Molecular Human Reproduction

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Molecular Human Reproduction, Vol. 8, No. 4, 326-332, April 2002
© 2002 European Society of Human Reproduction and Embryology

Testis and spermatogenesis

Inhibitors of receptor tyrosine kinases do not suppress progesterone-induced [Ca²⁺]_i signalling in human spermatozoa

J.C. Kirkman-Brown^1,3,6, L. Lefièvre², C. Bray³, P.M. Stewart⁴, C.L.R. Barratt^3,4,5 and S.J. Publicover¹

¹ School of Biosciences, University of Birmingham, ² Faculty of Medicine, McGill University, Montréal, Québec, Canada, ³ Reproductive Biology and Genetics Research Group and ⁴ Department of Medicine, The Medical School, University of Birmingham, B15 2TT and ⁵ Assisted Conception Unit, Birmingham Women's Hospital, Birmingham B15 2TG, UK

	Abstract

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Previous studies have implicated receptor tyrosine kinases in progesterone-induced [Ca²⁺]_i signalling, and consequent induction of the acrosome reaction, in human spermatozoa. We have investigated the effects of tyrosine kinase inhibition on [Ca²⁺]_i responses in large numbers of individual human spermatozoa. Genistein (5, 50 and 250 µmol/l), an inhibitor of receptor-linked tyrosine kinases, significantly inhibited the progesterone-induced acrosome reaction (P < 0.05). However, we could detect no effect of genistein on progesterone-induced [Ca²⁺]_i signalling. In control experiments, application of progesterone induced a significant transient [Ca²⁺]_i response in ~77% of cells and a sustained [Ca²⁺]_i ramp/plateau in ~48% of cells (n = 26; 5411 cells). In preparations pretreated with genistein (50 µmol/l), significant transient and sustained responses were detected in 69.5 and 39.1% of cells respectively (n = 5; 1109 cells). The amplitudes of both transient and sustained [Ca²⁺]_i responses were similar in control and genistein-pretreated preparations. Tyrphostin A47 (100 µmol/l), another receptor tyrosine kinase inhibitor, also failed to inhibit either the transient or sustained [Ca²⁺]_i response (n = 3; 468 cells). Assessment of tyrosine phosphorylation of two sperm proteins (p105/81) showed greatly increased levels of phosphotyrosine in response to capacitation but a negligible increase in response to progesterone stimulation. Pretreatment with genistein (50 and 250 µmol/l) decreased capacitation-induced tyrosine phosphorylation and resulted in a loss of phosphorylation in response to progesterone treatment. We conclude that neither the transient nor sustained phases of the progesterone-induced [Ca²⁺]_i response require receptor tyrosine kinase signalling. Previous reports of modulation of the progesterone-induced [Ca²⁺]_i signal by tyrosine kinase inhibition probably reflect inhibition of the acrosome reaction.

Ca²⁺/genistein/human spermatozoa/progesterone/tyrosine kinase

	Introduction

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
One of the best known examples of the non-genomic actions of steroid hormones is the induction of Ca²⁺ influx and the acrosome reaction (AR) of mammalian spermatozoa following exposure to micromolar concentrations of progesterone in vitro. Since progesterone is present in the cumulus oophorus at similar concentrations (Osman et al., 1989), this action of the hormone is likely to be of significance during fertilization in vivo (Fisher et al., 1998; Garcia and Meizel, 1999). Fluorimetric records of the responses [Ca²⁺]_i of human spermatozoa to progesterone are biphasic, consisting of a transient phase that lasts 1–3 min, followed by a sustained ramp or plateau which is activated soon after the transient peak (Blackmore et al., 1990; Yang et al., 1994; Bonnacorsi et al., 1995; Garcia and Meizel, 1996). We have recently shown that this biphasic pattern can also be detected in the posterior head of individually imaged human spermatozoa (Kirkman-Brown et al., 2000).

