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Regular Article |
a Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Sainte-Foy, Quebec, Canada G1K 7P4
b Montreal General Hospital Research Institute, Surgery (Urology Division) and Department of Experimental Medicine, McGill University, Montreal, Quebec, Canada H3G 1A4
ABSTRACT
Capacitation represents the final maturational steps that render mammalian sperm competent to fertilize, either in vivo or in vitro. Capacitation is defined as a series of events that enables sperm to bind the oocyte and undergo the acrosome reaction in response to the zona pellucida. Although the molecular mechanisms involved are not fully understood, sperm protein phosphorylation is associated with capacitation. The hypothesis of this study is that protein tyrosine phosphorylation and kinase activity mediate capacitation of porcine sperm. Fresh sperm were incubated in noncapacitating or capacitating media for various times. Proteins were extracted with SDS, subjected to SDS-PAGE, and immunoblotted with an antiphosphotyrosine antibody. An Mr 32 000 tyrosine-phosphorylated protein (designated as p32) appeared only when the sperm were incubated in capacitating medium and concomitant with capacitation as assessed by the ionophore-induced acrosome reaction. The p32 was soluble in Triton X-100. Fractionation of sperm proteins with Triton X-114 demonstrated that after capacitation, this tyrosine phosphoprotein is located in both the cytosol and the membrane. Enzyme renaturation of sperm proteins was conducted in gels with or without either poly glu:tyr (a tyrosine kinase substrate) or kemptide (a protein kinase A substrate). An Mr 32 000 enzyme with kinase behavior was observed in all gels but was preferentially phosphorylated on tyrosine, as assessed by phosphorimagery and by thin layer chromotography to identify the phosphoamino acids. Indirect immunolocalization showed that the phosphotyrosine residues redistribute to the acrosome during capacitation, which is an appropriate location for a protein involved in the acquisition of fertility.
gamete biology, kinases, signal transduction, sperm, sperm capacitation
INTRODUCTION
Sperm Capacitation
Capacitation, which represents the final maturational steps that render mammalian sperm competent to interact correctly with the oocyte, occurs in the female genital tract in vivo [1, 2]. In vitro, this phenomenon can also be reproduced in defined media [3]. Capacitation can be defined as a series of events that enables the sperm to bind with the oocyte and accomplish the acrosome reaction in response to the zona pellucida. The sperm membrane undergoes numerous modifications related to capacitation [4], including structural changes to protein-lipid organization [5, 6] and plasma membrane fluidization [7–9]. Other molecular events that coincide with capacitation are the activation of ion channels [10] and calcium uptake [11–14], the generation of cAMP [15, 16], and the production of reactive oxygen species [17, 18].
Signal Transduction Pathways During Capacitation
Protein phosphorylation plays an important role in regulating numerous cellular activities. Protein kinases, which phosphorylate specific targets to modulate their function, and protein phosphatases, which dephosphorylate the molecules, control protein phosphorylation. A major type of protein kinase includes those enzymes that catalyze the addition of the phosphate group on the amino acids serine (ser) or threonine (thr) of a protein substrate. The ser/thr kinases include (among others) cAMP-dependent protein kinase A (PKA), calcium-calmodulin regulated kinases, and phospholipid-activated protein kinases. A second category of kinases is the protein tyrosine kinases, which phosphorylate on tyrosine residues. This class of kinases is smaller than the ser/thr kinase group; however, the protein tyrosine kinases are very important mediators of diverse biological reactions [19], e.g., the function of several growth factors [20]. A third class of protein kinases has been recently characterized and is able to phosphorylate both tyrosine and ser/thr residues; these are the dual-specificity protein kinases [21].
Protein phosphorylation is known to regulate sperm function, such as motility [22, 23] and zona pellucida recognition [24–26]. Mammalian capacitation is also probably mediated by protein phosphorylation. Sperm phosphoproteins have been elucidated, although their roles and regulation with respect to capacitation are unclear. Incorporation of [
-32P]ATP into porcine and bovine sperm has revealed that protein phosphorylation during capacitation in the pig seems to be unrelated to sperm motility changes [27]. In contrast, phosphorylated bull sperm proteins may be involved in capacitation and/or associated motility changes [28]. Several specific signal transduction pathways probably are involved during sperm capacitation [29, 30]. Among species studied, mouse [29, 31], human [32, 33], and bovine [34] sperm capacitation appears to be associated with tyrosine phosphorylation of sperm proteins in a PKA-dependent manner. Porcine sperm proteins are also tyrosine phosphorylated during capacitation [35, 36]. Despite the importance of protein tyrosine phosphorylation during capacitation, the enzymes (kinases and phosphatases) and protein substrates implicated have not yet been fully characterized. The present study continues our research on the molecular mechanisms of capacitation of porcine sperm. To test the hypothesis that protein tyrosine kinase activity is involved, tyrosine phosphorylation of sperm proteins was identified and localized using anti-phosphotyrosine antibodies, and enzyme renaturation assays in the presence of tyrosine kinase substrates were performed under conditions that support capacitation. Our results revealed the tyrosine phosphorylation of an Mr 32 000 protein, the redistribution of phosphotyrosine residues to the membranes and in the sperm head, and tyrosine kinase-like activities that are associated with capacitation in pig sperm.
