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Journal of Andrology, Vol. 24, No. 5, September/October 2003
Copyright © American Society of Andrology

The Importance of Calcium in the Appearance of p32, a Boar Sperm Tyrosine Phosphoprotein, During In Vitro Capacitation

CHARLOTTE DUBÉ*, STEVE TARDIF*, PIERRE LECLERC{dagger} AND JANICE L. BAILEY*

From the Centre de Recherche en Biologie de la Reproduction, * Département des Sciences Animales and {dagger} Département d'Obstétrique et Gynécologie, Université Laval, Sainte-Foy, Québec, Canada.

Correspondence to: Dr Janice L. Bailey, Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Pavillon Paul Comtois, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4 (e-mail: janice.bailey{at}crbr.ulaval.ca).
Received for publication November 13, 2002; accepted for publication May 13, 2003.

   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
After ejaculation, mammalian sperm must undergo a preparation period known as "capacitation" to become capable of fertilizing the oocyte. Although physiological capacitation occurs in the female genital tract, the process can be reproduced in vitro by incubation in appropriate media. However, the signaling events regulating capacitation are poorly understood, especially in boar sperm. Calcium is thought to be of fundamental importance in capacitation. Our laboratory recently identified a tyrosine-phosphorylated protein of Mr 32 000 ("p32") from boar sperm, and its appearance is closely related to capacitation. The objective of this study was to understand the mechanism regulating the appearance of our p32 tyrosine phosphoprotein. Since calcium has been linked to sperm capacitation and protein tyrosine phosphorylation in other species, we hypothesized that extracellular calcium is involved in the appearance of the p32. Sperm were incubated in either noncapacitating medium (NCM) or capacitating medium (CM) for various times. Proteins were extracted with sodium dodecyl sulfate (SDS), separated by SDS-polyacrylamide gel electrophoresis (PAGE), and then immunoblotted with an antiphosphotyrosine antibody. To assess intracellular calcium levels, fresh sperm were loaded with the fluorescent calcium indicator indo-1, and relative fluorescence was measured by flow cytometry. Analysis demonstrated that relative intracellular calcium levels increased during incubation in capacitating conditions but not in NCM, which coincides with the appearance of the p32. The p32 tyrosinephosphorylated protein appeared only in the presence of calcium, and the calcium ionophore Br-A23187 accelerated its appearance. Consistent with our hypothesis, the appearance of the p32 was inhibited by extracellular calcium chelators (ethylene glycol-bis(2-aminoethylether)-N,N,N',N',-tetraacetic acid [EGTA], EDTA, and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid potassium salt [BAPTA-K+]), showing the importance of calcium in protein tyrosine phosphorylation related to capacitation in boar sperm.

     Key words: Tyrosine phosphorylation, chelator, A23187, indo-1


It has been more than 50 years since Austin (1951) and Chang (1951) independently discovered that sperm require a period of residence in the female tract, called "capacitation," in order to fertilize an oocyte. This process can also be reproduced in vitro by incubation in appropriate media, which generally contain bicarbonate, bovine serum albumin (BSA), and calcium (Visconti et al, 1998; Visconti and Kopf, 1998). Bicarbonate induces architectural modifications to the plasma membrane in boar sperm that are linked to capacitation (Harrison et al, 1996). Thus, in the boar, mouse, and bull, bicarbonate is hypothesized to activate a sperm adenylate cyclase and consequently to increase cyclic adenosine monophosphate levels to activate protein kinases and protein tyrosine phosphorylation (Galantino-Homer et al, 1997; Kalab et al, 1998; Visconti et al, 1999). The BSA is thought to act as a sink for the removal of cholesterol from the sperm plasma membrane (Cross, 1996, 1998), thereby increasing membrane fluidity. The role of calcium is less well known, but it is likely important in the signal transduction pathway of capacitation (Visconti et al, 1995a) and primes the sperm for acrosomal exocytosis (Arnoult et al, 1999).