Inhibition of receptor tyrosine kinases with genistein or herbimycin A has been reported to inhibit both the sustained phase of the [Ca²⁺]_i response (but not the initial transient phase) (Bonnacorsi et al., 1995; Mendoza et al., 1995; Tesarik et al., 1996) and the induction of the AR (Tesarik et al., 1993; Luconi et al., 1995). These findings suggest that the sustained [Ca²⁺]_i response is tyrosine kinase-dependent and is vital to successful activation of the AR by progesterone. Moreover, it is well established that an increase in protein tyrosine phosphorylation is associated with sperm capacitation in different mammalian species (Visconti et al., 1995; Galantino-Homer et al., 1997), including the human (Leclerc et al., 1996). However, the stimulation of protein tyrosine phosphorylation by progesterone during the AR is controversial. Some studies have shown a progesterone-associated increase (Tesarik et al., 1993; Luconi et al., 1995), while others show no differences in the levels of human sperm tyrosine phosphorylation (Aitken et al., 1996). We have therefore undertaken single-cell imaging to determine whether inhibition of the sustained phase of the [Ca²⁺]_i response to progesterone by receptor tyrosine kinase inhibitors can also be detected in individual cells, and whether this effect reflects modulation of the proportion of responsive cells and/or the amplitude of single cell responses. We have also investigated the effect of genistein on protein tyrosine phosphorylation during capacitation and the AR, induced by progesterone or ionophore A23187.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Preparation and capacitation of spermatozoa
All donors were recruited at the Birmingham Women's Hospital (Human Fertilisation and Embryology Authority centre number 0119), in accordance with the Human Fertilisation and Embryology Authority Code of Practice. Human ejaculated spermatozoa were obtained by masturbation from normal healthy donors of proven fertility. After semen liquefaction (~30 min), motile spermatozoa were harvested by swim-up (Mortimer, 1994). Briefly, 2 ml of supplemented Earle's balanced salt solution without phenol red (sEBSS; Gibco BRL, Paisley, Scotland; special order No: 041–94189H) + 0.3% bovine serum albumin (BSA; Pentex fraction V, pH 7; Pentex, Newbury, UK) was under-layered with 1 ml of liquefied semen in a 15 ml Blue Max 2095 tube (Becton Dickinson, Franklin Lakes, New Jersey, USA). The tube was then incubated at an angle of 45° for 1 h at 37°C and 5% CO₂. After 1 h the upper 1.75 ml of medium (containing the motile fraction of spermatozoa) was carefully removed using a sterile transfer pipette. The concentration of the collected spermatozoa was assessed using a Neubauer counting chamber according to World Health Organization methods (World Health Organization, 1999) and adjusted to 6x10⁶ cells per ml with sEBSS + 0.3% BSA. Aliquots of spermatozoa were capacitated (100 µl aliquots for AR experiments; 200 µl aliquots for confocal microscopy studies of calcium influx) for 6 h at 37°C, 5% CO₂.

Confocal imaging
Loading of cells with Calcium Green 1 (Molecular Probes, Cambridge Bioscience, Cambridge, UK) and confocal imaging were carried out as described previously (Kirkman-Brown et al., 2000).

All experiments consisted of a 5 min control period during which the imaging chamber was perfused with sEBSS followed by perfusion of the chamber with sEBSS containing 3.2 µmol/l progesterone (4-pregnene-3,20-dione; Sigma, Poole, UK). Images were captured at intervals of 15 s. Criteria for acceptance/rejection of data for analysis were as described previously (Kirkman-Brown et al., 2000). All data described in the results section are from experiments selected on these criteria. For experiments using receptor tyrosine kinase inhibitors (genistein and typhostin A47; Calbiochem, Nottingham, UK), these were prepared as concentrated stock solutions in phosphate-buffered saline (PBS) and were normally added at the same time as Calcium Green-1 (1 h before application of progesterone). In a few experiments, receptor tyrosine kinase inhibitors were added 10 min prior to exposure to progesterone. Inhibitors were also included, at the appropriate concentration, in the media used for superfusion during recording.