MATERIALS AND METHODS
Chemicals
Molecular weight standards and [-32P]ATP (3000 Ci/mmole) were obtained from Amersham International (Oakville, ON, Canada). Acrylamide N,N'-methylene bisacrylamide, ammonium persulfate, and tris (hydroxymethyl) aminoethane (Tris) were from BioRad Laboratories (Mississauga, ON, Canada). Monoclonal mouse anti-phosphotyrosine antibody was purchased from Upstate Biotechnology (clone 4G10; UBI, Lake Placid, NY), peroxidase-conjugated goat anti-mouse antibody was from BioRad Laboratories, and fluorescein-conjugated goat anti-mouse antibody was from Zymed Laboratories (South San Francisco, CA). Minicolumns (Sep-PakPlusQMA, Sep-PakPlusC-18) for phosphoamino acid separation were produced by Waters (Millipore, Milford, MA). Other chemical products were from the Sigma Chemical Company (St. Louis, MO).
Culture Media
The principal culture media used were based on Krebs Ringer bicarbonate [37]. Capacitating medium (CM) was composed of 4.8 mM KCl, 1.2 mM KH2PO4, 95 mM NaCl, 5.55 mM glucose, 25 mM NaHCO3, 2 mM CaCl2, 0.4% BSA, and 2 mM pyruvate (pH 7.4). The noncapacitating medium (NCM) was similar to CM but without calcium, bicarbonate, and BSA (2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, 137 mM NaCl, 5.55 mM glucose, and 2 mM pyruvate, pH 7.4).
Sperm Preparation
Semen was collected from fertile boars (30 different males) by the gloved hand method [38]. The sperm-rich fraction was diluted with Beltsville Thawing Solution [39] at the Centre d'Insémination Porcine du Québec (St. Lambert, PQ, Canada) and transported to the laboratory at 16°C within 20 min. The diluted semen was divided into two equal portions, centrifuged once (10 min, 22°C, 270 x g) and diluted (4 x 107 sperm/ml). The first portion was diluted in CM to induce capacitation, and the second portion was resuspended in NCM as a noncapacitating negative control. Sperm were then incubated at 39°C (the internal body temperature of the pig) in a humidified 5% CO2 atmosphere for up to 4.5 h.
Evaluation of Sperm Capacitation
Sperm capacitation was determined by the ability of the sperm to undergo the A23187-induced acrosome reaction as described previously [27]. Calcium (2 mM) was added to calcium-depleted medium (NCM) before induction with A23187. Acrosome-reacted sperm were determined using fluorescein-labeled Pisum sativum agglutinin (PSA-FITC) as previously described [27]. The number of viable sperm was determined by eosin-nigrosin exclusion staining [27]. Viability was also determined using a sperm LIVE-DEAD kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions.
Isolation of Pig Sperm Proteins
During incubation in either NCM or CM, sperm aliquots were taken (1–5 x 106 sperm) at different times. Sodium orthovanadate (0.2 mM) was added, and then the aliquots were centrifuged briefly to isolate a sperm pellet (2–5 min, 13 000 x g). The sperm pellet was resuspended in sample buffer [40] without ß-mercaptoethanol and boiled for 2–5 min. The sperm solution was recentrifuged (2–3 min, 13 000 x g), and the resulting supernatant was boiled in sample buffer with ß-mercaptoethanol (5%) for 2–5 min. The sperm protein sample was then subjected to SDS-PAGE.
To better localize the proteins of interest, sperm were also fractionated with detergents. Before and after incubation in CM (3 h), the pellet from 1 ml of prepared sperm suspension was resuspended and incubated (45 min, 4°C) with 0.5% Triton X-100 or 0.5% Triton X-114 containing an antiprotease cocktail (4 µg/ml pepstatin, 10 µg/ml aprotinin, 4 µg/ml leupeptin, 40 mM benzamidine, and 1 mM PMSF). During protein solublization with Triton, samples were occasionally shaken. Sperm were then centrifuged (2–5 min, 13 000 x g), and the Triton insoluble pellets were saved. The Triton X-100 supernatant also was reserved (soluble fraction), and the Triton X-114 supernatent was incubated for 5 min at 37°C and centrifuged (3 min, 500 x g). The upper and lower phases corresponded to the Triton X-114 cytosoluble and micellar (membranous) fractions, respectively. Before conducting SDS-PAGE, each sample was precipitated with acetone and resuspended in sample buffer (approximately 1 mg/ml of total protein).