In various mammalian species, protein phosphorylation is known to regulate sperm functions, such as motility (Tash and Means, 1983; Vijayaraghavan et al, 1997) and zona pellucida binding (Naz and Ahmad, 1994). Protein phosphorylation is also implicated in mammalian sperm capacitation (Visconti and Kopf, 1998; Tardif et al, 1999). It appears that several specific signal transduction pathways are involved during sperm capacitation (Visconti et al, 1995b; Baldi et al, 2002). In various species, such as the mouse (Visconti et al, 1995a,1995b), human (Luconi et al, 1996), bovine (Galantino-Homer et al, 1997), and pig (Kalab et al, 1998; Flesch et al, 1999), it has been well documented that capacitation appears to be associated with tyrosine phosphorylation of sperm proteins.

It is well established that calcium plays a crucial role in sperm function (Dragileva et al, 1999; Ho and Suarez, 2001). In 1915, Loeb was the first to demonstrate that extracellular calcium is required for fertilization in invertebrates. A similar requirement has been found for mouse sperm by Iwamatsu and Chang (1971). Since then, many studies have shown the importance of calcium for mammalian sperm capacitation (Handrow et al, 1989; DasGupta et al, 1993; Fraser et al, 1995) and for tyrosine phosphorylation of sperm proteins (Visconti et al, 1995a). We recently identified a tyrosine-phosphorylated protein "p32" in porcine sperm that seems to be closely related to capacitation (Tardif et al, 2001). As calcium is important for sperm capacitation and capacitation is a crucial phenomenon for fertilization, the major goal of this study was to elucidate the importance of calcium in the appearance of the p32 during capacitation in boar sperm. Our hypothesis was that the p32 is a calcium-dependent phosphoprotein related to boar sperm capacitation.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Chemicals

Monoclonal mouse antiphosphotyrosine antibody was obtained from Upstate Biotechnology (clone 4G10, UBI; Lake Placid, NY), and peroxidase-conjugated goat anti-mouse antibody was obtained from BioRad Laboratories (Mississauga, Ontario, Canada). Molecular-weight markers were obtained from Amersham International (Oakville, Ontario, Canada), and acrylamide N,N'-methylene bisacrylamide, ammonium persulfate, and tris (hydroxymethyl) aminoethane (TRIS) were obtained from BioRad. Indo-1 AM, pluronic F-127, 4,Br-A23187, and propidium iodide were purchased from Molecular Probes (Eugene, Ore), and other chemical products were obtained from Sigma Chemical Company (St Louis, Mo).

Culture Media

The principal culture media used were based on Kreb Ringer Bicarbonate (Toyoda and Chang, 1974). The capacitating medium (CM) was composed of 2.7 mM KCl, 1.2 mM KH2PO4, 8.1 mM Na2HPO4, 95 mM NaCl, 5.55 mM glucose, 25 mM NaHCO3, 2 mM CaCl2, 0.4% BSA (type V, free of fatty acids), and 2 mM pyruvate (pH 7.4; 230 mOsm). The noncapacitating medium (NCM) was similar to the CM but did not contain calcium, bicarbonate, or BSA (2.7 mM KCL, 1.2 mM KH2PO4, 8.1 mM Na2HPO4, 95 mM NaCl, 5.55 mM glucose, 2 mM pyruvate, pH 7.4, and 215 mOsm).

Sperm Preparation

Sperm preparation was as previously reported (Tardif et al, 2001). Briefly, semen was collected from fertile boars and diluted to 40 x 106 sperm/mL with Beltsville Thawing Solution (BTS) at the Centre d'Insémination Porcine du Québec (St-Lambert, Québec, Canada) and transported to the laboratory at 16°C to 18°C within 30 minutes. The diluted semen was divided into 2 equal fractions and centrifuged once (10 minutes, 270 x g, 25°C). The first fraction was resuspended in CM (40 x 106 sperm/mL) to induce capacitation, and the second portion in NCM (40 x 106 sperm/mL) was used as a noncapacitating negative control. Sperm were then incubated at 38.5°C, the internal body temperature of the pig, in a humidified 5% CO2 atmosphere.