Confocal data processing and analysis
Data was processed offline using Lucida software (Kinetic Imaging Ltd, UK) as described previously (Kirkman-Brown et al., 2000).

Raw intensity values were imported into Microsoft Excel and normalized using the equation R = [(F – F_rest )/F_rest ]x100%, where R is normalized fluorescence intensity, F is fluorescence intensity at time t and F_rest is the mean of at least 10 determinations of F taken during the control period.

At each time point, the normalized fluorescence intensity values (R) for each cell were compiled to generate the mean normalized head fluorescence (R_tot). The total series of R_tot were then plotted to give the mean normalized response of head fluorescent intensity for that experiment. For each cell, Excel was used to calculate the mean and 95% confidence interval of fluorescent intensity for (i) at least 10 images during the control period (C ± c); (ii) the four images spanning the time for the peak of the transient response [as assessed from R_tot for that experiment (T ± t)] and (iii) 12 images collected during the period 15–18 min after progesterone application [the sustained response (S ± s)]. The transient response was considered significant if T–t > C+c. The sustained responses was considered significant if S–s > C+c. Visual inspection of fluorescence–time plots for the cells in each of the categories confirmed that this technique resulted in successful sorting of the different response categories.

Frequency of occurrence of transient and sustained responses in control and pretreated preparations was compared, using Student's t-test, on arcsine-transformed data.

Response amplitude distributions for transient and sustained responses to progesterone were constructed using the single cell values T and S, calculated as described above. For every experiment the amplitude distributions for significant transient responses and significant sustained responses were obtained and then normalized (frequency in each amplitude class expressed as a proportion of all cells). Average distributions for control (23 experiments) and genistein-pretreatment (five experiments) were then obtained by plotting the mean normalized frequency ± SEM for each amplitude class.

Assessment of progesterone-induced AR
After capacitation, as described above, 100 µl aliquots of spermatozoa were stimulated with either 3.2 µmol/l progesterone, 10 µmol/l A23187 (Sigma) or solvent [0.05% dimethylsulphoxide (DMSO); Sigma). For experiments on the effect of receptor tyrosine kinase inhibitors on progesterone- or A23187-induced AR, drugs were added after 5 h capacitation, 60 min before addition of the agonist (as in the [Ca²⁺]_i-imaging studies). After addition of the agonist, cells were incubated for a further 60 min. At the end of the incubation period, spermatozoa were centrifuged briefly (300 g for 5 min), the supernatant removed and the spermatozoa resuspended in 0.5 ml of hypo-osmotic swelling (HOS) medium (0.74% sodium citrate, 1.35% fructose in double-distilled H₂O). After 45 min incubation in HOS media, the spermatozoa were centrifuged (300 g for 5 min) and re-suspended in a minimal volume of HOS media (20 µl), smeared on microscope slides (duplicate slides, previously coated with 10% poly-l-lysine solution) and air-dried.

Following permeabilization by immersion in methanol (2 min), cells were labelled with fluorescein isothiocyanate–Pisum sativum agglutinin (PSA) (50 µg/ml; Sigma) in PBS for 45 min in a moisture chamber at 37°C. Slides were then washed in a constant flow of mains (tap) water for 15 min before air drying and mounting with Fluoromount (BDH Merck, Poole, UK). Slides were stored refrigerated (4°C) and kept in darkness to prevent fading.

Fluorescence microscopy was used to evaluate acrosomal status; slides were scored blind and only viable (curly-tailed) spermatozoa were scored (Aitken et al., 1993). Acrosomal status was assessed as described elsewhere (Mendoza et al., 1992). A total of 200 spermatozoa were scored per treatment (100 per slide).

