of the calcium chelator was applied at the end of the second incubation (Fig. 5, top, ‘‘NP!Ca! anti-PY!hr’’). A complete blockage was also observed under these conditions, indicating that tyrosine-phosphorylated proteins are acting downstream of the release of intraacrosomal calcium (Fig. 5, top series). To distinguish between preexisting and de novo phosphorylation, genistein (185 AM) was used instead of 4G10. The results in Fig. 5 (bottom series) show that genistein blocks exocytosis, which is consistent with a kinase acting downstream the intra-acrosomal calcium release. A requirement for activation of PTK in the AR at this late step was further tested using two more PTK inhibitors, tyrphostin A47 (80 AM) and tyrphostin A51 (80 AM), using an identical strategy. Similar to 4G10, all three inhibitors blocked the AR regardless of the sequence of addition (Fig. 5, bottom series), suggesting that the AR relies on the phosphorylation of crucial phosphotyrosine residues by PTK on proteins whose role in fusion is played downstream

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of intra-acrosomal calcium release. Taken together, our data confirm the requirement of tyrosine phosphorylation involving PTK activities in the AR. Furthermore, the observations place a requirement for this posttranslational modification late in the signaling cascade leading to sperm membrane fusion. Enhanced human sperm protein tyrosine phosphorylation follows PTP inhibition by sodium pervanadate Although there has been progress in the last decade in dissecting tyrosine phosphorylation-related signaling pathways that are crucial for sperm function, surprisingly little attention has been paid to the dynamics of the process. PTP activity is of particular relevance since it can potentially control the strength of phosphotyrosine-related pathways. Therefore, we examined sperm PTP activity by analysis of phosphotyrosine-containing proteins during capacitation in the presence of a PTP inhibitor. To this end, we conducted immunoblot experiments using the specific antiphosphotyrosine antibody PY20. When capacitated sperm were exposed to different concentrations of vanadyl hydroperoxide (pervanadate)—a membrane-permeant derivative of the inhibitor sodium orthovanadate—for 3 h, we observed a stimulatory effect on protein tyrosine phosphorylation. With regard to PY levels, pervanadate displayed a biphasic dose – response curve, which was maximal at 100 AM and had returned to control or lower levels at 1 mM (Fig. 6A). Sperm viability was not affected (approximately 95% live cells were observed in the presence of 2 mM pervanadate; Fig. 6B). Motility was quantitatively and qualitatively affected only at pervanadate concentrations higher than 100 AM (approximately 50% ‘‘twitching’’ sperm with 0.5 mM and 100% immotility with 1 mM after 3 h of treatment; Fig. 6B). When the PTK inhibitor tyrphostin A47 was incorporated

Fig. 6. Enhanced protein tyrosine phosphorylation follows PTP inhibition by sodium pervanadate. (A) After incubation in HTF for 5 h, human sperm were exposed to 0 (lane 1), 10 (lane 2), 100 (lane 3), 500 (lane 4) and 1000 (lane 5) AM pervanadate in HTF for 3 h prior to processing for Western blot analysis using PY20 as probe. Protein corresponding to 1.6
106 cells was loaded per lane. The experiment was repeated three times, with similar results in each trial. Molecular weight standards (kDa) are indicated to the left of lane 1. (B) Effect of pervanadate on the total motility and viability of human sperm samples. Percentage of motility (circles) and viability (triangles) were scored for all samples illustrated in panel A immediately before processing for Western blot. (C) Human sperm were untreated (lane 1) or treated with 50 AM tyrphostin A47 (lane 2), 100 AM pervanadate (lane 3), or 50 AM tyrphostin A47 + 100 AM pervanadate (lane 4) in HTF for 3 h as described. Sperm were lysed, proteins extracted, and analyzed by Western blot using PY20 as probe. Protein from 106 cells was loaded per lane. Shown is a blot representative of three experiments.

