Mol. Hum. Reprod. Advance Access originally published online on January 31, 2008
Molecular Human Reproduction 2008 14(4):235-243; doi:10.1093/molehr/gan007
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Investigation of the role of SRC in capacitation-associated tyrosine phosphorylation of human spermatozoa
1Reproductive Science Group, Discipline of Biological Sciences, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia 2ARC Centre of Excellence in Biotechnology and Development, Discipline of Biological Sciences, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia
3 Correspondence address. Tel: +61-2-4921-2082; Fax: +61-2-4921-6308; E-mail: john.aitken{at}newcastle.edu.au
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Abstract |
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The process of capacitation is a pre-requisite for mammalian spermatozoa allowing them to gain the ability to fertilize an oocyte. A fundamental part of this mechanism is a dramatic increase in the level of tyrosine phosphorylation. Implicated in this process is a unique cAMP/protein kinase A (PKA)-mediated pathway involving an intermediate PKA-activated tyrosine kinase suggested to be pp60c-src (SRC) in the mouse. This study has verified the importance of SRC as a key intermediate kinase in promoting the tyrosine phosphorylation events associated with human sperm capacitation. The presence of SRC in human spermatozoa was confirmed immunocytochemically and the kinase was localized to subcellular domains compatible with a role in tyrosine phosphorylation. Additionally SRC co-immunoprecipitated with PKA and became activated by phosphorylation of the Y416 residue during human sperm capacitation. Furthermore, the suppression of PKA and SRC through the application of specific inhibitors led to a dramatic decrease in tyrosine phosphorylation. However, although the inhibition of PKA was also accompanied by a suppression of sperm motility, SRC inhibition did not induce a similar response.
Key words: spermatozoa/capacitation/tyrosine phosphorylation/SRC
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Introduction |
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Independent experiments by Chang and Austin in the 1950s demonstrated that spermatozoa must spend a period of time in the female reproductive tract in order to become functionally competent and express their full potential for fertilization (Austin, 1951; Chang, 1951). This process of sperm capacitation, unique to mammals, encompasses a number of physiological changes, many of which are not yet fully understood. These changes are, however, known to include modifications to membrane architecture, alterations in enzyme activity, an increased rate of respiration and a dramatic change in motility pattern to a hyperactivated state (Yanaginachi, 1988). Collectively, these modifications result in spermatozoa that possess the ability to bind to the zona pellucida, undergo acrosomal exocytosis, penetrate through to the perivitelline space and ultimately fuse with the oolemma and fertilize the oozyte (Yanagimachi, 1988).
Since it is widely accepted that spermatozoa are virtually transcriptionally and translationally silent (Engel et al., 1973; Hernandez-Perez et al., 1983; Gur and Breitbart, 2006), the acquisition of functional competence during capacitation must be largely dependent on post-translational modifications of pre-existing sperm proteins (Blaquier et al., 1988a,b; Ross et al., 1990). Analysis of the nature of these post-translational modifications has revealed that one of the most significant is a dramatic increase in the levels of tyrosine phosphorylation across a wide range of sperm proteins (Visconti et al., 1995a; Baker et al., 2004). Our recent studies in both human and mouse spermatozoa have revealed that majority of the tyrosine-phosphorylated proteins are localized to the tail (Baker et al., 2006; Mitchell et al., 2007). It is therefore likely that these phosphorylated proteins play some role in the attainment of a hyperactivated state (Lin et al., 2006). In addition, the increase in phosphotyrosine expression may also be involved in other aspects of fertilization including sperm–zona pellucida interaction. Thus, recent studies have revealed that mouse spermatozoa that have successfully bound to the zona are all completely tyrosine-phosphorylated, from the neck to the tail end piece compared with only 10–15% of the free swimming population (Urner et al., 2001; Asquith et al., 2004). Furthermore, the inhibition of phosphotyrosine expression compromises both the onset of hyperactivation and the ability of mouse spermatozoa to engage in sperm–zona pellucida interaction (Asquith et al., 2004; Baker et al., 2006).
Capacitation-associated tyrosine phosphorylation is thought to be primarily dependent upon an increase in intracellular levels of bicarbonate, resulting in the activation of a unique signaling pathway mediated by soluble adenylyl cyclase (sAC) and a concomitant increase in cyclic AMP generation (White and Aitken, 1989; Visconti et al., 1995b; Aitken et al., 1998). In this regard, it is well established that the addition of membrane permeable cAMP analogs to spermatozoa can augment the onset of capacitation and the levels of tyrosine phosphorylation attained by these cells (Aitken et al., 1995, Visconti et al., 1995b; Baker et al., 2004). Conversely, pharmacological inhibition of sAC suppressed intracellular cAMP levels and disrupted the induction of tyrosine phosphorylation during capacitation (Hess et al., 2005). Furthermore, male mice null for the sperm-specific catalytic subunit of PKA—a major, but not exclusive, mediator of cAMP action—exhibited infertility associated with impaired tyrosine phosphorylation (Nolan et al., 2004).