All calculations and statistical analysis were performed using the statistics module of Microsoft Excel for Macintosh. An arcsine transformation of AR percentage data was performed prior to testing for significance between treatment groups. Paired t-tests (two-tailed) were performed to test for significance. Statistical significance was set at P < 0.05.

Detection of tyrosine phosphorylation
Sperm treatments were carried out as for the AR protocol. Treatments were stopped by the addition of solubilization buffer [2% sodium dodecyl sulphate (SDS); 10% glycerol; 1.4% dithiothreitol; 62.5 mmol/l Tris–HCl, pH 6.8, 0.1 mmol/l vanadate]. Samples were heated at 100°C for 5 min and centrifuged at 12 000 g for 5 min. Proteins were separated by electrophoresis on SDS–polyacrylamide (10%) gels (Laemmli, 1970) and electrotransferred (Towbin et al., 1979) onto nitrocellulose membrane. Non-specific binding sites on the nitrocellulose membrane were blocked with 5% (w/v) dried skimmed milk in Tris-buffered saline (0.9% NaCl, 20 mmol/l Tris–HCl, pH 7.8) supplemented with Tween-20 (0.1%, v/v). The nitrocellulose membrane (0.22 µm pore size; Micron Separations Inc., Westboro, MA, USA) was incubated for 1 h at room temperature with the antiphosphotyrosine antibody (clone 4G10; Upstate Biotechnology Inc., Lake Placid, NY, USA), extensively washed in TTBS, incubated with goat anti-mouse immunoglobulin G conjugated with horseradish peroxidase (Gibco BRL, Burlington, ON, Canada) for 30 min, and again washed extensively with TTBS. Positive immunoreactive bands were detected by chemiluminescence using an enhanced chemiluminescence kit according to the manufacturer's instructions (Lumi-Light; Roche Molecular Biochemicals, Laval, PQ, Canada). Silver staining of the proteins transferred on the nitrocellulose membrane was performed (Jacobson and Karsnäs, 1990) after the detection of the tyrosine phosphorylated bands to confirm that the transferred protein patterns were similar for all samples. All other reagents employed in Western blotting were purchased from the Sigma Chemical Company.

	Results

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Genistein and progesterone-induced AR
Spontaneous AR occurred in 13.9 ± 5.7% (n = 4) of spermatozoa. Treatment with 3.2 µmol/l progesterone increased the rate of AR to 23.9 ± 7.0% (P < 0.05, paired t-test; n = 4). The vehicle for progesterone (0.05% DMSO) had no stimulatory effect. When cells were exposed to progesterone after pretreatment with genistein (5 or 50 µmol/l), the rate of AR was similar to the spontaneous rate (12.6 ± 5.8% and 13.9 ± 5.7% respectively; n = 4; Figure 1) and was significantly lower than in samples stimulated with progesterone in the absence of pre-treatment (P < 0.05, P < 0.03 for 5 and 50 µmol/l respectively; paired t-tests). In cells pretreated with 250 µmol/l genistein before exposure to progesterone the rate of AR was 16.3 ± 6.7%, not significantly different to non-pretreated preparations. Genistein treatment had no significant effect upon ionophore-induced AR rate (Figure 1).

View larger version (39K): [in this window] [in a new window]   Figure 1. Effect of genistein on progesterone-induced acrosome reaction. Data are mean ± SEM of four experiments. Bars above the Figure show the agonist [SC (solvent control – 0.05% DMSO); control (no agonist); 3.2 µmol/l progesterone; 10 µmol/l A23187] and labels below bars show the genistein pretreatment, applied 1 h before the agonist. *Significantly different from no agonist and solvent controls, Significantly different from effect of progesterone with no pretreatment.

