Download Progesterone-stimulated intracellular calcium increase in human

Molecular Human Reproduction vol.4 no.3 pp. 259–268, 1998
Progesterone-stimulated intracellular calcium increase in human
spermatozoa is protein kinase C-independent
L.Bonaccorsi, C.Krausz, P.Pecchioli, G.Forti and E.Baldi1
Dipartimento di Fisiopatologia Clinica, Unità di Andrologia, Università di Firenze, viale Pieraccini 6, I-50139 Firenze, Italy
1To
whom correspondence should be addressed
Indirect studies suggested that protein kinase C (PKC) has a role in sperm motility and the acrosome reaction.
Physiological inducers of the sperm acrosome reaction include progesterone, which can increase intracellular
calcium ([Ca21]i), tyrosine phosphorylation of proteins and chloride efflux in human spermatozoa. PKC may
be involved in progesterone-stimulated acrosome reaction, although controversial results have been obtained
concerning the effect of PKC inhibition on progesterone-stimulated [Ca21]i increase. In the present study, we
investigated the direct effect of progesterone on the activity of PKC, as well as the effect of a panel of PKC
inhibitors on progesterone-stimulated [Ca21]i increase and tyrosine phosphorylation of proteins. We found
that progesterone stimulates sperm PKC activity and that PKC inhibition with staurosporine and bisindolylmaleimide partially reversed the effect of progesterone on acrosome reaction, indicating an involvement of
the enzyme in the effect of the steroid. We next evaluated the effect of three different PKC inhibitors
(sangivamycin, staurosporine and bisindolylmaleimide) on progesterone-stimulated [Ca21]i increase. Neither
short-term (15 min) nor long-term (90 min) preincubation with any of the three compounds had a substantial
effect on the stimulatory effect of progesterone on sperm [Ca21]i. Nor was responsiveness to progesterone
affected by either short-term (determining activation of PKC) or long-term (determining down-regulation of
PKC) incubation with the tumour promoter phorbol myristate acetate (PMA), a known non-physiological
stimulator of PKC. These results indicate that progesterone-stimulated calcium influx is independent of PKC
activation. In addition, we found that preincubation with PKC inhibitors had a stimulatory effect per se on
tyrosine phosphorylation of sperm proteins. When compared with the appropriate control, the effect of
progesterone on tyrosine phosphorylation was slightly (but not significantly) reduced by the inhibitors,
sangivamycin, staurosporine and bisindolylmaleimide, but was significantly inhibited by calphostin C. These
results do not permit a final conclusion on the involvement of PKC in progesterone-stimulated tyrosine
phosphorylation of sperm proteins. However, the lack of effect of PMA on tyrosine phosphorylation indicates
that PKC stimulation is not sufficient to induce this effect. In conclusion, our results indicate that PKC plays
a role in progesterone-induced acrosome reaction and that progesterone-stimulated PKC activation is
downstream to stimulation of calcium influx by the steroid.
Key words: acrosome reaction/intracellular calcium/progesterone/protein kinase C/spermatozoa
Introduction
In recent years, accumulating evidence has indicated a role
for progesterone in the physiological stimulation of acrosome
reaction of capacitated mammalian spermatozoa (for review,
see Baldi et al., 1995). Progesterone, which is present in µM
concentrations in the cumulus matrix that surrounds the oocyte
at the time of fertilization, stimulates acrosome reaction by
interacting with sperm surface receptor(s) (Luconi et al., 1998)
which induce(s) a large influx of calcium (Thomas and Meizel,
1989; Blackmore et al., 1990; Baldi et al., 1991), phospholipid
hydrolysis (Thomas and Meizel, 1989; Roldan et al., 1994),
and stimulate(s) phosphorylation of proteins (Tesarik et al.,
1993; Luconi et al., 1995; Emiliozzi et al., 1996; O’Toole
et al., 1996) and chloride efflux from the cells (Meizel and
Turner, 1996). Interactions among the different signalling
pathways have been also demonstrated (Bonaccorsi et al.,
1995; Tesarik et al., 1996; Meizel and Turner, 1996; Luconi
et al., 1996).
© European Society for Human Reproduction and Embryology
An important component of the sequence of events ending
in fusion of the outer plasma membrane and acrosomal
membranes during the process of acrosome reaction (reviewed
in Breitbart and Spungin, 1997 and Aitken, 1997) could be
protein kinase C (PKC) which is known to play a modulatory
role during exocytotic responses in somatic cells
(Nishizuka,1992). Stimulation of phospholipid hydrolysis by
progesterone and other stimuli of acrosome reaction leads to
increased generation of diacylglycerol (DAG) (Roldan et al.,
1994), the main physiological stimulus of PKC in somatic
cells. DAG has been shown to play an important role in sperm
biology. Indeed, DAG and other agonists of PKC, such as
phorbol esters, increase acrosome reaction (De Jonge et al.,
1991; Breitbart et al. 1992; Rotem et al. 1992; O’Toole et al.,
1996) and motility of spermatozoa (Rotem et al. 1990).
