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Biology of Reproduction 67, 1538-1545 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Regulation of the Phosphotyrosine Content of Human Sperm Proteins by Intracellular Ca2+: Role of Ca2+-Adenosine Triphosphatases1

Véronique Dorvala, Maurice Dufourb, and Pierre Leclerc2,a

a Département d'Obstétrique/Gynécologie and Centre de recherche en Biologie de la Reproduction, Université Laval, and Centre de recherche du CHUQ, Québec, Québec, Canada G1L 3L5 b Centre de recherche du CHUL, Québec, Québec, Canada G1V 4G2


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An increase in the concentration of intracellular free Ca2+ and in the phosphotyrosine content of specific proteins characterizes human sperm capacitation. Whether tyrosine phosphorylation regulates the intracellular free Ca2+ concentration through modulation of Ca2+-ATPase activity or the phosphotyrosine content is under Ca2+ regulation was investigated using Ca2+-ATPase modulators and tyrosine kinase inhibitors. The presence of the Ca2+-ATPase-inhibitor thapsigargin during human sperm capacitation caused an increase in the cytoplasmic free Ca2+ concentration and was associated with an increase in the phosphotyrosine content of specific sperm proteins. Conversely, a decrease in protein tyrosine phosphorylation was observed when gingerol, a Ca2+-ATPase activator, was present during the incubation period. On the other hand, thapsigargin had no effect on the phosphotyrosine content or the cytoplasmic Ca2+ concentration when spermatozoa were incubated in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX). However, the effect of IBMX on phosphotyrosine-containing proteins appears to be a Ca2+-dependent phenomenon, because it was partly inhibited in spermatozoa pretreated with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) even though, by itself, BAPTA-AM caused an increase in sperm protein phosphotyrosine content. Tyrosine kinase inhibitors prevented the increase in the phosphotyrosine content without affecting the cytoplasmic free Ca2+ concentration. Based on these findings, the present study suggests that Ca2+-ATPases are involved in the filling of internal Ca2+ stores, such as the acrosome, and are inhibited later during capacitation. Their inhibition allows an increase in cytoplasmic free Ca2+, which is involved in the subsequent increase in the phosphotyrosine content of specific sperm proteins.

acrosome reaction, calcium, gamete biology, signal transduction, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
When human spermatozoa are freshly released from the male reproductive tract on ejaculation, they are unable to fertilize unless they undergo several physiological modifications, collectively called capacitation, to acquire the fertilizing potential [1]. Once capacitated, spermatozoa can bind to the egg's extracellular matrix, the zona pellucida; undergo the acrosome reaction, a specialized and regulated exocytotic event; penetrate the zona pellucida; and finally, fuse with the egg's plasma membrane.

Many biochemical and membranous modifications have been demonstrated during mammalian sperm capacitation [2], but precise molecular mechanisms are still under investigation. A decrease of membrane cholesterol [3], a rise in intracellular pH [4, 5] and cAMP concentrations [6], and a decrease in calmodulin [7] and calmodulin binding to specific proteins [8, 9] are some modifications previously reported.

An increase in the phosphotyrosine content of specific proteins is also observed during capacitation [10–14]. The mechanisms by which the increase in protein tyrosine phosphorylation occurs are still obscure. Although this phenomenon has been shown to involve a cAMP-dependent pathway, it is not known whether cAMP, through its cAMP-dependent Ser/Thr protein kinase, activates tyrosine kinases or inhibits tyrosine phosphatases [15]. Tyrosine kinase inhibitors prevent the acrosomal exocytosis [16], but the exact role of protein tyrosine phosphorylation during the process of sperm capacitation remains elusive. In human spermatozoa, p105 and p81 are the two major phosphotyrosine-containing proteins [11]. Their localization to the flagellar fibrous sheath [12] suggests a potential role in motility and is associated with sperm hyperactivation [17]. This is emphasized by the recent report of an 86-kDa flagellar protein that binds to Ca2+ on its capacitation-mediated tyrosine phosphorylation [18].

