Mol. Hum. Reprod. Advance Access originally published online on September 28, 2005
Molecular Human Reproduction 2005 11(9):683-691; doi:10.1093/molehr/gah226
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Bicarbonate and bovine serum albumin reversibly ‘switch’ capacitation-induced events in human spermatozoa
1School of Biosciences, 2Reproductive Biology and Genetics Research Group, The Medical School, University of Birmingham and 3Assisted Conception Unit, Birmingham Women’s Hospital, Birmingham, UK
4 To whom correspondence should be addressed at: School of Biosciences, University of Birmingham, UK. E-mail: s.j.publicover{at}bham.ac.uk
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Abstract |
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We have investigated the reversibility of biochemical and physiological changes that occur upon suspension of ejaculated human spermatozoa during in vitro capacitation. Cells were swum up in a simple HEPES-based saline [lacking bicarbonate and bovine serum albumin (BSA)], then resuspended either in supplemented Earle’s balanced salt solution (sEBSS) (25 mM bicarbonate) with 0.3% BSA (for in vitro capacitation) or in medium-lacking bicarbonate and/or BSA. Progesterone-induced acrosome reaction (AR) developed during in vitro capacitation (6 h). A progesterone-induced [Ca2+]i signal was detectable in cells maintained in the simple HEPES-based saline, but upon transfer to sEBSS, the response increased three- to four-fold, saturating within <30 min. Serine/threonine phosphorylation saturated within minutes of resuspension, but tyrosine phosphorylation developed over 3 h. Return of cells to non-capacitating conditions caused reversal of all capacitation-dependent changes. The [Ca2+]i signal reverted to its ‘uncapacitated’ size within <30 min. Protein phosphorylation reversed gradually and could be reinduced (kinetics resembling the first response) upon resuspension in sEBSS. The ability of cells to undergo progesterone-induced AR fell to levels similar to those in uncapacitated cells within 1 h of resuspension in medium not supporting capacitation. Loss of protein phosphorylation occurred only in the absence of both bicarbonate and BSA, but effects on [Ca2+]i signalling and AR could be seen after removal of only one of these factors. We conclude that key events in the capacitation of human spermatozoa are both reversible and repeatable.
Key words: calcium/cAMP/capacitation/sperm/phosphorylation
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Introduction |
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Freshly ejaculated mammalian sperm are not immediately capable of achieving fertilization (Yanagimachi, 1994
). During residence in the female tract, the cell undergoes a complex and poorly understood set of modifications which confer fertilization competence, a process collectively called capacitation (Baldi et al., 1996). Capacitation can be induced in vitro by incubation under appropriate conditions. The development of protocols for sperm capacitation in vitro has shown that there is a critical requirement for Ca2+, bicarbonate and a protein that can function as a cholesterol acceptor, usually a serum albumin (Visconti et al., 2002). The most widely accepted functional definition of capacitation is acquisition of ability to undergo acrosome reaction (AR) in response to a biological agonist (such as zona pellucida or progesterone). The events that must occur for successful induction of AR include agonist binding, activation of [Ca2+]i signalling (and further downstream signals) and ultimately fusion of the outer acrosomal membrane and plasmalemma.
Many changes have been observed in the biochemistry and physiology of cells exposed to capacitating conditions in vitro, which provide useful indicators of the occurrence of capacitation. These include an efflux of plasma membrane cholesterol, an increase in the activity of adenylate cyclase, both soluble and membrane localized, elevated levels of [cAMP] and Protein Kinase A (PKA) activity, a rise in pHi, hyperpolarization of membrane potential and increased serine/threonine and tyrosine phosphorylation of some proteins (Tash and Means, 1983; Leclerc et al., 1996; Cross, 1998; Osheroff et al., 1999; Lefièvre et al., 2002; Visconti et al., 2002; O’Flaherty et al., 2004; Fraser et al., 2005; Moseley et al., in press). However, it is still far from clear how these events relate to each other or whether all of them must occur for acquisition of fertilization competence. For instance, it has been shown recently that, in bovine spermatozoa, hyperactivation is dependent primarily upon [Ca2+]i and can occur separately to tyrosine phosphorylation (Marquez and Suarez, 2004) which is generally regarded as a good indicator of ability to undergo AR. Furthermore, though sperm probably possess the ability to regulate signalling pathways involved in capacitation, thus minimizing over-capacitation and premature AR (Fraser et al., 2005), it is not known to what extent the changes that have been observed in capacitating cells are reversible and whether they are capacitation endpoints or part of the capacitation process (such that reversal does not prevent subsequent induction of AR). It has recently been suggested that capacitation prepares the acrosome for fusion with the plasmalemma in a manner analogous to the docking process that occurs before Ca2+-mediated fusion of exocytotic vesicles (Tulsiani and Abou-Haila, 2004). In secretory systems, priming may take fusion to an ‘irreversible’ stage, such that Ca2+ is required only to remove the ‘brake’ on fusion (Sudhof, 2004).