However, although the presence of PKC in mammalian spermatozoa has been well documented (Rotem et al., 1990; Breitbart
et al., 1992; Lax et al., 1997), its activity is very low,
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L.Bonaccorsi et al.
representing ~3% of that detected in rat brain and 20% of that
measured in PC12 cells and in rat pituitary cells (Rotem et al.,
1990; Breitbart et al., 1992). The direct involvement of PKC
in the biological activity of DAG generated after stimulation
with progesterone or zona proteins in spermatozoa has not
been demonstrated. Indirect studies demonstrated that the PKC
inhibitors staurosporine and H-7 blunted the intracellular
calcium increase and the acrosome reaction induced by progesterone, suggesting a possible involvement of PKC in the influx
of calcium in response to progesterone (Foresta et al., 1995).
However, while very potent, these inhibitors lack specificity
for PKC (Kocher and Clementson, 1991; Wilkinson and
Hallam, 1994). Using different PKC inhibitors, the involvement
of PKC in progesterone-stimulated calcium influx has been
recently questioned (O’Toole et al., 1996). In the present study,
we re-examined the involvement of PKC in the effect of
progesterone in human spermatozoa by testing a panel of
different PKC inhibitors on progesterone-stimulated intracellular calcium ([Ca21]i), tyrosine phosphorylation of proteins and
acrosome reaction. In addition, we evaluated the effect of
progesterone and phorbol esters on human sperm PKC activity.
We demonstrated that PKC inhibition reduces acrosomal
exocytosis stimulated by progesterone without affecting calcium influx stimulated by the steroid. On the contrary, PKC
appears to be involved in progesterone-stimulated tyrosine
phosphorylation of sperm proteins.
Materials and methods
Reagents
Fura-2/AM, A23187, ionomycin, staurosporine and bisindolylmaleimide–HCl were obtained from Calbiochem (San Diego, CA, USA).
Sangivamycin was from National Institutes of Health Drug Synthesis
and Chemistry Branch (Bethesda, MD, USA). Progesterone and
fluorescein isothiocyanate (FITC)-labelled lectin, were obtained from
Sigma (St Louis, MO, USA). Human serum albumin (HSA)-free
Human Tubal Fluid (HTF) medium was from Irvine (Santa Ana,
CA, USA). Progesterone and A23187 were dissolved in dimethyl
sulphoxide (DMSO) at an initial concentration of 10 mg/ml and
10 mM respectively and further dilutions were made in HTF medium.
Preparation and incubation of spermatozoa
Human semen was obtained from normospermic men undergoing
semen analysis for couple infertility in our laboratory. Semen was
collected according to the World Health Laboratory (WHO, 1992)
recommended procedure by masturbation after 3–4 days of sexual
abstinence. Samples with a linear progressive motility of ,50% at
60 min and with leukocytes and/or immature germ cell concentration
.106/ml were not included in the study. For calcium studies,
determination of PKC activity and Western blot analysis of tyrosine
phosphorylation, semen samples were processed as previously
described (Baldi et al., 1991, 1993). Briefly spermatozoa were
separated on gradients of 40 and 80% Percoll, combined, washed in
HTF medium supplemented with 3 mg/ml bovine serum albumin
(BSA), and resuspended in a small volume of the same medium. For
the acrosome reaction, spermatozoa were prepared by swim-up
technique as previously described (Krausz et al., 1996). Spermatozoa
were counted and resuspended at a concentration of 103106/ml.
Samples were capacitated at 37°C in a 5% CO2 atmosphere for at
least 2 h before assessing [Ca21]i, tyrosine phosphorylation, PKC
activity and acrosome reaction in response to progesterone.
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Measurement of [Ca21]i
Spermatozoa were resuspended at the concentration of 53106/ml in
BSA-supplemented HTF, incubated at 37°C with fura-2/AM (2 µM)
for 45 min, washed, resuspended in FM buffer (125 mM NaCl,
10 mM KCl, 2.5 mM CaCl2, 0.25 mM MgCl2, 19 mM Na lactate,
2.5 mM Na pyruvate, 20 mM HEPES/NaOH, Thomas and Meizel,
1988) and further incubated for 15 min. [Ca21]i was measured as
previously described using a spectrofluorimetric method (Baldi et al.,
1991; Falsetti et al., 1993). Spermatozoa were transferred to a quartz
cuvette in a total volume of 2 ml containing a total of 53106
spermatozoa. Fluorescence was measured using a spectrofluorimeter
(University of Pennsylvania Biomedical Group, Philadelphia, PA,
USA) set at 340 nm excitation with emission at 510 nm. Spermatozoa
were stimulated with progesterone (1µg/ml) which was added directly
in the cuvette. Fluorescence measurements were converted to [Ca21]i
by determining maximal fluorescence (Fmax) with ionomycin (8 mM
final concentration) followed by minimal fluorescence (Fmin) with
10 mM EGTA, pH 10. [Ca21]i was calculated according to
Grynkiewicz et al. (1985) assuming fura-2 had a dissociation constant
for calcium of 224 nM. Addition of ionomycin yielded similar
increases in fluorescence in all the samples examined, indicating a
similar fura-2/AM incorporation. Autofluorescence of the cells was
assessed by measuring fluorescence of unloaded cells; autofluorescence subtraction did not modify basal or stimulated [Ca21]i as
previously reported (Falsetti et al., 1993). To evaluate the effect of
PKC inhibitors on progesterone-induced calcium influx, fura-2-loaded
spermatozoa were pre-treated with the inhibitors or control solvent
for the indicated times, centrifuged, resuspended in FM buffer and
analysed with the spectrofluorimeter as described above.