Even though the acrosomal exocytosis can be induced by acid-solubilized zonae pellucidae or activators of different protein kinases (PK), such as PKA, PKG, or PKC, in human spermatozoa incubated in Ca2+-free media [19], it is well accepted that extracellular calcium is required for spermatozoa to complete capacitation and to undergo the acrosome reaction [20, 21]. Although a net Ca2+ uptake is observed during sperm capacitation [22], a smaller increase in the cytoplasmic free Ca2+ concentration is measured [22, 23]. These observations suggest that part of the Ca2+ influx is stocked within intracellular stores or organelles, with one being the acrosome [24–27]. Different types of pumps, exchangers, and channels regulate the intracellular calcium concentration, promoting either its influx or its efflux from the cell. The Ca2+-ATPases are Ca2+ pumps that may be involved in maintaining a low cytoplasmic Ca2+ concentration. The presence of Ca2+-ATPase activity in the sperm head suggests a potential role in capacitation-related Ca2+ regulation [28–30]. This statement is emphasized by the fact that thapsigargin, a Ca2+-ATPase inhibitor, induces sperm acrosomal exocytosis and an increase in cytoplasmic Ca2+ concentration [24, 25, 27]. Because in different somatic cells the activity of Ca2+-ATPase has been reported to be regulated by cAMP [31–33], calmodulin [34], and tyrosine phosphorylation [35], which are effectors known to be modulated during capacitation, we wanted to determine whether sperm Ca2+-ATPase activity varied during this process. Therefore, the aim of the present study was to determine whether sperm protein tyrosine phosphorylation is under the control of intracellular free Ca2+ concentration or is a regulator of sperm intracellular Ca2+ content through the regulation of Ca2+-ATPases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Percoll used for washing spermatozoa and the enhanced chemiluminescence (ECL) kit were obtained from Amersham Pharmacia Biotech (Baie d'Urfé, PQ, Canada). Thapsigargin, progesterone, BSA, 3-isobutyl-1-methylxanthine (IBMX), monoclonal antitubuline antibody (clone B-5-1-2), and chemicals for the composition of the Biggers, Whitten, and Whittingham (BWW) medium were purchased from Sigma Chemical Company (St. Louis, MO). Gingerol and PP2 were supplied by Biomol Research Laboratories (Plymouth Meeting, PA). Monoclonal antiphosphotyrosine antibody (clone 4G10) was from Upstate Biotechnology (Lake Placid, NY), and horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Ig) G was purchased from Jackson Immunoresearch, Inc. (West Grove, PA). Nitrocellulose (pore size, 0.22 µm) was supplied by MSI, Inc. (Westborough, MA), and x-ray films were from Fuji (Tokyo, Japan). INDO-1/AM, Pluronic F-127, propidium iodide, and the cell permeant Ca2+-chelator BAPTA-AM (1,2-bis-[o-aminophenoxy]-ethane-N,N,N',N'-tetraacetic acid tetra-[acetoxymethyl]-ester) were purchased from Molecular Probes (Eugene, OR). All other chemicals were of analytical grade.

Preparation of Spermatozoa

Ejaculates were obtained by masturbation from healthy volunteers after 3 days of sexual abstinence. The semen was normal according to the criteria of the World Health Organization [36]. After liquefaction, the semen was layered on top of a discontinuous Percoll gradient composed of 2-ml fractions of 20%, 40%, and 65% and a 0.1-ml fraction of 95% (v/v) Percoll diluted in Hepes-buffered saline (25 mM Hepes, 130 mM NaCl, 4 mM KCl, 0.5 mM MgCl2, and 14 mM fructose; pH 7.6, 290–300 mOsm) and centrifuged (30 min at 1000 x g) to wash the sperm cells. Spermatozoa within the 95% Percoll fraction and at the 65%/95% interface, representing the highly motile sperm population, were collected, counted, and diluted to 20 x 106 sperm/ml in BWW medium slightly modified from the original formulation [37] (94.6 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25.1 mM NaHCO3, 5.6 mM glucose, 21.6 mM sodium lactate, 0.25 mM sodium pyruvate, 0.1 mg/ml phenol red, and 10 mM Hepes; pH 7.6) and supplemented with 3 mg/ml of BSA (BWW/BSA). The IBMX (500 µM), Rp-cAMPS (200 µM), Sp-cAMPS (200 µM), thapsigargin (1 µM), gingerol (50 µM), and PP2 (10 µM) were added in specific experiments as described in the text. In some experiments, spermatozoa were incubated for 30 min at 37°C in the presence of BAPTA-AM (50 µM) in a calcium-free BWW/BSA medium and washed in the same medium before incubation in Ca2+-containing BWW medium supplemented with BSA in the presence or absence of IBMX. Controls contained dimethyl sulfoxide (IBMX, thapsigargin, PP2, and BAPTA-AM) or ethanol (gingerol) used as the solvent for those agents. The sperm suspension was incubated at 37°C for 4 h (5% CO2 in air, 100% humidity).