Thus, while the central importance of cAMP and PKA in driving the tyrosine phosphorylation events associated with capacitation is beyond doubt, the PKA-activated tyrosine kinase that mediates this process is still, unresolved. Recent studies within our laboratory have revealed that in mouse spermatozoa the Src family kinase, pp60c-src (SRC), may fulfill this important intermediary function (Baker et al., 2006). A critical finding in these studies was that SRC co-immunoprecipitated with PKA in capacitated but not uncapacitated spermatozoa. Furthermore, incubation of mouse spermatozoa in the presence of SRC kinase inhibitors potently suppressed capacitation-associated tyrosine phosphorylation and the subsequent induction of hyperactivation (Baker et al., 2006). Interestingly, the use of similar inhibitors in human spermatozoa were also able to suppress the induction of tyrosine phosphorylation activated via a receptor mediated pathway involving platelet endothelial cell adhesion molecule (PECAM-1) (Nixon et al., 2005). In light of these findings, the purpose of this investigation was to determine whether SRC also represents a key intermediate kinase that contributes to the tyrosine phosphorylation signal transduction cascades associated with capacitation in human spermatozoa.
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Reagents and antibodies
Unless otherwise stated the chemicals used throughout this study were purchased from Sigma (St Louis, MO, USA) and were of research or molecular biology grade. HEPES, penicillin and streptomycin were purchased from Gibco (Paisley, UK). Sodium chloride, sodium hydrogen carbonate, calcium chloride dehydrate, potassium chloride, magnesium sulfate heptahydrate and D-glucose were purchased from Merck (Damstadt, Germany). Nitrocellulose was from GE Healthcare (Castle Hill, Australia).
Anti-phosphotyrosine antibodies (clone 4G10), anti-His Tag (clone 4D11) and anti-SRC were from Upstate Biotechnology (Lake Placid, NY, USA). Mouse anti-pY416 monoclonal, goat anti-rabbit (IgG) and rabbit anti-goat (IgG) horseradish peroxidase (HRP) conjugates were from Calbiochem (La Jolla, CA, USA). Anti-YES and anti-PKAc were purchased from BD Biosciences (San Jose, CA, USA). Anti-FYN and Anti-LYN were purchased from Abcam (Cambridge, UK). HRP-conjugated goat anti-mouse (IgG) was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Goat anti-rat (IgG) HRP conjugate was from Oncogene Research Products (San Diego, CA, USA). Anti-
-tubulin, all FITC-conjugated secondary antibodies and all sera were purchased from Sigma.
Preparation of human spermatozoa
Human semen samples were obtained from a panel of healthy normozoospermic volunteer donors assembled for the Reproductive Science Research Group at the University of Newcastle. After at least 48 h abstinence, semen samples were produced by masturbation and collected into sterile sample containers which were delivered to the laboratory within 1 h of ejaculation. Scientific use of these samples was approved by the University of Newcastle Human Ethics Committee.
Purification of human spermatozoa was achieved using a 44 and 88% discontinuous Percoll (GE Healthcare, Castle Hill, Australia) centrifugation gradient. For this procedure, Percoll (90 ml) was supplemented with 10 ml of 10 times Ham's F10 solution, 370 µl sodium lactate syrup, 3 mg sodium pyruvate, 210 mg sodium hydrogen carbonate and 100 mg polyvinyl alcohol (PVA). This isotonic Percoll solution was diluted with HEPES-buffered Biggers, Whitten and Whittingham medium (BWW) supplemented with 1 mg/ml PVA, maintained at an osmolarity of 300 mOsm/kg (Biggers et al., 1971) in order to create the discontinuous gradient. Up to 3 ml semen was layered on top of each gradient and centrifuged at 500 g for 30 min. Following centrifugation, the seminal plasma and Percoll were removed and discarded. Purified spermatozoa were recovered from the base of the 88% Percoll fraction and washed with BWW. They were then pelleted by centrifugation at 500 g for a further 15 min and finally resuspended at a concentration of 6 x 106cell/ml.