SDS-PAGE and Western Blotting
The sperm proteins were separated by SDS-PAGE on 12% polyacrylamide gels and transferred electrophorectically [41] (4–8 mA-1 h-1 cm2) to polyvinyldienne fluoride (PVDF) membranes. Nonspecific protein binding sites on the membrane were blocked with 5% dry nonfat milk in Tris-buffered saline (TBS: 25 mM Tris-HCl, pH 7.4, 150 mM NaCl). The PVDF membrane was incubated with anti-phosphotyrosine antibodies for 1 h at 1:20 000 in TTBS (TBS plus 0.5% Tween 20). After washing (three times for 10 min each), the blot was incubated with peroxidase-conjugated goat anti-mouse antibodies at 1:20 000 in TTBS for 45 min and washed again. Labeled proteins were revealed using enhanced chemiluminescence detection with the ECL Kit (Amersham) according to the manufacturer's instructions.
Enzyme Renaturation after SDS-PAGE
Protein samples were prepared as described above. Proteins were separated according to their molecular masses by SDS-PAGE with or without 100 µg/ml poly glu:tyr (4:1) or 15.4 µg/ml kemptide copolymerized in the gel matrix. Protein kinase renaturation was performed as described previously [42]. SDS was removed at room temperature (rt) from gels by one wash overnight in 20% propan-2-ol, 50 mM imidazole/28 mM iminodiacetic acid (buffer A), followed by another wash for 60 min (buffer A containing 10 mM ß-mercaptoethanol; rt). To unfold the proteins, gels were placed in plastic bags for 90 min (buffer A containing 8 M guanidine-HCl, 10 mM ß-mercaptoethanol). Gels then were incubated four times (and buffer refreshed) at 4°C with gentle agitation in buffer A containing 10% sucrose, 0.04% Tween 20, 10 mM ß-mercaptoethanol; the first and second incubations were for 90 min, the third was overnight, and the last was for 60 min. Gels then were equilibrated in buffer B (10 mM HEPES-NaOH, pH 7.4, 20 mM MgCl2, 5 mM MnCl2, 0.1 mM Na3PO4, 10 mM ß-mercaptoethanol) for 60 min at rt. Subsequently, kinase renaturation, activation, and substrate phosphorylation proceeded in plastic bags on a rotative agitator in buffer B including 100 µCi ATP--32P. Gels were successively washed with 5% trichloroacetic acid, 1% pyrophosphate, 10% phosphate (monobasic). As evaluated by Geiger counting, when the matrices were considered to be nonradioactive (i.e., in the corner of the gel away from the proteins), the gels were dehydrated in 10% methanol (1 h, rt) and dried. Finally, the gels were exposed on phosphorscreens, and substrate phosphorylation was quantified by phosphorimagery (PhosphorImager; Molecular Dynamics, Sunnyvale, CA). The band corresponding to the kinase-like enzyme of interest then was subjected to phosphoamino acid analysis to confirm at which residues the gel substrates were phosphorylated.
Phosphoamino Acid Analysis
The 32P-labeled band corresponding to the kinase of interest was further analyzed to identify the phosphorylated amino acids according to the substrates present within the gel matrix. The procedure for protein extraction, hydrolysis, and phosphoamino acid purification was essentially as described previously [43]. The equivalent of 10 bands of the 32P-kinase at Mr 32 000 were excised from the renaturating gels and placed in 1 ml of 12 N HCl and heated to 110°C for 3 h to partially hydrolyze the complexes. Hydrolysates were evaporated in a speed-vac and resuspended in 100 mM NH4OH. These polypeptides were placed in a C18 minicolumn, and the unretained fraction was then applied to an anionic exchange column (QMA cartridge). The column, which retained the phosphoamino acids, was washed with 10 ml of water. The phosphoamino acids then were eluted with 1 ml of 100 mM HCl and evaporated in a speed-vac. The phosphoamino acids were solubilized in 100 mM NH4OH, 5 µg of unlabeled phosphoserine, phosphothreonine, and phosphotyrosine were added as references, thin-layer chromatography was conducted as described previously [44], and phosphoamino acids were colored with 0.2% ninhydrine in acetone. The signals were visualized after 4 days of exposure to a phosphorscreen (Molecular Dynamics).
Indirect Immunofluorescence
Sperm in NCM or CM were centrifuged (10 min, 270 x g) and then incubated (1 h, 4°C) in 2% (v/v) formaldehyde in water. After a second centrifugation, cells were incubated in 2% (w/v) BSA-H2O overnight (4°C). The sperm were washed and resuspended in PBS, smeared onto a microscope slide, and allowed to air dry. For some experiments, sperm (on the slide) were permeabilized in absolute ethanol for 1 min. Slides were then incubated for 1 h at rt with anti-phosphotyrosine antibodies (clone 4G10, 1:10), rinsed with PBS, and incubated for an additional 1 h with fluorescein-conjugated goat anti-mouse antibodies. After rinsing with PBS, coverslips were mounted on the slides with 90% glycerol. Sperm were observed with a Nikon microscope equipped with fluorescent optics (excitation 450–490 nm: B2-A filter, 400x) for anti-phosphotyrosine antibody labeling.
Statistical Analyses
Differences in the percentages of capacitated and viable sperm due to treatment (NCM versus CM) or time were determined by analysis of variance using general linear model procedures [45]. The Fisher protected least significant difference (LSD) test was conducted when the main effect was significant (P < 0.05).
