Indirect studies suggested that protein kinase C (PKC) has a role in sperm ..... Effect of protein kinase C (PKC) inhibition on progesterone (P)- and phorbol ...
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, Unita` di Andrologia, Universita` 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, 259
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.
260
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 261
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. 263
L.Bonaccorsi et al.
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’Universita` 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.
References Aitken, R.J. (1997) Molecular mechanisms regulating human sperm function. Mol. Hum. Reprod., 3, 169–173. Aitken, R.J., Buckingham, D.W. and Fang, H.G. (1993) Analysis of response of human spermatozoa to A23187 employing a novel technique for assessing acrosome reaction. J. Androl., 14, 132–141. Al-Shami, A., Bourgoin, S.G. and Naccache, P.H. (1997) Granulocyte– macrophage colony stimulating factor-activated signaling pathways in human neutrophils. I tyrosine phosphorylation-dependent stimulation of phosphatidylinositol 3-kinase and inhibition by phorbol esters. Blood, 89, 1035–1044. Arnoult, C., Lemos, J.R. and Florman, H.M. (1997) Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation. EMBO J., 16, 1593–1599. Baldi, E., Casano, R., Falsetti, C. et al. (1991) Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa. J Androl, 12, 323–330. Baldi, E., Falsetti, C., Krausz, C., et al. (1993) Stimulation of plateletactivating factor synthesis by progesterone and A23187 in human spermatozoa. Biochem J. 292, 209–216. Baldi, E., Krausz, C. and Forti, G. (1995) Nongenomic action of progesterone on human spermatozoa. Trends Endocrinol. Metab., 6, 198–205. Baldi, E., Musial, A., and Kester, M. (1994) Endothelin stimulates phosphatydylcholine hydrolisis through both PLC and PLD pathways in mesangial cells. Am. J. Physiol, 266, F957–F965. Blackmore, P.F., Beebe, J., Danforth, D.R., and Alexander, N. (1990) Progesterone and 17α-hydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J. Biol. Chem., 265, 1376–1380. Bielfeld, P.R., Anderson, A., Mack, A.R. et al. (1994) Are capacitation or calcium ion influx required for the human sperm acrosome reaction? Fertil. Steril., 62, 1255–1261. Bonaccorsi, L., Luconi, M., Forti, G., and Baldi, E. (1995) Tyrosine kinase inhibition reduces the plateu phase of the calcium increase in response to progesterone in human sperm. FEBS Lett., 364, 83–86. Bonaccorsi, L., Luconi, M., Maggi, M. et al. (1997) Protein tyrosine kinase, mitogen-activated protein kinase and protein kinase C are involved in the mitogenic signaling of platelet-activating factor (PAF) in HEC-1A cells. Biochim. Biophys. Acta, 364, 83–86. Breitbart, H. and Spungin, B. (1997) The biochemistry of the acrosome reaction. Mol. Hum. Reprod., 3, 195–202. Breitbart, H., Lax, J., Rotem, R., and Naor, Z. (1992) Role of protein kinase C in the acrosome reaction of mammalian spermatozoa. Biochem. J., 281, 473–476. Breitbart, H., Shalev, Y., Marcus, S., and Shemesh, M. (1995) Modulation of prostaglandin synthesis in mammalian sperm acrosome reaciton. Hum. Reprod., 10, 2079–2084. Burks, D.J., Carballada, R., Moore, H.D.M., and Saling, P.M. (1995) Interaction of a tyrosine kinase from human sperm with the zona pellucida at fertilization. Science, 269, 83–86.