Capacitation of human spermatozoa
Following dilution in BWW, purified spermatozoa were incubated at 37°C under an atmosphere of 5% CO2:95% air. Non-capacitated cells were incubated in BWW prepared without NaHCO3 (BWW–HCO3–). Capacitated cells were incubated in BWW prepared without CaCl2 (BWW–Ca2+) but supplemented with 3 mM pentoxifylline (ptx), a phosphodiesterase inhibitor and 5 mM dibutryl cyclic adenosine monophosphate (dbcAMP), a membrane permeable cyclic AMP. These treatments have been shown to suppress or induce optimal levels of capacitation in human spermatozoa respectively (Mitchell et al., 2007). Incubations were conducted for a period of 3 h, after which the percentage of motile cells was assessed and the sperm were prepared for the various treatments outlined below.
Computer-assisted sperm analysis
Sperm motility parameters were evaluated using a Hamilton Thorne CASA System (Version 12; Hamilton Thorne Biosciences, Beverly, MA, USA). Human spermatozoa were suspended at 10 x 106 cell/ml and a volume of 2.5 µl was loaded onto a standard four chamber slide (Leja, The Netherlands). For this purpose, at least 200 cells were scored for each sample using standard settings for human spermatozoa (30 frames acquired at a frame rate of 60 Hz in 20 µm deep chambers at a constant temperature of 37°C). Each sample was analysed for the percentage of motile cells and those displaying progressive motility. Cells were counted as motile if they displayed an average path velocity greater than 5 µm/s and a progressive velocity greater than 11 µm/s. Cells were considered progressively motile if they displayed an average path velocity greater than 25 µm/s, and a straightness value greater than 80%. Given the inherent variability observed in the quality of human sperm samples, the data from each replicate was normalized against a population of spermatozoa incubated in complete BWW media.
Assessment of hyperactivation
A droplet of human sperm suspension was placed onto a microscope slide and viewed using phase microscopy. Two fields of view were video-taped and subsequently assessed for sperm displaying hyperactivated patterns of motility, characterized by sharply curved flagellar beats and a circular or erratic swimming trajectory. The results are expressed as a percentage of motile cells displaying hyperactivation.
SDS–PAGE and western blotting
Following incubation spermatozoa were collected by gentle centrifugation (500 g for 3 min) and protein extracts were prepared by solubilizing the cells in an SDS Extraction Buffer (2% SDS, 10% sucrose in 0.1875 M Tris pH 6.8) supplemented with a protease inhibitor cocktail (Roche, Manneheim, Germany) for 5 min at 100°C. Insoluble constituents were removed by centrifugation at 10 000 g for 15 min. Protein quantification of the isolated supernatant was determined using a bicinchoninic acid Protein Assay Kit (Pierce, Rockford, IL, USA) according to manufacturer's instructions.
Equivalent amounts of protein (2 µg) were boiled in SDS–PAGE sample buffer (SDS Extraction Buffer supplemented with 2% mercaptoethanol and bromophenol blue) for 5 min and resolved on 10% polyacrylamide gels (Laemmli, 1970). The proteins were then electro-transferred onto nitrocellulose membranes under a constant current of 300 mA for 1 h (Towbin et al., 1979).
Membranes were blocked for 1 h at room temperature with 3% bovine serum albumin (BSA) (Research Organics, Cleveland, OH, USA) in Tris-buffered saline (TBS) pH 7.4 supplemented with 0.1% polyoxyethylenesorbitan monolaurate (Tween 20). Membranes were rinsed in TBS and then probed with appropriate primary antibodies at 1:1000 dilution in 1% BSA, 0.1% Tween 20 in TBS for 2 h at room temperature. Following incubation, membranes were washed three times in TBS containing 0.01% Tween 20 (TBST) for 10 min. Membranes were then probed for 1 h with a 1:3000–1:5000 dilution of HRP-conjugated secondary antibody at room temperature. Following a further three washes in TBST, cross-reactive proteins were visualized using an enhanced chemiluminescence kit (GE Healthcare) according to the manufacturer's instructions. Membranes were then stripped and reprobed with anti--tubulin to ensure equivalent protein loading in each lane (Asquith et al., 2004).
2D gel electrophoresis
Following incubation spermatozoa were pelleted by gentle centrifugation and the supernatant was removed. Approximately 100 µl of 2D lysis buffer consisting of 50 mM Tris pH 8.5, 4% (w/v) (3-[(cholamidopropyl)dimethylammonio]-1-propansulfonate) CHAPS, 7 M urea and 2 M thiourea was used to resuspend the pellet. The sample was then incubated at 4°C for 1 h with vortexing every 10 min. Following this, the sample was centrifuged at 10 000 g for 15 min and the supernatant collected. The proteins within the supernatant fraction were purified by methanol/chloroform precipitation (Wessel and Flugge, 1984) and then resuspended in 100 µl of 2D lysis buffer. The protein concentration was determined using the EttanTM 2D Quant Kit (GE Healthcare) according to the manufacturer's instructions.