We have assessed progesterone-induced [Ca2+]i signalling and protein serine/threonine and tyrosine phosphorylation in capacitating cells to determine (i) their reversibility upon transfer of cells to medium that does not support capacitation and (ii) the effect of such reversal on the competence of cells to undergo AR upon subsequent challenge with progesterone. We report that serine/threonine phosphorylation and the ability to generate a Ca2+ signal in response to progesterone challenge saturates very rapidly after swim up into capacitating medium, but tyrosine phosphorylation and progesterone-induced AR develop much more slowly. All of these events, and the ability of cells to undergo AR, are reversed upon resuspension in non-capacitating medium (NCM) Ca2+ signalling, and AR being particularly sensitive to removal of bicarbonate.
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Materials and methods |
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Materials
Progesterone [4-pregnene-3, 20-dione], salts for the preparation of Earle’s balanced salt solution (EBSS), digitonin, EGTA, A23187 [GenBank] , dimethylsulphoxide (DMSO), lectin from Pisum Sativum [fluorescein isothiocyanate (FITC) labelled], HEPES were all obtained from Sigma-Aldrich, Poole, UK; Fura-2 AM from Molecular Probes (Cambridge Bioscience, Cambridge, UK); and bovine serum albumin (BSA) from JRH Biosciences, Andover, UK.
Serine–threonine phosphatase inhibitors calyculin A and okadaic acid were distributed by cell-signalling technology and Calbiochem (Merck Biosciences, Beeston, Nottingham, UK), respectively. Phospho-(Ser/Thr) PKA substrate antibody and anti-phosphotyrosine, recombinant 4G10, were obtained from New England Biolabs (Hitchin, Hertfordshire, UK) and Upstate (Dundee Technology Park, Dundee, UK), respectively. Secondary antibodies conjugated with horseradish peroxidase were purchased from Jackson Immuno Research Laboratories (distributed by Stratech Scientific, Soham, Cambs, UK) and LumiGLO, an enhanced chemiluminescence kit, by Insight Biotechnology, Wembley, Middlesex, UK. All other chemicals were purchased from Sigma.
Sperm preparation/capacitation
All donors were recruited at the Birmingham Women’s Hospital (HFEA centre number 0119), in accordance with the Human Embryology Authority Code of Practice. Human ejaculated spermatozoa were obtained from normal healthy donors of proven fertility by masturbation. After semen liquefaction (approximately 30 min), motile spermatozoa were harvested by swim up (Mortimer, 1994) in either sEBSS or NCM (see below). Briefly 1 ml of sEBSS or NCM was underlayed with 0.3 ml of liquefied semen in Falcon 2054 tubes. The tubes were then incubated for 1 h at 37°C, 5% CO2. After 1 h, the upper 0.7 ml of the medium (containing the motile fraction of spermatozoa) of all the tubes was collected into a 15 ml Blue max tube (Becton Dickinson, Franklin Lakes, NJ, USA) using a sterile transfer pipette. The concentration of the collected spermatozoa was assessed using a Neubauer counting chamber according to the World Health Organization (WHO) methods (World Health Organization, 1999) and adjusted to 6 x 106 cells per ml with the appropriate medium. About 2 ml aliquots of sperm suspension were made. Cells were spun at 500 g. Some of the aliquots were used immediately after swim up, whereas others were incubated for at least 6 h at 37°C, 5% CO2.
