Molecular Human Reproduction

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Molecular Human Reproduction, Vol. 9, No. 3, 125-131, March 2003
© 2003 European Society of Human Reproduction and Embryology

Article

Role of protein tyrosine phosphorylation in the thapsigargin-induced intracellular Ca²⁺ store depletion during human sperm acrosome reaction

Submitted on August 15, 2002; resubmitted on November 1, 2002. accepted on November 26, 2002

Véronique Dorval¹, Maurice Dufour² and Pierre Leclerc^1,3

¹ Département d’Obstétrique/Gynécologie and Centre de recherche en Biologie de la Reproduction, Université Laval, and Centre de recherche du CHUQ, Québec, G1L 3L5 and ² Centre de recherche du CHUL, Québec, Canada G1V 4G2

³ To whom correspondence should be addressed at: Endocrinologie de la Reproduction, Pav. St-François d’Assise, 10 de L’Espinay, Québec, PQ, Canada, G1L 3L5. e-mail: pierre.leclerc{at}crsfa.ulaval.ca

	ABSTRACT

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During human sperm capacitation, an increase in phosphotyrosine content of specific proteins results partially from an increase in the intracellular free Ca²⁺ concentrations. In the present study, the inter-regulation between protein phosphotyrosine content and the intracellular Ca²⁺ concentration during the thapsigargin treatment of capacitated human sperm was investigated. The involvement of a tyrosine kinase pathway in the thapsigargin-induced acrosome reaction was also investigated. In response to thapsigargin, two sperm subpopulations, called LR (low responsive) and HR (high responsive), according to their increase in intracellular Ca²⁺, were observed. In addition to their high increase in intracellular Ca²⁺, sperm from the HR population expressed a higher protein phosphotyrosine content, and a higher proportion (P < 0.05) of them underwent the acrosome reaction in response to thapsigargin, as compared with LR sperm. Although the tyrosine kinase inhibitor PP2 abolished the thapsigargin-induced increase in protein phosphotyrosine content, it did not affect the intracellular Ca ²⁺ concentration or the percentage of acrosome-reacted sperm. The inability of an src-related tyrosine kinase inhibitor to block the thapsigargin-mediated Ca²⁺ increase and acrosomal exocytosis suggests that, during the acrosome reaction, the signalling pathway mediated by src-related tyrosine kinases is involved upstream of the capacitative Ca²⁺ entry.

Key words: acrosome/calcium store/Ca ²⁺-ATPase/capacitation/phosphotyrosine

	Introduction

Top ABSTRACT Introduction Materials and methods Results Discussion REFERENCES

 
Freshly ejaculated sperm must undergo several biochemical and membranous modifications to acquire their fertilizing potential (Yanagimachi, 1994). Among these physiological modifications, an increase in the phosphotyrosine content of specific sperm proteins is observed (Leclerc et al., 1996, 1997, 1998; Emiliozzi and Fenichel, 1997). However, the role of tyrosine phosphorylation in sperm capacitation, as well as the mechanisms leading to the increase in protein phosphotyrosine content, remain unclear. Although a cAMP-dependent pathway was shown to be involved (Leclerc et al., 1996), it is not known whether a tyrosine kinase is activated or a tyrosine phosphatase is inhibited to promote the capacitation-related increase in protein phosphotyrosine content (Visconti et al., 2002). It was recently demonstrated that the increase in protein tyrosine phosphorylation results at least partially from an increase in the intracellular free Ca²⁺ concentration (Dorval et al., 2002), which is an important event in sperm capacitation (Handrow et al., 1989; Baldi et al., 1991).

The capacitation process is an obligatory prerequisite for sperm to undergo the acrosome reaction. This regulated exocytotic event takes place at the surface of the zona pellucida and allows the sperm cell to penetrate the oocyte-produced extracellular matrix and, finally, fertilize the oocyte. In humans, progesterone has been shown to be a natural and physiological inducer of the acrosome reaction (Osman et al., 1989). Several studies have reported an increase in the phosphotyrosine content of specific sperm proteins induced by this steroid (Tesarik et al., 1993; Luconi et al., 1995). Although tyrosine kinase inhibitors prevented the progesterone-induced acrosomal exocytosis (Luconi et al., 1995), the role of tyrosine phosphorylation during this process is still unclear. On the other hand, the increase in the intracellular free Ca²⁺ concentration during the acrosome reaction is a key regulatory event (Yanagimachi, 1994). Several observations suggest that this Ca ²⁺ elevation proceeds in several steps: (i) a transient phase resulting from an influx of extracellular Ca²⁺, and (ii) a sustained phase which involves a capacitative Ca²⁺ entry resulting from the depletion of an intracellular Ca²⁺ store (O’Toole et al., 2000; Rossato et al., 2001; Kirkman-Brown et al., 2002b). Several pumps and channels regulate the filling/depletion of the Ca²⁺ store and the Ca²⁺ influx from the extracellular medium. However, the mechanisms and their regulation throughout this process are not fully understood.