Aliquots containing 100 µg of protein were added to 1.3 µl immobilized pH-gradient (IPG) buffer (GE Healthcare) and 1.5 µl Destreak (GE Healthcare) and volumes made up to 130 µl with 2D lysis buffer. These aliquots were used to rehydrate IPG strips (pH 3–10, 7 cm, nonlinear, GE Healthcare). The IPG strips were then covered in mineral oil and left to rehydrate overnight at room temperature. Isoelectric focussing was performed using an IPGphor ceramic manifold (GE Healthcare) using the following program: 500 V (gradient) for 10 Vh, 4000 V (gradient) for 5600 Vh and 5000 V (step and hold) for 2500 Vh. Following focussing, the IPG strips were immediately incubated in equilibration buffer (30% (v/v) glycerol, 2% (w/v) SDS, 6 M urea, 50 mM Tris pH 8.8 and trace amounts of bromophenol blue) supplemented with 0.5% (w/v) dithiothreitol for 10 min at room temperature, followed by fresh equilibration buffer supplemented by 4.5% (w/v) iodoacetamide for a further 10 min at room temperature. The strips were then incubated in SDS-Running Buffer for 5 min at room temperature. The strips were placed on top of 10% polyacrylamide gels and held in place using 0.5% (w/v) agarose prepared in SDS-Running Buffer. Proteins were then resolved at constant voltage (120 V), electro-transferred onto nitrocellulose membranes and immunoblotted as previously described.
Immunolocalization on fixed spermatozoa
Following incubation, spermatozoa were fixed in 4% paraformaldehyde, washed three times with phosphate-buffered saline (PBS), aliquoted onto poly-L-lysine coated glass slides and air-dried. All subsequent incubations were performed in a humid chamber at 37°C. The cells were permeabilized with 0.2% Triton X-100 for 15 min, rinsed with PBS and blocked with 10% serum/3% BSA for 1 h. Slides were washed three times with PBS for 5 min and incubated in a 1:50 dilution of primary antibody at 4°C overnight. Slides were then subjected to 3 x 5 min washes with PBS and incubated in a 1:100 dilution of FITC-conjugated secondary antibody for 2 h at 37°C. Slides were again washed and mounted in 10% mowiol 4–88 (Calbiochem) with 30% glycerol in 0.2 M Tris (pH 8.5) with 2.5% 1,4-diazobicyclo-[2.2.2]-octane (DABCO). Cells were finally examined using either a Zeiss Axioplan 2 fluorescence microscope or an LSM510 laser scanning confocal microscope equipped with argon and helium/neon lasers. Control incubations in which spermatozoa were incubated in either an irrelevant primary antibody (anti-His Tag) or secondary antibody only were routinely included in all analyses.
Preparation of human sperm head and tail fractions
Human spermatozoa were isolated and capacitated as previously described. After incubation, the sperm samples were transferred into glass tubes and sonicated on ice at 60% power for 15 s to facilitate the separation of sperm heads and tails (Nixon et al., 2006). Following sonication, the samples were layered over a 75% Percoll cushion in 15 ml tubes, and centrifuged at 700 g for 15 min to isolate the heads and tails in separate fractions. The pellet that is formed contains the sperm heads, while the top layer contains the sperm membranes, and the tails reside at the interface between the two layers (Oko, 1988). The heads and tails were removed and diluted with BWW/PVA. The purity of each fraction was assessed by microscopy prior to proceeding to analysis. The head and tail samples were then recentrifuged at 400 g for 4 min, the supernatant removed and the remaining pellet SDS-extracted as described previously.
Co-immunoprecipitation studies
Approximately 60 µl (per treatment) magnetic Protein G-coated Dynabeads (Dynal Biotech ASA, Oslo, Norway) were washed three times in Washing and Binding Buffer (WBB)[5 mM Tris–HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl]. This was followed by conjugation with 4 µg anti-SRC antibody at 4°C overnight with constant mixing. A control sample of beads was left non-conjugated and was incubated with WBB only. Following conjugation the beads were washed two times in WBB. Non-capacitated and capacitated spermatozoa were then lyzed in IP lysis buffer [0.1% (v/v) Triton X-100, 300 mM NaCl, 20 mM Tris pH 7.4 supplemented with protease inhibitor cocktail and a 1:100 dilution of HALT complete phosphatase inhibitor cocktail (Pierce)]. Approximately 100 µg of soluble lysate was added to the pre-adsorbed beads and left to incubate at 4°C overnight with constant mixing. Following incubation, the beads were washed two times in WBB and resuspended in SDS Sample Buffer. The suspension was then boiled for 5 min, the beads removed and the precipitated proteins resolved on 10% polyacrylamide gels before being electro-transferred onto nitrocellulose membranes and immunoblotted as described previously. Control incubations were included where beads were incubated with sperm lysate in the absence of antibody, and also antibody-conjugated beads were incubated in the absence of cell lysate. These controls were processed as described above.