267
L.Bonaccorsi et al. Carrera, A., Moos, J., Ning, X.P., Gerton, G.L. et al. (1996) Regulation of protein phosphorylation in human sperm by a calcium/calmodulindependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. Dev. Biol., 180, 284–296. De Jonge, C., Han, H.L., Mack, S.R. and Zaneveld, I.D. (1991) Effect of phorboldiesters synthetic diacylglycerols and a protein kinase C inhibitor on the human sperm acrosome reaction. J. Androl., 12, 62–70. Emiliozzi, C., Cordonier, H., Guerin, J.F. et al. (1996) Effects of progesterone on human spermatozoa prepared for in vitro fertilization. Int. J. Androl., 19, 39–47. Falsetti, C., Baldi, E., Krausz, C. et al. (1993) Decreased responsiveness to progesterone of spermatozoa in oligozoospermic patients. J. Androl., 14, 17–22. Foresta, C., Rossato, M. and Di Virgilio, F. (1995) Differential modulation by protein kinase C of progesterone-activated responses in human sperm. Biochem. Biophys. Res. Commun., 206, 408–413. Furuya, S., Endo, Y., Osumi, K. et al. (1993) Effects of modulators of protein kinase C on human sperm capacitation. Fertil. Steril., 59, 1285–1290. Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) A generation of Ca indicators with greatly improved fluorescence properties. J. Biol. Chem., 260, 3440–3450. Gschwendt, M., Kittstein, W., and Marks, F. (1994) Tyrosine phosphorylation and stimulation of protein kinase delta from porcine spleen by src in vitro. Dependence on the activated state of protein kinase C delta. Febs Lett., 338, 218–220. Hakeda, Y., Shiokawa M., Mano, H., et al. (1997) Prostaglandin F2α stimulates tyrosine phosphorylation and mitogen-activated protein kinase in osteoblastic MC3T3-E1 cells via protein kinase C activation. Endocrinology, 138, 1821–1828. Hashimoto, S. and Hagino, A. (1989) Staurosporine-induced neurite outgrow in PC12 cells. Exp. Cell. Res., 184, 351–359. Hedberg, K.K., Birrell, G.B., Habliston, D.L. and Griffith, O.H. (1990) Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway. Exp. Cell. Res., 188, 199–208. Hepler, J.R., Earp, H.S. and Harden, T.K. (1988) Long-term phorbol ester treatment down regulates protein kinase C and sensitize the phosphoinositide signaling pathway to hormone and growth factor stimulation. J. Biol. Chem., 263, 7610–7619. Kester, M., Simonson, M.S., McDermott, R.G. et al. (1992) Endothelin stimulates phosphatidic acid formation in cultured rat mesangial cells: role of a protein kinase C-regulated phospholipase D. J. Cell. Physiol., 150, 578–585. King, W.G. and Rittenhouse, S.E. (1989) Inhibition of protein kinase C by staurosporine promotes elevated accumulations of inositol trisphosphates and tetrakisphosphate in human platelets exposed to thrombin. J. Biol. Chem., 264, 6070–6074. Kocher, M. and Clementson, K.J. (1991) Staurosporine both activates and inhibits serine/threonine kinases in human platelets. Biochem. J., 275, 301–306. Krausz, C., Bonaccorsi, L., Luconi, M. et al. (1996) Two functional assays of sperm responsiveness to progesterone and their predictive values in invitro fertilization. Hum. Reprod. 11, 1661–1667. Lax, Y., Rubinstein, S. and Breitbart, H. (1997) Subcellular distribution of protein kinase Cα and β1 in bovine spermatozoa, and their regulation by calcium and phorbol esters. Biol. Reprod., 56, 454–459. Leclerc, P., de Lamirande, E. and Gagnon, C. (1996) Cyclic adenosine 39,59 monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol. Reprod., 55, 684–692. Luconi, M., Bonaccorsi, L., Krausz, C. et al. (1995) Stimulation of protein tyrosine phosphorylation by platelet-activating factor and progesterone in human spermatozoa. Mol. Cell. Endocrinol., 108, 35–42. Luconi, M., Bonaccorsi, L., Maggi, M. et al. (1998) Identification and characterization of functional nongenomic progesterone receptors on human sperm mebrane. J. Clin. Endocrinol. Metab., in press. Luconi, M., Krausz, Cs., Forti, G. and Baldi, E. (1996) Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa. Biol. Reprod., 55, 207–216. Meizel, S. and Turner, K.O. (1996) Chloride efflux during the progesteroneinitiated human sperm acrosome reaction is inhibited by lavendustin A, a tyrosine kinase inhibitor. J. Androl., 17, 327–330. Nishizuka, Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science, 258, 607–614. O’Toole, C.M.B., Roldan, E.R.S., and Fraser, L.R. (1996) Protein kinase C
