Mol. Hum. Reprod. Advance Access originally published online on March 2, 2004
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Molecular Human Reproduction, Vol. 10, No. 5, pp. 355-363, 2004
© European Society of Human Reproduction and Embryology 2004
Phosphorylation of the Arginine-X-X-(Serine/Threonine) motif in human sperm proteins during capacitation: modulation and protein kinase A dependency
Urology Research Laboratory, Royal Victoria Hospital and Faculty of Medicine, McGill University, Montréal, Québec, Canada H3A 1A1
1 To whom correspondence should be addressed at: Urology Research Laboratory, H6.44, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1. e-mail: coflaher{at}yahoo.com
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ABSTRACT |
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Sperm capacitation is a complex process that involves a protein kinase A (PKA)-dependent tyrosine phosphorylation of proteins. We studied the time-course, the modulation and the cellular localization of the phosphorylation of the Arginine-X-X-(Serine/Threonine) motif, characteristic of PKA substrates, in sperm proteins during capacitation. There was an increased phosphorylation of 80 (p80) and 105 (p105) kDa protein bands in human sperm treated with different capacitation inducers. Phosphorylation of p80 and p105 induced by fetal cord serum ultrafiltrate or the combination of 3-isobutyl-1-methylxanthine and dibutyryl cAMP was prevented by H89 and Rp-adenosine-3',5'-cyclic monophosphorothionate, confirming the involvement of PKA in this effect. Inhibitors of protein kinase C, receptor type tyrosine kinase and mitogen-activated protein kinase kinase did not affect the Arginine-X-X-(Serine/Threonine) motif phosphorylation. Non-receptor type protein tyrosine kinase inhibitors, PP2 and herbimycin A, enzymatic antioxidants and a nitric oxide synthase inhibitor prevented the phosphorylation of p80 and p105 when sperm were incubated with fetal cord serum ultrafiltrate. The phosphorylated Arginine-X-X-Serine/Threonine motif was immunolocalized all along the flagellum and the fluorescent signal was higher in capacitating than in non-capacitating sperm. These results show for the first time the presence of a PKA-dependent phosphorylation of proteins in human sperm capacitation and its upstream modulation by reactive oxygen species and non-receptor type protein tyrosine kinase.
Key words: Key words: protein kinases/reactive oxygen species/spermatozoa/sperm capacitation/signal transduction
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Introduction |
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Mammalian sperm must complete a series of morphological and metabolic changes collectively termed as ‘capacitation’ in order to acquire fertilizing capacity (Yanagimachi, 1994; de Lamirande et al., 1997). This is a poorly understood and complex process that involves different signal transduction elements, such as protein kinases A (PKA), C (PKC), protein tyrosine kinase (PTK), and the extracellular signal-regulated kinase (ERK) signalling pathway (de Lamirande et al., 1997; de Lamirande and Gagnon, 2002; Thundathil et al., 2002; Visconti et al., 2002).
The presence and activity of PKA have already been reported in bovine (Garbers et al., 1973), mouse (Visconti et al., 1997) and human (Lefièvre et al., 2002) sperm. The inhibition of sperm capacitation and the related protein tyrosine phosphorylation by H89 and Rp-adenosine-3',5'-cyclic monophosphorothionate (Rp-cAMPS), both specific PKA inhibitors (Uguz et al., 1994; Leclerc et al., 1996; Galantino-Homer et al., 1997), suggested that PKA is involved in these processes. Conversely, treatment of sperm with phosphodiesterase inhibitors (ex: 3-isobutyl-1-methylxanthine (IBMX)] alone or in combination with a cell permeant analogue of cAMP (dibutyryl cAMP, dbcAMP) promoted sperm capacitation, confirming the importance of PKA in this process (Visconti et al., 1995; Leclerc et al., 1996). In human sperm treated with fetal cord serum ultrafiltrate (FCSu) as a capacitating agent, there was a 30% increase in PKA activity 30 min after the beginning of the incubation period (Lefièvre et al., 2002).
PKA is a ubiquitous tetrameric enzyme containing two regulatory (R) and two catalytic (C) subunits, and its activity is dependent on cAMP. PKA phosphorylates proteins on Serine (Ser) and Threonine (Thr) within the motif Arginine (Arg)-X-X-Ser/Thr (X represents any amino acid) (Bruce et al., 2002; Grøndborg et al., 2002). In human and mouse sperm, PKA was found in both the acrosomal cap and the flagellum (Pariset and Weinman, 1994; Visconti et al., 1997).