 
[Ca²⁺]_i responses to progesterone
As described previously (Kirkman-Brown et al., 2000), the response of calcium green 1-loaded human spermatozoa to progesterone (3.2 µmol/l) was an increase in fluorescence localized primarily in the posterior head. The [Ca²⁺]_i signal was composed of an initial transient elevation, which typically peaked within 1 min of progesterone application and decayed with a similar time course, and a sustained plateau or ramp, which became discernible during the falling phase of the transient response and persisted for at least 20 min. Both components were discernible in the responses of many individual cells to progesterone (Figure 2a inset). When the normalized data from all the cells imaged in an experiment were averaged (R_tot), we obtained smooth, biphasic responses to progesterone, similar to those seen in fluorimetric recordings (Kirkman-Brown et al., 2000; Figure 2a). Analysis of single cell records showed that 77.2 ± 2.5% of cells generated a significant transient increase in fluorescence and 49.9 ± 3.4% generated a significant sustained response (n = 23). The sustained response was strongly associated with the initial, transient response. Very few (3.8 ± 0.7%, n = 23) spermatozoa generated a sustained response that was not preceded by a significant transient response. Amplitude distributions for both transient and sustained responses showed a left skew (Figure 3).

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of genistein on the characteristics of progesterone-induced [Ca2+]i responses. (a) Control responses. (b) Responses of cells pretreated with 50 µmol/l genistein. In both panels the main graph shows the mean normalized response for all cells in one experiment (Rtot; control = 114 cells, genistein pretreated = 98 cells) and the inset shows the response of an individual cell from that experiment. Axes in inset are as for main graph. The population and single cell responses are clearly biphasic in both the control and genistein-pretreated cells.

 

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of genistein on amplitudes of progesterone-induced [Ca2+]i responses. (a) Mean normalized amplitude distributions (± SEM) for transient responses in control experiments (; 23 experiments) and genistein-pretreatment experiments (; five experiments). (b) Mean normalized amplitude distributions (± SEM) for sustained responses in control experiments (; 23 experiments) and genistein-pretreatment experiments (; five experiments). For both transient and sustained responses, the modal amplitude class and the shape of the amplitude distribution were unchanged.

 
Effect of genistein on the progesterone-induced [Ca²⁺]_i responses
Pretreatment with 50 µmol/l genistein for 60 min prior to progesterone addition produced the most significant inhibition of progesterone-induced AR (see above) and this protocol was therefore adopted for investigation of the effect of genistein on progesterone-induced [Ca²⁺]_i responses. Upon addition of progesterone (3.2 µmol/l) to the superfusing medium, we observed a biphasic increase in fluorescence, similar to that seen in control preparations. Both phases of the response were clearly discernible in single cell records. When the population response (R_tot) was derived by averaging the normalized responses of all spermatozoa in the field of view, smooth, biphasic responses were obtained (Figure 2b). Similar data were obtained in four other experiments (see below and Table I). In two experiments where cells were pretreated with 50 µmol/l genistein for just 10 min before application of progesterone, there was a similar lack of effect.

View this table: [in this window] [in a new window]   Table I. Effect of tyrosine kinase inhibitors on rate of occurrence of transient and sustained responses

 
Occurrence and characteristics of transient responses
As in control experiments, the majority of spermatozoa which had been pretreated with genistein for 60 min showed a significant, `transient' increase in [Ca²⁺]_i. In the five experiments that were suitable for detailed analysis, the proportion of cells showing the transient response was 69.5 ± 2.6% (Table I), a value similar to that in the control preparations (P > 0.10, t-test on arcsine transformed percentages). Analysis of the amplitudes of transient responses showed that the modal amplitudes and amplitude distributions for control and genistein-pretreated preparations were similar (Figure 3a).

Sustained responses
After pretreatment with genistein for 60 min, 39.1 ± 5.0% of cells (n = 5; Table I) showed a significant sustained rise in [Ca²⁺]_i, a response that was not significantly different from that in control preparations (P > 0.15, t-test on arcsine transformed percentages). The modal amplitudes and amplitude distributions for significant sustained responses (measured 15–18 min after progesterone application) were similar in control and genistein-pretreated preparations (Figure 3b).