Western blot analysis of tyrosine phosphorylated proteins
After the different incubations, the samples were processed for sodium
dodedyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)
as previously described (Luconi et al., 1995; Bonaccorsi et al., 1995).
Briefly, sperm samples, containing 33106 cells/ml were added with
1 mM Na3VO4, centrifuged at 400 g at 4°C for 10 min, washed in
HTF, and resuspended in 10 µl lysis buffer [20 mM Tris, pH 7.4,
150 mM NaCl, 0.25% NonidetP40 (NP40), 1 mM Na3VO4, 1 mM
phenylmethylsulphonyl fluoride (PMSF)]. After protein measurement
(see below), the sperm extracts, containing ~20 µg of proteins, were
diluted in equal volume of 23 SB (Laemmli’s sample buffer 5
62.5 mM Tris pH 6.8, containing 10% glycerol, 20% SDS, 2.5%
pyronin and 200 mM dithiothreitol), incubated at 95°C for 5 min and
loaded onto 8%, 10% polyacrylamide–bisacrylamide gels. After SDS–
PAGE, proteins were transferred to nitro-cellulose (Sigma) and stained
with Ponceau to verify equal protein loading. In some experiments,
equivalent protein loading was verified by staining parallel gels
with Coomassie Brilliant Blue (Sigma, St Louis, MO, USA). The
nitrocellulose was blocked in 5% BSA for 2 h in TTBS solution
(Tris-buffered saline containing 0.1% Tween 20, pH 7.4), washed
and then immunostained with peroxidase-conjugated monoclonal antiphosphotyrosine antibody (PY20, dilution 1:2000 in 2% BSA–TTBS).
The phosphotyrosine antibody-reacted proteins were revealed by
enhanced-chemiluminescence system (ECL; Amersham, Chalfont,
UK). The immunospecificity of PY20 was determined by preadsorbing the antibody with 40 mM of o-phospho-DL-tyrosine for 1 h
at room temperature (Luconi et al., 1995). Quantification of the bands
was made directly on the films by image analysis with a C3077/01
video camera, connected with the video frame grabber M4477
(Hamamatsu Photonics, Hamamatsu, Japan), a plug-in board used in
a Macintosh Ilsi PC (Apple, Cupertino, CA, USA). Acquisition of
images was carried out using Imagequest IQ Base software (Hamamatsu Photonics). Image processing and analysis was obtained by
PKC-independence of progesterone-stimulated calcium increase in spermatozoa
IMAGE free software, kindly provided by Wayne Rasband (NIMH,
Bethesda, MD, USA). For technical reasons, quantification was
performed in ECL low-exposed films.
Determination of PKC activity
PKC activity was determined essentially as described by Rotem et al.
(1990), with the difference that basal activity (representing calciuminsensitive PKC, whose value was subtracted from total activity) was
obtained by incubating the membranes in a calcium-free medium.
After washing free of seminal plasma, spermatozoa (4003106/sample)
were stimulated with phorbol myristate actetae (PMA, 1 µM) or
progesterone (10 µM) for 15 min at 37°C in BSA-supplemented HTF
medium, centrifuged and washed twice in a buffer (buffer 1) containing
20 mM Tris, 2 mM EDTA, 1 mM EGTA, 2 mM PMSF, 330 mM
sucrose, 50 mM 2βmercaptoethanol, 5% glycerol, 5 µg/ml leupeptin,
0.5 mg/ml bacitracin and 50 µg/ml soybean trypsin inhibitor. All
subsequent passages were performed on ice. Spermatozoa were
homogenized with a teflon/glass homogenizer and sonicated until a
homogeneous suspension was obtained. The suspension was then
centrifuged at 500 g for 5 min and the supernatant collected and
centrifuged at 100 000 g for 60 min at 4°C. The obtained pellet was
resuspended in buffer 1 containing 1% NP-40, homogenized in a
teflon/glass homogenizer, sonicated and incubated for 30 min at 4°C.
The particulate fraction was centrifuged at 500 g for 5 min and the
obtained supernatant applied to previously activated DEAE cellulose
columns equilibrated with buffer 1. The columns were washed three
times with buffer 1 without NP-40, and eluted with 2 ml of buffer 1
without NP-40 containing 200 mM NaCl. The eluate was concentrated
to a volume of ~60 µl with Centricon filters of 30000 exclusion
(Amicon, MA, USA). Protein concentration was determined by a
commercial kit (Bio-Rad protein assay; Bio-Rad, Munich, Germany).
PKC assay was carried out in a final volume of 100 µl of buffer 2
containing 1 mM CaCl2, 96 µg/ml phosphatidylserine, 10 mM MgCl2,
100 µM ATP, 3 µg/ml diolein, 1 mg/ml histone IIIS and 0.5 µCi 32PγATP. The reaction was initiated by adding protein suspension (40 µg/
tube). Basal activity was measured in buffer 2 devoid of CaCl2,
phosphatidylserine and diolein and containing 1 mM EGTA. The
assay was carried out for 30 min at 30°C. The reaction was then
stopped in ice and 50 µl of mixture was collected on membrane
filters (GF/C, Whatman). Filters were washed four times with phosphoric acid (75 mM). The samples were counted for radioactivity by
liquid scintillation. Blank values (obtained including all the reagents
but not membrane preparations) were subtracted from all experiments.