The cAMP/PKA system is needed for the tyrosine phosphorylation of 81 and 105 kDa proteins that occurs during capacitation induced by FCSu, bovine serum albumin (BSA), follicular fluid ultrafiltrate (FFu), progesterone, dbcAMP, and/or IBMX (Uhler et al., 1992; de Lamirande and Gagnon, 1995; Leclerc et al., 1996; Aitken et al., 1998; de Lamirande et al., 1998; Herrero et al., 2000). These proteins are from the fibrous sheath (Visconti et al., 1995; Aitken et al., 1998; Leclerc et al., 1998) and are antigenically related to A kinase anchoring proteins (AKAP) (Carrera et al., 1996).
There is accumulating evidence for the participation of reactive oxygen species (ROS) such as the superoxide anion (O2–•), hydrogen peroxide (H2O2) and nitric oxide (NO•) in human sperm capacitation (de Lamirande et al., 1997; Aitken et al., 1998; Herrero and Gagnon, 2001), and the associated triggering of the protein tyrosine phosphorylation (Leclerc et al., 1997; Aitken et al., 1998). Levels of cAMP are increased by NO• (Herrero et al., 2000), H2O2 (Aitken et al., 1998) and O2–• (Zhang and Zheng, 1996). However, superoxide dismutase (SOD, a O2–• scavenger) did not block the IBMX-induced capacitation, suggesting the participation of O2–• upstream of PKA during this process (de Lamirande et al., 1997).
Although there is consensus that PKA activity is associated with the increase of protein tyrosine phosphorylation (Visconti et al., 1995; Leclerc et al., 1996, 1997; Aitken et al., 1998), the events occurring between the early activation of PKA (Visconti et al., 1997; Lefièvre et al., 2002) and the late protein tyrosine phosphorylation remain unknown. It was hypothesized that PKA phosphorylates some enzymes/proteins as an intermediate step and that these phosphorylated PKA substrates are involved in the capacitation-related protein tyrosine phosphorylation. Therefore, our first objective was to determine whether the level of phosphorylated Arg-X-X-Ser/Thr motif, characteristic of PKA substrates, was modified in sperm proteins during capacitation. The second objective was to study the regulation of this phosphorylation by ROS and signal transduction kinases and determine its cellular localization.
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Materials
The following reagents were purchased from Sigma Chemical Company (USA): BSA (fatty acid-free), IBMX, dbcAMP (N6,2'-O-dibutyryl cAMP), progesterone, H89 {N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulphonamide}, L-NAME (NG-nitro-L-arginine methyl ester), D-NAME (NG-nitro-D-arginine methyl ester), phospho-Serine (phospho-Ser), phospho-Threonine (phospho-Thr) and phospho-Tyrosine (phospho-Tyr). DPI (diphenyliodonium chloride) was bought from Aldrich Chemical Company (USA). Spermine NONOate [N-(2-aminoethyl)-N-(2-hyrdoxy-3-methrosohydrazino)-1,2-ethylenedeamine] was bought from Cayman Chemical Company (USA). PD98059 (2'-amino-3'-methoxyflavone), chelerythrine, herbimycin A, PP2 {4-amino-5-(4-chloropenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine}, PP3 (4-amino-7-phenylpyrazol[3,4-d]-pyrimidine), tyrphostin A47, U126 [1,4-diamino-2,3-dicyano-1,4bis(2-aminophenylthio)butadiene], okadaic acid, bovine liver catalase (21 000 IU/mg), and Rp-cAMPS were purchased from Calbiochem (USA). SOD from bovine erythrocytes was bought from Roche Molecular Biochemicals (Canada). Percoll was purchased from Amersham Pharmacia Biotech (Canada). Nitrocellulose (0.22 µm pore size; Osmonics Inc., USA), the antibody raised against the phosphorylated Arg-X-X-Ser/Thr motif (anti-phospho PKA substrates antibody; Cell Signaling Technology, USA), donkey anti-rabbit IgG conjugated to horseradish peroxidase (Cedarlane Laboratories Ltd, Canada), an enhanced chemiluminiscence kit (Lumi-Light; Roche Molecular Biochemicals) and radiographic films (Fuji, Japan) were used for immunodetection of blotted proteins. The blocking agent of the anti-Arg-X-X-phospho (Ser/Thr) antibody was generously provided by Cell Signaling Technology, USA). Alexa Fluor 555 conjugate of streptavidin and Prolong Antifade kit were purchased from Molecular Probes (USA). All other chemicals were at least of reagent grade.