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into the capacitation media to suppress de novo phosphorylation, a marked general decrease in protein tyrosine phosphorylation was observed compared to the untreated control (Fig. 6C, lanes 1 and 2). In contrast, when PTP activity was abolished with 100 AM pervanadate, there was a significant increase in the phosphotyrosine content of sperm proteins (Fig. 6C, lane 3). The appearance of phosphotyrosine in this situation appears to depend upon an active PTK(s) since a combination of tyrphostin and pervanadate reversed protein tyrosine phosphorylation to levels similar to those in the control (Fig. 6C, lane 4). None of these treatments affected the percentage of live or motile cells (approximately 90%), although sperm treated with pervanadate exhibited less-vigorous motility. These data show that reversible phosphorylation and dephosphorylation takes place during human sperm capacitation. In addition, they substantiate the notion that responses to pervanadate are attributable to enhanced tyrosine phosphorylation. Finally, they identify robust PTP activity in human sperm since their inhibition can enhance protein tyrosine phosphorylation even in conditions when PTK activity is maintained at a minimum. PTP activity in human and mouse sperm In view of the functional role of PTP in sperm protein tyrosine phosphorylation, we undertook biochemical experiments to further characterize this activity. First, we searched for PTP activity in crude mixtures of whole human (Figs. 7A,B) and mouse (Figs. 7C – F) sperm extracts. Sperm lysates from both sources generated high PTP activity when measured with [32P]poly (Glu-Tyr) as substrate (Figs. 7A – F). PTP activity was 2.5-fold higher in lysates from noncapacitated compared to capacitated human sperm samples (Figs. 7A,B). The enhanced tyrosine phosphorylation observed in pervanadate-treated human sperm (Figs. 6 and 8) suggests that its effect is exerted through inhibition of PTP activity. To confirm this hypothesis, cells were treated with

pervanadate and the PTP activity extracted from these cells was determined. As seen in Fig. 7B, incubation of both noncapacitated and capacitated sperm with pervanadate completely inhibited PTP activity. Since previous reports on the presence of these enzymes in male gametes have been restricted to the identification of several cDNA sequences by PCR cloning in rodent libraries, we extended our analysis to include the determination of PTP activity in mouse sperm. As was the case in human sperm, PTP activity was 1.8-fold lower in capacitated than in noncapacitated mouse sperm (Figs. 7C,D). PTP activity partitioned into both the soluble and particulate compartments of capacitated and noncapacitated mouse sperm, perhaps reflecting the contribution of more than one PTP (Fig. 7F). Membranebound activity was 1.4-fold higher than that of the cytosolic fraction. The influence of capacitation followed the same pattern as that found in unfractionated sperm. As expected, human (Fig. 7B) and mouse (Fig. 7E) PTP activities were significantly inhibited by pervanadate. Random copolymers of glutamate and tyrosine—such as poly(Glu-Tyr), ratio 4:1, used throughout this study as PTP substrate—have been shown to be noncompetitive inhibitors of several PTP isoforms (Tonks et al., 1988b). Therefore, to maximize detection of all PTP activities in sperm, we turned to RCML as substrate. Similar to the previous results, extracts prepared from capacitated mouse sperm displayed PTP activity 5.1fold lower than that from noncapacitated cells (Fig. 7D). The characteristics of PTP activity measured with RCML followed the same pattern as with the poly(Glu-Tyr) substrate: activity was present in both the soluble and particulate fractions of mouse sperm with membrane-bound fractions demonstrating higher activity than the cytosolic, and capacitation decreased the activity in both fractions (data not shown). These results indicate that mammalian sperm exhibit substantial PTP activity and suggest that, in addition to differential kinase activity, PTP activity changes when cells are capacitated. To determine the presence of specific PTP isoforms in mammalian sperm, we performed