Detection of Phosphotyrosine Content of Sperm Proteins

After treatment, spermatozoa were washed by centrifugation (5 min at 500 x g) in PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4; pH 7.4), and proteins were extracted in sample buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 0.01% bromophenol blue) and heated for 5 min at 100°C. Sperm proteins were separated by electrophoresis on 7.5% SDS-polyacrylamide gel [38] and electrotransferred onto nitrocellulose [39]. Nonspecific-binding sites were blocked by incubating the membrane in TBSTW (154 mM NaCl, 20 mM Tris [pH 7.4], and 0.1% Tween 20) containing 5% (w/v) dry skimmed milk. The membrane was next incubated with an antiphosphotyrosine antibody for 1 h at room temperature, then for 45 min with a goat anti-mouse IgG conjugated to horseradish peroxidase. Between the two incubation periods and at the end, the membrane was extensively washed with several changes of TBSTW. Immunoreactive bands were detected using the ECL kit according to the manufacturer's instructions. The membrane was next reprobed using an anti-{alpha}-tubulin antibody to ensure that equivalent amounts of sperm protein were loaded on the gel for each treatment.

Evaluation of Intracellular Free Ca2+ Concentration

Washed spermatozoa were diluted to 25 x 106 sperm/ml in calcium-free BWW medium supplemented with 3 mg/ml of BSA and incubated in the presence of 2.5 µM INDO-1/AM and 0.00625% Pluronic F-127 for 30 min at room temperature [40]. Under these conditions, minimal intracellular compartmentalization of the Ca2+ probe, INDO-1, has been reported [41]. The sperm suspension was washed with the same calcium-free BWW medium to remove the noninternalized Ca2+ probe, then resuspended at 50 x 106 sperm/ml in Ca2+-containing BWW medium supplemented with BSA. In some experiments, IBMX, thapsigargin, gingerol, or PP2 were added as described above. Spermatozoa were incubated for 4 h at 37°C under 5% CO2. For the evaluation of cytoplasmic free Ca2+ concentration, spermatozoa were diluted to 1 x 106 sperm/ml in the BWW/BSA medium, and 5 µg/ml of propidium iodide were added to provide an indication of viability. On some occasions, 3 µM progesterone was added to the sperm suspension to evaluate the effect of thapsigargin and gingerol on the progesterone-induced intracellular Ca2+ increase. The measurements were performed by flow cytometry using an Epics Elite ESP (Beckman Coulter, Miami, FL) flow cytometer, equipped with a HeCd laser (Omnichrome Model 100, Omnichrome, Chino, CA) with an excitation wavelength of 325 nm. The violet (381 nm +Ca2+)/blue (525 nm -Ca2+) INDO-1 emission ratios were plotted versus time according to the method described in Current Protocols in Cytometry [41]. The kinetic analysis was performed using the shareware WinMDI 2.8 (available at http://facs.scripps.edu).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of Sperm Protein Phosphotyrosine Content by Intracellular Ca2+

An increase in the phosphotyrosine content of p105 and p81, the two major human sperm phosphotyrosine-containing proteins (named after their mass), is observed during sperm capacitation [11, 16]. Because an increase in the intracellular Ca2+ concentration also occurs during capacitation, experiments were designed to determine whether sperm protein tyrosine phosphorylation is regulated by intracellular free Ca2+. The effect of intracellular Ca2+ on protein phosphotyrosine content was investigated using the cell permeant BAPTA-AM to chelate the cytoplasmic calcium. Spermatozoa were incubated for 30 min in the presence of 50 µM BAPTA-AM to allow internalization of the Ca2+-chelator before the 4-h incubation in the absence or presence of IBMX. The effects of BAPTA-AM on protein tyrosine phosphorylation were different whether or not IBMX was present in the incubation medium to increase the phosphotyrosine content of sperm proteins. When spermatozoa were incubated in the absence of the phosphodiesterase inhibitor, the BAPTA-AM pretreatment caused an increase in the phosphotyrosine content of sperm proteins (Fig. 1A). On the other hand, a decrease in phosphotyrosine-containing proteins was observed when spermatozoa were treated with BAPTA-AM before the 4-h incubation with IBMX (Fig. 1B). These results show that modulation of intracellular Ca2+ concentration affects the phosphotyrosine content of specific sperm proteins, and they suggest that the cAMP-dependent increase in sperm protein phosphotyrosine content occurs, at least partially, through a Ca2+-dependent pathway.