Salines/media
The standard suspension medium was sEBSS, a capacitating medium containing NaCl (116.4 mM), KCl (5.4 mM), CaCl2 (1.8 mM), MgCl2 (1 mM), glucose (5.5 mM), NaHCO3 (25 mM), Na pyruvate (2.5 mM), Na lactate (19 mM), MgSO4 (0.81 mM) and 0.3% BSA, pH 7.4. sEBSS was modified to minimize capacitation by replacement of NaHCO3 with 10 mM HEPES and NaCl (sEBSS–bicarb), omitting BSA (sEBSS–BSA) or both replacement of NaHCO3 and omission of BSA (NCsEBSS). NCM, a simple HEPES-buffered, non-capacitating saline-contained NaCl (150 mM), KCl (5 mM), CaCl2 (2 mM), MgCl2 (1 mM), glucose (10 mM) and HEPES (10 mM) with a pH 7.4.
Fluorimetry
About 2 ml aliquots of uncapacitated and capacitated sperm suspensions were prepared for [Ca2+]i determination by labelling with acetoxy–methyl ester of fura-2 (1 µM final extracellular concentration) for 12 min at 37°C, 5% CO2. After dye loading, each 2-ml sample was centrifuged at 500 g for 5 min. The supernatant was discarded, and the pellets were resuspended in 2 ml appropriate medium. This was incubated for 17 min at 37°C, 5% CO2 to allow further de-esterification of the dye. Protocols used for incubation and resuspension are shown in Figure 1.
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Fluorimetric [Ca2+]i measurements were performed using an excitation wavelength pair of 340/380 nm and an emission wavelength of 510 nm. Fluorimetry was performed in a methylacrylate cuvette magnetically stirred and warmed to 37°C in a heated cuvette holder. Sufficient time (2–5 min) was allowed for the temperature of the sperm suspension to reach 37°C before measuring [Ca2+]i. Progesterone (3.2 µM, final) was added 400 s after the beginning of each experiment. About 20 µM digitonin and 47 µM of EGTA were added at approximately 800 s and 900 s, respectively, after the start of each experiment. The sequential addition of digitonin and EGTA were done to permit the determination of the calculated [Ca2+]i as previously described, using a Kd of 285 nM for fura-2 at 37°C.
Detection of phosphoserine/threonine- and phosphotyrosine-containing proteins
For the investigation of the reversibility of protein phosphorylation, spermatozoa were swum up in NCM and then incubated for a total of 9 h. Two sequences of incubation were used; either NCsEBSS (3 h) 
sEBSS (3 h) NCsEBSS (3 h) or sEBSS (3 h) NCsEBSS (3 h) sEBSS (3 h). Spermatozoa were washed once in the new medium at each saline change, and aliquots were removed at intervals for the investigation of protein phosphorylation. To verify the effects of albumin and/or bicarbonate on protein phosphorylation, after swim up, we incubated spermatozoa in sEBSS or sEBSS–bicarb or sEBSS–BSA or NCsEBSS for 6 h. Assessment of cell motility showed that these incubation protocols did not compromise viability compared with cells maintained in sEBSS.
Samples were washed to remove excess albumin (by centrifuging for 5 min at 600 g and resuspending in medium without BSA) before the addition of solubilization buffer. Solubilization buffer contained (final concentration): 2% (w/v) sodium dodecyl sulphate (SDS), 10% glycerol (v/v), 1.4% dithiothreitol (DTT) (w/v), 62.5 mM Tris–HCl, pH 6.8, 0.1 mM vanadate, 10 nM okadaic acid and 50 nM calyculin A. Samples were then boiled at 100°C for 5 min, sonicated and centrifuged at 14 000 g for 5 min. Proteins were separated by electrophoresis on SDS–polyacrylamide gel electrophoresis (SDS–PAGE) (12%) gels (Laemmli, 1970) and electrotransferred (Towbin et al., 1979) onto nitrocellulose membrane. Non-specific binding sites on the nitrocellulose membrane were blocked with either 5% BSA (w/v) or 5% (w/v) dry skim milk in Tris-buffered saline (0.9% NaCl (w/v), 20 mM Tris–HCl, pH 7.8) supplemented with 0.1 % (v/v) Tween-20 (TTBS) for the detection of phosphoserine/threonine and phosphotyrosine proteins, respectively. The nitrocellulose membranes were incubated overnight at 4°C or for 1 h at room temperature with the anti-phosphoserine/threonine PKA substrate or anti-phosphotyrosine antibodies, respectively. The membranes were then extensively washed with TTBS, incubated with corresponding secondary antibodies conjugated with horseradish peroxidase for 1 h and again extensively washed with TTBS. Positive immunoreactive bands were detected by chemiluminescence using LumiGLO, an enhanced chemiluminescence kit, according to the manufacturer’s instructions. Silver staining of the proteins transferred on the nitrocellulose membrane was performed after the detection to confirm that the transferred protein patterns were similar for all samples (Jacobson and Karnäs, 1990).