One of the roles of capacitation would be to fill intracellular Ca ²⁺ stores that will be emptied upon the right stimulus for the acrosomal exocytosis to proceed. The smaller increase in the intracellular free Ca²⁺ concentration (Handrow et al., 1989; Baldi et al., 1991) as compared with the net Ca²⁺ uptake (Handrow et al., 1989) observed during sperm capacitation strongly suggests that part of the cellular Ca²⁺ is stored internally within the sperm cell. The mitochondria, nucleus and cytoplasmic droplet have been presented as potential Ca²⁺ stores in sperm (Meizel and Turner, 1993; Naaby-Hansen et al., 2001). However, the presence of inositol 1,4,5-trisphosphate receptors (IP₃R) (Walensky and Snyder, 1995; Dragileva et al., 1999), and calreticulin (Nakamura et al., 1992; Naaby-Hansen et al., 2001), strongly suggest that the acrosome is an intracellular Ca²⁺ store. Moreover, the localization of thapsigargin-sensitive Ca²⁺-ATPases at the sperm acrosomal level suggests their involvement in the filling of that store (Spungin and Breitbart, 1996; Dragileva et al., 1999; Rossato et al., 2001).

In somatic cells, Ca²⁺-ATPases are regulated by tyrosine phosphorylation (Dean et al., 1997). The aim of this present study therefore was to investigate the filling of the thapsigargin-sensitive Ca²⁺ store during human sperm capacitation and the involvement of a tyrosine phosphorylation pathway during the thapsigargin-induced Ca²⁺ store depletion and acrosome reaction.

	Materials and methods

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Materials
Thapsigargin, bovine serum albumin (BSA), Pisum sativum agglutinin conjugated with fluorescein isothiocyanate (PSA–FITC), anti-bleaching agent 1,4-diazabicyclo[2.2.2]octane (Dabco), monoclonal anti-tubulin antibody (clone B-5-1-2) and chemicals for the composition of the Biggers–Whitten–Whittingham (BWW) medium were obtained from Sigma Chemical Company (St Louis, MO, USA). Indo-1/AM, Pluronic^® F-127 and propidium iodide were purchased from Molecular Probes (Eugene, Oregon, USA). The tyrosine kinase inhibitor, PP2, was supplied by Biomol Research Laboratories (Plymouth Meeting, PA, USA). The enhanced chemiluminescence kit (ECL) and Percoll used for washing sperm were purchased from Amersham Pharmacia Biotech (Baie d’Urfé, Canada). Monoclonal anti-phosphotyrosine antibody (clone 4G10) was from Upstate Biotechnology (Lake Placid, NY, USA) and horse-radish peroxidase (HRP)-conjugated goat anti-mouse IgG was purchased from Jackson Immunoresearch Inc (West Grove, PA, USA). Nitrocellulose 0.22 µm pore size was supplied by MSI Inc. (Westborough, MA, USA) and X-ray films were from Fuji (Tokyo, Japan). All other chemicals were of analytical grade.