Statistics
All experiments presented in this study were performed a minimum of three times using pooled semen samples from a number of different donors. Graphical data represents means ± SEM. Statistical differences between group means were determined using an analysis of variance (ANOVA) or paired t-test. P-values of <0.05 were considered significant.
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SRC family kinases are present in human spermatozoa
Previous studies within our laboratory have revealed SRC kinase acts as a key intermediary in the PKA-mediated increase in tyrosine phosphorylation observed during mouse sperm capacitation. Prior to investigating whether SRC kinase fulfils a similar function during the capacitation of human spermatozoa, we first sought to confirm the presence of this and other members of the Src family kinases in human spermatozoa. As illustrated in Fig. 1, cross-reactive proteins of the appropriate molecular weight for each of the Src family kinases examined (SRC, FYN, LYN and YES1) were detected in cell lysates extracted from both non-capacitated and capacitated human spermatozoa.
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Preliminary immunocytochemistry studies with each of the Src family kinase antibodies indicated that these reagents were unsuitable for reliable localization of the respective proteins within human spermatozoa. As an alternative strategy to ascertain the location of these proteins, we performed a subcellular fractionation to isolate sperm heads and tails to high-degree of purity (>95%, Fig. 2A). These preparations were then resolved by SDS–PAGE and prepared for immunoblotting. This approach revealed that each of the Src kinases examined are present in both the sperm head and the tail, however it can clearly be seen that they are all predominantly expressed in the tail (Fig. 2B), a site compatible with a role in mediating the capacitation-associated tyrosine phosphorylation cascade. The distribution of each protein was not significantly influenced by the capacitation status of the spermatozoa (Fig. 2B). Similarly the catalytic subunit of PKA itself was observed in both the sperm head and tail but predominated in the latter (Fig. 2B).
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Src co-precipitates with PKA in capacitated human spermatozoa
Having confirmed the existence of both PKA and SRC within the tail of human spermatozoa, we sought to examine whether SRC was responsible for coordinating the tyrosine phosphorylation cascade observed in these cells during capacitation. For this purpose, we initially investigated whether SRC is either constitutively or inducibly associated with the catalytic domain of PKA (PKA-c). Using a co-immunoprecipitation strategy it was shown that PKA-c could be isolated from soluble sperm lysates with anti-SRC antibodies (Fig. 3). Interestingly, this interaction appeared to be restricted to capacitated cells, as revealed by the presence of a single 40 kDa protein, corresponding to PKA-c, that was detected in the capacitated sperm sample but absent from the corresponding uncapacitated sample. This result is consistent with the hypothesis that PKA-mediated activation of SRC may form an integral part of the signaling cascade assembled during the capacitation of human spermatozoa.
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Src activity is increased during the capacitation of human spermatozoa
To substantiate the role of SRC in mediating capacitation-associated tyrosine phosphorylation, we next sought evidence for the functional activation of this kinase during human sperm capacitation. Although direct measurement of SRC kinase activity in spermatozoa is extremely difficult (Baker et al., 2006), we overcame this limitation by examining the phosphorylation status of SRC using an antibody (anti-pY416) that specifically recognizes SRC kinase, tyrosine-phosphorylated at position 416 (Cartwright et al., 1989; Katagiri et al., 1989; Bagrodia et al., 1993; Kralisz and Cierniewski, 2000). The rationale for this approach is based on the observation that, upon activation, SRC undergoes autophosphorylation of Y416 and thus phosphorylation of this residue can be taken as strong evidence for the presence of active SRC kinase (Boerner et al., 1996).
To investigate whether SRC activity increases as human spermatozoa undergo capacitation, 2D western blot analyses were performed with anti-pY416 antibodies to compare the phosphorylation status of SRC in populations of capacitated and non-capacitated cells. As shown in Fig. 4, the labeling intensity of the protein spot identified as SRC (isoelectric point 8.0 and molecular mass 60 kDa) was dramatically increased in capacitated spermatozoa compared to that detected in the non-capacitated sperm sample. The quantitative nature of this increase was confirmed by stripping the respective membranes and reprobing with anti-SRC antibody to ensure that equal amounts of the enzyme were present in each of the samples (Fig. 4).
