Molecular Human Reproduction, Vol. 8, No. 9, 811-816,
September 2002
© 2002 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Different signal transduction pathways are involved during human sperm capacitation induced by biological and pharmacological agents
1 Urology Research Laboratory, H6.44, Royal Victoria Hospital and McGill University, 687 Pine Avenue West, Montréal, Québec H3A 1A1, Canada
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Human sperm capacitation involves complex signal transduction mechanisms during which double phosphorylation of the threonine-glutamine-tyrosine motif (P-Thr-Glu-Tyr-P) occurs in some sperm proteins. The objective of this study was to investigate the regulation of this process. Fetal cord serum ultrafiltrate (FCSu), follicular fluid ultrafiltrate (FFu), progesterone and a combination of N6,2'-O-dibutyryl cAMP (dbcAMP; cell permeant analogue of cAMP) and 3-isobutyl-1-methylxanthine (IBMX; phosphodiesterase inhibitor) were used as inducers of capacitation alone or in combination with inhibitors of protein kinase A (H89), protein kinase C (chelerythrine), protein tyrosine kinase (tyrphostin A47, PP2) and of dual specificity kinase (MEK-like kinases; PD98059). The level of P-Thr-Glu-Tyr-P in sperm proteins of 80 and 105 kDa during capacitation induced by FCSu, FFu and progesterone was regulated by a similar signal transduction pathway and involved receptor type protein tyrosine kinase and dual specificity kinase (MEK or MEK-like) but not protein kinase A or C. However, the level of P-Thr-Glu-Tyr-P in these sperm proteins during capacitation induced by dbcAMP+IBMX was mainly mediated through protein kinase A and C and receptor type protein tyrosine kinase, but not by dual specificity kinase. In conclusion, human sperm capacitation induced by some biological and pharmacological agents is regulated through very different signal transduction pathways.
dual specificity kinase/protein kinases/protein phosphorylation/sperm/Thr-Glu-Tyr phosphorylation
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Ejaculated sperm must undergo a series of membranous, biochemical and metabolic modifications to attain fertilizing ability, which are collectively known as capacitation (de Lamirande et al., 1997
). Capacitation enables sperm to effectively participate in various steps of sperm–oocyte interaction (Yanagimachi, 1994; de Lamirande et al., 1997). Mechanisms that control sperm capacitation are not yet completely known (de Lamirande et al., 1997), but consensus is that this process is controlled by various signal transduction elements involving protein kinase A (PKA) (Leclerc et al., 1996; Visconti et al., 1997; Aitken et al., 1998; Visconti and Kopf, 1998), protein kinase C (PKC) (Furaya et al., 1993) and protein tyrosine kinases (PTK) (Leclerc et al., 1996, 1997). In humans, various proteins are tyrosine phosphorylated during capacitation, among which fibrous sheath proteins p81, p95 and p105 (Leclerc et al., 1997) are most abundant and related to A kinase anchoring proteins (AKAPs) (Carrera et al., 1996).
Various biological and pharmacological agents have been used to induce human sperm capacitation and study the underlying mechanisms. Capacitation induced by ultrafiltrates from fetal cord serum (FCSu), follicular fluid (FFu), progesterone (Foresta et al., 1992; de Lamirande and Gagnon, 1995; Leclerc et al., 1996; de Lamirande et al., 1998) and cell permeant analogues of cAMP, such as N6,2'-O-dibutyryl cAMP (dbcAMP), and/or phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX) (Leclerc et al., 1996, 1998) is associated with cAMP-dependent tyrosine phosphorylation of 105 and 81 kDa proteins. Although there is consensus that cAMP/PKA activity is associated with protein tyrosine phosphorylation (Leclerc et al., 1996, 1997; Aitken et al., 1998), the mechanisms leading to increased tyrosine phosphorylation remain unknown (de Lamirande et al., 1998). Since most of the effects of cAMP are mediated through the activation of PKA, it is hypothesized that PKA indirectly promotes tyrosine phosphorylation of proteins from the fibrous sheath by phosphorylation of serine/threonine residues (Ser/Thr) and activation of tyrosine kinases (Leclerc et al., 1996).