Preparation and capacitation of sperm
Ejaculates were obtained by masturbation from healthy volunteers after 3 days of sexual abstinence. All sperm donors gave informed written consent and ethical approval was obtained from the ethical committees of The St-François d’Assise hospital (CHUQ) and the Laval University. Only the samples with normal sperm parameters according to the World Health Organization (1999) criteria were used. After liquefaction, the semen was layered on top of a discontinuous Percoll gradient, composed of 2 ml fractions each of 20, 40, 65% and 0.1 ml fraction of 95% Percoll diluted in HEPES-buffered saline (HBS; 25 mmol/l HEPES, 130 mmol/l NaCl, 4 mmol/l KCl, 0.5 mmol/l MgCl₂, 14 mmol/l fructose, pH 7.6). The sperm cells were washed by centrifugation (30 min at 1000 g). The highly motile sperm, at the 65/95% interface and within the 95% Percoll fraction, were collected, counted, and diluted to 20x10⁶/ml in BWW medium slightly modified from the original formulation (Biggers et al., 1971) (94.6 mmol/l NaCl, 4.8 mmol/l KCl, 1.7 mmol/l CaCl₂, 1.2 mmol/l KH ₂PO₄, 1.2 mmol/l MgSO₄, 25.1 mmol/l NaHCO₃, 5.6 mmol/l glucose, 21.6 mmol/l sodium lactate, 0.25 mmol/l sodium pyruvate, 0.1 mg/ml phenol red and 10 mmol/l HEPES, pH 7.6) supplemented with 3 mg/ml BSA (BWW/BSA). In specific experiments, 10 µmol/l of PP2, a selective src-related tyrosine kinase inhibitor (Hanke et al., 1996), was added to the sperm suspension. The tyrosine kinase inhibitors PP1 and herbimycin A (both from Biomol) were also used in the same experiments. Sperm were incubated at 37°C (5% CO₂ in air, 100% humidity), and aliquots were collected at different times (0 to 4 h), as indicated in each experiment. The sperm suspension was divided to evaluate the phosphotyrosine content of sperm proteins and to assess the acrosomal status.

Detection of phosphotyrosine content of sperm proteins
Sperm were washed by centrifugation (5 min at 500 g) in phosphate-buffered saline (PBS; 137 mmol/l NaCl, 2.7 mmol/l KCl, 1.5 mmol/l KH₂PO₄, 8.1 mmol/l Na₂HPO₄, pH 7.4). Proteins were extracted in sample buffer (62.5 mmol/l Tris–HCl pH 6.8, 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, 0.01% bromophenol blue) and heated for 5 min at 100°C. Sperm proteins were separated by electrophoresis on 7.5% sodium dodecyl sulphate–polyacrylamide gel (Laemmli, 1970) and electrotransferred onto nitrocellulose (Towbin et al., 1979). Non-specific binding sites were blocked by incubating the membrane in Tris-buffered saline supplemented with Tween 20 (TBSTW; 154 mmol/l NaCl, 20 mmol/l Tris pH 7.4, 0.1% Tween 20) containing 5% (w/v) dry skimmed milk. The membrane was washed with TBSTW prior to the incubation with an anti-phosphotyrosine antibody for 1 h at room temperature. Again, the membrane was washed and then incubated with a goat anti-mouse IgG conjugated to HRP for 45 min. At the end, the membrane was extensively washed with more than five changes (5–10 min each) of TBSTW. Immunoreactive bands were visualized by enhanced chemiluminescence using an ECL kit, according to the manufacturer’s instructions. To ensure that equivalent amounts of sperm proteins were loaded on the gel for each treatment, the membrane was resubjected to immunodetection using a monoclonal anti--tubulin antibody.

Induction and assessment of the acrosome reaction
After different incubation periods, sperm were washed by centrifugation in PBS and diluted to 20x10⁶/ml in BWW/BSA medium supplemented with 10 µmol/l thapsigargin to induce the acrosome reaction. Sperm were incubated again at 37°C (5% CO₂ in air, 100% humidity) for different periods of time (0 to 60 min) as indicated in specific experiments. The sperm cells were then washed again and fixed/permeabilized on ice with methanol for 30 min. The sperm was then smeared on a slide and air-dried. To evaluate the acrosomal status, the fixed cells were incubated with 75 µg/ml PSA–FITC diluted in PBS for 30 min at room temperature, washed with water and covered with a coverslip. The anti-fading agent Dabco (0.22 mol/l prepared in 90% glycerol) was deposited on slides to prevent photobleaching. Sperm were observed by epifluorescence microscopy and the acrosomal status was scored according to the staining patterns previously described (Mendoza et al., 1992). More than 200 sperm cells were scored for each treatment in different experiments.