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To validate the results of the western blot data and confirm the increase in SRC phosphorylation during capacitation, populations of non-capacitated and capacitated human spermatozoa were prepared for immunocytochemical labeling using the anti-pY416 antibody. As illustrated in Fig. 5, the phosphorylated form of SRC was strongly detected along both the midpiece and principle piece of the tail of approximately 50% of capacitated human sperm cells (Fig. 5B). Interestingly, the intensity of anti-pY416 labeling varied considerably within this population of spermatozoa, and a number of cells (
10%) displayed relatively weak tail labeling. In contrast, only 5% of non-capacitated cells revealed any tail staining (Fig. 5A). The specificity of these results was confirmed by the inclusion of an irrelevant mouse monoclonal antibody (anti-His Tag) and a secondary antibody-only control, both of which revealed only minimal background fluorescence (Fig. 5C). These data further support the flagellar localization of this activated kinase, as suggested earlier by western blot analyses of the head and tail fractions from human sperm (Fig. 2). Additionally, these data are in keeping with the concept that tyrosine phosphorylation occurs mainly in the tail region during the capacitation of these cells (Baker et al., 2006; Mitchell et al., 2007).
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Inhibition of src kinase suppresses capacitation-associated tyrosine phosphorylation but not hyperactivation of human spermatozoa
In light of our previous evidence indirectly implicating SRC as a key intermediate of the tyrosine phosphorylation cascade associated with human sperm capacitation, we next investigated whether the direct pharmacological inhibition of this enzyme could suppress this signaling pathway. For this purpose, populations of human spermatozoa were incubated in the presence of the specific SRC-family kinase inhibitor, SU6656. Similar studies were also conducted with H89, a potent PKA inhibitor. The effects of each treatment were monitored by assessing the levels of tyrosine phosphorylation, SRC kinase activity (using anti-pY416) and hyperactivation in capacitating spermatozoa.
As anticipated, the addition of 25 µM H89 to capacitating human spermatozoa resulted in a modest decrease in the tyrosine phosphorylation status of these cells to a level that appeared comparable to that seen in the non-capacitated control population. Additionally, this was enhanced in populations of sperm incubated in media in which capacitation and tyrosine phosphorylation had been actively driven through the inclusion of dibutryl cAMP (a cell permeant analog of cAMP) and ptx (a phosphodiesterase inhibitor). The levels of phosphorylated SRC were similarly reduced following incubation of sperm with 25 µM H89 (Fig. 6A and C). An identical result was obtained when the tyrosine phosphorylation status of these cells was assessed by immunocytochemistry (Fig. 6B and D). Indeed, it is noteworthy that the suppression of phosphotyrosine expression appeared even more dramatic when assessed using immunocytochemistry compared with immunoblotting. This apparent discrepancy is explained by the fact that the SDS extraction protocol employed in the current study failed to solubilize all of the phosphorylated proteins associated with cytoskeletal elements of the sperm tail, such as the fibrous sheath. Indeed, this structure remained strongly labeled with anti-phosphotyrosine even after treatment with a stringent SDS extraction protocol.
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In order to better understand the functional impact of SRC activation during capacitation, we examined the effects of H89 and SU6656 on sperm motility. Sperm populations incubated in standard BWW media and those driven to capacitate by supplementing the BWW with 1 mM ptx and 1 mM dibutryl cyclic AMP, were generally characterized by
70% motile cells and 50% displaying forward progressive motility (the data for both motility and progressive motility from each replicate was normalized against a population of spermatozoa incubated in complete BWW media). In contrast, pre-incubation with H89 was correlated with a significant reduction in both the percentage of motile spermatozoa and those exhibiting progressive patterns of motility (Fig. 7). Interestingly, H89 treatment failed to suppress the induction of hyperactivated motility that was observed in approximately 20% of both the treated and untreated capacitated sperm populations.