Assessment of progesterone-induced AR
For assay of AR, cells were stimulated with progesterone (final concentration of 3.2 µM) or A23187
[GenBank]
(10 µM) or solvent control (0.05% DMSO) for an incubation period of 1 h. Cells were then centrifuged at 500 g for 5 min. The supernatant was removed, and the spermatozoa were resuspended in 0.5 ml of hypo-osmotic swelling (HOS) medium (0.74% sodium citrate, 1.35% fructose in double-distilled H2O). After 10 min of incubation in HOS media, the spermatozoa were centrifuged for 5 min at 500 g. The supernatant was removed leaving a minimum volume of HOS (30 µl) for resuspension. Resuspended pellets in remaining HOS were smeared on microscopic slides (duplicate slides) previously coated with 10% poly-L-lysine solution and air-dried. The cells were then permeabilized in methanol for 2 min. About 15 µl of FITC-labelled Pisum sativum agglutinin (FITC–PSA) in PBS was spread on each slide and incubated for 45 min in a humid chamber at 37°C. Slides were then washed in a constant flow of mains water for 15 min before air drying and mounting with fluoromount. 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 for each treatment (100 per slide). Progesterone AR was represented as the percentage of maximum (response to 10 µM ionophore A23187
[GenBank]
in that experiment), with minimum set as the response to DMSO, using the equation:
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Statistical analysis
All calculations and statistical analyses were performed using the statistics module of Microsoft Excel 97. The t-tests (two-tailed; paired where appropriate) were performed to test for significance.
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Results |
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Dependence of progesterone-induced AR on the duration of incubation in capacitating medium
When cells swum up in the simple HEPES-buffered saline (no bicarbonate or BSA; NCM) were then resuspended in sEBSS and incubated for 6 h before stimulation for 1 h with progesterone, the calculated rate of progesterone-induced AR was 13.6 ± 1.3 (% stimulation). However, if progesterone was applied to cells within 10 min of resuspension in sEBSS (incubation terminated after 1 h exposure), stimulation of AR was only 3.4 ± 1.6% (P < 0.0005; paired t-test), showing that capacitation (as assayed by progesterone-induced AR) developed during incubation in sEBSS (Figure 2). Because almost all progesterone-induced AR occurs during the first 10 min after progesterone stimulation (Sabeur and Meizel, 1995
; Harper, unpublished data), we investigated the effect of stimulation with progesterone for just 15 min, applied immediately after swim up. Under these circumstances, the calculated % stimulation of AR was –4.0 ± 1.0, indicating that during the first 15 min after resuspension, progesterone was unable to induce AR. (Figure 2). We conclude that the cells underwent capacitation (as assessed by progesterone-induced AR) upon incubation in sEBSS.
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AR. A 60 min exposure to progesterone at this time was sufficient to cause a small increase in AR, but after a 6 h incubation in sEBSS the effect of stimulation with progesterone for 60 min was far greater (P < 0.00025 compared with cells treated after 10 min). Each bar shows mean (±SEM) of seven experiments.
Dependence of progesterone-induced [Ca2+]i signalling on the duration of incubation in capacitating medium
The effects of progesterone on the progesterone-induced [Ca2+]i signal in populations of human spermatozoa that had been incubated for different durations in sEBSS were assessed by fluorimetry. When sperm were swum up (for 60 min) into sEBSS and then resuspended in the same medium, progesterone evoked a biphasic elevation of [Ca2+]i comprising an initial transient increase in [Ca2+]i followed by a sustained elevation (Figure 3). The resting [Ca2+]i and the amplitude and kinetics of the mean progesterone-activated [Ca2+]i, transient response were similar in the cells stimulated <30 min after swim up and in the cells incubated for >6 h (Figure 3; P > 0.05). Because any effects of capacitating medium on [Ca2+]i responses might begin during the swim-up period, we repeated these experiments, but cells were swum up into NCM and then resuspended in sEBSS. In cells transferred to sEBSS just 30 min before recording (incubation protocol shown in Figure 1a), the mean resting [Ca2+]i (87 ± 10 nM; n = 11) was significantly lower than in cells incubated for 6 h in sEBSS (113 ± 7 nM; n = 11; P < 0.01). However, the progesterone-induced [Ca2+]i transient was similar in both kinetics and amplitude (Figure 3) to that in cells maintained in sEBSS for more than 6 h (Figure 1b; P > 0.05). The sustained responses were similarly insensitive to the duration of incubation in capacitating medium (data not shown).