Tyrphostin A47 and progesterone-induced [Ca²⁺]_i responses
Since we detected no inhibitory effect of genistein on the progesterone-induced [Ca²⁺]_i signal, we looked at the effect of another inhibitor of receptor tyrosine kinases, tyrphostin A47 (100 µmol/l). Cells were pretreated for 60 min prior to progesterone addition. The proportion of cells showing significant transient responses was 65.9 ± 4.7% (n = 3; Table I), a response that was similar to that in control experiments (P > 0.15, t-test on arcsine transformed percentages). Significant, progesterone-induced, sustained [Ca²⁺]_i responses were seen in 65.0 ± 10.0% of spermatozoa (n = 3; P > 0.10 with respect to control, t-test on arcsine transformed percentages; Table I). The population response (R_tot) of all three experiments, derived by averaging the normalized responses of all cells in the field of view, was a smooth, biphasic response, similar to those seen in controls and in genistein-pretreated cells.

Western blot analysis of tyrosine phosphorylation
Sperm protein tyrosine phosphorylation is regulated by the cAMP/protein kinase A (PKA) pathway (Visconti et al., 1995; Leclerc et al., 1996) and the phosphotyrosine content of sperm proteins is increased both by dbcAMP, a membrane permeable cAMP analogue, and by 3-isobutyl-1-methylxanthine (IBMX), a non-selective cyclic nucleotide phosphodiesterase inhibitor (Visconti et al., 1995; Leclerc et al., 1996). We therefore employed IBMX as a positive control. After treatment with IBMX (500 µmol/l) for 7 h, the two major tyrosine phosphorylated proteins observed were of 105 and 81 kDa (p105/81), as described previously (Leclerc et al., 1996; Lefièvre et al., 2000; Figure 4, lane 2).

View larger version (29K): [in this window] [in a new window]   Figure 4. Effect of genistein on tyrosine phosphorylation during capacitation and acrosome reaction induced by progesterone or ionophore A23187. Lane 1 shows negligible phosphorylation of proteins from spermatozoa solubilized immediately after swim-up (negative control). Lane 2 shows tyrosine phosphorylation in spermatozoa incubated with 500 µmol/l IBMX (positive control) for 7 h. Lanes 3–11: spermatozoa were incubated for 5 h in sEBSS + 0.3% BSA at 37°C, 5% CO2. Cells were then treated with 50 µmol/l genistein (lanes 5, 8, 11), 250 µmol/l genistein (lanes 4, 7, 10) or vehicle (lanes 3, 6 and 9) for 1 h, after which progesterone (lanes 6–8), ionophore A23187 (lanes 9–11) or vehicle (lanes 2–5) was added and incubation was continued for an additional hour (total incubation time 7 h for all treatments).

 
The levels of tyrosine phosphorylation of p105/81 observed in spermatozoa incubated for 7 h in sEBSS + 0.3% BSA (capacitating conditions) were markedly higher than those in samples solubilized immediately after swim-up (Figure 4, lanes 1 and 3). Treatment with ionophore, during the final 60 min of incubation, clearly enhanced protein tyrosine phosphorylation above control levels (particularly p81), but the effect of progesterone was minimal (Figure 4, lanes 3, 6 and 9). The effects of treatment with genistein at 50 or 250 µmol/l were dose-dependent. Application of 50 µmol/l genistein, after 5 h, only slightly reduced the tyrosine phosphorylation that occurred during capacitation (Figure 4, lanes 3 and 5) and had little, if any, discernible effect on the level of tyrosine phosphorylation in cells treated with A23187 for the final 60 min of incubation (Figure 4, lanes 9 and 11). However, in preparations treated with progesterone in the presence of 50 µmol/l genistein, tyrosine phosphorylation (particularly of p81) was lower than that seen in preparations treated with 50 µmol/l genistein alone (Figure 4, lanes 5 and 8). Addition of 250 µmol/l genistein after 5 h of capacitation clearly reduced the levels of tyrosine phosphorylation compared with parallel controls (Figure 4, lanes 3 and 4). When cells were treated with ionophore or progesterone in the presence of 250 µmol/l genistein, not only was any agonist-induced tyrosine phosphorylation inhibited, but the overall tyrosine phosphorylation was reduced to levels lower than those seen in preparations which were treated with genistein without the addition of an agonist. This effect was particularly striking with progesterone (Figure 4, lanes 4 and 7).