RESULTS
A Tyrosine-Phosphorylated Protein Appears During Capacitation
Before incubation in either NCM or CM, several proteins isolated from freshly ejaculated boar sperm were already phosphorylated on tyrosine residues. These phosphoproteins (PP) were identified by Western blotting with a monoclonal anti-phosphotyrosine antibody and named PP60, PP44, and PP42. They were tyrosine phosphorylated throughout incubation, independent of time and medium (Fig. 1). A tyrosine phosphoprotein of Mr 32 000 (designated as p32) was detected when sperm were incubated in CM for a period of time. A weak band at Mr 32 000 at 4.5 h in NCM in Figure 1 is likely this same capacitation-associated p32. It occasionally appeared, probably because of "spontaneous" capacitation, and was consistently much fainter than the band from sperm in CM. The tyrosine-phosphorylated proteins recognized by the first antibody are specific; the labeling did not occur when the second antibody was used alone (data not shown).
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Figure 2 shows the distribution of p32 following Triton extraction. The p32 was solubilized with Triton X-100 (lanes 1 and 2) and was greatly enhanced after capacitation (3 h; Fig. 2B, lane 1). Fractionation of sperm proteins with Triton X-114 demonstrates that p32 is detectable in the cytosolic fractions before and after capacitation (lane 3) but is detectable in the membrane fraction only after capacitation (Fig. 2B, lanes 3 and 4).
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Incubation for 3 h in CM promoted sperm capacitation relative to incubation in NCM (Table 1; P = 0.001; n = 7). Sperm viability decreased during incubation in either medium (Table 2; P 
0.003; n = 10), but no significant difference was observed between media at 4.5 h (P = 0.34). A difference in the degree of agglutination was informally noted for sperm incubated in NCM and CM. Agglutination occurred only after incubation in CM for 3 h, and these sperm were viable, as determined by fluorescent staining with a viability kit of unfixed cells (data not shown).
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Sperm Tyrosine Kinase-Like Activity Concomitant with Capacitation
Kinase activities were detected by in-gel enzyme renaturation following SDS-PAGE of extracted sperm proteins. After capacitation (i.e., following incubation for 3 h in CM), protein tyrosine kinase-like activity of an Mr 32 000 enzyme was observed, as detected by the phosphorylation of the gel substrate, poly glu:tyr (Fig. 3A). Although not visible in Figure 3A, kinase activity of this Mr 32 000 enzyme was detectable by phosphorimagery even at 0 h in NCM (Fig. 4) and in all three substrate gels (Fig. 5). However, quantification of the signals confirmed that the maximum kinase activity occurred after 3 h in CM (P < 0.05; Fig. 4) and that substrate phosphorylation was highest in the gels containing poly glu:tyr (P < 0.05; Fig. 5).
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To characterize more fully the Mr 32 000 sperm kinase, the renaturations were also performed either without a specific kinase substrate (control) or with kemptide, a PKA substrate, in the gel matrix. These three gels (control, kemptide, or poly glu:tyr) were always processed simultaneously with the same sperm proteins to control for variation among ejaculates. At 3 h, the relative kinase activity of the Mr 32 000 enzyme in the control and kemptide gels was lower than that of the gels with poly glu:tyr (Fig. 5). Figure 3B shows that this difference was not associated with the quantity of protein on gel. These results suggest that the Mr 32 000 enzyme is a putative tyrosine kinase.
Analysis of 32P-labeled phosphoamino acids from this Mr 32 000 renatured kinase (excised from the gel in Fig. 3A) shows that the poly glu:tyr phosphorylation occurs on tyrosine residues and also on threonine and serine when the proteins were from sperm incubated for 3 h in CM (Fig. 6, lane C). The phosphorylation of capacitated sperm at Mr 32 000 that occurred in the control and kemptide gels was due to serine kinase activity because only serine was phosphorylated (Fig. 6, lanes A and B). Analysis of 32P-labeled phosphoamino acids at Mr 32 000 at 0 h was attempted; however, the quantity was too small for thin-layer chromatography.
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Indirect Immunolocalization of Boar Sperm Phosphotyrosine Proteins
Immunostaining of nonpermeabilized boar sperm with anti-phosphotyrosine antibodies revealed differential localization according to physiology. Sperm incubated in CM for 3 h, which is necessary to induce capacitation (Table 1), showed anti-phosphotyrosine labeling over the midacrosome and equatorial segment and faintly on the midpiece (Fig. 7D; nonpermeabilized cells). This specific signal on the acrosome occurred also in sperm incubated in NCM, but to a lesser degree than in those incubated in CM (Table 3; P = 0.001). The fluorescent pattern of capacitated sperm shown in Figure 7D was detectable in a minor subpopulation of sperm before incubation in either NCM or CM (Table 3; P > 0.05). However, before incubation, the majority of sperm showed faint fluorescence on the equatorial segment and midpiece (Fig. 7B).