Recent evidence indicates that components of the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinase (MAPK) are present in sperm and involved in sperm function (Naz et al., 1992; Ashizawa et al., 1997; Luconi et al., 1998a,b; Lu et al., 1999; de Lamirande and Gagnon, 2002). The basic assembly of all MAPK pathways involves a module conserved from yeast to humans and in which three kinases are sequentially activated; a module includes a MAPK kinase kinase (for Ser/Thr), a MAPK kinase and a MAPK (Kolch, 2000). The MAPK kinases are dual specificity kinases (Dhanasekaran and Reddy, 1998) and phosphorylate Thr and Tyr residues present in Thr-X-Tyr motifs. Some MAPK kinases, such as MEK, phosphorylate the Thr and Tyr residues within the Thr-Glu-Tyr motif which is present not only in ERK 1 and 2, but also in ERK 5 (big MAPK) (Zhou et al., 1995), ERK 7 (Yan et al., 2001) and other important signal transduction elements such as MOK (Miyata and Nishida, 1999). In a recent study, de Lamirande and Gagnon reported that FCSu-induced capacitation of human sperm is associated with a progressive increase in the level of double phosphorylation of the Thr-Glu-Tyr (P-Thr-Glu-Tyr-P) motif of sperm proteins other than ERK 1 and 2 (de Lamirande and Gagnon, 2002). This finding suggested that different dual specificity kinases (MEK or MEK-like) and their substrates are involved in human sperm capacitation.
The objective of the present study was to investigate the regulation of the P-Thr-Glu-Tyr-P motif present in sperm proteins during capacitation induced by biological agents (Leclerc et al., 1996; de Lamirande et al., 1998; FCSu, and FFu and progesterone respectively) and pharmacological agents (Leclerc et al., 1996; dbcAMP+IBMX) using inhibitors of MEK-like dual specificity kinases (PD98059), PKA (H89), non-receptor type PTK (PP2), PKC (chelerythrine) and receptor type PTK (tyrphostin A47). We report that the double phosphorylation of the Thr-Glu-Tyr motif present in p80 and p105 during capacitation induced by biological and pharmacological agents is regulated through very different signal transduction pathways.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Materials
The following reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA): IBMX, progesterone, dbcAMP, lysophosphatidylcholine and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulphonamide (H89). Percoll was obtained from Amersham Pharmacia Biotech (Baie d’Urfé, Québec, Canada). 2'-amino-3'-methoxyflavone (PD98059), chelerythrine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), U0126 and tyrphostin A47 were purchased from Calbiochem (LaJolla, CA, USA). Upstate Biotechnology (Lake Placid, NY, USA) was the supplier of anti-phosphotyrosine (clone 4G10) antibody. The polyclonal antibody raised against the P-Thr-Glu-Tyr-P motif was bought from New England Biolabs Ltd (Missisauga, Ontario, Canada). 3-cyclohexylamino-1-propane sulphonic acid (CAPS) was purchased from Fisher Scientific (Nepean, Ontario, Canada). Nitrocellulose (0.2 µm pore size; Osmonics Inc, Westborough, MA, USA), goat anti-rabbit IgG and goat anti-mouse IgG both conjugated to horseradish peroxidase (Amersham), an enhanced chemiluminescence kit (Lumi-Light; Roche Molecular Biochemicals, Laval, Québec, Canada) and radiographic films (Fuji, Minami-Ashigra, Japan) were used for immunodetection of blotted proteins. All other chemicals were of at least reagent grade.
Fetal cord blood was collected at the birthing centre of the Royal Victoria Hospital (Montréal, Québec, Canada). Human follicular fluid was collected from preovulatory 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 hospital approved the present study. Fetal cord blood and follicular fluid samples were centrifuged (1000 g, 30 min, 4°C), pooled and frozen (–20°C) until use. FCSu and FFu were prepared from at least 15 individual samples using YM3 membranes (exclusion limit: 3 kDa; Amicon, Oakville, Ontario, Canada) (de Lamirande and Gagnon, 1995).
Inhibitors to be tested with sperm were dissolved in distilled 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.
Sperm preparation and capacitation
Semen samples from healthy volunteers were washed on 4-layer (95–65–40–20%) Percoll gradients 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). The 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 200x106 cells/ml with the 95% Percoll solution. Only samples with progressive motility >70% were used. Sperm were additionally diluted 5-fold in Biggers, Whitten and Whittingham medium (BWW; pH 8) (Biggers et al., 1971) devoid of bicarbonate and bovine serum albumin (BSA) and containing 1 mmol/l CaCl2.
Sperm capacitation was evaluated after 3.5 h of incubation in the presence of FCSu (10%, v/v) or dbcAMP (1 mmol/l) + IBMX (0.1 mmol/l), by the induction of the acrosome reaction with lysophosphatidylcholine (LPC) as previously described (de Lamirande et al., 1997). In brief, at the end of the 3.5 h incubation period, sperm were washed with HEPES-balanced saline to remove capacitation inducers and/or inhibitors, resuspended in BWW medium containing 3 mg/ml BSA and 100 µmol/l LPC and incubated for a further 30 min at 37°C. Sperm were washed again in HEPES-balanced saline and then fixed in ethanol. The acrosomal status of these LPC-treated sperm was determined using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (Cross et al., 1986). On each slide, the acrosomal status of >200 sperm was evaluated and the proportion of sperm undergoing the acrosome reaction was determined as a measure of capacitation. None of the chemicals and biological agents tested affected the percentage of sperm motility at the concentrations used in this study and over a period of at least 4 h. Differences in the level of capacitation obtained after various treatments of sperm were evaluated by analysis of variance (two-tailed, paired values).