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Consistent with the results obtained following PKA inhibition, the addition of SU6656 to capacitating human spermatozoa also led to a dramatic reduction in the overall level of phosphotyrosine expression within these cells (Fig. 6C). As expected, this reagent also suppressed the levels of phosphorylated SRC within these cells. However, despite these overt effects, SU6656 treatment failed to significantly suppress either the percentage of motile spermatozoa, the percentage of sperm displaying forward progressive motility or the induction of hyperactivated motility (
21% hyperactivated, data not shown) in capacitated spermatozoa. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary dataFundingReferences |
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Mammalian spermatozoa undergo a series of maturational events as they progress through the male and female reproductive tracts before acquiring the ability to fertilize an oocyte. Emerging evidence indicates that one of the most important changes associated with the capacitation-dependent stages of this maturation process, is the induction of a unique signal transduction pathway that culminates in the tyrosine phosphorylation of a number of sperm proteins (Visconti et al., 1995a,b). This time-dependent increase in tyrosine phosphorylation is, in turn, held to be essential for many facets of sperm function including the induction of hyperactivation and the ability to engage in sperm–zona binding and initiate fertilization (Asquith et al., 2004; Baker et al., 2006).
The stimulation of capacitation-associated tyrosine phosphorylation appears to involve the interplay of a number of signaling pathways. For instance, a number of studies have raised the possibility that phosphotyrosine expression is regulated in part by redox-cycling, a notion supported by the demonstration that this event can be either directly or indirectly driven by the generation of reactive oxygen species such as superoxide anion and/or hydrogen peroxide (Aitken et al., 1995, 1998; O'Flaherty et al., 2006). It is also evident that spermatozoa possess a unique signaling cascade that incorporates a novel, soluble form of adenylyl cyclase (sAC) (Esposito et al., 2004; Xie et al., 2006). The activation of sAC leads to an abundant production of the second messenger, cAMP, and the concomitant stimulation of the cAMP-dependent protein kinase, PKA (White and Aitken, 1989; Visconti et al., 1995b; Aitken et al., 1998). As a consequence of cAMP interaction with the regulatory subunits of PKA, the tetrameric holoenzyme dissociates, liberating the two catalytic subunits from the inhibitory action of the two regulatory subunits. Despite overwhelming recognition of the central role of PKA in capacitation-associated sperm signaling, the fact that this kinase modulates the activity of its effectors via serine/threonine phosphorylation precludes its direct involvement in phosphotyrosine expression. Rather it must orchestrate this cellular response indirectly through activation of a promiscuous tyrosine kinase, the identity of which remains the subject of active debate. Whereas previous candidates include mitogen-activated protein kinases (MAPKs) (Luconi et al., 1998) and PI 3-kinase (Luconi et al., 2004), in this study we present several lines of evidence that SRC may act as one of the key mediators of PKA-activated tyrosine kinase activity in human spermatozoa, as has also recently been reported in the mouse (Baker et al., 2006).
Although PKA has broad specificity, the phosphorylation of its target substrates is regulated in part by the subcellular distribution of the kinase. This, in turn, is defined by A-kinase anchor proteins (AKAPs), a family of distinct but functionally conserved proteins that tether PKA to specific subcellular compartments. In addition, AKAPs also interact with additional signaling proteins, including SRC, in other cell types including carcinoma cell lines (Tao et al., 2007) and thus may serve as scaffolding proteins that coordinate the spatial and temporal activity of signaling pathways downstream of PKA (Burton and McKnight, 2007). This notion is consistent with the fact that we have shown that both PKA and SRC display a highly polarized distribution, with highest expression within the sperm flagellum, as has previously been reported for SRC (Kumar and Meizel, 2005) and several sperm AKAPs (Carrera et al., 1996; Mandal et al., 1999). Furthermore, we provide evidence for the physical interaction of PKA and SRC in capacitated, but not in uncapacitated spermatozoa, through an immunoprecipitation strategy. However, we cannot preclude the possibility that SRC is also capable of interacting directly with AKAP proteins independent of PKA; thus the participation of AKAPs in the formation of this signaling complex awaits further investigation.
Previous findings from our laboratory have demonstrated that tyrosine phosphorylation of human spermatozoa can be elicited in a rapid, dose-dependent and lectin-specific manner via incubation in wheat germ agglutinin (WGA). This response to WGA challenge is however abrogated by pre-incubation of spermatozoa with either the PKA inhibitor, H89, or the broad spectrum SRC family kinase inhibitors, PP1 and PP2, in addition to the more specific inhibitor SU6656 (Nixon et al., 2005). Commensurate with these findings, we have shown in the present study that capacitation-associated phosphotyrosine expression is also susceptible to similar pharmacological inhibition.