Evaluation of intracellular free Ca²⁺ concentration and cell sorting
Percoll-washed sperm were diluted to 25x10⁶/ml in calcium-free BWW medium (BWW medium without added CaCl₂) supplemented with 3 mg/ml BSA and incubated for 30 min at room temperature in the presence of 2.5 µmol/l Indo-1/AM and 0.00625% Pluronic ^® F-127 as previously described (Collin et al., 2000). The sperm suspension was centrifuged (10 min at 1000 g) with calcium-free BWW/BSA medium to remove the non-internalized Ca²⁺ probe. The sperm cells were then resuspended at 50x10⁶/ml in complete BWW/BSA medium. In some experiments, PP2 was added to the suspension. Sperm were capacitated for different periods of time (0 to 4 h) at 37°C, under 5% CO₂. For the evaluation of intracellular free Ca²⁺ concentration, sperm were diluted to 10⁶ cells/ml in the BWW/BSA medium. As an indicator of sperm viability, 5 µg/ml propidium iodide (PI) was added. Thapsigargin (10 µmol/l) was added to the sperm suspension as indicated in specific experiments. Measurements were performed by flow cytometry, using an Epics Elite ESP (Beckman Coulter, Miami, FL, USA) flow cytometer, equipped with a HeCd laser (Omnichrome Model 100; Omnichrome, Chino, CA, USA) with an excitation wavelength of 325 nm. The violet (381 nm Ca²⁺ -bound)/blue (525 nm Ca²⁺-unbound) Indo-1 emission ratios were plotted versus time, as indicated in the Current Protocols in Cytometry (June et al., 1997). More than 10 000 sperm were analysed for each treatment in different experiments. The kinetic analysis was performed using the shareware WinMDI 2.8 (http://facs.scripps.edu).

In a set of experiments, sperm were sorted out according to their different violet/blue Indo-1 emission ratios following the addition of thapsigargin. A total of 1x10⁶ sperm cells from each population was collected in PBS and washed by centrifugation (10 min at 1000 g). Sperm proteins were extracted in sample buffer, separated by electrophoresis, electrotransferred and subjected to evaluation of the phosphotyrosine content, as described earlier. In addition, for each population, 3000 sperm were collected and directly smeared onto a microscope slide and air-dried. The cells were fixed/permeabilized with methanol at 4°C and their acrosomal status was assessed using PSA–FITC as indicated above.

Statistical analysis
Statistical analyses were carried out using analysis of variance (ANOVA) and multiple comparison tests. P < 0.05 was considered to be statistically significant. Results are expressed as means ± SEM.

	Results

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Protein phosphotyrosine content during sperm capacitation and thapsigargin treatment
When sperm were incubated in the BWW/BSA medium for up to 4 h, a gradual increase in the phosphotyrosine content of sperm proteins, including the two major phosphotyrosine-containing proteins p105 and p81, was observed (Figure 1). At the end of this 4 h incubation, sperm were challenged with the Ca²⁺-ATPase inhibitor thapsigargin for different periods of time. Sperm protein phosphotyrosine content rapidly increased following the addition of thapsigargin, with a maximum reached after 15 min of incubation (Figure 2). Associated with the increase in protein tyrosine phosphorylation, an increase in acrosome reaction was observed in sperm treated with thapsigargin, rising from 2.3% ± 0.4, before the addition of thapsigargin, to 6.1% ± 1.3 after a 30 min treatment with the Ca²⁺ -ATPase inhibitor (P < 0.05, n = 4). When the tyrosine kinase inhibitor PP2 was present throughout the initial 4 h incubation period, a significant decrease in sperm protein tyrosine phosphorylation was observed (Figure 2, at 0 min thapsigargin). In addition, PP2 completely inhibited the thapsigargin effect on sperm phosphotyrosine-containing proteins (Figure 2 ). However, this tyrosine kinase inhibitor had no effect on sperm acrosome reaction either before (2.4% ± 0.9 versus 2.6 ± 0.4, n = 4) or after 1 h of the thapsigargin challenge (5.6% ± 1.8 versus 6.6% ± 2.6, n = 4). Similar effects on sperm phosphotyrosine content and acrosome reaction were obtained using the src-related tyrosine kinase inhibitors PP1 or herbimycin A (data not shown).

View larger version (69K): [in this window] [in a new window]   Figure 1. Phosphotyrosine content of sperm proteins during capacitation. Sperm were incubated at 37°C in calcium-containing Biggers–Whitten–Whittingham/bovine serum albumin medium. Aliquots were collected at different times during the incubation as indicated. The proteins were solubilized, separated by SDS–PAGE and transferred onto nitrocellulose. The membrane was probed using a monoclonal anti-phosphotyrosine antibody, as described in Materials and methods. Molecular weight markers (kDa) are indicated on the left. An experiment representative of three with similar results is shown.