SDS–PAGE and immunoblotting
Sperm proteins were electrophoresed on 12% polyacrylamide gels and electrotransferred (using 10 mmol/l CAPS buffer, pH adjusted to 11, and containing 10% methanol) to nitrocellulose membranes. The membranes were incubated with a solution of skimmed milk (5%, w/v) in Tris (20 mmol/l, pH 7.8)-buffered saline containing Tween 20 (0.1%, v/v; TTBS), and then with the primary antibody overnight at 4°C. The antibody against the P-Thr-Glu-Tyr-P motif was diluted 1:1000 in TTBS supplemented with 25 mg/ml BSA and 0.1% (w/v) sodium azide and incubated with the membrane overnight at 4°C. The anti-phosphotyrosine (anti-P-Tyr) antibody was diluted 1:1000 in TTBS supplemented with sodium azide (0.1%, w/v) and incubated with the membranes for 1 h at 20°C. After washing with TTBS, membranes were incubated with the secondary antibody (goat anti-rabbit IgG or goat anti-mouse IgG conjugated with horseradish peroxidase) for 45 min at 20°C and washed again with TTBS. Positive immunoreactive bands were detected using the Lumi-Light chemiluminescence kit. At the end of 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.
Comparison of tyrosine phosphorylation and double phosphorylation of the Thr-Glu-Tyr motif in sperm proteins during capacitation induced by dbcAMP+IBMX
The objective of this experiment was to compare the labelling pattern of anti-P-Thr-Glu-Tyr-P and anti-P-Tyr antibodies to ascertain that the proteins recognized by these antibodies are different. Percoll-washed sperm were resuspended in BWW at a final concentration of 40x106/ml and incubated without (control) or with dbcAMP+IBMX (1 and 0.1 mmol/l respectively) for 2 h. Sperm proteins were electrophoresed, electrotransferred and immunoblotted as described above with anti-P-Tyr and anti-P-Thr-Glu-Tyr-P antibodies.
Time course study of phosphorylation of the Thr-Glu-Tyr motif following induction of capacitation by FCSu or dbcAMP +IBMX
Sperm in BWW medium were incubated at 37°C without (control) or with FCSu or dbcAMP+IBMX as inducer of capacitation. Sperm samples were drawn at time 0 (control; before addition of capacitation inducer) and after 5, 15, 30, 60 and 120 min of incubation and supplemented with electrophoresis sample buffer containing vanadate (0.1 mmol/l) and ß-glycerolphosphate (20 mmol/l) as phosphoprotein phosphatase inhibitors.
Regulation of double phosphorylation of the Thr-Glu-Tyr motif during capacitation induced by FCSu, FFu, progesterone or dbcAMP+IBMX
Sperm preparations (40x106/ml in BWW medium) were preincubated with or without PD98059 (100 µmol/l), H89 (10 µmol/l), PP2 (10 nmol/l), chelerythrine (10 µmol/l) or tyrphostin A47 (100 µmol/l) for 30 min at 37°C and then supplemented or not (control; BWW alone) with FCSu (10%, v/v), FFu (10%, v/v), progesterone (30 µmol/l) or a combination of dbcAMP+IBMX (1 and 0.1 mmol/l respectively) as capacitation inducers and incubated for an additional 2 h at 37°C. None of the inhibitors affected sperm motility at the concentrations used and over the course of a 3.5 h incubation. Electrophoresis sample buffer was then added and the sperm samples were processed for immunoblotting with the anti-P-Thr-Glu-Tyr-P antibody.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Protein tyrosine phosphorylation and double phosphorylation of the Thr-Glu-Tyr motif are different during capacitation induced by dbcAMP +IBMX
The objective of this experiment was to ascertain that the proteins recognized by anti-P-Thr-Glu-Tyr-P and anti-P-Tyr antibodies are different. The labelling patterns (Figure 1
) demonstrated that these antibodies recognized different proteins. The increase in protein tyrosine phosphorylation in dbcAMP+IBMX-treated sperm was limited almost to only p80 and p105 (Figure 1A; exposure time: 1 s). With a longer exposure time (Figure 1A; 5 s), when intensity of p80 and p105 was saturated, other bands limited mostly to higher molecular mass protein bands (p200 to p67) could be observed. The amount of sperm protein needed for immunoblotting with anti-P-Thr-Glu-Tyr-P antibody was 10-fold greater than that needed for a similar experiment performed with anti-P-Tyr antibody. The P-Tyr-Glu-Thr-P content in dbcAMP+IBMX-treated sperm increased in p80 and p105 as well as in various proteins extending from 200 to 21 kDa (Figure 1B; exposure time: 60 s). This time, bands other than p80 and 105 could be observed without saturation of p80 and p105.