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When the sperm were permeabilized with ethanol, the specificity of this distribution pattern was lost, and all sperm showed labeling over the acrosome and faintly on the equatorial segment and mitochondrial region. The equatorial fluorescence, however, was so pale that is was not clearly visible in the photomicrographs. This fluorescent pattern was observed similarly in sperm from both NCM and CM at 0 and 3 h. Figure 8 depicts the labeling of ethanol-permeabilized sperm in CM at 0 and 3 h.
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DISCUSSION
A Tyrosine-Phosphorylated Sperm Protein Appears During Capacitation
In this study, porcine capacitation was associated with both the tyrosine phosphorylation of an Mr 32 000 sperm protein, p32, that may possess tyrosine kinase-like activity and with the redistribution of phosphotyrosine residues to the acrosome. The differential phosphorylation patterns of the sperm proteins between sperm incubated in NCM and and those incubated in CM suggest that tyrosine phosphorylation plays an important role in porcine capacitation. Others have suggested that capacitation is directly related to sperm tyrosine phosphorylation in various mammalian species [31, 32, 34]. Our culture conditions induced sperm capacitation in vitro within 3 h in CM (Table 1), which is similar to observations by others [27, 46–49]. In addition, when the sperm were incubated in CM, considerable head-to-head agglutination was evident (data not shown), which is indicative of head plasma membrane modifications due to capacitation [50]. During boar sperm capacitation, a membrane sialoprotein originating from epididymal and seminal fluids is lost, favoring agglutination [51].
During in vitro capacitation (in CM), the phosphotyrosine content increased on p32, and once phosphorylated, its apparent level of tyrosine phosphorylation remained relatively constant. This behavior is not inconsistent with what is known about tyrosine phosphorylation. The receptors for epidermal growth factor and platelet-derived growth factor are tyrosine kinases, and their activities are ligand mediated. Thus, upon binding to its ligand, receptor-protein tyrosine kinase activity and function are established [52]. The tyrosine phosphorylation of p32 may occur sometime during and before the completion of capacitation (Fig. 1, 1.5 h). Capacitation is a slow process both in vivo and in vitro [3]. The completion of capacitation in porcine sperm is likely driven by earlier essential events such as the tyrosine phosphorylation of p32.
Boar sperm p32 is not likely associated with the cytoskeleton, because it was easily extracted by Triton, a nonionic detergent (Fig. 2). In contrast, in sperm from other species capacitation-associated tyrosine phosphoproteins have not been solublized by Triton X-100 and are thus associated with cytoskeletal structures [53, 54]. The appearance of p32 at 0 h (before capacitation; Fig. 2A, lanes 1 and 3) is likely due to the large quantities of sperm used for the Triton extractions; 40 x 106 sperm were used compared with only 5 x 106 sperm during the normal SDS extraction (as in Fig. 1). However, the presence of p32 was considerably enhanced after capacitation (Fig. 2b, lanes 1 and 3). After capacitation, p32 had affinity for both the hydrophilic (cytosolic) and hydrophobic (micellar or membranous) environments (Fig. 2B, lanes 3 and 4). Although the mechanism for this redistribution to the membrane is unknown, capacitation-mediated events such as increased membrane fluidity may be a signal for its relocalization. Regardless, the net quantity of the tyrosine phosphoprotein increased after capacitation, whether it was extracted using SDS, Triton X-100, or Triton X-114.
This p32 appears to be a novel sperm protein associated with porcine capacitation. In 1999, Flesch et al. [36] observed a 34-kDa tyrosine phosphorylated protein of boar sperm (which could be p32) that appeared concomitant with capacitation. In contrast, Kalab et al. [35] reported earlier that several boar sperm proteins, p34, p38, p40, and p44, were constitutively tyrosine phosphorylated and that the tyrosine phosphorylation of p93, p175, and p220/230 was upregulated in the presence of cAMP-elevating agents. The p34 tyrosine phosphorylated protein identified by Kalab et al. probably is the same as that subsequently identified by Flesch et al. [36] and in our laboratory. However, the pattern of tyrosine phosphorylated proteins shown in Kalab's earlier study differs from that reported here and by Flesh and collaborators [36]. More relevant is the fact that Kalab et al. [35] did not observe increased tyrosine phosphorylation on p32/p34 during capacitation, thus pointing to the possibility that methodological differences might be responsible for the conflicting results. Flesch and collaborators [36] suggested that differing anti-phosphotyrosine antibodies and the type of transfer membranes may account for differences between their study and that by Kalab et al. [35]. Our methods more closely resembled those of Kalab et al., yet our observations of the Mr 32 000 tyrosine phosphorylated protein (p32) support those of Flesch et al. [36]. Another methodological difference is that Kalab et al. [35] used a Percoll gradient to select the sperm before experimentation. Percoll washing could be sufficient to permit premature phosphorylation of p32/p34 before incubation to induce capacitation, possibly by removing decapacitation factors from the sperm membrane. Berger and coworkers [55] demonstrated that fresh pig sperm efficiently penetrated an oocyte after Percoll washing, suggesting the sperm were capacitated. Tanphaichitr et al. [56] also reported that washing ejaculated human semen through Percoll selected capacitated cells.