 

View larger version (66K): [in this window] [in a new window]   Figure 2. Effect of the Ca2+ -ATPase inhibitor thapsigargin on human sperm phosphotyrosine-containing proteins. Sperm were capacitated for 4 h at 37°C in the absence or presence of the tyrosine kinase inhibitor PP2 (10 µmol/l). The sperm cells were then washed and incubated in the presence of 10 µmol/l thapsigargin for different periods of time, as indicated. Sperm proteins were processed as described in Materials and methods. Molecular weight markers (kDa) are indicated on the left. An experiment representative of four is shown.

 
Free intracellular Ca²⁺ concentration during capacitation and thapsigargin treatment
The intracellular free Ca²⁺ concentration was next studied during both sperm capacitation and a thapsigargin challenge on capacitated cells. Sperm were capacitated in the BWW/BSA medium for up to 4 h as described earlier. At different indicated times, sperm were processed for the evaluation of the intracellular free Ca²⁺ concentration by flow cytometry using the Ca²⁺ probe Indo-1. Sperm intracellular Ca²⁺ concentration during capacitation was higher at 2 h and remained at that level until the end of the 4 h incubation period (Figure 3).

View larger version (14K): [in this window] [in a new window]   Figure 3. Free intracellular Ca2+ concentration () and thapsigargin-induced increase in intracellular Ca 2+ () during human sperm capacitation. Sperm were loaded with the internal Ca2+ probe Indo-1/AM, as described in Materials and methods, and incubated for up to 4 h. Aliquots were collected at different times and processed for the flow cytometry. At each time, some sperm were challenged with 10 µmol/l thapsigargin (arrow in the right panel). A typical response to thapsigargin is shown in the right panel. The mean intracellular free Ca2+ concentrations measured before and after the addition of thapsigargin (at 4 min) are expressed as the ratio between the intensity of fluorescence emitted by the Ca2+ -bound Indo-1 (violet)/Ca2+-unbound Indo-1 (blue) and plotted versus time of incubation. Values represent the mean ± SEM of four different experiments. *Significantly different (P < 0.05) from the intracellular Ca2+ concentration at the beginning of the incubation. Significantly different (P < 0.05) from the mean response to thapsigargin at the beginning of sperm incubation.

 
The Ca²⁺-ATPase inhibitor thapsigargin is known to induce an increase in intracellular free Ca²⁺ concentration by inhibiting Ca²⁺ storage. Therefore, the effect of thapsigargin on sperm intracellular Ca²⁺ concentration was measured at different times during capacitation as an indication of the time necessary for sperm to fill their thapsigargin-sensitive intracellular Ca²⁺ stores. The thapsigargin-induced increase in intracellular free Ca ²⁺ was significant after 1 h of capacitation compared with the levels reached in non-incubated sperm (Figure 3). However, at any time during capacitation, the intracellular free Ca²⁺ concentration reached after the thapsigargin treatment was always higher (P < 0.05) than those of the incubated untreated sperm.

Relationship between the protein phosphotyrosine content, intracellular Ca²⁺ concentration and acrosome reaction upon thapsigargin treatment
The flow cytometer allows the determination of intracellular Ca²⁺ concentration in individual cells. As shown in Figure 4, some sperm undergo a small increase in their intracellular Ca²⁺ concentration in response to thapsigargin, the ‘low responsive’ sperm (LR), while a higher increase is observed in other cells, the ‘high responsive’ sperm (HR). Further experiments were performed to better characterize the sperm cells from the two subpopulations obtained upon thapsigargin treatment. In order to investigate the behaviour of these two subpopulations during capacitation, sperm were incubated for different periods of time prior to the thapsigargin challenge. The number of sperm within each different thapsigargin-responsive population, LR and HR, was determined throughout the 4 h capacitation period. The percentage of sperm cells in the HR population remained stable during the first hour of incubation and started to rise thereafter. The HR subpopulation of sperm increased from 34.6% at the beginning to 53.9% at the end of the 4 h incubation period (Figure 5).

View larger version (56K): [in this window] [in a new window]   Figure 4. Effect of thapsigargin on the intracellular Ca2+ concentration of individual sperm. Indo-1/AM loaded sperm were incubated for 4 h at 37°C and then processed for flow cytometry to evaluate intracellular Ca2+ levels. The arrow indicates the addition of 10 µmol/l thapsigargin. The relative Ca2+ concentration is indicated as described in Figure 3. About 34 000 sperm are shown. LR = low responsive population. HR = high responsive population. The mean responses to thapsigargin of each population, LR and HR, are displayed on the right of the panel. A typical experiment representative of 12 is shown.