|
Double phosphorylation of the Thr-Glu-Tyr motif in sperm during capacitation induced by FCSu or dbc AMP +IBMX follows different time courses
A study was conducted to determine the time course of Thr-Glu-Tyr phosphorylation during capacitation induced by FCSu or a combination of dbcAMP+IBMX (Figure 2
). As previously observed (de Lamirande and Gagnon, 2002), there was a progressive increase in the phosphorylation of the Thr-Glu-Tyr motif during FCSu-induced capacitation. The P-Thr-Glu-Tyr-P content in p80 and p105 (according to molecular mass) was higher in FCSu-treated than in control (BWW medium alone) sperm 1 h after the beginning of incubation and a further increase in P-Thr-Glu-Tyr-P was observed during the next 1 h of incubation (Figure 2A). However, when capacitation was induced with dbcAMP+IBMX, the P-Thr-Glu-Tyr-P content of p80 and p105 increased at as early as 15 min and was more pronounced during the next 2 h of incubation (Figure 2B). Other proteins (66–21 kDa) were detected by the anti-P-Thr-Glu-Tyr-P antibody but the changes in the content of P-Thr-Glu-Tyr-P in these proteins over a period of 2 h of incubation (Figure 2A–C) were mild and transient as compared with those observed for p80 and p105.
|
The faster increase in, and higher levels of, Thr-Glu-Tyr phosphorylation were observed with dbcAMP+IBMX in all experiments performed. Furthermore, when sperm samples submitted to both treatments were analysed together (same electrophoresis gel and immunoblot; Figure 2C
), the content of P-Thr-Glu-Tyr-P was always higher in sperm treated with dbcAMP+IBMX than with FCSu (30 min and 2 h incubation; Figure 2C). Finally, when the film exposure was increased so that the 80 and 105 kDa bands of FCSu-treated sperm were as intense as those observed in sperm treated with dbcAMP+IBMX, there was no difference between sperm treated or not with FCSu before 1 h incubation (data not shown). Therefore, treatment of sperm with dbcAMP+IBMX induced a faster and more pronounced phosphorylation of Thr-Glu-Tyr than treatment with FCSu. Because of the progressive increase in the P-Thr-Glu-Tyr-P level in p80 and p105 as capacitation proceeds and because of the difference in level and kinetics of the double phosphorylation of the Thr-Glu-Tyr motif in these two capacitation systems, a study was conducted on the regulation of this phenomenon. A 2 h incubation period was chosen because it allows better differentiation between control and capacitating sperm.
Regulation of the double phosphorylation of the Thr-Glu-Tyr motif during capacitation induced by FCSu, FFu or progesterone
As the role of PTK and PKC in human sperm capacitation has sometimes been controversial because the inhibitors tested are not specific (Furaya et al., 1993; staurosporine for PKC) or used without pretreatment (Leclerc et al., 1997; tyrphostin A47), we first determined whether chelerythrine (PKC inhibitor), PP2 and tyrphostin A47 (two PTK inhibitors) could prevent FCSu- or dbcAMP+IBMX-induced capacitation as measured by the LPC-induced acrosome reaction. The results presented in Table I support a role for PTK and PKC in capacitation.
|
Experiments were performed to study the regulation of double phosphorylation of Thr-Glu-Tyr in sperm during capacitation induced by three biological agents, FCSu, FFu and progesterone. The effect of inhibitors of signal transduction elements that were found to prevent human sperm capacitation such as PD98059 (100 µmol/l; inhibitor of MEK) (Luconi et al., 1998a
; de Lamirande and Gagnon, 2002), H89 (10 µmol/l; inhibitor of PKA) (Leclerc et al., 1996), PP2 (10 nmol/l; inhibitor of non-receptor type PTK), chelerythrine (10 µmol/l; inhibitor of PKC) and tyrphostin A47 (100 µmol/l; inhibitor of receptor type PTK) were evaluated.
The increase in P-Thr-Glu-Tyr-P content in p80 and p105 of sperm treated with FCSu (Figure 3A) was prevented by PD98059 and tyrphostin A47. However, H89, PP2 and chelerythrine did not have such an effect (Figure 3A). U0126 (0.3 µmol/l), an inhibitor of dual specificity kinases such as MEK that acts by a different mechanism than PD98059 and inhibits capacitation (de Lamirande and Gagnon, 2002), had the same effect as PD98059 (data not shown). FFu caused an increase in P-Thr-Glu-Tyr-P of p80 and p105, but also of a 60 kDa protein (Figure 3B). As observed with FCSu, the increase in P-Thr-Glu-Tyr-P content in p60, p80 and p105 of sperm treated with FFu was prevented by PD98059 and tyrphostin A47 but not by H89, PP2 and chelerythrine (Figure 3B). Progesterone caused a similar increase in P-Thr-Glu-Tyr-P content as FCSu or FFu (Figure 3C). The rise in P-Thr-Glu-Tyr-P content due to progesterone was also prevented by PD98059 and tyrphostin A47 but not by H89, PP2 and chelerythrine (Figure 3C). These results indicate that the increase and regulation of double phosphorylation of Tyr-Glu-Thr during capacitation induced by FCSu, FFu and progesterone is similar and involves a dual specificity kinase (MEK or MEK-like) and receptor type PTK.