Fetal cord blood was collected at the birthing centre of the Royal Victoria Hospital (Montréal, QC, Canada) and follicular fluid was collected from pre-ovulatory follicles after gonadotrophin stimulation at the IVF centre of the Royal Victoria Hospital. In both cases, informed consent was obtained from the patients and the ethics board of the Royal Victoria Hospital approved the present study. Fetal cord blood and follicular fluid samples were centrifuged (1000 g, 30 min, 4°C), and supernatants were pooled and frozen at –20°C until used. The FCSu and FFu were prepared from three pools of 14–24 different samples using YM3 membranes with an exclusion limit of 3 kDa (Amicon, Canada) (de Lamirande and Gagnon, 1995). Silver stain indicated that no protein was present in 10% polyacrylamide gels loaded with FCSu or FFu alone. FCSu was used as capacitation inducer as it was shown to trigger capacitation and hyperactivated motility to similar levels and with similar kinetics as other agents such as BSA (de Lamirande et al.., 1997). Furthermore, as observed in other systems, FCSu-induced capacitation is associated with a cAMP-dependent tyrosine phosphorylation of fibrous sheath proteins (Leclerc et al., 1997), the generation of ROS (de Lamirande and Gagnon, 1995; Herrero et al., 2000), activation of PKA (Lefièvre et al., 2002), and modifications of the sulphydryl content of Triton-soluble proteins (de Lamirande and Gagnon, 2003).
Inhibitors and activators used in this study were dissolved in water or dimethylsulphoxide (DMSO). The concentration of DMSO in the incubation media never exceeded 1% (v/v), a condition that does not affect sperm capacitation. None of the chemicals tested caused a decrease in sperm motility over a 3.5 h incubation period at 37°C.
Sperm preparation
Semen samples from 12 healthy volunteers used in this study were normal according to World Health Organization (1992) criteria. Semen samples were washed on four-layer (95–65–40–20%) Percoll gradient buffered in HEPES-balanced saline (115 mmol/l NaCl, 4 mmol/l KCl, 0.5 mmol/l MgCl2, 14 mmol/l fructose, 25 mmol/l HEPES, pH 8.0). Samples were centrifuged for 30 min at 2300 g, and sperm at the 65–95% Percoll interface and in the 95% Percoll layer were pooled and diluted to 250x106 cells/ml with the 95% Percoll solution. Only samples in which progressive motility was >70% were used. Sperm were further diluted to 50x106 cells/ml in Biggers–Whitten–Whittingham medium (BWW) (Biggers et al., 1971) devoid of bicarbonate and BSA and containing 1 mmol/l CaCl2 and 25 mmol/l HEPES (pH 8.0).
SDS–PAGE and immunobloting
At the end of each experiment, treated samples were supplemented with electrophoresis buffer containing vanadate (100 µmol/l), ß-glycerolphosphate (20 mmol/l), sodium fluoride (5 mmol/l), and okadaic acid (10 nmol/l), incubated at 100°C for 5 min and then centrifuged at 21 000 g for 5 min. Sperm proteins were electrophoresed on 10% polyacrylamide gels and electrotransferred to nitrocellulose membranes using 10 mmol/l CAPS (3-cyclohexylamino-1-propane sulphonic acid) buffer (pH 11) containing 10% methanol. The membranes were incubated with a solution of skim milk (5%, w/v) in Tris (20 mmol/l, pH 7.8)-buffered saline containing Tween 20 (0.1%, v/v) (TTBS) for 20 min. We used an antibody that detects a phosphorylated motif (Arg-X-X-Ser/Thr) characteristic of PKA substrates. It was diluted 1:1000 (v/v) in TTBS supplemented with 25 mg/ml BSA and 0.1% (w/v) sodium azide and then incubated with the membrane overnight at 4°C. After washing with TTBS, membranes were incubated with donkey anti-rabbit IgG conjugated with horseradish peroxidase [diluted 1:2500 (v/v) in TTBS] for 45 min at 20°C and washed again with TTBS. Positive immunoreactive bands were detected using the Lumi-Light chemiluminiscence kit. At the end of all the experiments, blots were rinsed in distilled water and silver-stained (Jacobson and Karsnas, 1990) to ascertain that the amount of protein loaded in each well was the same.
The anti-Arg-X-X-phospho-(Ser/Thr) antibody was preadsorbed with the blocking agent, as recommended by Cell Signaling Technology for 2 h at 20°C, to confirm the specificity of the antibody (Figure 1B). The anti-Arg-X-X-phospho-(Ser/Thr) antibody was also preadsorbed with a combination phospho-Ser, phospho-Thr and phospho-Tyr (each of them at a molar concentration 10 000-fold higher than that of the antibody) to confirm that the antibody did not non-specifically recognize any phosphorylated amino acid.