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  		FIG. 1. Effect of intracellular calcium on tyrosine phosphorylation of human sperm proteins. Fifty micromolar BAPTA-AM was incorporated into spermatozoa as described in Materials and Methods. Spermatozoa were next incubated at 37°C for 4 h in calcium-containing BWW/BSA medium in the absence (A) or presence (B) of 500 µM IBMX. Sperm proteins were solubilized, separated by SDS-PAGE, transferred onto nitrocellulose, and probed using a monoclonal antiphosphotyrosine antibody as described in Materials and Methods. Molecular weight markers (kDa) are indicated on the left. An experiment representative of seven is shown

The tyrosine kinase-inhibitor PP2 caused a decrease in protein tyrosine phosphorylation (Fig. 2A) when present throughout the incubation. This inhibitory effect of PP2 was observed even when spermatozoa were incubated in the presence of IBMX (Fig. 2A). However, this inhibition mediated by PP2 might not occur through a Ca2+-dependent pathway, because this tyrosine kinase inhibitor had no effect on the cytoplasmic free Ca2+ concentration (Fig. 2B). Similar results were obtained when two other tyrosine kinase inhibitors, PP1 and Herbimycine A, were used (data not shown).



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  		FIG. 2. Effect of the tyrosine kinase-inhibitor PP2 on human sperm phosphotyrosine-containing proteins and intracellular free Ca2+ concentration. A) Spermatozoa were incubated in the absence or presence of 10 µM PP2 at 37°C for 4 h in calcium-containing BWW/BSA medium in the absence (n = 8) or presence (n = 5) of 500 µM IBMX. Sperm proteins were processed as described in Figure 1. Molecular weight markers (kDa) are indicated on the left. B) INDO-1/AM was incorporated into spermatozoa as described in Materials and Methods. Spermatozoa were next incubated in calcium-containing BWW/BSA medium. The evaluation of the intracellular free Ca2+ concentration was processed by flow cytometry. The relative intracellular Ca2+ concentration is expressed as the ratio between the intensity of fluorescence emitted by Ca2+-bound (violet)/Ca2+-unbound (blue) INDO-1 probe according to the method described in Current Protocols in Cytometry [41]. An experiment representative of 10 with similar results is shown

Regulation of Human Sperm Intracellular Ca2+ Concentration by Ca2+-ATPase

Although a Ca2+-dependent increase in cAMP concentration has been reported in progesterone-treated human spermatozoa [42], an increase in cytoplasmic free Ca2+ concentration was observed when spermatozoa were incubated for 4 h in the presence of the phosphodiesterase-inhibitor IBMX to prevent cAMP catabolism (Fig. 3). Similar results were obtained with the cell permeant cAMP-analogues Sp-cAMPS (Fig. 3) and dbcAMP (data not shown). Conversely, Rp-cAMPS, the Sp-cAMPS stereoisomer that binds to and inhibits cAMP-dependent protein kinase, had no effect on sperm cytoplasmic Ca2+ concentration. Considered together, these results suggest that the IBMX-mediated increase in sperm cytoplasmic Ca2+ occurs through the cAMP-dependent signaling pathway.