 

View larger version (12K): [in this window] [in a new window]   Figure 5. Dynamic of the percentage of sperm in the high response (HR) population. Indo-1/AM-loaded sperm were incubated up to 4 h at 37°C, aliquots were collected at different times and processed for the evaluation of the intracellular free Ca2+ concentration by flow cytometry. The percentage of sperm in the HR population upon the thapsigargin treatment is plotted versus the time of incubation. Values represent the mean ± SEM of four different experiments. *Significantly different (P < 0.05) from the percentage of sperm in the HR population at the beginning of the incubation.

 
Since the number of sperm in each of the two sperm populations (LR and HR) changed during capacitation, our next attempt was to study whether the increase in the intracellular Ca²⁺ concentration varied in the two subpopulations upon thapsigargin treatment. In response to the Ca²⁺-ATPase inhibitor, the intracellular Ca²⁺ concentration in sperm from the LR population increased slightly during capacitation (Figure 6). After 1 h of incubation in BWW/BSA prior to the thapsigargin challenge, sperm intracellular Ca²⁺ concentration was significantly higher (P < 0.05) than that from non-incubated cells. However, throughout the 4 h incubation period, the intracellular Ca²⁺ concentration in sperm from the LR population was never significantly different from that measured in sperm prior to the thapsigargin challenge. On the other hand, the intracellular Ca²⁺ concentration of the HR sperm clearly increased during the capacitation period. The intracellular Ca²⁺ levels in sperm from the HR subpopulation were significantly higher than those from non-incubated cells even after a 15 min incubation under capacitating conditions (Figure 6). However, their response to the Ca ²⁺-ATPase inhibitor reached a maximum after 1 h of incubation. Throughout the 4 h capacitation period, the intracellular Ca²⁺ levels in the HR population upon thapsigargin treatment were higher (P < 0.001) than the intracellular Ca²⁺ levels in both the LR population and the incubated sperm that were not challenged with thapsigargin.

View larger version (15K): [in this window] [in a new window]   Figure 6. Intracellular Ca2+ concentration in sperm from low responsive (LR) () and high responsive (HR) () populations in response to thapsigargin during capacitation. Indo-1/AM-loaded sperm were processed as described in Figure 4. Values represent mean ± SEM of four different experiments. *Significantly different (P < 0.05) from the Ca 2+ concentration of each respective population at t = 0. The basal intracellular free Ca2+ concentration before thapsigargin treatment, reproduced from Figure 3, is indicated by the dotted line.

 
Since only the HR subpopulation of the sperm suspension underwent a significant increase in intracellular free Ca²⁺ concentration in response to thapsigargin, an experiment was designed to determine whether the phosphotyrosine content and the percentage of acrosome reactions differed between the LR and HR subpopulations. Moreover, since the tyrosine kinase inhibitor PP2 (or PP1 and herbimycin A, not shown) prevented the thapsigargin-mediated increase in protein phosphotyrosine content, the involvement of a tyrosine kinase pathway was also studied. Sperm from the LR and HR subpopulations were sorted according to their intracellular Ca²⁺ concentration in response to thapsigargin after a 4 h incubation in the absence or presence of PP2. A higher phosphotyrosine content was observed in the HR population compared with the LR population (Figure 7A). A smaller increase in protein tyrosine phosphorylation was also observed in the HR population of sperm incubated in the presence of the tyrosine kinase inhibitor (Figure 7A). In fact, the phosphotyrosine content of sperm proteins was lower in both populations when PP2 was present during the 4 h incubation period. As for the protein phosphotyrosine content, the HR population was characterized by a significantly higher percentage of acrosome-reacted sperm compared with the LR population (19.6% ± 5.0 versus 2.8% ± 1.6; Figure 7B). Interestingly, as for the intracellular Ca²⁺ concentration in both populations, the tyrosine kinase inhibitor PP2 had no effect on the acrosome reaction in either LR or HR sperm populations.