Data are expressed as pM phosphate transferred/min/µg of protein.
Determination of acrosome reaction
Acrosome-reacted spermatozoa were evaluated using FITC-labelled
Arachis hypogea (peanut) lectin, as described previously (Aitken
et al., 1993; Krausz et al., 1996). Briefly, aliquots (125 µl) of
spermatozoa (103106/ml) recovered after swim-up procedure, were
incubated with an equal volume of progesterone (10 µM) or PMA
(1 µM) in the presence or absence of the indicated inhibitors or
DMSO control solvent for 1 h at 37°C, centrifuged and further
incubated in 0.5 ml hyposmotic swelling medium for 1 h. After
centrifugation, spermatozoa were finally suspended in 50 µl ice-cold
methanol, layered on a slide, stained with fluorescent lectin and
scored using a fluorescent microscope (Leitz Wetzlar, Germany). The
acrosome reaction was evaluated on a total of 200 spermatozoa/slide.
Only curly tailed spermatozoa were scored. The difference between
percentage progesterone- or PMA-induced and percentage spontaneous acrosome reacted spermatozoa in the presence or absence of
inhibitors, was defined as the percentage of spermatozoa in the
population capable of responding to progesterone or PMA [and
Table I. Basal, phorbol myristate acetate (PMA)- and progesteronestimulated proein kinase C (PKC) activity (pmol/µg protein/min) in human
spermatozoa
Expt. no.
Control
PMAa
Progesteroneb
1
2
3
0.037
0.131
0.104
0.147
–
–
–
0.216
0.146
a1
µM, 5 min stimulation.
µM, 15 min stimulation.
b10
thus called respectively, acrosome reaction following progesterone
challenge (ARPC) and acrosome reaction following PMA challenge
(ARPMAC)].
Statistical analysis
Data are expressed as mean 6 SEM. Statistical analysis of the data
was performed by unpaired t-test.
Results
Effect of PMA and progesterone on membrane PKC
activity
PKC activity in human spermatozoa was determined by
measuring the transfer of the phosphate group from [γ-32P]ATP to the histone III-s as described in material and methods.
Due to the low PKC activity present in spermatozoa (Rotem
et al., 1990) and the consequently large number of spermatozoa
needed to evaluate it (Rotem et al., 1990), we used pooled
samples from 10–20 subjects for each experiment. We
measured basal, PMA (1 µM, 5 min)- and progesterone (10 µM,
15 min)-stimulated PKC activity in partially purified human
sperm membrane preparation. As shown in Table I, basal PKC
activity varied from 0.037 to 0.131 (mean value: 0.09) pmol/
µg protein/min in the three pooled samples used in our
experiments. This value is lower than the average 0.22 pmol/
min/µg protein detected by Rotem et al. (1990); however, the
fact that calcium-independent PKC activity was subtracted
from our values, may account for this slight difference. On
the other hand, in other mammalian species, sperm membrane
PKC activity was found to vary from 0.1 in the bull to 0.28
pmol/min/µg protein in the ram (Breitbart et al., 1992). PMA
produced a 4.5-fold stimulation of PKC activity, in agreement
with the 3-fold stimulation observed in bovine sperm membranes (Lax et al., 1997). Progesterone was much less potent
than the phorbol ester, but still stimulated membrane PKC
activity by 1.4–1.6-fold (Table I), indicating that DAG generation in response to progesterone may be sufficient to stimulate
PKC activity. This result prompted us to evaluate the effects
of PKC inhibitors on the different biological effects of progesterone on human spermatozoa.
Effect of PKC inhibition on progesterone-stimulated
acrosome reaction
We first tested the effects of the PKC inhibitors staurosporine
and bisindolylmaleimide, on progesterone (10 µM)- and
PMA(1 µM)-stimulated acrosome reaction. As shown in Figure
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L.Bonaccorsi et al.
Figure 1. Effect of protein kinase C (PKC) inhibition on progesterone (P)- and phorbol myristate acetate (PMA)-stimulated acrosome
reaction of human spermatozoa. Capacitated spermatozoa were incubated for 1 h with progesterone (10 µM) and PMA (1 µM) in the
presence of staurosporine (sta, 0.1 µM), bisindolylmaleimide (bis, 0.1 µM) or control solvent (dimethylsulphoxide, 0.01%). y axis: %
acrosome reacted spermatozoa in response to the agonist minus % acrosome reacted spermatozoa in respective control. The number of
experiments is indicated inside each column; results are expressed as mean 6 SEM. *P ,0.05 compared with progesterone; **P , 0.005
compared with PMA.