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  		FIG. 3. Role of cAMP in sperm intracellular free Ca2+ concentration. The INDO-1/AM-loaded spermatozoa were incubated at 37°C for 4 h in calcium-containing BWW/BSA medium in the absence or presence of the phosphodiesterase-inhibitor IBMX (A; 500 µM, n = 6) or in the presence of 200 µM Rp-cAMPS or Sp-cAMPS, an inhibitor and activator of PKA, respectively (B; n = 3). The relative intracellular Ca2+ concentration is expressed as in Figure 2. The relative intracellular Ca2+ concentration of the control cells (left) is indicated by the dotted line

Because the concentration of intracellular Ca2+ results from the equilibrium between the influx and efflux processes, and because the capacitation-related increase in intracellular Ca2+ is hypothesized to result from the inactivation of Ca2+-ATPase, the involvement of these latter enzymes during sperm capacitation was investigated. An increase in the cytoplasmic free Ca2+ concentration was observed when spermatozoa were incubated in the presence of the Ca2+-ATPase-inhibitor thapsigargin during the 4-h incubation period (Fig. 4A). However, this inhibitor had no effect on the cytoplasmic Ca2+ concentration when the sperm cells were incubated in the presence of IBMX (Fig. 4B). On the other hand, the concentration of sperm cytoplasmic free Ca2+ appeared not to be affected by the Ca2+-ATPase-activator gingerol during the 4-h incubation period. Using our current procedure, gingerol was inefficient at modulating the cytoplasmic Ca2+ concentration both in the absence and in the presence of IBMX (Fig. 5).



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  		FIG. 4. Effect of the Ca2+-ATPase-inhibitor thapsigargin on sperm intracellular free Ca2+ concentration. The INDO-1/AM-loaded spermatozoa were incubated at 37°C for 4 h in the absence or presence of 1 µM thapsigargin in calcium-containing BWW/BSA medium either alone (A; n = 7) or supplemented with 500 µM IBMX (B; n = 4). The relative intracellular Ca2+ concentration is expressed as in Figure 2



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  		FIG. 5. Effect of the Ca2+-ATPase-activator gingerol on sperm intracellular free Ca2+ concentration. The INDO-1/AM-loaded spermatozoa were incubated at 37°C for 4 h in the absence or presence of 50 µM gingerol in calcium-containing BWW/BSA medium either alone (A; n = 7) or supplemented with 500 µM IBMX (B; n = 4). The relative intracellular Ca2+ concentration is expressed as in Figure 2

Progesterone is known to cause an increase in cytoplasmic free Ca2+ concentration when added to a capacitated sperm suspension [23, 43]. The ability of Ca2+-ATPase modulators to regulate intracellular Ca2+ storage was evaluated using progesterone, which induces an increase in sperm cytoplasmic free Ca2+ concentration that is mediated by the depletion of internal Ca2+ stores on a primary uptake from the extracellular medium [44]. A smaller cytoplasmic Ca2+ increase induced by progesterone was observed when spermatozoa were incubated for 4 h with thapsigargin (Fig. 6A). No effect, however, was observed in the time lapse between the addition of the progesterone and the onset of the Ca2+ increase. Thapsigargin affected the cytoplasmic Ca2+ levels during both the transient and the sustained phases of the Ca2+ increase. This result is in agreement with a minimal contribution of the internal stores to the Ca2+ increase triggered by progesterone. On the other hand, a greater increase in the cytoplasmic free Ca2+ concentration induced by progesterone was observed when spermatozoa were previously incubated in the presence of the Ca2+-ATPase-activator gingerol (Fig. 6B), with both the transient and sustained phases of the Ca2+ increase being affected. This result also indicates that gingerol was effective at filling intracellular Ca2+ stores through the activation of Ca2+-ATPase.



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  		FIG. 6. Effect of Ca2+-ATPase modulators on the sperm intracellular free Ca2+ concentration in response to progesterone. The INDO-1/AM-loaded spermatozoa were incubated at 37°C for 4 h in calcium-containing BWW/BSA medium in the absence or presence of 1 µM thapsigargin (A) or 50 µM gingerol (B) before being challenged with 3 µM progesterone. Zero time represents the time at which progesterone was added to the sperm suspension. Evaluation of the intracellular free Ca2+ was processed by flow cytometry as described earlier. Experiments representative of seven with similar results are shown. The relative intracellular Ca2+ concentration is expressed as in Figure 2