Isolation of Pig Sperm Proteins

Sperm proteins from pig sperm were isolated essentially as described previously (Tardif et al, 2001). Briefly, aliquots (1-5 x 106 sperm) of sperm in either NCM or CM were taken before and during incubation at different times. Sodium orthovanadate (0.2 mM final concentration) was added, and the samples were centrifuged to obtain a sperm pellet (4 minutes, 16 060 x g, room temperature), which was resuspended in sample buffer without ß-mercaptoethanol and heated for 1 minute at 95°C. The sperm solution was recentrifuged (4 minutes, 16 060 x g), and ß-mercaptoethanol (5%) was added to the resulting supernatant. At this point, the samples were stored immediately at -80°C until the day of electrophoresis. The sperm protein samples were heated for 1 minute at 95°C. Sperm proteins were then ready for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

SDS-PAGE and Western Blotting

As described in Tardif et al (2001), the sperm proteins were loaded on 12% SDS-polyacrylamide gels, and separated proteins were transferred electrophoretically (overnight, 200 mA) to polyvinylidene fluoride membranes. Nonspecific protein-binding sites on the membrane were blocked with 5% dry nonfat milk in TRIS-buffered saline (TBS: 25 mM TRIS-HCl and 150 mM NaCl). Blots were incubated with antiphosphotyrosine antibodies (1:20 000) for 1 hour in TTBS (0.1% Tween 20, 25 mM TRISHCl, and 150 mM NaCl) at room temperature, washed 3 times for 10 minutes with fresh TTBS, and then incubated with peroxidase-conjugated goat anti-mouse antibodies (1:20 000 in TTBS, 45 minutes, room temperature). Membranes were again washed 3 times for 10 minutes with fresh TTBS. Labeled tyrosine phosphoproteins were visualized with a chemiluminescence detection kit (ECL, Amersham) according to the manufacturer's instructions.

Measurement of Intracellular Calcium Level

Sperm calcium levels were assessed following a protocol described by Collin et al (2000) that was modified to exclude dead or damaged sperm on the basis of propidium iodide incorporation. Briefly, sperm diluted in BTS were centrifuged (10 minutes, 270 x g, 25°C) and resuspended to 40 x 106 sperm/mL in NCM. For each sample tested, indo-1-AM was diluted in NCM and added to the sperm solution for a final concentration of 2.5 µM. The sperm and indo-1-AM suspension was then gently mixed and incubated for 30 minutes at 25°C in the dark. Sperm were washed twice by centrifugation (10 minutes, 350 x g, 25°C) to remove excess indo-1-AM, resuspended in either CM or NCM at 40 x 106 sperm/mL, and incubated at 39°C in a 5% CO2 humidified atmosphere for up to 3 hours. For flow cytometric analysis, 20 µL of indo-loaded sperm preparation was diluted in 980 µL of either CM or NCM, which included 3 µL of propidium iodide (2.4 mM) to exclude dead or damaged sperm and 20 µL of protamine sulfate (1 mg/mL) to prevent agglutination. Flow cytometric analysis was performed using an EPICS ELITE ESP (Beckman Coulter, Miami, Fla). The flow cytometer detected indo-1 bound to calcium (violet fluorescence) with a 440 DL filter (381 nm wavelength) and calcium-free indo-1 (blue fluorescence) with a 550 DL filter (525 nm wavelength). The relative intracellular calcium level of each sperm cell was expressed as the ratio of violet/blue fluorescence. Analysis was conducted at 37°C, and a total of 10 000 cells were scored for each aliquot. Debris and cell aggregates were excluded from the analysis by gating out nonfluorescent cells or particles. Only live sperm were assessed, as propidium iodide-labeled sperm were excluded from the analyses.

Statistical Analyses

Differences in calcium levels due to treatment (NCM vs CM) or time were determined by analysis of variance using General Linear Model procedures (Statistical Analysis Systems, 1990). The Fisher protected least significant difference test was conducted when the main effect was found to be significant (P < .05).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Calcium Favors the Appearance of the p32

Figure 1 shows the importance of calcium for the appearance of the p32 tyrosine-phosphorylated sperm protein. At 0 hours of incubation, the p32 is not present in CM either with or without calcium. The p32 tyrosine phosphoprotein appears after 3 hours of incubation in CM (which contains calcium) and in NCM supplemented with calcium. In "normal" NCM, which is without calcium, the p32 does not appear. The appearance of the p32 in NCM supplemented with calcium at 0 hours is somewhat variable and seems to depend on the boar. It is noteworthy that other proteins are tyrosine phosphorylated. Proteins present at approximately Mr 45 000, Mr 48 000, and, with a weaker intensity, Mr 66 000 are phosphorylated on tyrosine in all samples and are not markedly influenced by the treatments, although they sometimes appear to be slightly down-regulated in the presence of calcium after 3 hours of incubation. An interesting protein of approximately Mr 23 000 appeared in CM with or without calcium after 3 hours of incubation. The regulation of this protein is unknown, although calcium appears not to be important.