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Assessment of reversibility on the progesterone-induced [Ca2+]i signal
The progesterone-induced [Ca2+]i signal was apparently near maximal within 30 min of the transfer of cells, after swim up, from NCM to capacitating medium (sEBSS, see preceding section). We therefore examined the responses to progesterone in cells resuspended and maintained in NCM (Figure 1c) or sEBSS (Figure 1b) for 6 h after swimming up in NCM. Following the 6 h incubation, the cells were loaded with fura-2 and then resuspended in the same medium for fluorimetry. The progesterone-induced [Ca2+]i-transient response was reduced in amplitude by >70% (P < 0.01) in the cells incubated in NCM (Figure 4). However, if cells incubated in for 6 h, NCM were resuspended in sEBBS for just 17 min (during de-esterification of fura-2; Figure 1d), the amplitude of the progesterone-induced [Ca2+]i-transient ‘recovered’ to control levels (Figure 4; not significant compared with cells maintained in sEBSS) and was significantly (P < 0.01) greater than in cells run in parallel but maintained in NCM. The kinetics of the [Ca2+]i response were indistinguishable from those of cells maintained in sEBSS (data not shown). Conversely, when cells incubated for 6 h in sEBBS were resuspended in NCM for 17 min (Figure 1e), the transient of the progesterone-activated [Ca2+]i increase was almost 70% smaller than in cells run in parallel but maintained in sEBSS (Figure 4; P < 0.01) and resembled that seen in cells maintained in NCM throughout incubation (statistically not significant). Analysis of the size of the sustained [Ca2+]i response at 200, 300 or 400 s after the application of progesterone showed a pattern similar to those for the amplitude of the [Ca2+]i transient. As with the transient [Ca2+]i response, the sustained response to progesterone could be ‘converted’ rapidly upon resuspension in a new medium (not shown). Assessment of cells by computer-assisted semen analysis (CASA) revealed no effect of these incubation protocols on cell viability, % motility being maintained at levels equivalent to those seen in cells maintained in sEBSS.
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A pattern of response similar to that described was observed when the simple HEPES-buffered saline (NCM) was replaced by sEBSS lacking bicarbonate and BSA (NCsEBSS), showing that the rapid changes in response to progesterone were because of the presence or removal of these capacitating factors (data not shown). When sperm were swum up into sEBSS (rather than NCM) medium before commencement of experiments, the pattern of response was, again, as described above (data not shown).
Assessment of the reversibility of serine/threonine and tyrosine phosphorylation of sperm proteins
We have investigated tyrosine phosphorylation of two major proteins of 105 and 81 kDa (Leclerc et al., 1996; Lefièvre et al., 2000; Kirkman-Brown et al., 2002) and the previously described PKA-dependent serine/threonine phosphorylation of two proteins of similar molecular weights (O’Flaherty et al., 2004; Moseley et al., 2005).
After swim up in NCM, levels of both phosphoserine/threonine (Figure 5a) and phosphotyrosine (Figure 5b) protein remained low in spermatozoa when incubated in sEBSS-lacking bicarbonate and BSA (NCsEBSS) for 3 h (Figure 5a and b). When transferred into capacitating condition (NCsEBSS sEBSS) for a further 3 h, levels of phosphorylation significantly increased (Figure 5a and b). Transfer to sEBSS had an effect on serine/threonine phosphorylation within 1 min (Figure 5a), whereas tyrosine phosphorylation reached a maximum at only 2–3 h (Figure 5b). To investigate whether this increase in phosphorylation could be reversed, spermatozoa were washed and returned to their original non-capacitating conditions (NCsEBSS sEBSS NCsEBSS). Serine/threonine and tyrosine phosphorylation gradually decreased to nearly undetectable levels, similar to those observed during the first 3 h of incubation (Figure 5a and b).