1, both agonists induced a significant increase in the percentage
of acrosome reacted spermatozoa. To our surprise, the PKC
inhibitors staurosporine and bisindolylmaleimide had a stimulatory effect on the basal acrosome reaction (% acrosome reacted
spermatozoa: 6.5 6 1.4 in control, 12.4 6 2.7 in staurosporine,
P , 0.05 and 10.0 6 2.4 in bisindolylmaleimide-treated spermatozoa, P , 0.05, n 5 6). Compared with the appropriate control,
the stimulatory effect of progesterone and PMA on acrosome
reaction were significantly inhibited by the two PKC inhibitors
(Figure 1). These results suggest that progesterone- and PMAstimulated acrosome reaction involves PKC activation.
Effect of PKC inhibitors on progesterone-induced
calcium influx
As previously demonstrated by our and other groups, the
addition of nano- to micromolar concentrations of progesterone
to fura-2-loaded spermatozoa induces a large, dose-dependent
influx of calcium (Blackmore et al. ,1990; Baldi et al., 1991).
Such influx is characterized by a rapid increase of [Ca21]i
followed by a long-lasting sustained phase (typical Ca21 waves
in response to progesterone are shown in Figure 4). We
evaluated the effect of short-term (15 min) preincubation with
the different PKC inhibitors on progesterone-induced calcium
influx. Such incubation time was chosen on the basis of the
PKC inhibitory effect of these compounds in somatic cells
(King and Rittenhouse, 1989; Tuollec et al., 1991; Kester
et al., 1992; Baldi et al., 1994). As shown in Figure 2A–C,
the three PKC inhibitors staurosporine (0.01–1µM, panel A),
bisindolylmaleimide (0.001–1 µM, panel B) and sangivamycin
(0.001–1 µM, panel C) did not affect progesterone-stimulated
[Ca 21 ]i in human spermatozoa at any tested concentration.
Foresta et al. (1995) showed that a longer (90 min) preincubation with staurosporine and H-7 was able to inhibit the
262
progesterone-stimulated calcium increase in human spermatozoa in a dose-dependent manner. Although both staurosporine
and H-7 do not require, at least in somatic cells (King and
Rittenhouse 1989; Toullec et al., 1991; Kester et al., 1992;
Baldi et al., 1994), such a long incubation to inhibit PKC
activity, it is possible that a long incubation is required in the
case of spermatozoa. To test this possibility, spermatozoa were
incubated with the three PKC inhibitors for 90 min. Such a
treatment did not affect either motility, evaluated with a sperm
motion analyser (Hamilton-Thorn, Danvers, MA, USA), or
viability, evaluated with eosin staining according to the WHO
(1992) procedure (data not shown). As shown in Figure 3
progesterone-stimulated [Ca21]i increase was not significantly
affected by a lengthy treatment with 0.1 or 1 µM concentrations
of sangiovamycin or bisindolylmaleimide, whereas was slightly
but significantly reduced by staurosporine. Typical calcium
waves in fura-2-loaded spermatozoa stimulated with progesterone after short term (panel A) or long term (panel B) preincubation with the different PKC inhibitors obtained in typical
experiment are shown in Figure 4. As can be observed, no
substantial changes in calcium waves were observed with the
different treatments. The lack of effect of the PKC inhibitors
cannot be due to degradation of the compounds, since in
parallel experiments conducted in a different cell system, their
inhibitory activity has been clearly demonstrated by our group
(Bonaccorsi et al., 1997).
In somatic cells, calcium and phospholipase C-mediated
signalling stimulated by different agonists may be affected by
PKC activation or PKC down-regulation with tumour promoters such as PMA (Sibley et al., 1987). In particular, shortterm incubation with PMA, which leads to PKC activation,
inhibits, whereas long-term incubation with PMA, which
down-regulates PKC activity, potentiates agonist-stimulated
PKC-independence of progesterone-stimulated calcium increase in spermatozoa
Figure 2. Effect of protein kinase C (PKC) inhibition (short-term incubation) on progesterone-stimulated intracellular calcium ([Ca21]i)
increase in human spermatozoa. Capacitated, fura-2-loaded spermatozoa were preincubated for 15 min with increasing concentrations of
staurosporine (sta, panel A), bisindolylmaleimide (bis, panel B) and sangivamycin (san, panel C) and [Ca21]i increase in response to
progesterone (1 µg/ml) evaluated by a spectrofluorimetric method. y axis: % [Ca21]i increases in response to progesterone in the presence of
the inhibitors versus progesterone alone (taken as 100% and identified by a horizontal line in each panel). Results are expressed as mean 6
SEM.
signalling (Hepler et al., 1988; Pfeilschifter et al., 1989). We
evaluated the effect of short-term and long-term treatment of
spermatozoa with 1 µM PMA on progesterone-stimulated
[Ca21]i increase in human spermatozoa. As shown in Figure
5, which reports mean fold increase of [Ca21]i in response to
progesterone at its peak level in different conditions, the effect
of progesterone was not modified by either treatment. Similar
results were obtained with 0.1 µM PMA (not shown). No
modifications with short- or long-term PMA treatment were
observed in the plateau phase (not shown). Taken together,
our results indicate that progesterone-stimulated calcium influx
in human spermatozoa is PKC-independent.