268
activation during progesterone-stimulated acrosomal exocytosis in human spermatozoa. Mol. Hum. Reprod., 2, 921–927. Pfeilschifter, J., Ochsner, M., Whitebread, S., and De Gasparo, M. (1989) Down-regulation of protein kinase C potentiates angiotensin II-stimulated polysphoinositide hydrolysis in vascular smooth-muscle cells. Biochem. J., 262, 285–291. Roldan, E.R.S., Murase, T., and Shi, Q.-X. (1994) Exocytosis in spermatozoa in response to progesterone and zona pellucida. Science, 266, 1578–1581. Rollet E., Caon, A.C., Roberge, C.J. et al. (1994) Tyrosine phosphorylation in activated human neutrophils. J. Immunol., 153, 353–363. Rotem, R., Paz, G.F., Homonnai, Z.T. et al. (1990) Protein kinase C is present in human sperm: possible role on flagellar motility. Proc. Natl. Acad. Sci. USA, 87, 7305–7308. Rotem, R., Paz, G.F., Homonnai, Z.T. et al. (1992) Ca21-independent induction of acrosome reaction by protein kinase C in human sperm. Endocrinology, 131, 2235–2243. Sako, T., Tauber, A.I., Jeng, A.Y. et al. (1988) Contrasting actions of staurosporine, a protein kinase C inhibitor, on human neutrophils and primary mouse epidermal cells. Cancer Res., 48, 4646–4650. Sengoku, K., Tamate, K., Takuma, N. et al. (1996) Involvement of protein kinases in platelet-activating factor-induced acrosome reaction of human spermatozoa. Mol. Hum. Reprod., 2, 401–404. Sibley, D.R., Benovic, D.L., Caron, M.G., and Lefkowitz, R.F. (1987) Regulation of transmembrane signal by receptor phosphorylation. Cell, 48, 913–922. Spungin, B., and Breitbart, H. (1996) Calcium mobilization and influx during sperm exocyotsis. J. Cell Sci., 109, 1947–1955. Tesarik, J., Carreras, A. and Mendoza, C. (1996) Single cell analysis of tyrosine kinase dependent and independent Ca21 fluxes in progesterone induced acrosome reaction. Mol. Hum. Reprod., 2, 225–232. Tesarik, J., Moos, J. and Mendoza, C. (1993) Stimulation of protein tyrosine phosphorylation by a progesterone receptor on the cell surface of human sperm. Endocrinology, 133, 328–335. Thomas, P. and Meizel, S. (1989) Phosphatidylinositol 4,5-bisphosphate hydrolysis in human sperm stimulated with follicular fluid or progesterone is dependent upon Ca21 influx. Biochem. J., 264, 539–546. Thomas, P. and Meizel, S. (1988) An influx of extracellular calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid. Gamete Res., 20, 397–411. Toullec, D., Pianetti, P., Coste, H. et al. (1991) The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinse C. J. Biol. Chem., 266, 15771–15781. Watanabe, T., Nakao, A., Emarling, D. et al. (1994) Prostaglandin F2α enhances tyrosine phosphorylation and DNA synthesis through phospholipase C-coupled receptor via Ca-dependent intracellular pathway in NIH-3T3 cells. J. Biol. Chem., 269, 17619–17625. Wilkinson, S.E. and Hallam, T.J. (1994) Protein kinase C: is its pivotal role in cellular activation over stated? Trends Pharmacol. Sci., 15, 53–57. World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interactions. Cambridge University Press, Cambridge, UK. Zhang, C., Hirasawa, N. and Beaven, M.A. (1997) Antigen activation of mitogen-activated protein kinase in mast cells through protein kinase C-dependent and independent pathways. J. Immunol., 158, 4968–4975. Received on September 11, 1997; accepted on January 9, 1998