|
Regulation of the double phosphorylation of the Thr-Glu-Tyr motif during capacitation induced by a combination of dbcAMP +IBMX
The increase in P-Thr-Glu-Tyr-P content in p80 and p105, as compared with what was observed in control (BWW medium alone) sperm, was higher in sperm treated with dbcAMP+IBMX (Figure 3D
) than with FCSu, FFu or progesterone (Figure 3A–C). This increase was prevented by H89, chelerythrine and tyrphostin A47 but not by PD98059 or PP2, suggesting that the regulation of this phenomenon involves PKA, PKC and receptor type PTK but not dual specificity kinases (MEK or MEK-like) and non-receptor type PTK (Figure 3D). |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Our previous study demonstrated that capacitation is associated with an increased phosphorylation of Thr and Tyr residues within a Thr-Glu-Tyr motif in various proteins in human sperm and suggested that a dual specificity kinase with specificity similar to MEK is responsible for this phosphorylation (de Lamirande and Gagnon, 2002
). In the present study, we investigated the regulation of this process during capacitation induced by FCSu, FFu, progesterone and dbcAMP+IBMX.
We used an antibody specific for the P-Thr-Glu-Tyr-P motif. Quality control tests done by the manufacturer (New England Biolabs) indicate that the antibody does not react with phospho-threonine (P-Thr), P-Tyr or P-Thr-X-Tyr-P (X being an amino acid other than Glu). Furthermore, the anti-P-Thr-Glu-Tyr-P antibody recognized phosphorylated ERK 1 and 2 (bacterially expressed and treated with MEK) but not the non-phosphorylated form of the same kinases (controls provided by New England Biolabs). We conducted a study to compare immunolabelling with anti-P-Tyr and anti-P-Thr-Glu-Tyr-P antibodies in human sperm. Although both antibodies recognized protein bands at p80 and p105 kDa, results were very different with respect to the intensity of bands at 80 and 105 kDa and molecular mass of other proteins detected, clearly indicating that the two antibodies have different labelling patterns (Figure 1). Because P-Thr-Glu-Tyr-P motifs present in p80 and p105 include P-Tyr, part of the signals detected by anti-P-Tyr antibody are from the P-Thr-Glu-Tyr-P motif present in these proteins. p80 and p105, the most abundant P-Tyr-containing proteins, are present in the fibrous sheath of human sperm flagellum (Leclerc et al., 1997) and are related to AKAPs (Carrera et al., 1996). Based on the finding that the P-Thr-Glu-Tyr-P motif was found in proteins of similar molecular masses, we hypothesize that fibrous sheath proteins also contain the P-Thr-Glu-Tyr-P motif or that this motif is present in proteins other than from fibrous sheath but having similar molecular masses.
The kinetics of phosphorylation of the Thr-Glu-Tyr motif during capacitation induced by FCSu were different from those induced by dbcAMP+IBMX (Figure 2). An increase in the content of P-Thr-Glu-Tyr-P in proteins of 80 and 105 kDa was evident after 15 min of incubation with dbcAMP+IBMX. However, a similar increase was evident only after 60 min of incubation when capacitation was induced by FCSu. In addition, the P-Thr-Glu-Tyr-P content in p80 and p105 was much higher at all times in dbcAMP+IBMX- than in FCSu-treated sperm. The shorter incubation (15 min) required for an increase and the much higher content of P-Thr-Glu-Tyr-P after 120 min of incubation in dbcAMP+IBMX-treated as compared with FCSu-treated sperm indicated a difference in kinetics. These findings suggested that different signal transduction pathways may be involved in the regulation of Thr-Glu-Tyr phosphorylation during capacitation induced by biological and pharmacological agents.