The experiment shown in Figure 4 was carried out on three occasions, the results being qualitatively similar in each case.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Our finding that pre-incubation with genistein inhibited progesterone-induced AR in human spermatozoa is consistent with previous reports (Tesarik et al., 1993; Luconi et al., 1995) and confirms the importance of tyrosine kinase activity in this vital process. Treatment of cells with genistein (50 and 250 µmol/l) for the final 2 h of incubation resulted in a clearly dose-dependent inhibition of the p105/81 phosphorylation that occurred during capacitation. A similar inhibition, by genistein, of capacitation-dependent tyrosine phosphorylation has been reported previously (Aitken et al., 1996). We could detect little, if any, progesterone-induced enhancement of phosphotyrosine levels and stimulation with progesterone during inhibition of receptor tyrosine kinases, by 50 or 250 µmol/l genistein, resulted in a reduction in levels of tyrosine phosphorylation. A failure of progesterone to increase tyrosine phosphorylation has been reported previously (Aitken et al., 1996). These authors further observed that, in the presence of the tyrosine phosphatase inhibitor vanadate, some stimulatory effect of progesterone on phosphotyrosine levels could be observed. Orthovanadate was present in the in-vitro kinase assay used by Tesarik et al. to investigate progesterone-induced tyrosine phosphorylation (Tesarik et al., 1993) and in some of the experiments described by Luconi et al. where progesterone-induced tyrosine phosphorylation was reported (e.g. Figure 3) (Luconi et al., 1995). We speculate that progesterone treatment may stimulate activity of both tyrosine kinases and tyrosine phosphatases, such that inhibition of kinases by genistein may shift the balance of activity leading to detectable dephosphorylation of some proteins and inhibition of phosphatases by vanadate may reveal kinase activity. Tyrosine kinase inhibition may, therefore, inhibit the AR both by effects on capacitation-related tyrosine phosphorylation (Aitken et al., 1996; Visconti and Kopf, 1998) and also by a direct effect on events occurring in response to progesterone (Tesarik et al., 1993; Luconi et al., 1995).

Data from previous studies suggest that an important effect of tyrosine kinase inhibitors on progesterone-induced AR is suppression of the sustained phase of Ca²⁺ influx. Bonnacorsi et al. using fluorimetric measurement of [Ca²⁺]_i in human spermatozoa, observed a reduction in the amplitude of the sustained response to progesterone of ~90% in preparations pretreated with 1–10 µmol/l genistein (Bonnacorsi et al., 1995). A smaller (25–30%) inhibition was seen in preparations pretreated with 30 µmol/l herbimycin A. Tesarik et al. imaged fluo-3-labelled human spermatozoa and reported a complex response to progesterone composed of the initial, characteristic [Ca²⁺]_i transient followed by a second, discrete peak which occurred 2–10min after progesterone application (Tesarik et al., 1996). The second of these responses was strongly inhibited by 370 µmol/l genistein or 30 µmol/l herbimycin A. However, we found that pretreatment with genistein or tyrphostin A47 (both potent broad range receptor tyrosine kinase inhibitors) (Akiyama et al., 1987; O'Dell et al., 1991; Levitzki and Gazit, 1995) at doses at the top of their usable range, had no significant effect on the [Ca²⁺]_i signal. The amplitude of single cell responses was unaffected (Figure 3) and the proportion of responsive cells, though slightly decreased by genistein (Table I), was not significantly different (P > 0.15). Though the small reduction in the proportion of responsive cells might become discernible in population measurements made by fluorimetry, our data are not consistent with the strong inhibitory effect described previously.