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Immunolocalization of the Arg-X-X-phospho-(Ser/Thr) motif in capacitating sperm
Sperm suspensions were incubated without or with FCSu (10% v/v) at 37°C for 30 min and prepared for immunocytochemistry. Smears were prepared on Superfrost Plus slides (Fischer Scientific, Canada). After permeabilization by methanol and rehydration, smears were treated with 5% goat serum in PBS for 30 min, washed with PBS containing 0.1% Triton X-100 (PBS-T), and incubated with the anti-Arg-X-X-(phospho-Ser/Thr) antibody (dilution 1:100) for 2 h at 20°C. Smears were then washed with PBS-T and incubated with biotinylated goat anti-rabbit antibody (dilution 3:1000) for 1 h, and then with an Alexa Fluor 555 conjugate of streptavidin (1:500 w/v) in PBS-T. Smears were mounted with Prolong Antifade and observed under a Carl Zeiss (Germany) Axiophot microscope (exciter filter BP450–490) at x1000 magnification. As controls, smears were incubated with the anti-Arg-X-X-(phospho-Ser/Thr) antibody preadsorbed with the blocking agent or with the biotinylated goat anti-rabbit antibody alone or with the Alexa Fluor 555 conjugate of streptavidin alone.
Phosphorylation of the Arg-X-X-Ser/Thr motif during capacitation: time-course and involvement of PKA
Sperm samples in BWW were incubated without (control) or with the capacitation inducer FCSu (10%, v/v) and aliquots were taken at 0, 5, 15, 30, 60 and 120 min. Because the phosphorylation reached a plateau after 15–30 min incubation and because the maximal PKA activity of FCSu-treated sperm occurs at 30 min (Lefièvre et al., 2002), the following experiments were performed with a 30 min incubation time. Sperm were also incubated with progesterone (10 µmol/l), FFu (10% v/v), IBMX (0.5 mmol/l), the combination IBMX (0.1 mmol/l) + dbcAMP (1 mmol/l) or BSA (3 mg/ml). All incubations were performed at 37°C. Only for BSA-treated sperm were cells submitted to a one-step wash over a 20% Percoll layer (2000 g, 5 min) to remove most the BSA present in the incubation medium; sperm were collected by aspiration of the loose pellet through the Percoll layer as previously described (de Lamirande et al., 1998).
To confirm that PKA is involved in the phosphorylation of proteins recognized by the antibody used, sperm were incubated with FCSu (10% v/v) or IBMX (0.1 mmol/l) + dbcAMP (1 mmol/l) in the presence of H89 (10 µmol/l), Rp-cAMPS (200 µmol/l) or the combination of H89 + Rp-cAMPS.
A Triton X-100 extraction was also performed. Control and capacitating sperm were concentrated to 200x106 cells/ml. After the addition of vanadate (100 µmol/l), ß-glycerolphosphate (20 mmol/l), sodium fluoride (5 mmol/l), and okadaic acid (10 nmol/l), sperm were incubated with Triton X-100 (0.2%, v/v) for 10 min on ice, and then centrifuged (12 000 g). The Triton-soluble and -insoluble (resuspended to the original volume with HEPES-balanced saline containing vanadate, ß-glycerolphosphate, sodium fluoride, okadaic acid and Triton X-100 at the same concentrations used above) fractions were used for immunoblotting as described above.
Phosphorylation of the Arg-X-X-Ser/Thr motif during capacitation: regulation by ROS and kinases
The role of ROS on the phosphorylation of the Arg-X-X-Ser/Thr motif in human sperm was first studied by the addition of 50 µmol/l H2O2 or 100 µmol/l spermine-NONOate (sp-NONOate) to sperm. The effect of catalase (0.1 mg/ml), SOD (0.1 mg/ml), DPI (100 µmol/l), L-NAME (1 mmol/l) or D-NAME (1 mmol/l) on the phosphorylation of the Arg-X-X-(Ser/Thr) motif induced by FCSu or IBMX + dbcAMP was also studied. In another set of experiments, the effect of SOD + catalase + L-NAME on sperm treated with IBMX + dbcAMP was studied.
To determine the role of kinases in the phosphorylation of the Arg-X-X-Ser/Thr motif, sperm were supplemented or not with FCSu (10%, v/v), in the absence or presence of chelerythrine (10 µmol/l), tyrphostin A47 (10 µmol/l), PD98059 (100 µmol/l) or U126 (0.3 µmol/l), herbimycin A (10 µmol/l), PP2 (10 nmol/l) or PP3 (10 nmol/l). All the inhibitors used in this study were added to sperm 30 min before the capacitation inducers.