Dual specificity kinases, such as MEK, are usually responsible for Thr-Glu-Tyr phosphorylation (Dhanasekaran and Reddy, 1998). This is the case for phosphorylation and consequent activation of important signal transduction elements such as ERK 1 and 2 (Luconi et al., 1998a; Widmann et al., 1999), ERK 5 (Mody et al., 2001), ERK 7 (Zhou et al., 1995) and MOK (Miyata and Nishada, 1999). However, double phosphorylation of the Thr-Glu-Tyr motif through MEK-independent pathways has also been observed in the prolonged activation of ERK 1 and 2 mediated through phosphatidylinositol-3-kinase and PKC in Swiss 3T3 fibroblasts during platelet-derived growth factor signalling (Grammer and Blenis, 1997). Therefore, it is hypothesized that the Thr and Tyr residues of the Thr-Glu-Tyr motif could also be phosphorylated through MEK-independent pathways. Therefore, we compared the signal transduction pathways involving dual specificity kinase (MEK or MEK-like), PKA, PKC and PTK in the regulation of the double phosphorylation of the Thr-Glu-Tyr motif during capacitation induced by three biological agents, FCSu, FFu and progesterone and by dbcAMP+IBMX.
Double phosphorylation of the Thr-Glu-Tyr motif during capacitation induced by FCSu, FFu and progesterone involved receptor type PTK and dual specificity kinase (MEK or MEK-like) but not PKA, non-receptor type PTK or PKC. Tyrphostin A 47, at 100 µmol/l, could also inhibit non-receptor type PTK. However, PP2, an inhibitor of non-receptor type PTK at 10 nmol/l, did not prevent the increase of P-Thr-Glu-Tyr-P during sperm capacitation.
The enzyme responsible for the double phosphorylation of the Thr-Glu-Tyr motif in sperm capacitated with biological fluids is presently unknown. Although immunoblotting data have indicated the presence of MEK1 in human sperm (Luconi et al., 1998a), other dual specificity kinases such as MEK2, MEK5 (Dhanasekaran and Reddy, 1998) and others still unidentified (Mody et al., 2001) also exist and phosphorylate the same motif. Our present observation that PD98059 (Figure 3) and U0126 (n = 4; data not shown) prevents the increase in phosphorylation of P-Thr-Glu-Tyr-P motif during capacitation induced by biological agents suggest that a dual specificity kinase (MEK or MEK-like) is involved in this process.
Contrary to that observed with biological agents, phosphorylation of the Thr-Glu-Tyr motif during capacitation induced by dbcAMP+IBMX appeared to be mainly mediated through PKA and PKC without involving dual specificity kinase (MEK or MEK-like). In physiological conditions, compartmentalized pools of cAMP and anchoring of PKA by AKAPs ensures that PKA is exposed to localized changes in cAMP (Pawson and Scott, 1997). The 1 mmol/l concentration of dbcAMP generally used to capacitate human sperm (Leclerc et al., 1996; de Lamirande et al., 1997) is 100- to 200-fold higher than that of intracellular cAMP in sperm. It can be hypothesized that treatment of sperm with this high concentration of dbcAMP will lead to an increase in cAMP to an extent that could favour non-physiological activation and/or interaction of PKA with different signal transduction pathways. Subcellular targeting of PKC is also mediated through anchoring proteins, such as AKAP79, which co-localizes PKC with PKA (Pawson and Scott, 1997). Activation of PKA could lead to phosphorylation induction of PTK, as previously suggested (Leclerc et al., 1996). Inhibition of Thr-Glu-Tyr phosphorylation by tyrphostin A47 during capacitation induced by dbcAMP+IBMX is consistent with this mechanism. Therefore, although cAMP/PKA can inhibit Raf-1 (Kolch, 2000), a MEK-independent phosphorylation of the Thr-Glu-Tyr motif resulting from cross-talk between PKA, PKC and PTK may be possible in sperm.
In conclusion, we demonstrated double phosphorylation of the Thr-Glu-Tyr motif in human sperm proteins of 80 and 105 kDa during capacitation induced by various biological and pharmacological agents. This phenomenon appears to be regulated through dual specificity kinases (MEK or MEK-like) and receptor type PTK during capacitation induced by biological agents. However, PKA and PKC and receptor type PTK were mainly involved in this process when capacitation was induced by pharmacological agents. Therefore, even though FCSu, FFu, progesterone and dbcAMP+IBMX induce capacitation in sperm to a similar extent, there is clear evidence that very different signal transduction pathways are involved and that caution must be exercised in interpreting the results of signal transduction studies when using these pharmacological agents.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
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. J.T. was supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. This work was supported by a grant from the Canadian Institutes of Health Research to C.G.
|
Notes |
|---|
1 To whom correspondence should be addressed. E-mail: jthundathil{at}hotmail.com
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Aitken, R.J., Harkiss, D., Knox, W., Paterson, M. and Irvine, D.S. (1998) A novel signal transduction cascade in capacitating human spermatozoa characterized by a redox-regulated cAMP-mediated induction of tyrosine phosphorylation. J. Cell Sci., 111, 645–656.[Abstract]
Ashizawa, K., Hashimoto, K., Higashio, M. and Tsuzuki, Y. (1997) The addition of mitogen-activated protein kinase and p34cdc2 kinase substrate peptides inhibits the flagellar motility of demembranated fowl spermatozoa. Biochem. Biophys Res. Comm., 240, 116–121.[ISI][Medline]
Biggers, J.D., Whitten, W.K. and Whittingham, D.G. (1971) The culture of mouse embryos in vitro. In Daniel, J.C. (ed.) Methods in Mammalian Embryology. W.H. Freeman, San Francisco, USA, pp. 86–116.