Our attempts to directly measure SRC kinase activity proved unsuccessful because of the limited amount of enzyme present in spermatozoa. However, previous studies in T-cells, carcinoma cell lines, embryo fibroblasts and platelets have highlighted the fact that upon activation, SRC undergoes a process of autophosphorylation at the tyrosine residue 416 (Cartwright et al., 1989; Katagiri et al., 1989; Bagrodia et al., 1993; Kralisz and Cierniewski, 2000). We were therefore able to exploit this property to assess SRC activity, albeit indirectly, in capacitating sperm populations. As anticipated, this approach revealed a significant increase in SRC activity during capacitation. Similar to global phosphotyrosine expression, this activity was also suppressed through the action of the H89 and SU6656 inhibitors (Fig. 6). Although we did not examine this in the present study, it is of interest that SRC activity can also be positively modulated by hydrogen peroxide (Suzaki et al., 2002), consistent with a role for this kinase in the redox regulation of the signaling cascades that drive capacitation (Aitken et al., 1995, 1998).
Consistent with the distribution of PKA and SRC, the sperm flagellum appears to be the major target for tyrosine phosphorylation in most species examined, including the human (Carrera et al., 1996; Leclerc et al., 1997; Mitchell et al., 2007). In keeping with with these findings, proteomic analyses have revealed that a number of the phosphorylated proteins in human sperm are in fact predominantly or exclusively expressed in the flagella. These include: fibrous sheath AKAPs (AKAP3 and AKAP4) (Carrera et al., 1996; Mandal et al., 1999), CABYR (calcium-binding and tyrosine phosphorylation-regulated protein; Naaby-Hansen et al., 2002), tubulin (Kadam et al., 2007), dynein, and chaperones such as heat shock protein (HSP)90 (Ecroyd et al., 2003), HSP40, HSP60 and HSP70 (Mitchell et al., 2007). It is of considerable interest that a number of these proteins (such as HSP90 and tubulin) are known substrates for SRC.
The restricted pattern of phosphotyrosine expression is compatible with a physiological role for this process in the regulation of sperm motility. Our studies confirmed that PKA inhibition induced a concomitant reduction in the percentage of motile human sperm and those displaying patterns of progressive motility, in keeping with the central role proposed for adenylyl cyclase and PKA in the regulation of sperm movement (Skalhegg et al., 2002; Hess et al., 2005). However, despite its apparent role in the regulation of tyrosine phosphorylation, we observed no overt alterations in motility levels following SRC inhibition with SU6656. Although the reason for this apparent discrepancy is not immediately apparent, it is possible that since SU6656 is a competitive inhibitor of ATP binding to SRC, the protein may retain enough residual kinase activity to maintain normal motility levels. Alternatively, it is possible that SRC substrates do not form part of the biochemical machinery responsible for directly regulating flagellar function or that it may fulfill a supportive, not obligatory, role. Indeed, the fact that SRC inhibition did not completely eliminate tyrosine phosphorylation suggests that other signal transduction pathways are likely to be working simultaneously. In this context, it is noteworthy that we also identified a number of additional SRC family kinases in human spermatozoa, including: LYN, YES, and FYN, all of which shared a similar pattern of flagellar localization to SRC. It is therefore possible that there is some redundancy in the molecular mechanisms driving tyrosine phosphorylation in human spermatozoa. Tyrosine phosphorylation itself seems to be an indispensable element in the induction of hyperactivated movement (Bajpai et al., 2003; Buffone et al., 2004). However, phosphotyrosine expression is probably not the only factor; other conditions might have to be met before this pattern of movement is enabled. This conclusion is consistent with a recent study indicating that the composition of incubation media can have a profound effect on the hyperactivation of human spermatozoa via mechanisms that are quite independent of PKA (Moseley et al., 2005). An obvious candidate for this additional factor is the regulated availability of intracellular calcium via the CatSper complex (Suarez and Ho, 2003; Ishijima et al., 2006; Qi et al., 2007) working in concert with tyrosine phosphorylation mediated by SRC-family kinases to induce a hyperactivated state.
In conclusion, this study has provided clear evidence for the involvement of a non-receptor tyrosine kinase, SRC, in regulating the tyrosine phosphorylation cascade associated with sperm capacitation. The fact that inhibition of SRC did not completely suppress tyrosine phosphorylation or perturb the expression of hyperactivated movement suggests that this is but one component of a complex signal transduction pathway that exhibits a degree of functional redundancy in order to ensure levels of tyrosine phosphorylation compatible with the attainment of a capacitated state.
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Supplementary data are available at http://molehr.oxfordjournals.org/.
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The authors wish to acknowledge the financial support from the Australian Research Council, Centre of Excellence in Biotechnology and Development and the New South Wales Department of State and Regional Planning.
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Submitted on October 31, 2007; resubmitted on January 13, 2008; accepted on January 25, 2008.
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