Fig. 7. PTP activity in human and mouse sperm. (A) Noncapacitated (NC, grey circles) and capacitated (C, black circles) human sperm were homogenized and PTP activity measured in aliquots of the crude lysates using [32P]poly(Glu-Tyr) as substrate. Data represent mean from five experiments conducted in triplicate. (B) Noncapacitated (NC, grey bars) and capacitated (C, black bars) human sperm were preincubated with (pV) or without (control) 1 mM sodium pervanadate before homogenization. PTP activity was assessed as in A. Data (106 cells/assay) are normalized with respect to the activity exhibited by noncapacitated sperm without pervanadate treatment (100%) and represent the mean F SEM from three experiments conducted in duplicate. (C) Noncapacitated (NC, grey circles) and capacitated (C, black circles) mouse sperm were homogenized and PTP activity measured in aliquots of the crude lysates using [32P]poly(Glu-Tyr) as substrate. Data represent mean from ten experiments conducted in duplicate. (D) Noncapacitated (NC, grey bars) and capacitated (C, black bars) mouse sperm samples were homogenized and PTP activity measured in aliquots of the crude lysates using [32P]poly(Glu-Tyr) (G-T)n and [32P]RCML (RCML) as substrates. Data (106 cells/assay) are normalized respect to the activity exhibited by noncapacitated sperm (100%) and represent the mean F SEM from two experiments conducted in duplicate. (E) PTP activity from noncapacitated mouse sperm lysates (106 cells/assay) was assessed with [32P]poly(Glu-Tyr) in the presence of increasing concentrations of pervanadate. Data are normalized with respect to activity in the absence of pervanadate (100%) and represent the mean from two experiments conducted in triplicate. (F) Noncapacitated (NC, grey bars) and capacitated (C, black bars) mouse sperm (106 cells/assay) were separated into soluble (sol) and particulate (part) fractions and tested for PTP activity with [32P]poly(Glu-Tyr). Data are normalized with respect to the activity exhibited by the particulate fraction of noncapacitated sperm (100%) and represent the mean F SEM from four experiments conducted in triplicate. (G) Proteins from noncapacitated (lanes 1 and 3) and capacitated (lanes 2 and 4) mouse sperm cytosol (5
106cells/lane) and an unfractionated human sperm extract (106 cells, lane 5) were run in 10% SDS-gels, transferred and probed with the anti-PTP1B antibodies clone 15 (lanes 1 and 2) and FG6 (lanes 3 – 5). Electrophoretic migration of PTP1B is indicated by an arrow. Molecular weight standards (kDa) are indicated to the left of lane 1. (H) Human sperm were fixed, permeabilized, and immunostained with a mouse monoclonal anti-PTP1B antibody (clone FG6) followed by a TRITC-labeled goat antimouse antibody. Shown is an epifluorescence micrograph of three typically stained cells (bar = 6 Am).

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Fig. 8. PTP activity is required for human sperm AR. (A) Human sperm were capacitated, SLO-permeabilized, and incubated for 15 min at 37jC in the presence of 350 nM His6-BoNT/E-LC to block exocytosis. Aliquots were incubated F30 AM sodium pervanadate (pV) or F0.5 mM CaCl2 (10 AM free ion, Ca) for 15 min at 37jC. Afterward, cells were processed for indirect immunofluorescense using 10 Ag/ml PY20 as primary antibody. The percentage of labeled heads was calculated as described in the legend to Fig. 2. The data represent the mean F SEM of two independent experiments. (B) Human sperm were capacitated, SLO-permeabilized, and incubated for 15 min at 37jC in the presence of 350 nM His6-BoNT/E-LC to block exocytosis. One aliquot was untreated (control) and another was incubated with 30 AM sodium pervanadate (pV) for 15 min at 37jC. Sperm were lysed, and proteins were extracted and analyzed by Western blot using PY20 as probe. Protein from 2.2
106 cells was loaded per lane. Molecular weight standards (kDa) are indicated to the right. Shown is a blot representative of three experiments. (C) SLO-permeabilized human sperm were treated for 15 min at 37jC in the presence of 30 AM pervanadate (pV), 10 AM phenylarsine oxide (PAO), 40 AM DMHV (DMHV), or 40 AM ethyl-3,4-dephostatin (dephost) plus 0.5 mM CaCl2 (10 AM free ion). Acrosomal exocytosis was evaluated by FITC – PSA binding. The values were normalized as explained in Materials and methods. Actual percentages of reacted sperm for negative (control, 0 mM CaCl2) and positive (Ca, 0.5 mM CaCl2, no further additions) controls ranged between 5 – 12% and 15 – 22%, respectively. The data represent the mean F SEM of at least three independent experiments.