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  		Figure 1. Western blots of boar sperm proteins labeled with antiphosphotyrosine antibodies. Fresh boar sperm were incubated for 0 and 3 hours either in noncapacitating medium (NCM) without (lanes 1 and 5) or with 2 mM calcium (lanes 2 and 6) or in capacitating medium (CM) without (lanes 3 and 7) or with calcium (lanes 4 and 8). Note that CM without calcium (lanes 3 and 7) also contained 2 mM ethylene glycolbis(2-aminoethylether)-N,N,N' ,N'-tetraacetic acid (EGTA). The tyrosine phosphoprotein p32 (arrow) appears only in the presence of calcium and is greatly enhanced after 3 hours of incubation in calcium-containing media. This experiment was repeated more than 10 times, and a representative gel is shown.

 

To confirm that calcium is necessary for the appearance of the p32, different chelators were added to the sperm incubation media, and the results are shown in Figure 2. Since EDTA chelates both Mg2+ and Ca2+, we also tested ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid potassium salt (BAPTA-K+), which predominantly chelate Ca2+. As expected, in NCM with or without chelators at 0 and 3 hours of incubation, the p32 is not visible. As seen previously, when porcine sperm are incubated in CM, the tyrosine phosphoprotein p32 appears after 3 hours of incubation (Tardif et al, 2001). The addition of 6 mM EGTA, 6 mM EDTA, and 6 mM BAPTA-K+ (determined by dose response, data not shown) almost totally reduces the appearance of the tyrosine-phosphorylated protein, confirming the hypothesis that calcium is essential for p32 appearance.



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  		Figure 2. Western blots of boar sperm proteins labeled with antiphosphotyrosine antibodies. Fresh boar sperm were incubated for 0 hours (Panel A) and 3 hours (Panel B) in noncapacitating medium (NCM) without chelators (lane 1), with 6 mM ethylene glycol-bis(2-aminoethylether)N,N,N',N'-tetraacetic acid (EGTA) (lane 3), with 6 mM EDTA (lane 5), and with 6 mM 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid potassium salt (BAPTA-K+) (lane 7) or in capacitating medium (CM) without chelators (lane 2), with 6 mM EGTA (lane 4), with 6 mM EDTA (lane 6), and with 6 mM BAPTA-K+ (lane 8). Note that p32 (arrow) appears after 3 hours in CM without chelators (lane 2). p32 faintly appears also in CM with EGTA but not with EDTA or BAPTA-K+. This experiment was repeated 3 times, and a representative gel is shown.

 

To further investigate the role of calcium in the appearance of the p32, we used the nonfluorescent calcium ionophore Br-A23187, which promotes calcium influx into cells. We added 10 µM Br-A23187 after 1 hour of incubation, and sperm proteins were extracted at 0, 0.5, and 1.5 hours post-A23187 of incubation. Results are shown in Figure 3. The effects of the addition of the calcium ionophore are evident at 0.5 hours post-A23187, as the p32 is clearly enhanced by incubation with Br-A23187 in CM and, to a lesser degree, in NCM. The enhancement is even more obvious at 1.5 hours post-A23187. A tyrosine phosphoprotein also appears at Mr 21 000 in a time-dependent manner and with the addition of calcium ionophore; this is likely the same protein (Mr 23 000) that was observed in Figure 1. As well, the tyrosine phosphoproteins present at approximately Mr 45 000 and Mr 48 000 appear to be slightly down-regulated in the presence of A23187, but only in CM after 1.5 and 3 hours.



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  		Figure 3. Western blots of boar sperm proteins labeled with antiphosphotyrosine antibodies. Fresh boar sperm were incubated for 0, 1.5, and 3 hours in noncapacitating medium (NCM) or capacitating medium (CM). Ionophore Br-A23187 (10 µM) was added after 1 hour of incubation. Lanes 1 and 2 of each panel represent, respectively, NCM without and with 10 µM Br-A23187. Lanes 3 and 4 of each panel represent, respectively, CM without and with 10 µM Br-A23187. Note that the inclusion of Br-A23187 during incubation markedly enhances the appearance of p32. This experiment was repeated 3 times, and a representative gel is shown.