Tyrosine Kinase Activity Concomitant with Capacitation
The kinase activity of an Mr 32 000 enzyme also increased during incubation in CM but not during incubation in NCM. Similar in-gel assays have been previously used to detect various kinases, e.g., microtubule-associated protein 2 kinase [57], histone kinase [58], and prostatic tyrosine kinase [59]. To our knowledge, the present research represents the first demonstration of a putative tyrosine kinase (or enzyme with tyrosine kinase-like activity) associated with capacitation in mammalian sperm. Berruti and Martegani [60] identified three major polypeptides of 36, 40, 43 kDa in pig sperm that possessed tyrosine kinase activity and could correspond to the enzymes visualized between Mr 34 000 and 45 000 (Fig. 3A; poly glu:tyr). We did not assess whether these kinases were activated during capacitation, but their molecular masses seem sufficiently different from the Mr 32 000 kinase to indicate that they are not likely to be the same.
This Mr 32 000 sperm kinase preferentially phosphorylated the poly glu:tyr substrate during capacitation, which is expected for a protein with tyrosine kinase activity. An unexpected observation was of the appearance of phosphothreonine from the Mr 32 000 kinase in poly glu:tyr, which may reflect enzyme autophosphorylation or the phosphorylation of a substrate having a very similar molecular mass. Poly glu:tyr contains 80% glutamic acid and 20% tyrosine polymer and is a good substrate for detecting the insulin receptor tyrosine kinase [61]. Other sperm kinases were renatured by this technique (Mr 35 000–58 000; Fig. 3A), but their activities were not related to capacitation. Some of these kinases are likely those previously identified by Berruti and Martegani [60]. The Mr 32 000 kinase also phosphorylated kemptide, a PKA substrate. Kemptide is a hexapeptide (arg-arg-ala-ser-leu-gly) and has been used as a substrate for sperm PKA activity [62]. Nevertheless, the Mr 32 000 kinase showed a certain degree of specificity. The capacitation-related kinase activity was high in the poly glu:tyr gels (assessed by relative 32P-labeling in Fig. 5), whereas no difference was observed between the control and kemptide gels. Thus, tyrosine was preferentially phosphorylated (versus kemptide) during capacitation.
The Mr 32 000 kinase may act on a substrate other than the p32 identified by Western blotting (Fig. 1). However, the kinase activity by the Mr 32 000 renatured enzyme in the absence of a specific substrate (control) suggests that autophosphorylation may occur. Protein tyrosine kinases are typically autophosphorylated on tyrosine residues when active [63, 64]. However, the Mr 32 000 kinase in the control and kemptide gels phosphorylated on serine residues, as determined by phosphoamino acid analysis. The poly glu:tyr gels were also intriguing, where the 32P-labeled phosphoamino acids after phosphorylation by the Mr 32 000 enzyme were identified to be serine, threonine, and tyrosine. This putative Mr 32 000 kinase would be included in the category of dual-specificity protein kinases, which phosphorylate on ser/thr and tyrosine amino acids [21]. Pig sperm capacitation may be associated with tyrosine and/or ser/thr phosphorylation, although analyses of the kinase substrates clearly showed that the phosphorylation level concomitant with capacitation was more influenced by tyrosine kinase activity.
Phosphotyrosine Residues Relocate over the Acrosome During Capacitation
Indirect phosphotyrosine immunolocalization (Fig. 7) supports the relevance of tyrosine phosophoproteins during capacitation of pig sperm. The phosphotyrosine proteins relocalized during capacitation to the midacrosome, which is appropriate should the tyrosine phosphorylation be implicated in capacitation and/or the acrosome reaction. Previously, similar indirect immunofluorescence experiments revealed prominent phosphotyrosine proteins on the midpiece and principal piece of capacitated mouse and human sperm, with negligible labeling on the head [53, 54]. With capacitated boar sperm, however, only faint signal occurred on the midpiece. The phosphotyrosine proteins of mouse and human sperm are associated with the fibrous sheath, suggesting they may be involved in hyperactivated motility, which is associated with capacitation. In contrast, the absence of flagellar labeling on boar sperm also tends to support the importance of these tyrosine phosphoproteins in capacitation and/or acrosome reaction.
Only nonpermeabilized sperm demonstrated this redistribution of tyrosine phosphoproteins to the acrosome during capacitation. When permeabilized, both capacitated and noncapacitated sperm showed acrosomal localization (Fig. 8). This observation suggests that tyrosine phosphoproteins become associated with the external membranes of the acrosomal region during capacitation. It is unknown whether the acrosomal tyrosine phosphoproteins in Figure 7D actually correspond to p32 or to other membrane tyrosine phosphoproteins (Fig. 2B, lanes 1 and 4) and whether the proteins are associated with the membrane prior to tyrosine phosphorylation or whether they translocate from the sperm interior. These questions require closer investigation, particularly considering the fact that a p32 is present in the cytosol before capacitation but appears in the membrane only after capacitation (Fig. 2).