Western blot screening with an array of anti-PTP antibodies specific for transmembrane and soluble enzymes. We obtained a positive signal when probing with two mouse monoclonal antibodies raised against human PTP1B expressed in E. coli. FG6 detected a 50 kDa protein in noncapacitated and capacitated mouse sperm cytosol and an unfractionated human sperm extract (Fig. 7G, lanes 3, 4 and 5, respectively). Similarly, PTP1B was detected in the cytosol of noncapacitated (Fig. 7G, lane 1) and capacitated (Fig. 7G, lane 2) mouse sperm by a Transduction Laboratories antibody (clone 15). Neither antibody detected PTP1B in the particulate fraction of either noncapacitated or capacitated sperm (data not shown). Indirect immunofluorescence experiments conducted with the FG6 monoclonal antibody indicate that PTP1B localizes to the midpiece of the flagellum and the equatorial segment of human sperm (Fig. 7H). This study represents, to the best of our knowledge, the first report describing PTP presence and activity at the protein level in human sperm. Furthermore, our findings support the notion that capacitation might modulate tyrosine phosphorylation through the inhibition of one or more PTPs. PTP activity is required for calcium-triggered human sperm AR The data presented above demonstrate that protein phosphorylation plays a fundamental role in regulating acrosomal exocytosis in human sperm. In addition, they

indicate the sensitivity of human sperm PTPs to pervanadate. Since considerable evidence suggests that PTPs can exert both positive and negative effects on a signaling pathway, we therefore set out to investigate the requirement of PTP in the AR. First, we inspected the subcellular localization of tyrosine phosphorylation in cells where PTP activity was suppressed by the use of pervanadate. When capacitated human sperm were SLO-permeabilized, treated with His6-tagged BoNT/E-LC to prevent exocytosis, and subjected to indirect immunofluorescence with the PY20 antiphosphotyrosine antibody, a high percentage of cells displayed strong, bright fluorescence restricted to the principal piece of the flagellum. Fluorescence was observed on the sperm head in a small subpopulation (7%) of cells (Fig. 8A, ‘‘control’’). As before, treatment with calcium induced a 4-fold increase (27%) in the percentage of sperm depicting head-associated fluorescence (Fig. 8A, ‘‘Ca’’). Similarly, when sperm PTPs were inhibited by a 15-min treatment with sodium pervanadate, bright staining could be detected on the head region in addition to the principal piece in 26% of the cells (Fig. 8A, ‘‘pV’’). Simultaneous treatment with calcium and pervanadate did not increase the percentage of stained cells any further (data not shown), perhaps reflecting saturation of the system. The increase in protein tyrosine phosphorylation in SLO-permeabilized cells accomplished by a brief PTP inhibition with pervanadate was also detected by Western blot in a variety of proteins (Fig. 8B). These data suggest that sperm possess one or more active tyrosine kinases and

