Molecular Human Reproduction Vol.9, No.3 pp. 125±131, 2003
DOI: 10.1093/molehr/gag017
Role of protein tyrosine phosphorylation in the
thapsigargin-induced intracellular Ca2+ store depletion
during human sperm acrosome reaction
VeÂronique Dorval1, Maurice Dufour2 and Pierre Leclerc1,3
1
DeÂpartement d'ObsteÂtrique/GyneÂcologie and Centre de recherche en Biologie de la Reproduction, Universite Laval,
and Centre de recherche du CHUQ, QueÂbec, G1L 3L5 and 2Centre de recherche du CHUL, QueÂbec, Canada G1V 4G2
3
To whom correspondence should be addressed at: Endocrinologie de la Reproduction, Pav. St-FrancËois d'Assise, 10 de L'Espinay,
QueÂbec, PQ, Canada, G1L 3L5. E-mail: [email protected]
During human sperm capacitation, an increase in phosphotyrosine content of speci®c 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
Freshly ejaculated sperm must undergo several biochemical and
membranous modi®cations to acquire their fertilizing potential
(Yanagimachi, 1994). Among these physiological modi®cations, an
increase in the phosphotyrosine content of speci®c 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 cAMPdependent 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, ®nally,
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 speci®c sperm proteins induced by this
ã European Society of Human Reproduction and Embryology
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 in¯ux 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 ®lling/depletion of the Ca2+ store and the
Ca2+ in¯ux 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 ®ll 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
125
V.Dorval, M.Dufour and P.Leclerc
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 ®lling 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 ®lling 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.
1979). Non-speci®c 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 ®ve 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-a-tubulin antibody.
Materials and methods
Induction and assessment of the acrosome reaction
Materials
Thapsigargin, bovine serum albumin (BSA), Pisum sativum agglutinin
conjugated with ¯uorescein 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, Pluronicâ 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'UrfeÂ, Canada). Monoclonal antiphosphotyrosine 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 mm pore size was supplied by MSI Inc.
(Westborough, MA, USA) and X-ray ®lms 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-FrancËois
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 203106/ml in BWW
medium slightly modi®ed 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
speci®c experiments, 10 mmol/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% b-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.,
126
After different incubation periods, sperm were washed by centrifugation in
PBS and diluted to 203106/ml in BWW/BSA medium supplemented with 10
mmol/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 speci®c experiments. The sperm cells were then
washed again and ®xed/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 ®xed cells were incubated with 75 mg/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
epi¯uorescence 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 253106/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 mmol/l
Indo-1/AM and 0.00625% Pluronic â 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 503106/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 mg/ml propidium iodide (PI) was added. Thapsigargin (10 mmol/l) was added
to the sperm suspension as indicated in speci®c experiments. Measurements
were performed by ¯ow cytometry, using an Epics Elite ESP (Beckman
Coulter, Miami, FL, USA) ¯ow 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 13106 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 ®xed/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
signi®cant. Results are expressed as means 6 SEM.
Ca2+ store depletion and tyrosine phosphorylation
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 antiphosphotyrosine 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.
Results
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% 6 0.4, before the addition of thapsigargin, to 6.1% 6
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 signi®cant 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% 6 0.9 versus 2.6 6 0.4, n = 4)
or after 1 h of the thapsigargin challenge (5.6% 6 1.8 versus 6.6% 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).
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 ¯ow
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).
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 mmol/l).
The sperm cells were then washed and incubated in the presence of 10 mmol/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.
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 ®ll their
thapsigargin-sensitive intracellular Ca2+ stores. The thapsigargininduced increase in intracellular free Ca 2+ was signi®cant 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 ¯ow 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 ®rst 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).
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
127
V.Dorval, M.Dufour and P.Leclerc
Figure 3. Free intracellular Ca2+ concentration (j) and thapsigargin-induced increase in intracellular Ca 2+ (m) 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 ¯ow cytometry. At each time, some sperm were challenged with 10 mmol/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 ¯uorescence emitted by the Ca2+ -bound Indo-1 (violet)/Ca2+-unbound Indo-1 (blue) and plotted versus time of
incubation. Values represent the mean 6 SEM of four different experiments. *Signi®cantly different (P < 0.05) from the intracellular Ca2+ concentration at the
beginning of the incubation. ²Signi®cantly different (P < 0.05) from the mean response to thapsigargin at the beginning of sperm incubation.
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 ¯ow cytometry to evaluate intracellular Ca2+ levels.
The arrow indicates the addition of 10 mmol/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.
was signi®cantly 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 signi®cantly
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
signi®cantly 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
128
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 ¯ow cytometry. The percentage
of sperm in the HR population upon the thapsigargin treatment is plotted
versus the time of incubation. Values represent the mean 6 SEM of four
different experiments. *Signi®cantly different (P < 0.05) from the percentage
of sperm in the HR population at the beginning of the incubation.
both the LR population and the incubated sperm that were not
challenged with thapsigargin.
Since only the HR subpopulation of the sperm suspension
underwent a signi®cant 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
Ca2+ store depletion and tyrosine phosphorylation
Figure 6. Intracellular Ca2+ concentration in sperm from low responsive
(LR) (j) and high responsive (HR) (h) populations in response to
thapsigargin during capacitation. Indo-1/AM-loaded sperm were processed as
described in Figure 4. Values represent mean 6 SEM of four different
experiments. *Signi®cantly 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.
(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 signi®cantly higher percentage of acrosome-reacted
sperm compared with the LR population (19.6% 6 5.0 versus 2.8% 6
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.
Discussion
As observed in previous reports (Leclerc et al., 1996, 1997, 1998;
Emiliozzi and Fenichel, 1997), an increase in the phosphotyrosine
content of speci®c 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 ®rst 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 speci®c 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
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 mmol/l PP2. Following the addition of 10
mmol/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±¯uoroscein, as described in Materials and methods. *A
signi®cant difference (P < 0.05) was observed between the percentage of
acrosome-reacted sperm in LR versus HR populations in sperm preincubated in the absence of PP2.
intracellular Ca2+ store. The length of time necessary to ®ll 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 suf®cient to
®ll sperm intracellular Ca2+ stores. This also suggests that the role of
acrosomal Ca2+-ATPase is to ®ll 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 observedÐLR 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 signi®cantly 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
129
V.Dorval, M.Dufour and P.Leclerc
progressively acquire the ability to undergo a net Ca2+ in¯ux 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
speci®cally the tyrosine kinases from the src family (Hanke et al.,
1996), markedly attenuated the basal as well as the thapsigargininduced 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 ®lling 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 ®nding 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 progesteroneinduced 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+
in¯ux from the extracellular medium. Whether or not voltageoperated 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 (KirkmanBrown et al., 2002b). Only the sustained phase of progesteroneinduced 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+ in¯ux.
In our study, the tyrosine kinase PP2 did not affect the thapsigargininduced 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 ®rst steps of the acrosome reaction induced by
progesterone, between the initial transient Ca2+ in¯ux 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 Cg (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
130
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 ®lm 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 ®lled 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 ®lling/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 aÁ la Recherche (to V.D.) and a
scholarship from Fonds de la Recherche en Sante du QueÂbec (to P.L.)
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Submitted on August 15, 2002; resubmitted on November 1, 2002; accepted on
November 26, 2002
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