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Figure 3. Effect of protein kinase C (PKC) inhibition (long-term incubation) on progesterone-stimulated intracellular calcium ([Ca21]i)
increase in human spermatozoa. Capacitated, fura-2-loaded spermatozoa were preincubated for 90 min with the indicated concentrations of
sangivamycin (san), staurosporine (sta) and bisindolylmaleimide (bis) and [Ca21]i increase in response to progesterone (1 µg/ml) evaluated
by a spectrofluorimetric method. y axis: % [Ca21]i increases in response to progesterone in the presence of the inhibitors versus
progesterone alone (taken as 100% and identified by an horizontal line) are reported. Results are expressed as mean 6 SEM. *Significantly
different from controls (P , 0.05).
Effect of PKC inhibitors on progesterone-stimulated
tyrosine phosphorylation
Recently, it has been reported that PKC inhibition reduces
progesterone-mediated stimulation of total protein phosphorylation of human spermatozoa (O’Toole et al., 1996). In addition,
it has been shown that the poorly selective PKC inhibitor
staurosporine but not the more selective chelerythrine, inhibits
the spontaneous increase of tyrosine phosphorylation that
occurs during capacitation probably due to an aspecific effect
(Carrera et al., 1996). To examine whether PKC is involved in
the progesterone-induced increase in tyrosine phosphorylation,
spermatozoa were preincubated with the PKC inhibitors sangivamycin, staurosporine and bisindolylmaleimide (at the final
concentration of 0.1 µM) for 30 min and then stimulated with
progesterone (10 µM) for 5 min. As shown in Figure 6A,
progesterone stimulates tyrosine phosphorylation of an array
of proteins, including one of ~97 kDa, which has been shown
to be the main tyrosine-phosphorylated protein in detergent
extracts of human spermatozoa (Tesarik et al., 1993; Luconi
et al., 1995; Burks et al., 1995; Emiliozzi et al., 1996). Since
in most of the experiments preincubation with the three
PKC inhibitors had a stimulatory effect per se on tyrosine
phosphorylation of proteins of capacitated spermatozoa (mean
fold increases of tyrosine phosphorylation of p97 in four
different experiments: 1.9 5 6 0.35 for sangivamycin, 1.7 6
0.31 for staurosporine and 1.66 6 0.32 for bisindolylmaleimide), the effect of progesterone on tyrosine phosphorylation
in the presence of the PKC inhibitors was compared to the
respective control. As shown in Figure 6B, the mean fold
increase in tyrosine phosphorylation of p97 stimulated by
progesterone was partially, but not significantly, reduced by
pretreatment with staurosporine, sangivamycin and bisindolylmaleimide.
O’Toole et al. (1996) demonstrated that the PKC inhibitor
calphostin C was able to inhibit progesterone-induced acrosome
reaction. We therefore evaluated also the effect of this inhibitor
on progesterone-stimulated tyrosine phosphorylation. As
264
shown in Figure 7A, calphostin C (0.15 µM, 30 min pretreatment) caused an increase in tyrosine phosphorylation of sperm
proteins, as observed for the other inhibitors. Compared with
the appropriate control, the effect of progesterone was reduced
by treatment with calphostin C (Figure 7A) (fold increase
progesterone stimulation of p97 vs respective control: 1.4 6
0.16 in the absence and 0.74 6 0.09 in the presence of
calphostin, n 5 4, P ,0.05). Quantification of p97 band of
the experiment shown in Figure 7A is represented in Figure
7B. We confirmed also that calphostin C inhibits progesteroneinduced acrosome reaction (% ARPC in a representative
experiment: 12 in the absence of the inhibitor, 6 in the presence
of calphostin C).
If PKC is involved in tyrosine phosphorylation of proteins,
direct activation of the enzyme with PMA should result in
increased tyrosine phosphorylation. In agreement with previous
results by Leclerc et al. (1996), PMA did not affect tyrosine
phosphorylation of sperm proteins in our experimental conditions (Figure 7). This result has been confirmed in three
different experiments performed in different sperm samples
(mean 6 SEM tyrosine phosphorylation in arbitrary units:
65.3 6 10.0 in control, 62.4 6 13.04 in PMA-stimulated
spermatozoa).
Discussion
The rapid, non-genomic effects of progesterone on human
spermatozoa have been reported in several studies (for review,
see Baldi et al., 1995). These studies indicate that progesterone
can be considered, together with the oocyte zona pellucida
protein ZP3, a physiological inducer of acrosome reaction. In
the present study we provide evidence that activation of protein
kinase C (PKC), a family of threonine- and serine-specific
protein kinases which play a crucial role in signal transduction
mechanisms, may be involved in the induction of acrosome
reaction by progesterone. In addition, we demonstrate that
PKC activation is unlikely to be involved in the increase of
[Ca21]i stimulated by progesterone.
PKC-independence of progesterone-stimulated calcium increase in spermatozoa
Figure 4. Typical intracellular calcium ([Ca21]i) waves in response to progesterone (P, 1 µg/ml) in fura-2-loaded spermatozoa after shortterm (15 min, panel A) and long-term (90 min, panel B) preincubation with different concentrations of the protein kinase C (PKC) inhibitors
staurosporine (sta), sangivamycin (san) and bisindolylmaleimide (bis). C: [Ca21]i response to progesterone in the absence of PKC inhibitors.
The arrows indicate the addition of progesterone. Results are expressed as mean 6 SEM.