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Results |
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Phosphorylation of the Arg-X-X-Ser/Thr motif increases in sperm incubated under capacitating conditions
The antibody raised against the Arg-X-X-(phospho-Ser/Thr) motif recognized few sperm proteins bands of 40, 80, 105, 116 and 140 kDa (Figure 1A). When sperm were incubated under capacitating conditions (FCSu), there was an increased phosphorylation of the 80 (p80) and 105 (p105) kDa protein bands as compared with that noted in sperm in BWW alone. This FCSu-related increase was observed as early as 5 min after the beginning of the incubation, plateaued at 15 and 30 min and then decreased (Figure 1A). In sperm incubated with BWW alone there was a basal phosphorylation which was always lower than that observed in capacitating sperm, and even decreased with time. Because of this early phosphorylation, and because the maximal PKA activity of FCSu-treated sperm occurs at 30 min (Lefièvre et al., 2002), the following experiments were performed with a 30 min incubation time. Only the two protein bands p80 and p105 will be presented since the changes in the Arg-X-X-(phospho-Ser/Thr) motif related with capacitation were observed only in these proteins. We also observed a time-dependent diminution of the phosphorylation of the Arg-X-X-(Ser/Thr) motif in proteins of 40 and 140 kDa, but there was no difference between sperm incubated in the presence of FCSu or BWW alone and the phosphorylation decreased with time. Blots of sperm proteins incubated with the second antibody alone did not present any bands (Figure 1A).
The two protein bands p80 and p105 were not detected when the anti-Arg-X-X-(phospho-Ser/Thr) antibody was preadsorbed with the blocking agent, confirming the specificity of the antibody. (Figure 1B). Furthermore, the anti-Arg-X-X-(phospho-Ser/Thr) antibody preadsorbed with a mixture of phospho-Ser, phospho-Thr and phospho-Tyr was as efficient as the untreated antibody in binding to p80 and p105 (Figure 1B), confirming that the antibody did not recognize unspecifically all phospho amino acid. To confirm that the same amount of protein was loaded in all the wells, blots were always silver-stained at the end of the experiments (Figure 1A).
The proteins p80 and p105 carrying the Arg-X-X-(phospho-Ser/Thr) motif were found in the sperm Triton-insoluble fraction (Figure 2). The protein band at 40 kDa recognized by the antibody in whole sperm samples and not modified by capacitation conditions (Figure 1A) was found in the Triton-soluble fraction (Figure 2).
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Other inducers used to study in vitro capacitation, FFu, progesterone (Uhler et al., 1992; de Lamirande and Gagnon, 1995; de Lamirande et al., 1998; Leclerc et al., 1998), IBMX, IBMX + dbcAMP (Visconti et al., 1995; Leclerc et al., 1996) and BSA (Aitken et al., 1998; Herrero et al., 2000) were also tested. The effects of different biological fluids and pharmacological compounds used in this study on human sperm capacitation are summarized in Table I. There was an increase in the phosphorylation of the Arg-X-X-Ser/Thr motif in all the cases (Figure 3). These data indicated that the effect observed was related to the capacitation process and was not specific for FCSu. Moreover, the levels of phosphorylation were low in sperm incubated in BWW alone and the effect of FCSu was reproducible as the data presented in Figure 3 are from three different donors.
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PKA is involved in the phosphorylation of the Arg-X-X-Ser/Thr motif
The Arg-X-X-(Ser/Thr) motif is characteristic of PKA substrates. The pharmacological increase of intracellular cAMP by treatment with IBMX or IBMX + dbcAMP induces sperm capacitation (Leclerc et al., 1996; Aitken et al., 1998) and increases in Arg-X-X-(Ser/Thr) phosphorylation (Figure 4A), suggesting a role for PKA. Conversely, the phosphorylation of the Arg-X-X-Ser/Thr motif in p80 and p105 induced by FCSu was partially prevented by H89, (a specific PKA inhibitor) or Rp-cAMPS (a cell-permeant inactive analogue of cAMP), but the inhibition was complete when both compounds were present (Figure 4B). Additional indication of the involvement of PKA in the phosphorylation of the Arg-X-X-(Ser/Thr) motif was obtained when pharmacological conditions, IBMX + dbcAMP, were used to promote sperm capacitation. The prevention of the phosphorylation of these bands was partial with Rp-cAMPS and complete with H89 or the combination of H89 + Rp-cAMPS (Figure 4A).