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that phosphoprotein accumulation is normally prevented by vigorous concomitant phosphatase activity. Furthermore, enhanced protein tyrosine phosphorylation secondary to inhibition of PTP occurs during the restricted time frame when sperm are primed to undergo the AR. Last, we explored the possibility that PTP, as well as PTK, might be involved in the AR. To this end, we calculated the IC50 of four different PTP inhibitors in our assay, which were 6.64 AM for pervanadate, 0.7 AM for phenylarsine oxide (PAO), 3.74 AM for bis(N,N-dimethylhydroxoamido)hydroxovanadate (DMHV), and 7.16 AM for ethyl-3,4-dephostatin (data not shown). When permeabilized human sperm were incubated with 30 AM pervanadate and simultaneously challenged with Ca2+, a marked inhibition of sperm exocytosis was observed (Fig. 8C, ‘‘pV’’). Because pervanadate inhibits other enzymes in addition to PTP (Simons, 1979), we tested the effect of the more specific PTP inhibitors PAO (10 AM), DMHV (40 AM), and ethyl-3,4-dephostatin (40 AM). Treatment of permeabilized sperm with all these inhibitors caused an almost complete inhibition of the calcium-triggered AR (Fig. 8C). These results suggest that PTP activity is necessary for calcium to elicit acrosomal exocytosis. Taken together, our data suggest that dynamic protein tyrosine phosphorylation—catalyzed by PTK and PTP—has an essential role in human sperm acrosomal exocytosis.

Discussion Fertilization depends, among other things, upon the regulated fusion of the acrosome with the spermatozoon’s plasma membrane, leading to the exocytotic process termed the AR. In several mammalian species, secretion is triggered during gamete contact by ZP3, a glycoprotein component of the zona pellucida (reviewed in McLeskey et al., 1998; Wassarman, 1999; Wassarman et al., 2001). ZP3 produces a sustained increase of the internal Ca2+ concentration, which subsequently leads to the AR (Bailey and Storey, 1994; O’Toole et al., 2000). Similarly, one of the nongenomic effects of the steroid hormone progesterone is induction of calcium influx and the AR of mammalian spermatozoa (Aitken et al., 1996; Kirkman-Brown et al., 2000; Mendoza et al., 1995). Protein phosphorylation plays a central role in regulating many cellular processes in eukaryotes. In particular, phosphorylation on tyrosyl residues has been shown to modulate several aspects of sperm function. Although several laboratories have been concerned with unveiling the interplay between calcium and tyrosine phosphorylation during acrosomal exocytosis, the issue remains unresolved and many contradictory findings are unexplained. For instance, modulation of T-type calcium channels in murine spermatogenic cells by PTKs and PTPs has been reported (Arnoult et al., 1997). In mouse sperm, tyrphostin A48 inhibits the ZPinduced AR as well as the increase in intracellular calcium

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(Bailey and Storey, 1994). In contrast, treatment with genistein or tyrphostin A47 has been shown to inhibit the progesterone-induced AR of human sperm. Yet, these PTK inhibitors have no effect on progesterone-triggered calcium influx (Aitken et al., 1996; Kirkman-Brown et al., 2002; Mendoza et al., 1995), suggesting that in this system PTKs exert their effects on the AR at a site downstream of calcium mobilization. On the other hand, src-related PTKs are involved in the AR upstream of the capacitative calcium entry (Dorval et al., 2003). In the present study, we investigate the involvement of protein tyrosine phosphorylation during the calcium-dependent acrosomal exocytosis in human sperm. We make use of a permeabilization protocol developed in our laboratory which permits entry of antibodies, recombinant proteins, Ca2+, and other molecules into the cell (Diaz et al., 1996; Yunes et al., 2000). This system is readily manipulated, reflects the in vivo organization of the cell, and has been useful to demonstrate roles for Rab3A (Yunes et al., 2000), NSF (Michaut et al., 2000), synaptotagmin VI (Michaut et al., 2001), the SNARE complex (Tomes et al., 2002), and calmodulin (Yunes et al., 2002) in the Ca2+-dependent AR of human sperm. Furthermore, it has the important advantage of dissociating capacitation from exocytosis-linked changes. Previous studies regarding the role of protein tyrosine phosphorylation on the AR were restricted to inhibition of ZP- or progesteroneinduced sperm exocytosis by PTK inhibitors, some of them not highly specific (reviewed in Baldi et al., 2002). Because permeation of most of these inhibitors through cells’ plasma membrane is slow, extensive incubation times are required for intracellular diffusion. Consequently, it is difficult to unequivocally ascertain whether a given inhibitor affects capacitation or exocytosis. In contrast, the entrance of inhibitors in permeabilized sperm is instantaneous and incubations proceed in conditions that do not support capacitation. When our experiments required the use of inhibitors, we were careful to apply them in conditions designed to minimize possible unspecific effects. To this end, we performed dose –response curves and calculated the IC50 for each PTK and PTP inhibitor in calcium-triggered AR assays in SLO-permeabilized sperm. Applying this criterion, we selected the minimum concentration of each inhibitor that caused a maximal biological effect. These concentrations were always V 10 IC50. Thus, we used 185 AM genistein (IC50 = 85 AM), 80 AM tyrphostin (IC50 = 29 AM), 30 AM pervanadate (IC50c7 AM), 10 AM PAO (IC50c1 AM), 40 AM DMHV (IC50c0.4 AM), and 40 AM ethyl-3,4-dephostatin (IC50 = 7 AM). Our chosen concentrations of genistein, tyrphostins, pervanadate, and PAO were similar or lower than those reported by others working with mammalian sperm (Arnoult et al., 1997; Leclerc et al., 1997; Leyton et al., 1992; Luconi et al., 1995; Mendoza et al., 1995). Since our study constitutes the first report on the use of ethyl-3,4-dephostatin and DMHV on sperm, we refer to the IC50 reported by their manufacturer (3.2 and 22 – 27 AM, respectively).