View larger version (25K): [in this window] [in a new window]   Figure 7. Protein phosphotyrosine content and the percentage of acrosome reactions in sperm from the low responsive (LR) and high responsive (HR) populations upon thapsigargin treatment. Sperm were loaded with Indo-1/AM and capacitated for 4 h in calcium-containing Biggers–Whitten–Whittingham/bovine serum albumin medium in the absence (–) or presence (+) of 10 µmol/l PP2. Following the addition of 10 µmol/l thapsigargin, sperm within LR and HR populations were sorted according to their intracellular Ca2+ concentrations, as described in Material and methods. (A) The phosphotyrosine content was evaluated as described in Materials and methods. Molecular weight markers (kDa) are indicated on the left. An experiment representative of four is shown. (B) The acrosomal status of sperm from the LR and HR populations was evaluated using Pisum sativum agglutinin–fluoroscein, as described in Materials and methods. *A significant difference (P < 0.05) was observed between the percentage of acrosome-reacted sperm in LR versus HR populations in sperm pre-incubated in the absence of PP2.

 

	Discussion

Top ABSTRACT Introduction Materials and methods Results Discussion REFERENCES

 
As observed in previous reports (Leclerc et al., 1996, 1997, 1998; Emiliozzi and Fenichel, 1997), an increase in the phosphotyrosine content of specific human sperm proteins occurs during capacitation, an effect that is prevented by src-related tyrosine kinase inhibitors (Luconi et al., 1995; Leclerc et al., 1997; Dorval et al., 2002). An increase in sperm protein tyrosine phosphorylation is also known to occur during the progesterone-induced acrosome reaction (Tesarik et al., 1993; Luconi et al., 1995). This sperm exocytotic event is also induced by the Ca²⁺-ATPase inhibitor thapsigargin (Meizel and Turner, 1993). In the present study, an increase in protein tyrosine phosphorylation of capacitated human sperm upon a thapsigargin treatment is demonstrated for the first time. Although it did not prevent the acrosomal exocytosis, the tyrosine kinase inhibitors herbimycin A, PP1, or PP2 abolished the increase in tyrosine phosphorylation that occurs during sperm capacitation as well as the one induced by thapsigargin. This effect is different from the one reported using the tyrosine kinase inhibitor erbstatin which prevented both sperm acrosome reaction and protein tyrosine phosphorylation induced by progesterone (Luconi et al., 1995).

Thapsigargin is a specific inhibitor of Ca²⁺-ATPase from the sarcoplasmic and endoplasmic reticulum (Thastrup et al., 1990; Lytton et al., 1991). It has recently been demonstrated that thapsigargin binds to the acrosomal region of sperm (Rossato et al., 2001) and inhibits acrosomal Ca ²⁺-ATPase (Spungin and Breitbart, 1996; Rossato et al., 2001), which suggests that the acrosome is an intracellular Ca²⁺ store. The length of time necessary to fill this thapsigargin-sensitive Ca ²⁺ store during capacitation was investigated by the addition of the Ca²⁺-ATPase inhibitor at different times during sperm capacitation. The thapsigargin-induced increase in intracellular Ca²⁺ concentration reached a maximum after 1 h of capacitation, suggesting that 1 h of incubation under such conditions is sufficient to fill sperm intracellular Ca²⁺ stores. This also suggests that the role of acrosomal Ca²⁺-ATPase is to fill the acrosome during capacitation to ensure that sperm have enough Ca²⁺ stored to undergo the acrosome reaction.

Upon the addition of thapsigargin to a sperm suspension, two subpopulations were observed—LR and HR sperm. Sperm from the LR population showed a weak increase in the intracellular free Ca²⁺ concentration in response to thapsigargin but the levels reached were never significantly different from those measured prior to the thapsigargin treatment. Whether these sperm cells were capacitated or not remains to be established. The number of sperm within the HR population increases during capacitation, indicating that sperm progressively acquire the ability to undergo a net Ca²⁺ influx in response to thapsigargin during capacitation. Even though the intracellular Ca²⁺ concentration is elevated in all the sperm cells within the HR population, not all sperm (20%) experienced an exocytosis of the acrosome upon thapsigargin treatment.

In capacitated sperm, thapsigargin induced an increase in the intracellular free Ca²⁺ concentration, which was associated with an increase in the phosphotyrosine content and, ultimately, with the acrosomal exocytosis. On the other hand, the presence of the tyrosine kinase inhibitors herbimycin A, PP1, or PP2 during capacitation did not affect the thapsigargin-induced increase in Ca²⁺ concentration (not shown). The tyrosine kinase inhibitor PP2, which inhibits more specifically the tyrosine kinases from the src family (Hanke et al., 1996), markedly attenuated the basal as well as the thapsigargin-induced increase in the protein phosphotyrosine content of sperm cells within the HR population (Figure 7A). This supports the hypothesis that the increase in protein tyrosine phosphorylation observed during sperm capacitation is caused by an src-related tyrosine kinase, and would suggest that protein tyrosine phosphorylation is not involved in Ca²⁺-ATPase activity and thus filling of intracellular Ca²⁺ stores. On the other hand, the percentage of acrosome-reacted sperm in both the LR and HR population were not affected by PP2 in the incubation medium.