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When cells were resuspended in sEBSS after swim up (in NCM), serine/threonine phosphorylation was observed at the earliest time point (within minutes after resuspension; Figure 5c; O’Flaherty et al., 2004; Moseley et al., 2005), and this was maintained during the 3 h of incubation. This rapid response was observed in all experiments. In contrast, a time-dependent increase, reaching a maximum at up to 3 h, was observed for tyrosine phosphorylation of the 105 and 81 kDa proteins (Figure 5d; Moseley et al., 2005). The increased levels of phosphorylation that were obtained with sEBSS greatly reduced when spermatozoa were transferred into a medium that did not support capacitation (sEBSS NCsEBSS) for 3 h (Figure 5c and d). To investigate whether human spermatozoa could undergo a second wave of phosphorylation, spermatozoa were then returned to sEBSS for a further 3 h (sEBSS NCsEBSS sEBSS). Levels of both serine/threonine and tyrosine phosphorylation increased and were sustained throughout the additional 3 h of incubation (Figure 5c and d), the phoshorylation of tyrosine residues again being more gradual and time-dependent than serine/threonine phosphorylation. These patterns of reversible phosphorylation were seen in all experiments (data not shown). Assessment of motility revealed no effect of these incubation protocols on cell viability.
Effect of BSA and bicarbonate on the progesterone-induced [Ca2+]i signal
Because the rapid effects of incubation medium on the progesterone-induced [Ca2+]i response were owing to presence or absence of BSA and bicarbonate, we investigated the effects of selective removal of just one of these factors from sEBSS. After swim up in NCM, cells were suspended for 6 h in sEBSS or in medium lacking bicarbonate (sEBSS–bicarb), BSA (sEBSS–BSA) or both (NCsEBSS), before recording the response to progesterone. Omission of either bicarbonate or BSA caused a reduction in the amplitude of the progesterone-induced transient response (P < 0.05), but the effect of omitting bicarbonate was much more marked and consistent. The responses to progesterone of cells suspended in medium lacking both bicarbonate and BSA was similar to that in bicarbonate-free medium (Figure 6a, left panel).
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To confirm that the effects of omission of bicarbonate (sEBSS–bicarb) on the progesterone-induced [Ca2+]i signal were reversible, we incubated cells swum up in NCM in sEBSS–bicarb for 6 h and then suspended in sEBSS for 17 min. This was sufficient to allow complete recovery of the response to progesterone compared with cells maintained in sEBBS for >6 h (Figure 6a, right panel).
Effect of BSA and bicarbonate on the serine/threonine and tyrosine phosphorylation of sperm proteins
To assess the role of bicarbonate and/or albumin in serine/threonine and tyrosine phosphorylation in our experiments, spermatozoa were incubated in media deficient in one or both of these ingredients. As expected, spermatozoa incubated in the absence of both bicarbonate and albumin (NCsEBSS) showed low levels of phosphorylation. The presence of albumin (sEBSS–bicarb) was sufficient to increase serine/threonine and tyrosine phosphorylation of sperm proteins to levels similar to those in sEBSS (Figure 6b). In seven, experiments with bicarbonate-only (sEBSS–BSA) incubation consistently caused increased phosphorylation compared with that seen in NCsEBSS but levels varied, such that in 3/7 experiments, phosphorylation appeared greater than in complete medium (sEBSS) (Figure 6b).
Assessment of the reversibility of progesterone-induced AR
Previous work using inhibitors of tyrosine kinase has shown that activity of this enzyme (and tyrosine phosphorylation) is necessary for progesterone-induced AR (Tesarik et al., 1993; Luconi et al., 1995; Visconti and Kopf, 1998 Kirkman-Brown et al., 2002). However, because medium containing either BSA or bicarbonate (but not both) is sufficient to support serine/threonine and tyrosine phosphorylation (Figure 6b; Osheroff et al., 1999), we investigated the effects of salines lacking just one of these components on progesterone-induced AR. When cells were incubated in sEBSS for 7 h before progesterone stimulation (1 h), the rate of AR was much higher than in cells incubated for an equivalent period in sEBSS–bicarb (Figure 7a). However, if cells were transferred from sEBSS to sEBSS–bicarb after 6 h (and then incubated for a further 1 h), the ability to undergo progesterone-induced AR was greatly reduced. Removal of BSA from the medium after incubation in sEBSS was similarly effective. When cells were transferred from sEBSS to sEBSS–BSA after 6 h (and then incubated for a further 1 h), similarly to the effect of bicarbonate removal, the ability to undergo progesterone-induced AR was greatly reduced compared with cells maintained in sEBSS (Figure 7b). Cells incubated in bicarbonate-free medium for 6 h, then transferred to sEBSS for a further 1 h responded to progesterone with a greatly increased level of AR compared with those that were maintained in bicarbonate-free medium (Figure 7a).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We have investigated the induction and reversibility of many capacitation-related events in human spermatozoa. When cells were swum up into medium deficient in bicarbonate and albumin, levels of serine/threonine phorphorylation and tyrosine were low, and the progesterone-induced [Ca2+]i transient was small (typically 50–100 nM). In agreement with previous studies, transfer of the cells to a medium-supporting capacitation (sEBSS) caused enlargement of the progesterone-induced [Ca2+]i signal (typically 300–400 nM) and development of both serine/threonine and tyrosine phosphorylation of sperm proteins and of the ability of cells to undergo progesterone-induced AR. Subsequent transfer of cells to NCM had effects on all of these factors. These effects were independent of cell viability as assessed by motility and also as reflected in the maintained amplitude of the [Ca2+]i signal (a near-ubiquitous response; Harper et al., 2003
) when measured in sEBSS.