Carrera, A., Moos, J., Ning, X.P., Gerton, G.L., Tesarik, J., Kopf, G.S. and Moos, S.B. (1996) Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: Identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. Dev. Biol., 180, 284–296.[ISI][Medline]
Cross, N.L., Morales, P., Overstreet, J.W. and Hanson, F.W. (1986). Two simple methods for detecting the acrosome reaction of human sperm. Gamete Res., 15, 29–40.
de Lamirande, E. and Gagnon, C. (1995) Capacitation-associated production of superoxide anion by human spermatozoa. Free Rad. Biol. Med., 18, 487–496.[ISI][Medline]
de Lamirande, E. and Gagnon, C. (2002) The extracellular signal-regulated kinase pathway is involved in human sperm function and modulated by the superoxide production. Mol. Hum. Reprod., 8, 124–135.
de Lamirande, E., Leclerc, P. and Gagnon, C. (1997) Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization. Mol. Hum. Reprod., 3, 175–194.
de Lamirande, E., Harakat, A. and Gagnon, C. (1998) Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion. J. Androl., 19, 215–225.
Dhanasekaran, N. and Reddy, E.P. (1998) Signalling by dual specificity kinases. Oncogene, 17, 1447–1445.[ISI][Medline]
Foresta, C., Rossato, M., Mioni, R. and Zorzi, M. (1992) Progesterone induces capacitation in human spermatozoa. Andrologia, 24, 33–35.[ISI][Medline]
Furaya, S., Endo, Y., Osumi, K., Oba, M., Nosawa, S. and Suzuki, S. (1993) Effect of modulators of protein kinase C on human sperm capacitation. Fertil. Steril., 59, 1285–1290.[ISI][Medline]
Grammer, T.C. and Blenis, J. (1997) Evidence for MEK-independent pathways regulating the prolonged activation of the ERK-MAP kinases. Oncogene, 14, 1635–1642.[ISI][Medline]
Jacobson, G. and Karsnas, P. (1990) Important parameters in semi-dry electrophoresis transfer. Electrophoresis, 11, 46–52.[ISI][Medline]
Kolch, W. (2000) Meaningful relationships: the regulation of the Ras/Raf/Mek/Erk pathway by protein interactions. Biochem. J., 351, 289–305.
Leclerc, P., de Lamirande, E. and Gagnon, C. (1996) Cyclic adenosine 3',5' monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol. Reprod., 55, 684–692.[Abstract]
Leclerc, P., de Lamirande, E. and Gagnon, C. (1997) Regulation of protein tyrosine phosphorylation and human sperm capacitation by reactive oxygen species derivatives. Free Rad. Biol. Med., 22, 643–656.[ISI][Medline]
Leclerc, P., de Lamirande, E. and Gagnon, C. (1998) Interaction between Ca2+, cyclic 3',5' adenosine monophosphate, the superoxide anion, and tyrosine phosphorylation pathways in the regulation of human sperm capacitation. J. Androl., 19, 434–443.
Lu, Q., Sun, Q.Y., Breitbart, H. and Chen, D.Y. (1999) Expression and phosphorylation of mitogen-activated protein kinases during spermatogenesis and epididymal maturation in mice. Arch. Androl., 43, 55–66.[ISI][Medline]
Luconi, M., Barni, T., Vanelli, G.B., Crausz, C., Marra, F., Benedetti, P.A., Evangelista, V., Francavilla, S., Poperzi, G. and Baldi, E. (1998a) Extracellular signal regulated kinases modulate capacitation of human spermatozoa. Biol. Reprod., 58, 1476–1489.
Luconi, M., Krausz, C., Barni, T., Vannelli, G.B., Forti, G. and Baldi, E. (1998b) Progesterone stimulates p42 extracellular signal-regulated kinase (p42erk) in human spermatozoa. Mol. Hum. Reprod., 4, 251–258.
Miyata, Y. and Nishida, E. (1999) Distantly related cousins of MAP Kinase: Biochemical properties and possible physiological functions. Biochem. Biophys Res. Comm., 266, 291–295.[ISI][Medline]
Mody, N., Leitch, J., Armstrong, C., Dixon, J. and Cohen, P. (2001) Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway. FEBS Lett., 502, 21–24.[ISI][Medline]
Naz, R.K., Ahmad, K. and Kaplan, P. (1992) Expression and functions of ras proto-oncogene protein in human sperm cells. J. Cell Sci., 102, 487–494.