Regulation of Sperm Protein Phosphotyrosine Content by Ca2+-ATPase

To further investigate whether a decrease in Ca2+-ATPase activity occurs during sperm capacitation, experiments were designed to determine whether Ca2+-ATPases are involved in the regulation of protein phosphotyrosine content in addition to intracellular Ca2+ levels. The Ca2+-ATPase-inhibitor thapsigargin induced an increase in the phosphotyrosine content of sperm proteins when these cells were incubated in BWW medium containing BSA (Fig. 7A). Both this effect and the increase in cytoplasmic Ca2+ concentration (Fig. 4) were not due to the exocytosis of the acrosome by thapsigargin, because the 4-h treatment with the Ca2+-ATPase inhibitor had no effect on the spontaneous acrosome reaction (1.5 ± 0.5% versus 1.2 ± 0.3%, n = 8). When spermatozoa were incubated in the presence of IBMX, which increases protein tyrosine phosphorylation, thapsigargin did not further increase protein phosphotyrosine content (Fig. 7B). On the other hand, gingerol, the Ca2+-ATPase activator, slightly decreased sperm protein tyrosine phosphorylation when spermatozoa were incubated in the BSA-containing medium (Fig. 8A). However, a significant decrease in protein phosphotyrosine content was observed when gingerol was present in the sperm incubation medium containing IBMX (Fig. 8B).



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  		FIG. 7. Effect of thapsigargin on human sperm phosphotyrosine-containing proteins. Spermatozoa were incubated in the absence or presence of 1 µM thapsigargin at 37°C for 4 h in calcium-containing BWW/BSA medium either alone (A; n = 9) or supplemented with 500 µM IBMX (B; n = 7). Sperm proteins were processed as described in Figure 1. Molecular weight markers (kDa) are indicated on the left



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  		FIG. 8. Effect of gingerol on human sperm phosphotyrosine-containing proteins. Spermatozoa were incubated in the absence or presence of 50 µM gingerol at 37°C for 4 h in calcium-containing BWW/BSA medium alone (A; n = 9) or supplemented with 500 µM IBMX (B; n = 5). Sperm proteins were processed as described in Figure 1. Molecular weight markers (kDa) are indicated on the left


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of the present study was to determine whether the phosphotyrosine content of sperm proteins is regulated by intracellular Ca2+ and is affected by the activity of Ca2+-ATPases. Despite a net decrease in the phosphotyrosine content of p105 and p81, the two major human sperm phosphotyrosine-containing proteins, PP2, an src-related tyrosine kinase inhibitor, had no effect on the cytoplasmic free Ca2+ concentration. On the other hand, the modulation of Ca2+-ATPase activity affects the intracellular free Ca2+ concentration and the phosphotyrosine content of sperm proteins. Thapsigargin is an irreversible inhibitor of Ca2+-ATPases specific to endoplasmic and sarcoplasmic reticulum, which ultimately results in the depletion of intracellular Ca2+ stores [45]. The thapsigargin-induced elevation in cytoplasmic Ca2+ concentration was associated with an increase in the phosphotyrosine content of sperm proteins. A similar increase in protein tyrosine phosphorylation induced by thapsigargin has been reported in human platelets [46] and endothelial cells [47]. This result is in agreement with the effect of the cell permeant Ca2+-chelator BAPTA-AM, which, once internalized, depletes the cytosol of its free Ca2+ content and results in internal Ca2+ store depletion. An increase in tyrosine phosphorylation has been reported when rat liver epithelial cells were pretreated with BAPTA-AM [48]. Different mechanisms might be involved in the BAPTA-AM-mediated increase in sperm protein tyrosine phosphorylation. A decrease in cytoplasmic Ca2+ concentration would inactivate Ca2+/calmodulin-dependent phosphodiesterases in spermatozoa [49, 50], leading to an increase in cAMP levels, which is well known to promote an increase in sperm protein phosphotyrosine content [11, 14]. The effects of calmodulin antagonists on the phosphotyrosine-containing proteins of human spermatozoa [13, 51] are in complete agreement with such a mechanism. Internal Ca2+ store depletion can also be achieved by incubating the cells in the presence of an extracellular Ca2+ chelator. Under those conditions, an increase in human sperm protein phosphotyrosine content has been reported [13, 51, 52]. The effect of BAPTA-AM on phosphotyrosine-containing proteins can also occur through an increase in the activity of the src-related tyrosine kinase Fyn and a decrease in protein tyrosine phosphatase activity, as reported in rat liver cells [48]. Therefore, an increase in protein phosphotyrosine content can be obtained either through the depletion of internal Ca2+ stores or through an increase in cytoplasmic Ca2+ concentration [46]. This statement is supported by the increase in sperm phosphotyrosine-containing proteins induced by progesterone [16] or the ionophore A23187 [13, 53], which are known to promote a Ca2+ uptake from the extracellular medium.