 

Sperm Calcium Increases During Capacitation

To investigate the relationship between extracellular calcium, intracellular calcium, and p32 appearance, indo-1-loaded sperm were suspended in either NCM or CM (Figure 4A) and in NCM plus or minus Ca2+ (2 mM) or CM plus or minus Ca2+ (2 mM) before incubation (Figure 4B). Calcium levels were detected by flow cytometry, and the level of calcium was expressed as the ratio of violet/blue fluorescence (RU indicates relative units). At 0 hours, there was no significant difference in intracellular calcium levels between sperm incubated in NCM or CM (9.92 RU ± 1.4 vs 13.10 RU ± 2.0, respectively; P > .05), which represent approximately 28 and 34 nM calcium, respectively (according to the calculation of Grynkiewicz et al, 1985). After 3 hours of incubation, the level of intracellular calcium increased markedly in sperm incubated in CM (33.58 RU ± 6.3; approximately 128 nM). The level of intracellular calcium in NCM at this time of incubation was not significantly different from that at 0 hours of incubation (15.04 RU ± 4.3; P > .05).



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  		Figure 4. Flow cytometry measurement of intracellular calcium levels of sperm incubated in normal noncapacitating medium (NCM) or capacitating medium (CM) (A) (n = 5) and in the presence or absence of calcium in the media (B) (n = 4). Porcine sperm were incubated with 2.5 µM indo-1-AM before suspension in media with or without 2 mM calcium. Calcium levels were assessed at 0 and 3 hours of incubation. At least 10 000 sperm were evaluated, and those staining positively with propidium iodide were excluded from the analysis.

 

At 0 hours, the addition of calcium to the media (Figure 4B) showed no significant effect in media without calcium (NCM without calcium: 11.65 RU ± 3.9, CM without calcium: 11.35 RU ± 1.2; P > .05) or with calcium (NCM with calcium: 15.75 RU ± 8.2, CM without calcium: 12.35 RU ± 1.5; P > .05). In contrast, after 3 hours of incubation, there was a significant difference between NCM without and with calcium (12.67 RU ± 3.5 vs 21.32 RU ± 3.8, respectively; P < .05) and between CM without and with calcium (13.45 RU ± 4.9 vs 25.22 RU ± 2.7, respectively; P < .05).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The Appearance of the p32 Depends on the Presence of Extracellular Calcium

We have previously reported that the capacitation of pig sperm is associated with the tyrosine phosphorylation of the p32, a protein of Mr 32 000 (Tardif et al, 2001). The two media used for this study, NCM and CM, induced different tyrosine phosphorylation profiles of sperm proteins, suggesting that tyrosine phosphorylation plays an important role in porcine capacitation. In this study, we investigated the modulation of the p32 by calcium during capacitation to better understand the implication of this phosphoprotein and the regulation of its tyrosine phosphorylation during capacitation.

When calcium was added to the negative control, NCM, the p32 appeared after 3 hours of incubation (Figure 1), as in capacitating conditions (CM), supporting the hypothesis that the p32 (and thereby capacitation) requires calcium for its appearance. The presence of a tyrosine-phosphorylated protein of Mr 32 000 in NCM supplemented with calcium could also indicate that the p32 does not require BSA and bicarbonate to appear. These components have been reported to be essential for protein tyrosine phosphorylation and capacitation observed in mouse sperm (Visconti et al, 1995a). Occasionally, an Mr 32 000-tyrosine phosphoprotein was observed at 0 hours in NCM supplemented with calcium, but this seems to depend on the boar. Our calcium-free treatments confirmed the importance of calcium for p32 appearance: the p32 does not appear in NCM or CM without calcium at any time. Taken together, these results clearly demonstrate that calcium is essential for the appearance of the p32.