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ROS modulate the phosphorylation of the Arg-X-X-Ser/Thr motif in capacitating human sperm
The role of ROS, such as O2–•, H2O2 and NO• in human sperm capacitation is now well established (de Lamirande et al., 1997; Aitken et al., 1998; Herrero et al., 2001). As ROS were shown to affect intracellular cAMP levels (Zhang and Zheng, 1996; Aitken et al., 1998; Herrero et al., 2000) and therefore PKA activity, the possible role of ROS in the modulation of the phosphorylation of the Arg-X-X-Ser/Thr motif detected in previous experiments was evaluated. SOD and catalase (scavengers of O2–• and H2O2, respectively) and L-NAME (a NO• synthase inhibitor) totally (on p80) and partially (on p105) prevented the increase in Arg-X-X-Ser/Thr phosphorylation of p80 and p105 bands in sperm treated with FCSu (Figure 5A); D-NAME (the inactive analogue of L-NAME) did not prevent the phosphorylation. The combination of SOD, catalase and L-NAME completely prevented the phosphorylation in both p80 and p105 (Figure 5A). On the other hand, when sperm were incubated with IBMX + dbcAMP in the presence of SOD, catalase or L-NAME, alone or in combination, no inhibition of the phosphorylation of the Arg-X-X-Ser/Thr motif on p80 and p105 was observed (Figure 5B). Exogenous addition of ROS using sp-NONOate (NO• donor) and H2O2 caused an increase in the intensity of the p80 and p105 bands (Figure 5C). The combination of xanthine and xanthine oxidase as a source of O2–• could not be used since it also requires addition of catalase to remove the H2O2 generated, an enzyme that prevents the effect of FCSu (Figure 5A). However, DPI—an inhibitor of flavin-containing enzymes such as NADPH oxidases (Hancock and Jones, 1987; O’Donnell et al., 1993) that inhibits the O2–• production in capacitating human sperm (de Lamirande et al., 1997)—caused a decrease in the phosphorylation of the Arg-X-X-Ser/Thr motif both in capacitating and non capacitating sperm, suggesting an involvement of O2–• in the modulation of the phosphorylation of this motif during capacitation with FCSu (Figure 5A).
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PTK but not PKC or mitogen-activated protein kinase kinase (MEK) is involved in the modulation of the phosphorylation of the Arg-X-X-Ser/Thr motif in capacitating sperm
Herbimycin A and PP2 (inhibitors of non-receptor type PTK), but not tyrphostin A47 (inhibitor of receptor type PTK), chelerythrine (inhibitor of PKC) or PD98059 (100 µmol/l) and U126 (0.3 µmol/l) (inhibitors of MEK) totally prevented the phosphorylation of the Arg-X-X-Ser/Thr motif in FCSu-treated sperm (Figure 6A). Moreover, tyrphostin A47 and chelerythrine produced a slight increase in the phosphorylation pattern. PP3 (the inactive analogue of PP2) did not inhibit the phosphorylation of this motif (data not shown). On the other hand, the phosphorylation of the Arg-X-X-(Ser/Thr) motif in sperm treated with IBMX + dbcAMP was not prevented but rather increased by hermymicin A or PP2 (Figure 6B).
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Immunolocalization of the Arg-X-X-(phospho-Ser/Thr) motif in human sperm
Immunocytochemistry with the anti-Arg-X-X-(phospho-Ser/Thr) antibody indicated that sperm proteins containing this motif were all along the flagellum and brighter in the mitochondrial helix region (Figure 7A). The proportion of sperm labelled with the antibody was the same in capacitating as in non-capacitating sperm but the fluorescence was more intense in capacitating than in non-capacitating sperm (Figure 7A). As observed with immunoblots, preadsorption of the antibody with its blocking peptide reduced the signal to that observed with the second antibody alone (Figure 7B).
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Discussion |
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The data presented above indicated that the phosphorylation of the Arg-X-X-(Ser/Thr) motif is increased in p80 and p105 during capacitation induced by different physiological (FCSu, FFu, progesterone, and BSA) and pharmacological (IBMX, dbcAMP alone or in combination) agents and depends, directly or indirectly, on PKA activity. This phosphorylation of p80 and p105 appeared to be modulated by ROS and a non-receptor type PTK, both acting upstream of PKA.