A plausible explanation for the differences between our findings and those outlined above is that, in other studies, progesterone-induced AR contributed to the `[Ca²⁺]_i response'. Inhibition of the AR by inhibitors of receptor tyrosine kinases could then result in an apparent inhibition of late [Ca²⁺]_i signalling events. It has been argued previously that the sustained phase of the progesterone-induced [Ca²⁺]_i response (measured fluorimetrically) is an artefact caused by escape of fluorochrome into the extracellular medium upon the AR (Garcia and Meizel, 1996, 1999). We have demonstrated, using single cell imaging, that a genuine sustained [Ca²⁺]_i response is induced by progesterone treatment of human spermatozoa (Kirkman-Brown et al., 2000). However, it is likely that fluorimetric recordings include an artefactual component caused by escape of the fluorochrome. The apparent inhibitory effect of receptor tyrosine kinase inhibitors on the sustained component of the [Ca²⁺]_i signal reported by Bonnacorsi et al. might thus reflect an effect on the AR (Bonnacorsi et al., 1995). In the single cell imaging study by Tesarik et al. fluo-3 fluorescence was distributed throughout the sperm heads, indicating significant loading of the fluorochrome into the acrosome (Tesarik et al., 1996). The secondary response to progesterone, which was genistein- and herbimycin A-sensitive, was not the ramp/plateau which has been observed in other studies, but took the form of a discrete peak of variable latency and duration, which ended abruptly with fluorescence then falling below the initial resting level. This loss of fluorescence was interpreted as loss of acrosomal fluo-3 during the AR (Tesarik et al., 1996). In zona-pellucida (ZP)-stimulated hamster spermatozoa, where fluorescence from the acrosome and post-acrosomal regions can be clearly distinguished, a rapid loss of fluorescence from the acrosome is sometimes seen and has been interpreted similarly. The cytoplasmic [Ca²⁺]_i signal, from the post-acrosomal region, is not affected (Shirakawa and Miyazaki, 1999). It is conceivable that fluo-3 labelling of the acrosome resulted in monitoring of a tyrosine kinase-regulated, intra-acrosomal [Ca²⁺] response that was not detected in this study. However, the simplest interpretation of these data is that the second progesterone-induced fluorescence peak observed by Tesarik et al. reflects influx of extracellular Ca²⁺ into the fluo-3 loaded acrosome during initial fusion events prior to the AR (Tesarik et al., 1996). The inhibitory effects of genistein and herbimycin A on this response thus reflect inhibition of progesterone-induced AR.

If the effects of receptor tyrosine kinase inhibitors on the AR do not reflect suppression of Ca²⁺ influx then the effect is exerted in another way, probably at a site that is of importance for events downstream of Ca²⁺ mobilization. Such an effect could occur during capacitation (Aitken et al., 1996; Visconti and Kopf, 1998; see above), only becoming apparent upon activation of stimulus-secretion coupling, or could occur downstream of the progesterone-induced Ca²⁺ influx. It has been suggested that progesterone stimulation of tyrosine phosphorylation acts via two non-genomic mechanisms, one of which is dependent upon the presence of Ca²⁺ in the medium and therefore is presumably such a downstream event (Martinez et al., 1999).

	Acknowledgements

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
This work was supported by grants from BBSRC (J.K.-B./S.P.) and NHS (programme Grant #0205 to C.L.R.B.). Our thanks to the laboratory staff of the ACU for assistance with donor recruitment and support.

	Notes

 
⁶ To whom correspondence should be addressed at: School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK. E-mail: j.kirkmanbrown{at}bham.ac.uk

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Submitted on December 29, 2000; resubmitted on October 15, 2001; accepted on December 20, 2001.

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