Pawson, T. and Scott, J.D. (1997) Signaling through scaffold, anchoring, and adaptor proteins. Science, 278, 2075–2079.
Visconti, P.E. and Kopf, G.S. (1998) Regulation of protein phosphorylation during sperm capacitation. Biol. Reprod., 59, 1–6.
Visconti, P.E., Johnson, L.R., Oyaski, M., Fornes, M., Moss, S.B., Gerton, G.L. and Kopf, G.S. (1997) Regulation, localization, and anchoring of protein kinase A subunits during mouse sperm capacitation. Dev. Biol., 192, 351–363.[ISI][Medline]
Widmann, C., Gibson, S., Jarpe, M.B. and Johnson, G.L. (1999) Mitogen-activated protein kinase: Conservation of a three-kinase module from yeast to human. Physiol. Rev., 79, 143–180.
Yan, C., Luo, H., Lee, J-D., Abe, J. and Berk, B.C. (2001) Molecular cloning of mouse ERK5/BMK1 splice variants and characterization of ERK5 functional domains. J. Biol. Chem., 276, 10870–10878.
Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neil J.D. (eds) The Physiology of Reproduction. Vol 2. Raven Press Ltd, New York, USA, pp. 189–317.
Zhou, G., Bao, Z.Q. and Dixon, J.E. (1995) Components of a new human protein kinase signal transduction pathway. J. Biol. Chem., 270, 12665–12669.
Submitted on December 6, 2001; resubmitted on April 15, 2002; accepted on June 12, 2002.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y.-F. Li, W. He, K. N. Jha, K. Klotz, Y.-H. Kim, A. Mandal, S. Pulido, L. Digilio, C. J. Flickinger, and J. C. Herr FSCB, a Novel Protein Kinase A-phosphorylated Calcium-binding Protein, Is a CABYR-binding Partner Involved in Late Steps of Fibrous Sheath Biogenesis J. Biol. Chem., November 23, 2007; 282(47): 34104 - 34119. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. White, E. de Lamirande, and C. Gagnon Protein kinase C is an important signaling mediator associated with motility of intact sea urchin spermatozoa J. Exp. Biol., November 15, 2007; 210(22): 4053 - 4064. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Bastian, A. Zepeda-Bastida, S. Uribe, and A. Mujica In spermatozoa, Stat1 is activated during capacitation and the acrosomal reaction Reproduction, September 1, 2007; 134(3): 425 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.N. Jha, A.M. Salicioni, E. Arcelay, O. Chertihin, S. Kumari, J.C. Herr, and P.E. Visconti Evidence for the involvement of proline-directed serine/threonine phosphorylation in sperm capacitation Mol. Hum. Reprod., December 1, 2006; 12(12): 781 - 789. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Baker, L. Hetherington, and R. J. Aitken Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa J. Cell Sci., August 1, 2006; 119(15): 3182 - 3192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. O'Flaherty, E. de Lamirande, and C. Gagnon Reactive Oxygen Species and Protein Kinases Modulate the Level of Phospho-MEK-Like Proteins During Human Sperm Capacitation Biol Reprod, July 1, 2005; 73(1): 94 - 105. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Liguori, E. de Lamirande, A. Minelli, and C. Gagnon Various protein kinases regulate human sperm acrosome reaction and the associated phosphorylation of Tyr residues and of the Thr-Glu-Tyr motif Mol. Hum. Reprod., March 1, 2005; 11(3): 211 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Nauc, E. De Lamirande, P. Leclerc, and C. Gagnon Inhibitors of Phosphoinositide 3-Kinase, LY294002 and Wortmannin, Affect Sperm Capacitation and Associated Phosphorylation of Proteins Differently: Ca2+-Dependent Divergences J Androl, July 1, 2004; 25(4): 573 - 585. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R E Spindler, Y Huang, J G Howard, P Wang, H Zhang, G Zhang, and D E Wildt Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda Reproduction, May 1, 2004; 127(5): 547 - 556. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. O'Flaherty, E. de Lamirande, and C. Gagnon Phosphorylation of the Arginine-X-X-(Serine/Threonine) motif in human sperm proteins during capacitation: modulation and protein kinase A dependency Mol. Hum. Reprod., May 1, 2004; 10(5): 355 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Minelli, L. Liguori, I. Bellazza, R. Mannucci, B. Johansson, and B. B. Fredholm Involvement of A1 Adenosine Receptors in the Acquisition of Fertilizing Capacity J Androl, March 1, 2004; 25(2): 286 - 292. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Baker, L. Hetherington, H. Ecroyd, S. D. Roman, and R. J. Aitken Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation J. Cell Sci., January 15, 2004; 117(2): 211 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Kirkman-Brown Editorial Commentary J Androl, September 1, 2003; 24(5): 734 - 735. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||