The phosphodiesterase-inhibitor IBMX stimulates tyrosine phosphorylation of human sperm proteins through a cAMP pathway [11, 13], and this effect is partly inhibited by the Ca2+-chelator BAPTA-AM that has been previously internalized. Because the increase in sperm cAMP levels is a Ca2+-dependent event [42, 54], this partial blockade of the IBMX-induced increase in phosphotyrosine-containing proteins might occur through the inhibition of a Ca2+/calmodulin-dependent adenylyl cyclase [55]. Nevertheless, in the absence of intracellular Ca2+, the phosphotyrosine content of sperm proteins was still higher in the presence than in the absence of IBMX. On the other hand, because IBMX as well as the cell permeant cAMP-analogues Sp-cAMPS or dbcAMP (data not shown) induced an increase in sperm cytoplasmic Ca2+ concentration, these results suggest that the cAMP-dependent increase in protein phosphotyrosine content occurs, at least partially, in a Ca2+-dependent manner. Because thapsigargin had no effect on the cytoplasmic free Ca2+ concentration or the sperm protein tyrosine phosphorylation when the cells were incubated in the presence of IBMX, both cAMP and thapsigargin may affect sperm cytoplasmic Ca2+ and phosphotyrosine-containing proteins through a similar mechanism, but without additive or synergistic effects. Whether cAMP inhibits sperm Ca2+-ATPases remains to be investigated.

Gingerol, a specific activator of sarcoplasmic reticulum Ca2+-ATPase [56], decreased the phosphotyrosine content of sperm proteins when spermatozoa were incubated for 4 h in the presence of the phosphodiesterase-inhibitor IBMX and, to a lower extent, when the cells were incubated in the absence of IBMX. However, this Ca2+-ATPase activator had no effect on the cytoplasmic free Ca2+ concentration when spermatozoa were incubated in either the absence or the presence of IBMX. The absence of an effect of gingerol on the Ca2+ concentration might be explained by the compartmentalization of the Ca2+ probe INDO-1 into the acrosome. A decrease in cytosolic Ca2+ induced by gingerol would be masked or compensated by an increase of Ca2+ into that store. However, the INDO-1/AM loading protocol used in the present study minimizes internalization of the dye, making this hypothesis less appealing. A lower temperature (room temperature instead of 37°C), a shorter incubation period (30 min instead of 1 h), and the presence of Pluronic F-127 (to ensure loading homogeneity and complete hydrolysis of the dye) all greatly reduce, but do not prevent, intracellular compartmentalization of the Ca2+ probe INDO-1 [41]. Another explanation would be that the Ca2+ pumped by gingerol-activated Ca2+-ATPase is rapidly released from the acrosome to maintain the intracellular Ca2+ homeostasis. Whether the enzymes, kinases, or phosphatases involved in the increase in sperm protein phosphotyrosine content are present in the acrosome or associated with the acrosomal membranes, and which are affected by Ca2+, remain to be established. An antagonism between cytosolic and intraluminal Ca2+ on platelet phosphotyrosine-containing proteins has been reported [46], and the opposite effects of thapsigargin and gingerol (present study) are in perfect agreement with this mechanism. Recently, c-yes, an src-related tyrosine kinase, has been localized at the acrosomal level of human spermatozoa, and its activity has been found to be inhibited by Ca2+ [57].

Progesterone, which is known as an acrosome reaction inducer [23, 43], causes an increase in cytoplasmic Ca2+ concentration. This increase in Ca2+ concentration occurs in a biphasic way [58]: a rapid and transient phase resulting from an influx of calcium from the external medium, followed by a sustained phase that possibly results from a Ca2+ release from an internal store, such as the acrosome [24, 25], as stated in a recent review [44]. Inhibition of Ca2+-ATPase by thapsigargin decreased the Ca2+ concentration reached during both the transient and the sustained phases of the Ca2+ increase induced by progesterone. Conversely, the Ca2+ levels reached in response to progesterone were further increased by the Ca2+-ATPase-activator gingerol, and this effect was observed in both the transient and the sustained phases of the Ca2+ increase. This strongly supports the idea that one of the roles of capacitation is to fill the intracellular stores with Ca2+, possibly through the action of Ca2+-ATPase. On the other hand, when thapsigargin is given to previously incubated spermatozoa, a rapid increase in cytoplasmic Ca2+ concentration is observed (data not shown) [24, 25]. It is not likely, however, that thapsigargin induces the release of the internal calcium store directly on its own. Instead, it prevents Ca2+ internalization, whereas the Ca2+ release from cellular stores might result from the activation of inositol trisphosphate (IP3) or ryanodine receptors that are associated with Ca2+ channels. The presence of IP3 receptors in spermatozoa has previously been reported [26, 59].