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Following the SLO-permeabilization approach, we were able to determine a substantial increase in protein tyrosine phosphorylation on the head of unreacted human sperm because of the activation by calcium of the signaling pathways leading to acrosomal exocytosis (Fig. 2A). Acrosomal localization is what would be expected of a protein modification with a putative role in the AR. Phosphorylation augmented by Ca2+ appears to be due to stimulation of PTKs rather to inhibition of PTP, since tryphostin A47 reverses it completely. Whether calcium activates PTKs directly or indirectly is currently unknown, as are the kinases involved in the process. Interestingly, a calciumdependent tyrosine kinase, known variously as PYK2, CAKb, or RAFTK, has recently been described in mouse spermatozoa (Chieffi et al., 2003). We have previously shown that the acrosome behaves as a calcium storing organelle (De Blas et al., 2002). Release of intravesicular calcium takes place after tethering of the acrosome to the plasma membrane and is necessary for human sperm AR. By using a photosensitive calcium chelator in combination with a PTK inhibitor, we have been able to reveal the existence of two components of tyrosine phosphorylation: an early phase accomplished by addition of external calcium, and a secondary phase that requires efflux of intra-acrosomal calcium (Fig. 4). We hypothesize that the first would be a consequence of the increase in calcium that follows opening of voltage-sensitive and storeoperated channels in the plasma membrane. The second phase would take place only after this increase in cytosolic calcium elicits the efflux of more calcium from the acrosome, and might reflect reactions that only take place in a particular microenvironment characterized by highly localized, elevated calcium concentrations. Our functional assay shows that calcium-induced protein tyrosine phosphorylation is essential for the human sperm AR. The first line of evidence comes from the use of antiphosphotyrosine antibodies. Treatment with 10 Ag/ml 4G10 resulted in inhibition of Ca2+-induced acrosomal release (Fig. 3A), indicating a need for tyrosine-phosphorylated proteins in the AR. This result also highlights the high sensitivity of our assay, since in the only other related study published, microinjection of 5 mg/ml of an antiphosphotyrosine antibody was necessary to inhibit exocytosis of GLUT4 transporters (Chen et al., 1997a). In human sperm, the small GTPase Rab3 stimulates the AR to levels similar to those attained with calcium (Yunes et al., 2000). Rab3stimulated secretion is also inhibited effectively by 4G10 (Fig. 3A), indicating that tyrosine-phosphorylated proteins play a role downstream of the Rab3-triggered tethering of the acrosome to the plasma membrane. The second line of evidence on the role of protein tyrosine phosphorylation in the AR comes from the use of PTK inhibitors. As shown in Fig. 3B, loading of SLO-permeabilized sperm with genistein, tyrphostin A47, or tyrphostin A51 reduced the sperm’s exocytotic response to Ca2+. Since the identity of the kinases involved is unknown, we chose to use a variety of