Protein phosphorylation
Tyrosine phosphorylation is a well-established marker of cells undergoing capacitation (Visconti et al., 1995a,b; Leclerc et al., 1996; Osheroff et al., 1999) and was detected in cells incubated in sEBSS, showing a time-dependent increase over a 3 h incubation (Figure 8, yellow line). Upon resuspension in medium that did not support capacitation (NCsEBSS), tyrosine phosphorylation clearly reversed (Figure 5d) and, after reversal, rephosphorylation occurred upon resuspension in sEBSS (Figure 5d). Phosphorylation of serine/threonine and tyrosine residues occurred in the presence of either BSA alone or bicarbonate (25 mM) alone (Figure 6b), a finding consistent with the observations of Osheroff et al. (1999). Omission of BSA sometimes caused an apparent increase in tyrosine phosphorylation compared with cells in sEBSS (Figure 6b), but this effect was inconsistent. Previous studies have shown that the inhibition of tyrosine kinases can lead to loss of tyrosine phosphorylation in human spermatozoa, suggesting that, in capacitating cells, levels of phosphotyrosine may be determined by the balance of phosphorylation and dephosphorylation (Aitken et al., 1996; Kirkman-Brown et al., 2002; Tomes et al., 2004). If levels of tyrosine kinase activity are regulated by factors in capacitation-supporting medium, withdrawal of these factors would reveal the effects of ‘stable’ tyrosine phosphatase activity. Tyrosine phosphorylation is believed to be initiated by activity of cAMP-regulated PKA, a serine/threonine kinase activated both by bicarbonate and by removal of membrane cholesterol (Visconti et al., 1995b; 1999a,b; Sinclair et al., 2000). Parallel assessment of serine/threonine phosphorylation showed a similar reversible effect, but the induction of phosphoserine/threonine was much more rapid than tyrosine phosphorylation (Figures 5 and 8, green line). This suggests that that generation of cAMP and activation of PKA occurs immediately upon suspension of cells in medium-supporting capacitation but that the consequent increase in tyrosine phosphorylation is more gradual (Moseley et al., 2005), perhaps because this is achieved against a background of persistent tyrosine-phosphatase activity. This finding is consistent with the rapid PKA-mediated effects of bicarbonate on mouse sperm (Wennemuth et al., 2003; see next section). It is noteworthy that removal of membrane cholesterol by BSA (which must be a non-reversible process) does not prevent subsequent reversal of protein phosphorylation in non-capacitating media, suggesting that this process is an adequate but not a primary determinant of protein phosphorylation.
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Progesterone-activated [Ca2+]i signalling
In agreement with previous studies (Blackmore et al., 1990; Aitken et al., 1996), we observed that the progesterone-induced [Ca2+]i response was maximal in cells swum up in NCM and transferred to sEBSS for <30 min and did not develop during a subsequent 6 h incubation (Figure 8, blue line), despite the increase in progesterone-induced AR that occurred over this period (Figure 8, red line). Clearly the time-dependent increase in progesterone-induced AR did not reflect a greater Ca2+ influx. However, transfer of cells, from sEBSS to NCM or NCsEBSS caused a rapid reduction in the amplitude of the [Ca2+]i response that could be reversed, equally rapidly, upon return to sEBSS, an effect that was primarily because of the removal or addition of bicarbonate, removal of BSA being less effective (Figure 6a). Application of bicarbonate to epididymal mouse spermatozoa causes increased flagellar beat and enhanced activity of voltage-operated Ca2+ channels within a period of seconds to minutes, an effect dependent upon cAMP/PKA. Reversal occurs within 10 min. Our data show that, in human spermatozoa, bicarbonate has an effect on the channel(s) that mediate the progesterone-induced [Ca2+]i signal in <20 min. Although regulation of the channel by PKA is clearly a possibility, an alternative interpretation of these observations is that pHi of spermatozoa falls in the absence of extracellular bicarbonate and that this pH change inhibits progesterone-induced Ca2+ influx. Such an effect has been described in human spermatozoa incubated in bicarbonate-free conditions for 3 h (Aitken et al., 1998). Regulation of the progesterone-activated Ca2+-influx pathway requires further investigation.