During sperm capacitation, intracellular Ca2+ levels might be regulated in a stepwise fashion, as shown in the model depicted in Figure 9: 1) the intracellular stores are filled with Ca2+; 2) the internalized Ca2+ interacts with Ca2+-binding proteins such as calreticulin, which is present in spermatozoa [26, 60] and is known to inhibit Ca2+-ATPases at high intracellular store Ca2+ concentrations [61]; and 3) this inhibition in Ca2+-ATPase activity results in an increase in cytoplasmic free Ca2+ concentration, with the latter causing an increase in the phosphotyrosine content. The higher cytoplasmic Ca2+ concentration would then promote an increase in cAMP level, which is known to increase, through an undefined mechanism, the phosphotyrosine content. Thereafter, on the appropriate signal, the intracellular pool of Ca2+ is rapidly released to allow capacitated spermatozoa to undergo the acrosome reaction (Fig. 9).



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  		FIG. 9. Model of the regulation of the phosphotyrosine content by intracellular Ca2+. 1) The Ca2+ uptake from the external medium during human sperm capacitation. 2) The Ca2+-ATPases maintain a low cytoplasmic Ca2+ concentration by pumping these cations out of the cell (a; plasma membrane) and by filling the acrosome (b; outer acrosomal membrane). 3) The Ca2+-binding-protein calreticulin, which is present in the acrosomal Ca2+ store, would inhibit Ca2+-ATPases of the outer acrosomal membrane, resulting in an increase in the cytoplasmic Ca2+ concentration. 4) The higher Ca2+ concentration induces an increase in the phosphotyrosine content of specific sperm proteins. 5) Cytoplasmic Ca2+ induces an increase in intracellular cAMP levels. These latter are involved in an elevation of the cytoplasmic Ca2+ concentration. 6) The cAMP/PKA pathway increases, by an unknown mechanism, the phosphotyrosine content. Whether cAMP inhibits Ca2+-ATPases remains to be established

Such a regulation in Ca2+ concentration during sperm capacitation explains the inability of thapsigargin to induce the acrosome reaction in noncapacitated cells [24, 25]. The inhibitory effect of thapsigargin on progesterone-induced Ca2+ increase (Fig. 6A) could also be realized according to this model, because no or little Ca2+ can be stored when the Ca2+-ATPase inhibitor is present from the beginning of the incubation period.

Considered together, our results support the presence of Ca2+-ATPases at the acrosomal level of human spermatozoa. The modulation of the pump activity affects human sperm phosphotyrosine-containing proteins. The cytosolic free Ca2+ induces an increase in sperm protein tyrosine phosphorylation, whereas the intracellular stored Ca2+ has the opposite effect. Whether these Ca2+ localization-dependent effects are mediated by the activation or the inhibition of tyrosine kinases and/or tyrosine phosphatases as well as the intracellular localization of these enzymes remain to be elucidated.


   ACKNOWLEDGMENTS
 
The authors are thankful to Dr. Janice Bailey for her help and discussion throughout the study and to Dr. Robert Sullivan for his careful revision of the manuscript. Special thanks also are given to all the volunteers who contributed to this work.


   FOOTNOTES
 
1 Supported by a grant from Canadian Institutes of Health Research (to P.L.), a studentship from Fonds pour la Formation de Chercheurs et Aide à la Recherche (to V.D.), and a scholarship from Fonds de la Recherche en Santé du Québec (to P.L.). Back

2 Correspondence: Pierre Leclerc, Endocrinologie de la Reproduction, Pav. St-François d'Assise, 10 de L'Espinay, Québec, PQ, Canada G1L 3L5.> FAX: 418 525 4195; pierre.leclerc{at}crsfa.ulaval.ca Back

Received: 1 February 2002.

First decision: 26 February 2002.

Accepted: 19 June 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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