inhibitors because each appears to be selective for subsets of enzymes rather than generic for all PTKs (Casnellie, 1991). Furthermore, we selected drugs with different mechanisms of action. Thus, genistein is an inhibitor competitive with ATP whereas tyrphostins are competitive with protein and peptide substrates (Casnellie, 1991). The fact that two independent methods to block tyrosine phosphorylation prevent ARs provides compelling evidence that calcium utilizes a tyrosine kinase pathway to mediate acrosomal exocytosis. The third line of evidence comes from the combination of NP –EGTA – AM with antiphosphotyrosine antibodies or PTK inhibitors. Our results indicate that phosphotyrosine-containing proteins are required at or after release of calcium from the acrosome (Fig. 5). It is worth noting that the possibility of phosphotyrosine-containing proteins playing a role in earlier steps during the AR is not disputed by our data. These findings pose the exciting question of whether these features are a signature of sperm’s exocytotic cascade or represent a widespread, and as of yet unexplored, phenomenon. Putative candidates for this late role in fusion that have been shown to undergo tyrosine phosphorylation in sperm and other systems include the NSF analog valosin-containing protein (Ficarro et al., 2003), the SNARE protein SNAP-25 (Zhou and Egan, 1997), and synaptophysin (Pang et al., 1988). Like most normal cells, resting sperm contain comparatively low levels of tyrosine-phosphorylated proteins, presumably reflecting the finding that overall PTP activity is many orders of magnitude higher than overall PTK activity. Indeed, increasing concentrations of pervanadate result in an increase in phosphotyrosine on a number of intracellular substrates in capacitating sperm (Fig. 6). This finding also suggests that one or more PTK is constitutively active in these cells. In fact, tyrphostin A47 significantly decreased basal tyrosine phosphorylation as well as that stimulated by pervanadate (Fig. 6C). Taken together, these results emphasize the importance of PTP in the maintenance of tyrosine phosphorylation during sperm capacitation. After capacitation and SLO permeabilization, inhibition of PTP with a low dose of pervanadate applied during a short time led to a significant increase in protein tyrosine phosphorylation (Figs. 8A,B), reflecting the presence of vigorous PTP activity under these conditions. Although indirect evidence concerning the presence of PTP activity in sperm, assessed by an increase in tyrosine phosphorylation following treatment with vanadate, has been reported by others (see for instance Aitken et al., 1995; Berruti and Martegani, 1989; Leclerc et al., 1997; Luconi et al., 1996), direct measurement of this activity is lacking in the literature, except for work from Shivaji’s laboratory, concerned mainly with hamster sperm motility (Devi et al., 1999; Patil et al., 2002). Several PTPs have been cloned from rat (Mizoguchi and Kim, 1997) and mouse (Kaneko et al., 1993; Miyasaka and Li, 1992a,b; Ohsugi et al., 1997; Park et al., 2000) testis cDNA libraries; for some of them, spermatogenic cell localization has been suggested. Here, we report the detec-