The involvement of PKA during capacitation has been demonstrated by activating this kinase with dbcAMP (direct effect) and IBMX (indirect effect through inhibition of phosphodiesterases), or by using PKA inhibitors such as H89 and Rp-cAMPS (Leclerc et al., 1996; Visconti et al., 1995; Aitken et al., 1998). Furthermore, PKA activity peaks in human sperm 30 min after the beginning of FCSu-induced capacitation (Lefièvre et al., 2002) and it is needed for the next steps (such as tyrosine phosphorylation increasing after 1–2 h of capacitation) of capacitation to proceed (de Lamirande et al., 1997). Although the participation of PKA is well established in sperm capacitation, there are only few reports on the Ser/Thr phosphorylation of sperm proteins during capacitation. Human sperm treated with radioactive phosphate showed an increased phosphorylation of proteins of 78, 71, 56 and 57.5 kDa during capacitation (Furuya et al., 1993). Immunoblotting experiments using anti-phospho-Ser and anti-phospho-Thr antibodies indicated an increase in Ser/Thr phosphorylation in 18, 35 43–55, 94, 110 and 190 kDa Triton-soluble proteins during capacitation (Naz, 1999); however, the effect of kinase inhibitors was not tested, so that no conclusion on the participation of a specific kinase could be drawn.
The antibody against the phosphorylated Arg-X-X-(Ser/Thr) motif was shown to be a powerful tool to study PKA substrates. In a mass spectrometry-based proteomic approach for identification of Ser/Thr-phosphorylated proteins in HeLa and 293T cells, a novel PKA substrate named ‘Frigg’ with a phospho-Ser/Thr with an Arg in the -3 position was recognized using this antibody. There was an increase in the phosphorylation of the Arg-X-X-(Ser/Thr) motif in ‘Frigg’ when this protein was incubated with the catalytic subunit of PKA (Grøndborg et al., 2002). Moreover, forskolin increased the phosphorylation of the Arg-X-X-(Ser/Thr) motif, as evaluated with the anti-Arg-X-X-(phospho-Ser/Thr) antibody, in the inositol 1,4,5-triphosphate receptors of parotid acinar cells; H89 and Rp-cAMPS inhibited this effect (Bruce et al., 2002), confirming the involvement of PKA and the use of the antibody to determine PKA substrates in cells. Here we used the antibody against the Arg-X-X-(phospho-Ser/Thr) motif, in combination with agents that stimulate (directly with dbcAMP, indirectly with IBMX) or inhibit (H89 and Rp-cAMPS) PKA, to study the PKA-dependent Ser/Thr phosphorylation in capacitating human sperm. It is not possible at this time to demonstrate that p80 and p105 are directly phosphorylated by PKA. However, these proteins appeared to include the motif Arg-X-X-(phospho-Ser/Thr) characteristic of PKA substrates and recognized by the antibody. Furthermore, the early phosphorylation of the Arg-X-X-Ser/Thr motif observed in this study is coincident with the maximal PKA activity measured in FCSu-induced capacitation (Lefièvre et al., 2002). Also, the fact that H89 and Rp-cAMPS prevented the increase in phosphorylation of p80 and p105 in FCSu- and IBMX + dbcAMP-treated sperm (Figure 4) strongly suggests that this phenomenon is PKA dependent.
The inducers FCSu, FFu, progesterone, BSA, IBMX and dbcAMP previously shown to trigger capacitation (Uhler et al., 1992; de Lamirande and Gagnon, 1995; Visconti et al., 1995; Leclerc et al., 1996; Aitken et al., 1998; de Lamirande et al., 1998; Herrero et al., 2000) and associated phosphorylation events (Leclerc et al., 1996; de Lamirande and Gagnon, 2002; Thundathil et al., 2002) promoted the Arg-X-X-(Ser/Thr) phosphorylation of p80 and p105 (Figure 3), confirming that the effect observed was dependent on the capacitation process rather than on a specific agent.
Although a significant increase in phosphorylation was always observed on p80 and p105 (Figure 1), there was sometimes a mild increase in the phosphorylation of a 116 kDa protein (Figure 2) that was variable between donors and between samples from the same donor.