Progesterone-induced AR
The ability of the cells to undergo progesterone-induced AR was undetectable immediately after swim up into NCM but developed during a subsequent 6 h incubation (Figure 8, red line). We have shown previously (Moseley et al., 2005) that incubation in sEBSS for 30 min, followed by stimulation with progesterone, results in negligible levels of AR. The progesterone-induced [Ca2+]i signal is maximal within <30 min of suspension in medium supporting capacitation, so events downstream of Ca2+ mobilization (probably including tyrosine phosphorylation; Figure 8 yellow line) are the limiting factor at this point. In contrast, when cells incubated in capacitating medium were resuspended in sEBSS–bicarb for 1 h, the ability to undergo AR was greatly reduced (Figure 7a). A similar, though less striking effect was observed when cells were transferred from sEBSS to sEBSS–BSA (Figure 7b), again showing that removal of cholesterol by BSA, though necessary, does not have irreversible effects. We observed significant tyrosine phosphorylation in cells incubated in either sEBSS–bicarb or sEBSS–BSA (Figure 6b). Rapid loss of competence to undergo AR in cells incubated under similar conditions has also been observed by Sabeur and Meizel (1995). This suggests either that tyrosine phosphorylation in the acrosomal region (Ficarro et al., 2003; Sakkas et al., 2003; Asquith et al., 2004) not detected in this study—see Results] is crucial for AR and is reversed under these conditions or that in these experiments the rapid effects of bicarbonate on [Ca2+]i signalling are the controlling factor, emphasizing the dependence of AR on a series of events, all of which are subject to regulation. Figure 8 shows the way in which AR competence would be determined by the kinetics of tyrosine phosphorylation during capacitation and by decay of the progesterone-induced [Ca2+]i signal during decapacitation.
In summary, we have shown that in human spermatozoa, protein tyrosine phosphorylation, protein serine/threonine phosphorylation, progesterone-induced AR and progesterone-induced [Ca2+]i signalling are all reversibly regulated by the incubation medium. The presence of bicarbonate is particularly significant, and removal of cholesterol by BSA (which must be irreversible) is not sufficient to cause irreversible changes in any of the factors measured in this study. The kinetics of the responses vary considerably. Under the conditions used in this study, serine/threonine phosphorylation and [Ca2+]i-signalling ‘switch’ between maximum and minimum in minutes. In contrast, acquisition of competence to undergo progesterone-induced AR takes at least 90 min for completion (Moseley et al., 2005), and the occurrence of tyrosine phosphorylation of tail proteins requires up to 3 h to reach maximum. We have recently shown that, under appropriate conditions (use of IVF medium), in vitro capacitation can be greatly accelerated (Moseley et al., 2005). It is already known that key events in capacitation are subject to tight regulation (Visconti et al., 2002; Ecroyd et al., 2004; Fraser et al., 2005). The findings reported here suggest that, in the female tract, human spermatozoa are capable of repeated and reversible cycles of many of the events that occur in response to capacitating conditions and that the cells may therefore have a high degree of plasticity and adaptability in their responses to events which signal ovulation. This ability to ‘switch off’ may be pivotal to the ability of cells to remain viable for extended period in the female tract (Wilcox et al., 1995).
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The authors acknowledge Bayard Storey for critical reading of this article. K.B.-A. was in receipt of support from the Ghanaian High Commission. This work was supported by the Birmingham Women’s Healthcare NHS Trust (C.L.R.B.) and Fonds de recherche en santé du Québec to (L.L.).
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* The authors equally contributed to this work.
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Submitted on June 13, 2005; revised on July 28, 2005; accepted on August 18, 2005
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