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tion of PTP activity in whole cell extracts from mouse and human sperm (Fig. 7). The observation that sperm incubation in capacitating conditions results in marked inhibition of PTP activity is particularly attractive, since it provides a plausible explanation for the increased protein tyrosine phosphorylation observed by several laboratories in a number of species. We detected nontransmembrane PTP-1B by Western blot in mouse and human sperm (Fig. 7G) and by indirect immunofluorescence in human sperm (Fig. 7H). Interestingly, there is partial overlapping in the localization between PTP1B and its putative targets, tyrosine-phosphorylated proteins (Fig. 1D, arrowheads). Although we do not yet have evidence regarding the physiological role of this enzyme in sperm, it is conceivable to speculate on a role of this protein in secretion since it is enriched in secretory granules (Wimmer et al., 1999) and modulates glucose transporter GLUT4 translocation (Chen et al., 1997b). Next, we investigated whether PTP activity plays a direct role in the acrosomal exocytosis. As is the case with PTK, the actual PTP is unknown. Therefore, we selected compounds with different mechanisms of action to inhibit sperm PTP activity. The hallmark of PTPs is an essential cysteine residue at the catalytic site, which forms a thiol-phosphate intermediate during catalysis (Guan and Dixon, 1991). Since reducing conditions usually maintained by thiol reagents are necessary for keeping the enzyme active, thiol-oxidizing agents are potent PTP inhibitors. PAO forms a stable ring complex with vicinal dithiols (Rokutan et al., 2000), whereas pervanadate irreversibly promotes the oxidation of the cysteine group to cysteic acid (Huyer et al., 1997). Pervanadate is the product of a mixture of orthovanadate and hydrogen peroxide, which constitutes a more potent inhibitor of PTP than orthovanadate, and penetrates cells more readily (Kadota et al., 1987). Both PAO and pervanadate were effective in inhibiting the AR (Fig. 8C). In addition to pervanadate and PAO, we tested the recently described, more specific PTP inhibitors DMHV (Cuncic et al., 1999) and ethyl-3,4-dephostatin (Suzuki et al., 2001) and found that they abolish the calcium-triggered AR as well (Fig. 8C), supporting the suggestion that PTPs are involved in sperm exocytosis. The fact that both PTK and PTP activities are necessary for acrosomal exocytosis is intriguing. One possible explanation is that one or more tyrosine residues on the proteins required for exocytosis need to cycle between a phosphorylated and a dephosphorylated state to function properly. Alternatively, certain tyrosine residues on protein members of the fusion machinery might need to be phosphorylated while others might need to be dephosphorylated to elicit exocytosis. Furthermore, note that, in addition to controlling the phosphorylation states of PTK substrates, PTPs can also directly modulate PTK activity (Haj et al., 2003; Zhang et al., 2002b). Conversely, PTKs have been shown to regulate PTP activity (Zhang et al., 2003). Interestingly, serine and threonine—but not tyrosine—dephosphorylation have been shown to participate in the late phases of membrane fusion in yeast (Bryant and James, 2003;

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Marash and Gerst, 2001; Peters et al., 1999). Which, if any, of these processes is involved in the AR is currently under investigation in our laboratory. In short, sperm’s highly differentiated structure and function renders it a likely candidate for the application of a range of informative analyses to directly assess the role of proteins in the exocytosis of preformed granules. In this context, the beauty of our AR model is two-fold. Specifically, it allows the use of biochemical and molecular biological techniques to understand fundamental aspects of sperm biology that have not—so far—been successfully addressed. And more generally, it may disclose widespread pathways that have proven difficult to investigate in cells that, in addition to exocytosis, exhibit other types of membrane trafficking events such as endocytosis, membrane recycling, and ER to Golgi transport.

Acknowledgments The authors thank Athy Robinson and Tirso Sartor for excellent technical assistance, Drs. Silvia Belmonte and Roberto Yunes for critical reading of the manuscript, Drs. Philip Stahl, Marı´a I. Colombo, and Thomas Binz for plasmids, and Drs. Keith Burridge and Nicholas Tonks for the PTP substrates as well as assay procedures. Dr. Tonks also provided assorted anti-PTP antibodies. CNT and CMR are thankful to The Pew Foundation, The Andrew Mellon Foundation, and Carrillo-On˜ativia for fellowships. This work was supported by an International Research Scholar Award from the Howard Hughes Medical Institute, a Carrillo-On˜ativia Scholar Award (Argentina), and grants from Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de Argentina to LSM and from the NIH (HD 18201) to PMS.

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