The concept that ROS, at low levels, have a positive role in human sperm capacitation has grown in recent years (de Lamirande et al., 1997; Aitken et al., 1998; Herrero et al., 2000). ROS are involved in different capacitation-related events such as the increase in cAMP levels and activation of the cAMP/PKA-dependent pathway (Zhang and Zheng, 1996; Leclerc et al., 1997; Aitken et al., 1998; Herrero et al., 2000), the tyrosine phosphorylation of fibrous sheath proteins (Carrera et al., 1996; Leclerc et al., 1997) and the double phosphorylation of the Thr-Glutamine-Tyr motif in sperm proteins of low (de Lamirande and Gagnon, 2002) and high (Thundathil et al., 2003) molecular masses. The phosphorylation of the Arg-X-X-(Ser/Thr) motif in p80 and p105 was prevented by a combination of ROS scavengers (SOD and catalase) and the NOS inhibitor L-NAME, as well as by an inhibitor of sperm O2–• generation (DPI) during incubation with FCSu (Figure 5A), therefore suggesting a role for ROS in this process. Interestingly, the three ROS, O2–•, H2O2 and NO• that induce sperm capacitation and the related protein tyrosine phosphorylation through the cAMP/PKA-dependent pathway (Leclerc et al., 1996; Aitken et al., 1998; Herrero et al., 2000) also appear to be involved in the phosphorylation of the Arg-X-X-(Ser/Thr) motif. The participation of ROS appears to be upstream of PKA activation since SOD, catalase and L-NAME (Figure 5B) did not prevent the phosphorylation of the Arg-X-X-(Ser/Thr) motif of p80 and p105 in sperm incubated with IBMX + dbcAMP, a condition that bypasses the first steps of sperm capacitation allowing the direct activation of PKA (Visconti et al., 1995; Leclerc et al., 1996).
Herbimycin A and PP2, but not tyrphostin A47, chelerythrine, PD98059 and U126, prevented the phosphorylation of the Arg-X-X-(Ser/Thr) motif, suggesting the participation of non-receptor type PTK (but not MEK or PKC) in the regulation of the PKA-dependent phosphorylation of p80 and p105. Moreover, the lack of inhibition by herbimycin A and PP2 on the protein Arg-X-X-(Ser/Thr) phosphorylation of sperm incubated with IBMX + dbcAMP strongly suggests that a tyrosine kinase acts upstream of PKA activation. This would also imply that at least two different PTK are involved in capacitation; one would act at the beginning of capacitation and be related to PKA activation and the other would be related to the protein tyrosine phosphorylation of fibrous sheath proteins. It is presently not known how the early activation of a PTK could trigger PKA during capacitation. However, a PTK was shown to induce an increase of intracellular cAMP in porcine heart vascular smooth muscle cells (El-Mowafy and White, 1998) and in HT4.7 neural cells (Stringfield and Morimoto, 1997).
Protein tyrosine phosphorylation is associated with capacitation and increases progressively from 1 h after the beginning of incubation under capacitating conditions (Visconti et al., 1995; Leclerc et al., 1996; Aitken et al., 1998). The early PKA-related phosphorylation of the Arg-X-X-(Ser/Thr) motif observed in this study would also be in agreement with the cAMP/PKA dependency of protein tyrosine phosphorylation (Visconti et al., 1995; Leclerc et al., 1996; Aitken et al., 1998).
The molecular masses (Figure 1) and the Triton-insoluble nature (Figure 2) of the proteins recognized by the anti-Arg-X-X-Ser/Thr antibody could suggest that these proteins belong to the fibrous sheath of sperm. However, immunocytochemistry experiments indicated that proteins carrying the Arg-X-X-(phospho-Ser/Thr) motif are located all along the flagellum (Figure 7A) and not strictly on the principal piece as it was observed for proteins carrying phosphorylated Tyr (Leclerc et al., 1997) or double phosphorylated Thr-Glu-Tyr (Thundathil et al., 2003). The fluorescence was more intense in capacitating than in non-capacitating sperm (Figure 7A) but the proportion of sperm labelled with the antibody was the same in the two sperm populations. This observation is important because it indicates another difference between proteins carrying the Arg-X-X-(phospho-Ser/Thr) motif and those carrying phosphorylated Tyr or double-phosphorylated Thr-Glu-Tyr; in the latter cases, capacitation was associated with an increase in the proportion of sperm with bright fluorescence rather than an increase in the level of fluorescence on the whole population of sperm. The nature of the proteins carrying the Arg-X-X-(phospho-Ser/Thr) remains to be elucidated.
In conclusion, this study shows, for the first time, that the phosphorylation of the Arg-X-X-(Ser/Thr) motif, characteristic of PKA substrates, is increased during human sperm capacitation and that this phosphorylation is dependent on PKA and modulated by ROS and non-receptor type protein tyrosine kinase both acting upstream of PKA. These (Ser/Thr)-phosphorylated proteins could be the link between the early increase of PKA activity and the late protein tyrosine phosphorylation during human sperm capacitation.
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Acknowledgements |
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We thank Drs Alice Benjamin and Lucie Morin for collecting fetal cord blood. We also thank the staff of the IVF centre at the Royal Victoria Hospital, McGill University for providing follicular fluid. We wish to acknowledge the participation of volunteers in this study. C.G. was supported by a grant of the Canadian Institutes for Health Research (CIHR), Canada.
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Submitted on December 18, 2003; resubmitted on January 16, 2004; accepted on January 25, 2004.
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