This finding differs from previous reports using progesterone, where a decrease in the percentage of acrosome-reacted sperm induced by the steroid was observed when sperm cells were previously capacitated in the presence of tyrosine kinase inhibitors (Luconi et al., 1995). Whether or not tyrosine kinase inhibitors block the progesterone-induced increase in sperm intracellular Ca²⁺ remains controversial (Bonaccorsi et al., 1995; Kirkman-Brown et al., 2002a). In the process of the acrosome reaction, progesterone causes an initial transient Ca²⁺ influx from the extracellular medium. Whether or not voltage-operated Ca²⁺ channels are involved in this cation uptake remains an open issue (Kirkman-Brown et al., 2002b). This initial increase in intracellular Ca²⁺ concentration depletes Ca²⁺ stores possibly through the action of IP3, the concentration of which is increased upon progesterone treatment (Thomas and Meizel, 1989). This in turn might activate the IP3 receptors located at the acrosomal level (Walensky and Snyder, 1995; Naaby-Hansen et al., 2001). This Ca²⁺ store depletion promotes the opening of store-operated Ca²⁺ channels (SOCC), together causing the sustained Ca²⁺ elevation (Kirkman-Brown et al., 2002b). Only the sustained phase of progesterone-induced increase in sperm intracellular Ca ²⁺ concentration is blocked by tyrosine kinase inhibitors (Bonaccorsi et al., 1995; Tesarik et al., 1996), in agreement with the involvement of the tyrosine kinase src in store-operated Ca²⁺ uptake (Babnigg et al., 1997). Thapsigargin immediately induces, although indirectly, Ca²⁺ store depletion followed by SOCC activation (Rossato et al., 2001), bypassing the initial transient Ca²⁺ influx.

In our study, the tyrosine kinase PP2 did not affect the thapsigargin-induced increase in Ca²⁺ concentration (not shown), in agreement with the previously reported effects of the tyrosine kinase inhibitor genistein (Bonaccorsi et al., 1995). In contrast to progesterone effects, the thapsigargin-induced acrosome reaction is not affected by tyrosine kinase inhibitors, which suggests that sperm tyrosine kinases are involved in the first steps of the acrosome reaction induced by progesterone, between the initial transient Ca²⁺ influx and the Ca²⁺ store depletion. In the mouse, it has been shown that tyrosine kinase inhibitor blocked the zona pellucida-induced activation of sperm phospholipase C (Tomes et al., 1996), an enzyme responsible for the generation of IP3. Therefore, the thapsigargin-mediated increase in sperm protein phosphotyrosine content most likely occurs after the increase in intracellular Ca²⁺, via a pathway that may not be involved in acrosomal exocytosis. In the present study, however, attention was focused on the major phosphotyrosine-containing proteins which could mask the minor proteins on the film upon Western blot analysis. Whether or not this thapsigargin-induced increase in protein phosphotyrosine content of capacitated sperm is involved in the cytoskeletal changes that occur during the acrosome reaction (Zaneveld et al., 1991; Breitbart, 2002) remains elusive.

Taken together, our results suggest that a thapsigargin-sensitive internal Ca²⁺ store in sperm, probably the acrosome, is filled during capacitation. Only sperm cells able to undergo a thapsigargin-induced depletion of Ca²⁺ stores and the capacitative Ca²⁺ entry showed an increase in the phosphotyrosine content and underwent the acrosome reaction. Since herbimycin A, PP1 and PP2 did not affect the increase in the intracellular Ca²⁺ concentration or the percentage of acrosome reactions, tyrosine phosphorylation mediated by src-related tyrosine kinases does not appear to be involved in the acrosome reaction triggered by thapsigargin. This suggests that tyrosine kinases from the src-family are involved in a pathway upstream of, and perhaps involved in, Ca²⁺ store filling/depletion.

	Acknowledgements

 
The authors are thankful to Drs Robert Sullivan and Janice Bailey for their careful revision of the manuscript. Special thanks are also due to all the volunteers who participated in this study. This work was supported by a grant from the 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.)

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