Figure 5. Effect of short-term (5 min) and long-term (24 h)
exposure to phorbol myristate acetate (PMA, 1 µM) on intracellular
calcium [Ca21]i) increase in response to progesterone in fura-2loaded spermatozoa. For short-term exposure, PMA was added
directly to fura-2-loaded spermatozoa in the cuvette. y axis: fold
increase sperm [Ca21]i in response to 1 µg/ml progesterone.
Our results confirm that PKC activity in human spermatozoa
is extremely low (Rotem et al., 1990). However, we report
here that PKC activity may be enhanced not only by PMA,
an exogenous synthetic activator of PKC, but also, although
to a much lesser extent, by a short treatment with progesterone,
a physiological inducer of the acrosome reaction. These results
indicate a direct involvement of PKC in the induction of
acrosome reaction by the steroid, confirming indirect studies
using various PKC inhibitors (Foresta et al., 1995; O’Toole
et al., 1996; present study). Controversial results have been
reported concerning the involvement of PKC in calcium
signalling stimulated by progesterone (Foresta et al., 1995;
O’Toole et al., 1996). We re-examined this issue by using a
panel of three different PKC inhibitors, staurosporine, bisindolylmaleimide and sangivamycin. Both short-term (15 min)
and long-term (90 min) preincubation with increasing concentrations of the three compounds did not substantially affect
the progesterone-stimulated [Ca21]i increase in human spermatozoa. The increase in [Ca21]i mediated by progesterone
was only slightly affected by long-time pretreatment with
micromolar concentrations of staurosporine. However, staurosporine is a less specific PKC inhibitor than the other two
inhibitors employed in our study and it is known to inhibit
other protein kinases besides PKC (Kocher and Clementson,
1991; Gschwendt et al., 1994). It is thus possible that the slight
inhibitory effect of staurosporine on progesterone-stimulated
calcium influx in human spermatozoa observed with a long
incubation time is due to non-specific effects of this compound.
In a recent paper, Tesarik et al. (1996) demonstrated the
involvement of tyrosine kinases in late Ca21 fluxes stimulated
by progesterone. We have recorded fura-2 fluorescence for
265
L.Bonaccorsi et al.
Figure 6. Effect of protein kinase C (PKC) inhibition on
progesterone stimulated tyrosine phosphorylation of sperm proteins.
Panel A: representative experiment. Capacitated spermatozoa were
stimulated for 5 min with progesterone (progesterone, 10 µM) in
the presence or absence (C) of 0.1 µM concentration of the PKC
inhibitors bisindolylmaleimide (bis), staurosporine (sta) and
sangivamycin (san). The obtained protein extracts were separated
by 8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE) and probed with peroxidase-conjugated PY20
antiphosphotyrosine antibody. The major sperm tyrosine
phosphorylated protein (p97) is indicated by the arrow. Molecular
weight markers (3 103 kDa) are indicated to the right of the blot.
Panel B: mean (6SEM) fold increase tyrosine phosphorylation of
p97 vs respective control, following stimulation with progesterone
in the presence or absence of the PKC inhibitors. Quantification of
the bands was obtained as described in the Materials and methods
section. Results are expressed as mean 6 SEM; no significant
differences were observed with the three inhibitors.
only 1 min after addition of progesterone and thus these late
fluxes and their eventual sensitivity to treatment with PKC
inhibitors could have escaped our observation. However, these
fluxes appear to occur only in a small subpopulation of
spermatozoa (Tesarik et al., 1996), rendering difficult their
detection with our method. The lack of involvement of PKC
in progesterone-stimulated calcium influx was confirmed by
short-term (leading to PKC activation) and long-term (leading
to PKC down-regulation) preincubation of spermatozoa with
the phorbol diester PMA, known to stimulate PKC in vivo and
in vitro. Such treatments did not affect progesterone-stimulated
calcium influx, indicating that PKC activation is not required
for calcium signalling. These results are in contrast with those
of Foresta et al. (1995), who demonstrated that two poorly
selective PKC inhibitors, staurosporine and H-7, and prolonged
sperm incubation with PMA, abolish [Ca21]i increase stimulated by progesterone. On the contrary, O’Toole et al. (1996)
demonstrated that the inclusion of nifedipine, a dihydropyridine
Ca21 channel antagonist, blunted progesterone-stimulated
266
Figure 7. Effect of the protein kinase C (PKC) inhibitor calphostin
C on progesterone-stimulated tyrosine phosphorylation of proteins.
Capacitated spermatozoa were stimulated for 5 min with
progesterone (P, 10 µM) and phorbol myristate acetate (PMA,
1 µM) in the presence or absence of 0.15 µM calphostin C (Calph,
30 min preincubation). The obtained protein extracts were separated
by 8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE) and probed with peroxidase-conjugated PY20
antiphosphotyrosine antibody. The major sperm tyrosine
phosphorylated protein (p97) is indicated by the arrow; RF 5
buffer front. Molecular weight markers (3 103 kDa) are indicated
to the right of the blot. Panel B: quantification of p97 band
(expressed as arbitrary units) of the experiment shown in panel A.
phosphorylation of proteins mediated by PKC, indicating,
although indirectly, that the influx of calcium is necessary for
activation of PKC and not vice versa. In addition, Arnoult
et al. (1997), recently demonstrated that basal calcium currents
in mouse dissociated spermatogenetic cells are not affected by
H-7 and H-9. In agreement with O’Toole et al. (1996), our
results support the possibility that calcium influx is upstream
to the activation of PKC. Along with this evidence, it has been
recently shown that PKC activity is greatly stimulated by
calcium in bull spermatozoa (Lax et al., 1997). It must be
mentioned that it is not clear, at the moment, if the stimulatory
effects of PMA in spermatozoa are Ca21-dependent or not.