How Can an Mr 32 000 Kinase Regulate Capacitation?
In this study, p32, a protein from porcine sperm, was phosphorylated on tyrosine residues during conditions that support capacitation in vitro. This protein is Triton-soluble and is located in the membrane after capacitation as well as in the cytosol. Concomitant with capacitation, a novel enzyme of Mr 32 000 with tyrosine kinase-like activity also was identified. Immunolocalization experiments revealed that the sperm phosphotyrosine residues redistributed to the acrosome during capacitation. Whether these events actually mediate capacitation or are a consequence of capacitation is not yet clear. However, based on these data, it is tempting to speculate that when the sperm are incubated in conditions that support capacitation, p32 translocates to the pig sperm plasma membrane. Such redistribution would then induce or accompany other events related to capacitation, possibly including calcium influx and sperm-zona binding. A 34-kDa cytosolic tyrosine kinase in neutrophils translocates and is anchored by glycosylphosphatidylinositol to the plasma membrane, where its subsequent activation regulates cell migration and adherence [65]. Experiments are currently underway to identify the pig sperm protein(s) of Mr 32 000 and to elucidate its role in the capacitation pathway.
ACKNOWLEDGMENTS
We thank Drs. Marc-André Sirard and Pierre Leclerc for critically reviewing this manuscript, Sylvain Tessier for technical help, and the Centre d'Insémination Porcine du Québec for generously donating fresh porcine semen.
FOOTNOTES
First decision: 19 March 2001.
1 This research was funded by the Natural Sciences and Engineering Research Council of Canada and the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche. 
2 Correspondence: Janice L. Bailey, Centre de Recherche en Biologie de
la Reproduction, Département des Sciences Animales, Pavillon Paul-Comtois,
Université Laval, Sainte-Foy, PQ, Canada G1K 7P4. FAX: 481 656 3766; janice.bailey{at}crbr.ulaval.ca
Accepted: April 17, 2001.
Received: February 16, 2001.
REFERENCES
-32P. Mol Reprod Dev 1999; 54:292-302[CrossRef][Medline]This article has been cited by other articles:
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  L. Gonzalez-Fernandez, C. Ortega-Ferrusola, B. Macias-Garcia, G.M. Salido, F.J. Pena, and J.A. Tapia Identification of Protein Tyrosine Phosphatases and Dual-Specificity Phosphatases in Mammalian Spermatozoa and Their Role in Sperm Motility and Protein Tyrosine Phosphorylation Biol Reprod, June 1, 2009; 80(6): 1239 - 1252. [Abstract] [Full Text] [PDF]
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|
|
|
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M. Kong, E. S. Diaz, and P. Morales Participation of the Human Sperm Proteasome in the Capacitation Process and Its Regulation by Protein Kinase A and Tyrosine Kinase Biol Reprod, May 1, 2009; 80(5): 1026 - 1035. [Abstract] [Full Text] [PDF]
|
|
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 P. Grasa, C. Colas, M. Gallego, L. Monteagudo, T. Muino-Blanco, and J. A. Cebrian-Perez Changes in content and localization of proteins phosphorylated at tyrosine, serine and threonine residues during ram sperm capacitation and acrosome reaction Reproduction, April 1, 2009; 137(4): 655 - 667. [Abstract] [Full Text] [PDF]
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A. R. Ramirez, M. A. Castro, C. Angulo, L. Ramio, M. M. Rivera, M. Torres, T. Rigau, J. E. Rodriguez-Gil, and I. I. Concha The Presence and Function of Dopamine Type 2 Receptors in Boar Sperm: A Possible Role for Dopamine in Viability, Capacitation, and Modulation of Sperm Motility Biol Reprod, April 1, 2009; 80(4): 753 - 761. [Abstract] [Full Text] [PDF]
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Y. Seita, S. Sugio, J. Ito, and N. Kashiwazaki Generation of Live Rats Produced by In Vitro Fertilization Using Cryopreserved Spermatozoa Biol Reprod, March 1, 2009; 80(3): 503 - 510. [Abstract] [Full Text] [PDF]
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M. L. Vadnais, D. N. Foster, and K. P. Roberts Molecular Cloning and Expression of the CRISP Family of Proteins in the Boar Biol Reprod, December 1, 2008; 79(6): 1129 - 1134. [Abstract] [Full Text] [PDF]
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A. Barbonetti, M.R.C. Vassallo, B. Cinque, C. Antonangelo, F. Sciarretta, R. Santucci, A. D'Angeli, S. Francavilla, and F. Francavilla Dynamics of the Global Tyrosine Phosphorylation During Capacitation and Acquisition of the Ability to Fuse with Oocytes in Human Spermatozoa Biol Reprod, October 1, 2008; 79(4): 649 - 656. [Abstract] [Full Text] [PDF]
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C. Lawson, S. Goupil, and P. Leclerc Increased Activity of the Human Sperm Tyrosine Kinase SRC by the cAMP-Dependent Pathway in the Presence of Calcium Biol Reprod, October 1, 2008; 79(4): 657 - 666. [Abstract] [Full Text] [PDF]