As shown in Figure 2, extracellular calcium seems to be crucial for the appearance of the p32 in capacitating conditions. We used calcium chelators (EGTA, EDTA, and BAPTA-K+) to investigate whether the p32 depends on calcium by inhibiting its appearance. As expected, chelation of extracellular calcium by EDTA and BAPTA-K+ eliminated the p32. BAPTA-K+ is more selective for calcium than are EGTA and EDTA, although EDTA and BAPTA-K+ also chelate magnesium. EGTA is predominantly a calcium chelator. A faint p32 band appeared in the presence of EGTA, but we attribute this observation to the possible implication of magnesium in p32 appearance; however, this hypothesis remains to be tested. Conversely, by using calcium ionophore Br-A23187 (Figure 3), we demonstrated that increased calcium entry into the sperm promotes the appearance of the p32. We added calcium ionophore after 1 hour of incubation because we presumed that the elevated membrane fluidity due to the onset of capacitation facilitated the action of calcium ionophore. In fact, it is obvious on the Western blot at 1.5 and 3 hours that the inclusion of Br-A23187 in the incubation medium enhances the appearance of the p32. These results imply that an artificial entry of calcium into the sperm cell by Br-A23187 accelerates p32 appearance by increasing the calcium influx or bypassing the membrane mechanism necessary for calcium influx. Therefore, we could suggest that extracellular calcium is essential for the signaling events leading to tyrosine phosphorylation of the p32. The signaling pathway implicated in the appearance of the p32, however, remains unknown. As discussed in Tardif et al (2001), the p32 may be comparable to the Mr 33 000 protein reported by Flesch et al (1999). Indeed, the p32 is likely the same as that reported by Green and Watson (2001). The minor differences in the reported molecular weights could be due to the protocol. However, it should also be mentioned that a preliminary investigation by our laboratory indicates that a cohort of various tyrosine-phosphorylated proteins appears around Mr 32 000, although the identities of the capacitation-associated proteins remain to be confirmed (Dubé et al, 2002).

Increased Sperm Intracellular Calcium During Capacitation Coincides With the Appearance of the p32

The results obtained by flow cytometry (Figure 4A) indicate that the level of cytosolic calcium increases markedly in sperm incubated in conditions known to support capacitation (3 hours of incubation in CM). These results are similar to observations by Zhou et al (1990), who reported that intracellular calcium increases during capacitation in boar sperm. The intracellular calcium levels increased in CM supplemented with calcium after an incubation of 3 hours (Figure 4B), coinciding with the appearance of the p32 tyrosine phosphoprotein visualized by Western blotting (Figure 1). In the absence of calcium in the medium, the level of intracellular calcium did not rise in the sperm. Flow cytometry analysis shows that extracellular calcium in the medium is essential to increase intracellular levels and that this corresponds to p32 appearance. So, the appearance of the p32 phosphoprotein and the elevation of the intracellular calcium level in boar sperm both occur during capacitation and could be linked. Our negative control NCM, which contains no calcium, bicarbonate, or BSA, does not support capacitation (Tardif et al, 1999). In noncapacitating conditions, there is no increase in the intracellular calcium level between 0 and 3 hours, and the p32 does not appear. These results suggest a strong correlation between the increase in intracellular calcium level and the appearance of the p32.

It is notable that in human sperm, increased extracellular calcium and the addition of calcium ionophore to the medium resulted in the dephosphorylation of phosphotyrosine-containing human sperm proteins (Carrera et al, 1996; Luconi et al, 1996; Leclerc and Goupil, 2002). Similarly, we observed that the boar sperm tyrosine phosphoproteins of Mr 45 000 and Mr 48 000 also seem to be down-regulated in the presence of calcium (Figures 1 and 3). In contrast, however, calcium seems to act differently in tyrosine phosphorylation in boar sperm for the appearance of the tyrosine phosphoprotein p32.


   Conclusion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
In conclusion, the p32 is a tyrosine-phosphorylated protein, which appears in porcine sperm after 3 hours of incubation in conditions known to support capacitation. The appearance of the p32 coincides with an elevation of intracellular calcium in capacitating conditions. Furthermore, the appearance of p32 and the elevation of cytosolic calcium occur only if there is calcium in the medium. The importance of calcium for the appearance of the p32 was clearly established by experiments with calcium chelators. The use of calcium ionophore Br-A23187 also supported the hypothesis that the p32 is modulated by calcium. Now, our aim is to identify, sequence, and purify the p32 and elucidate its role in the capacitation pathway in conjunction with extracellular calcium as a second messenger.


   Acknowledgments
 
We thank the Centre d'insémination porcine du Québec for supplying the boar semen and Dr Maurice Dufour for performing the cytometric assays.


   Footnotes
 
Supported by NSERC of Canada and FCAR of Québec.


   References
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 Materials and Methods
 Results
 Discussion
 Conclusion
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