Early studies by Rotem et al. (1992) and Furuya et al. (1993)
suggested that this effect is Ca21-independent, while other
authors (De Jonge et al., 1991; Spungin and Breitbart, 1996)
have demonstrated that extracellular Ca21 is required. In our
hands PMA was unable to stimulate an increase of [Ca21]i in
human spermatozoa (unpublished observations), indicating that
PKC-independence of progesterone-stimulated calcium increase in spermatozoa
the stimulatory effect of PMA on the acrosome reaction can
be obtained in the absence of a detectable increase in [Ca21]i.
We have demonstrated previously that progesterone promotes tyrosine phosphorylation of several sperm proteins
(Bonaccorsi et al., 1995; Luconi et al., 1995). In somatic cells,
both PKC-dependent and -independent stimulation of tyrosine
phosphorylation have been demonstrated (Rollet et al., 1994;
Watanabe et al., 1994; Hakeda et al., 1997; Zhang et al.,
1997). We show here that these inhibitors have a stimulatory
effect per se on tyrosine phosphorylation of proteins of
capacitated spermatozoa. Such an effect might explain the
observed stimulatory effect of bisindolylmaleimide and staurosporine on the acrosome reaction. The stimulation by staurosporine of kinase activity has been also demonstrated in
human platelets (Kocher and Clementson, 1991). In addition,
staurosporine has been demonstrated to have PKC-independent
stimulatory effects in several cell systems (Sako et al., 1988;
Hashimoto and Hagino, 1989; Hedberg et al., 1990). It is also
possible that non-specific effects of the the PKC inhibitors on
tyrosine phosphatases are responsible for the increase of
tyrosine phosphorylation. In addition, PKC might negatively
regulate tyrosine kinases, as has been observed in human
neutrophils (Al-Shami et al., 1997), although the absence of
effect of PMA on sperm tyrosine phosphorylation seems to
exclude this possibility. On the other hand, in capacitating
spermatozoa, Carrera et al. (1996) demonstrated that staurosporine, but not the more selective PKC inhibitor chelerythrine,
reduced the spontaneous increase in tyrosine phosphorylation
which occurs during capacitation, concluding that PKC is not
involved in tyrosine phosphorylation of sperm proteins at least
during the process of capacitation. We show here that tyrosine
phosphorylation of proteins in response to progesterone is only
slightly, but not significantly, reduced in the presence of
staurosporine, sangivamycin and bisindolylmaleimide, but is
significantly reduced by calphostin C if the effect of progesterone is compared with the appropriate control. However, we
cannot exclude the possibility that the stimulatory effect of
the PKC inhibitors on tyrosine phosphorylation observed
in our experiments might aspecifically mask the effect of
progesterone. On the other hand, the lack of effect of PMA
on tyrosine phosphorylation, which confirms previous results
by Leclerc et al. (1996), seems to exclude the possibility that
PKC activation is sufficient to stimulate tyrosine kinase activity.
A similar situation has been described by Watanabe et al.
(1994) in NIH-3T3 cells, where the stimulatory effect of
prostaglandin F2α (PGF2α) on tyrosine phosphorylation,
although sensitive to both staurosporine and H7, appears to
be PKC-independent. At the moment, our results do not permit
a final conclusion to be drawn about the involvement of
PKC in progesterone-stimulated tyrosine phosphorylation of
sperm proteins.
Other possible roles for PKC in human sperm acrosomal
exocytosis have been proposed. Among these, PKC-mediated
activation of phospholipase A2 (PLA2), appears to be involved
in prostaglandin E2 (PGE2)-induced acrosome reaction
(Breitbart et al., 1995). It has been demonstrated that arachidonic acid is liberated by spermatozoa after stimulation with
progesterone (Baldi et al., 1993). Since exogenous arachidonate
stimulates acrosomal exocytosis (Breitbart et al., 1995), it is
possible that PKC inhibition blunts the effect of progesterone
on arachidonate release, resulting in inhibition of acrosome
reaction. Such inhibition, however, was not complete (Figure
1). This result is not surprising, since other kinases and other
intracellular effectors are stimulated by progesterone besides
PKC (Baldi et al., 1995). Similarly, the stimulatory effect of
other effectors on acrosome reaction is only partially reversed
by PKC inhibition (Bielfeld et al., 1994; Sengoku et al., 1996).
Acknowledgements
This work was supported by grants from Ministero dell’Università e
della Ricerca Scientifica e Tecnologica (MURST) and Regione
Toscana. Dr Bonaccorsi is recipient of a grant from Associazione
Italiana Ricerca sul Cancro (AIRC, Milano). We wish to thank Prof.
Mario Serio for helpful suggestions.
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Received on September 11, 1997; accepted on January 9, 1998