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R. Jones, P. S. James, D. Oxley, J. Coadwell, F. Suzuki-Toyota, and E. A. Howes The Equatorial Subsegment in Mammalian Spermatozoa Is Enriched in Tyrosine Phosphorylated Proteins Biol Reprod, September 1, 2008; 79(3): 421 - 431. [Abstract] [Full Text] [PDF]
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I M Aparicio, M J Bragado, M C Gil, M Garcia-Herreros, L Gonzalez-Fernandez, J A Tapia, and L J Garcia-Marin Porcine sperm motility is regulated by serine phosphorylation of the glycogen synthase kinase-3{alpha} Reproduction, September 1, 2007; 134(3): 435 - 444. [Abstract] [Full Text] [PDF]
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 M. L. Vadnais and K. P. Roberts Effects of Seminal Plasma on Cooling-Induced Capacitative Changes in Boar Sperm J Androl, May 1, 2007; 28(3): 416 - 422. [Abstract] [Full Text] [PDF]
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P. Grasa, J. A. Cebrian-Perez, and T. Muino-Blanco Signal transduction mechanisms involved in in vitro ram sperm capacitation. Reproduction, November 1, 2006; 132(5): 721 - 732. [Abstract] [Full Text] [PDF]
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C. Dube, P. Leclerc, T. Baba, C. Reyes-Moreno, and J. L. Bailey The Proacrosin Binding Protein, sp32, Is Tyrosine Phosphorylated During Capacitation of Pig Sperm J Androl, July 1, 2005; 26(4): 519 - 528. [Abstract] [Full Text] [PDF]
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 K. L. Asquith, R. M. Baleato, E. A. McLaughlin, B. Nixon, and R. J. Aitken Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition J. Cell Sci., July 15, 2004; 117(16): 3645 - 3657. [Abstract] [Full Text] [PDF]
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S. Shadan, P. S. James, E. A. Howes, and R. Jones Cholesterol Efflux Alters Lipid Raft Stability and Distribution During Capacitation of Boar Spermatozoa Biol Reprod, July 1, 2004; 71(1): 253 - 265. [Abstract] [Full Text] [PDF]
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K S Sidhu, K E Mate, T Gunasekera, D Veal, L Hetherington, M A Baker, R J Aitken, and J C Rodger A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula) Reproduction, January 1, 2004; 127(1): 95 - 103. [Abstract] [Full Text] [PDF]
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H. Harayama Viability and Protein Phosphorylation Patterns of Boar Spermatozoa Agglutinated by Treatment With a Cell-Permeable Cyclic Adenosine 3',5'-Monophosphate Analog J Androl, November 1, 2003; 24(6): 831 - 842. [Abstract] [Full Text] [PDF]
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C. Dube, S. Tardif, P. Leclerc, and J. L. Bailey The Importance of Calcium in the Appearance of p32, a Boar Sperm Tyrosine Phosphoprotein, During In Vitro Capacitation J Androl, September 1, 2003; 24(5): 727 - 733. [Abstract] [Full Text] [PDF]
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T.C. McCauley, W.C. Buhi, G.M. Wu, J. Mao, J.N Caamano, B.A. Didion, and B.N. Day Oviduct-Specific Glycoprotein Modulates Sperm-Zona Binding and Improves Efficiency of Porcine Fertilization In Vitro Biol Reprod, September 1, 2003; 69(3): 828 - 834. [Abstract] [Full Text] [PDF]
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N. Cormier and J. L. Bailey A Differential Mechanism Is Involved During Heparin- and Cryopreservation-Induced Capacitation of Bovine Spermatozoa Biol Reprod, July 1, 2003; 69(1): 177 - 185. [Abstract] [Full Text] [PDF]
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A. C. Pommer, J. Rutllant, and S. A. Meyers Phosphorylation of Protein Tyrosine Residues in Fresh and Cryopreserved Stallion Spermatozoa under Capacitating Conditions Biol Reprod, April 1, 2003; 68(4): 1208 - 1214. [Abstract] [Full Text] [PDF]
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H. Harayama, K. Okada, and M. Miyake Involvement of Cytoplasmic Free Calcium in Boar Sperm: Head-to-Head Agglutination Induced by a Cell-Permeable Cyclic Adenosine Monophosphate Analog J Androl, January 1, 2003; 24(1): 91 - 99. [Abstract] [Full Text] [PDF]
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V. Fournier, P. Leclerc, N. Cormier, and J. L. Bailey Implication of Calmodulin-Dependent Phosphodiesterase Type 1 During Bovine Sperm Capacitation J Androl, January 1, 2003; 24(1): 104 - 112. [Abstract] [Full Text] [PDF]
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S. Tardif, C. Dube, and J. L. Bailey Porcine Sperm Capacitation and Tyrosine Kinase Activity Are Dependent on Bicarbonate and Calcium but Protein Tyrosine Phosphorylation Is Only Associated with Calcium Biol Reprod, January 1, 2003; 68(1): 207 - 213. [Abstract] [Full Text] [PDF]
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