Molecular Human Reproduction, Vol. 8, No. 2, 124-135,
February 2002
© 2002 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
The extracellular signal-regulated kinase (ERK) pathway is involved in human sperm function and modulated by the superoxide anion
Urology Research Laboratory, Royal Victoria Hospital and McGill University, Montréal, Québec, Canada
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Abstract |
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Our aim was to ascertain the role of the extracellular signal-regulated protein kinase (ERK) pathway in human sperm capacitation induced by fetal cord serum ultrafiltrate (FCSu) and its regulation by the superoxide anion (O2-·). Immunoblotting indicated the presence of Shc, Grb2, Rasp21, Raf and ERK1 and 2 (ERK1/2) in spermatozoa. Grb2, Rasp21, Raf and MEK inhibitors dose-dependently prevented sperm capacitation and protein tyrosine phosphorylation, without modifying sperm O2-· production. Therefore, the whole ERK cascade plays a role in capacitation, downstream of O2-· but upstream of protein tyrosine phosphorylation. Upon incubation with FCSu, the early (5 min) increase in ERK1/2 activity (as shown by double phosphorylation of the Thr-Glu-Tyr motif) was followed by an important decrease over the next 2 h; superoxide dismutase did not change this pattern. The phosphorylation of the Thr-Glu-Tyr motif present in other sperm proteins (16–33 kDa) also increased (5 min incubation with FCSu) and then progressively decreased, and this effect was regulated by O2-·, MEK and cAMP. The phospho-Ser/Thr-Pro content (characteristic of ERK1/2 substrates) of Triton-insoluble proteins (75 and 80 kDa) increased during capacitation and also appeared to be regulated by O2-· and the ERK pathway. Inhibition of ERK1/2 activation reduced lysophosphatidylcholine-induced acrosome reaction and the associated protein tyrosine phosphorylation. These results support a role for the ERK pathway in human sperm function.
acrosome reaction/dual specificity kinase/mitogen-activated protein kinase/signal transduction/tyrosine phosphorylation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Capacitation is defined as the series of transformations that spermatozoa normally undergo during their transit in the female genital tract in order to reach, and bind to, the zona pellucida, undergo the acrosome reaction and fertilize the oocyte (Yanagimachi, 1994
; de Lamirande et al., 1997). It is a complex and finely tuned process in which membranous, ionic and metabolic alterations occur at specific times and locations and in which several components of signal transduction pathways are involved (Yanagimachi, 1994; de Lamirande et al., 1997). Capacitation involves an increase in cAMP (Parinaud and Milhet, 1996), a Ca2+ influx (de Lamirande et al., 1997) and the phosphorylation of proteins on serine (Ser) and threonine (Thr) (Naz, 1999) which could be due to activation of protein kinases A (PKA) (Leclerc et al., 1996; Visconti et al., 1997) and C (PKC) (de Lamirande et al., 1997). Protein tyrosine kinases also play a role in capacitation since it is prevented by inhibitors of these enzymes (herbimycin A, genistein, tyrphostin A47) in mouse and human spermatozoa (de Lamirande et al., 1997; Leclerc et al., 1997).
Capacitation is associated with the generation of increased amounts of reactive oxygen species (ROS) such as the superoxide anion (O2-·), hydrogen peroxide (H2O2) and nitric oxide (NO·) by spermatozoa (de Lamirande and Gagnon, 1995; de Lamirande et al., 1997,1998a, b; Herrero et al., 2000). ROS, cAMP and Ca2+ regulate the time-dependent increase in tyrosine (Tyr) phosphorylation of sperm proteins that occurs during capacitation (Aitken et al., 1995; Visconti et al., 1995; Leclerc et al., 1996,1997,1998; Luconi et al., 1996; Galantino-Homer et al., 1997; de Lamirande et al., 1998a). In the human, this tyrosine phosphorylation affects mainly proteins from the fibrous sheath; three of these, p81, p95 and p105 (according to their molecular masses) are related to A kinase anchoring proteins (AKAP) (Carrera et al., 1996; Leclerc et al., 1998; Mandal et al., 1999; Herrero et al., 2000).
Because of its importance in fertilization, it is very likely that redundant mechanisms control human sperm capacitation and that cross-talks between different pathways occur during this process (de Lamirande et al., 1997). Beside the transduction mechanisms described above, recent evidence indicates that components of the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases (MAPK) are present in spermatozoa and involved in motility and capacitation (Naz et al., 1992; Ashizawa et al., 1997; Luconi et al., 1998a,b; Lu et al., 1999). The basic assembly of all MAPK pathways is a module conserved from yeast to human and in which three kinases are sequentially activated (Figure 1) (Windmann et al., 1999; Kolch, 2000). The ERK module includes Raf, as MAPK kinase kinase (for Ser/Thr), MEK, as MAPK kinase (dual specificity for Ser/Thr and Tyr) and ERK1 and 2 (ERK1/2, p42/p44), as MAPK (for Ser/Thr). Whereas the action of most MAPK modules (e.g. p38, JNK) is directed mainly towards activation of transcription factors, that of the ERK module is also on the triggering of cytoplasmic events, via phosphorylation of proteins, such as the kinase p90rsk, phospholipase A2, PKA, Sos and microtubule-associated proteins (Figure 1) (Graves et al., 1996; Windmann et al., 1999; Kolch, 2000). Upstream of the ERK module is a cascade of factors that includes: small GTP-binding proteins of the Ras or Rho family, such as Rasp21, which interact with Raf and participate in its activation; Sos, a guanidine exchange factor; Grb2, an adaptor protein with SH2 and SH3 domains; and Shc, an adaptor protein with SH2, SH3 and protein tyrosine binding domains (Figure 1) (Windmann et al., 1999; Kolch, 2000).
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The role of the ERK pathway in sperm function is probably complex. The expression and activation of ERK1/2 varies during spermatogenesis in mice and it is suggested that these kinases could contribute to the mitotic proliferation of primary spermatogonia and later to the acquisition of sperm motility (Lu et al., 1999
). ERK1/2 could mediate the phosphorylation of axonemal and/or accessory cytoskeletal proteins and be involved in the regulation of flagellar motility as observed in fowl spermatozoa (Ashizawa et al., 1997). Finally, in humans, ERK1/2 appear to be involved in sperm capacitation but their role in the acrosome reaction remains unclear (Luconi et al., 1998a,b).
Bovine serum albumin (BSA)-induced capacitation is associated with a time-dependent (over the course of 24 h) increase in tyrosine phosphorylation of two protein bands of 42 and 44 kDa that are immunoreactive to an anti-ERK1/2 antibody. This increase in tyrosine phosphorylation correlates with elevated enzyme activity as measured by an in-gel kinase assay using myelin basic protein as substrate (Luconi et al., 1998a). Furthermore, PD98059, an inhibitor of ERK1/2 activation by MEK (Alessi et al., 1995; Dudley et al., 1995) prevents sperm capacitation as well as the increases in both the tyrosine phosphorylation and kinase activity of p42/p44 (Luconi et al., 1998a). Immunocytochemistry data suggest that ERK1/2 are relocalized from the post-acrosomal to the equatorial region of the sperm head during the induction of the acrosome reaction with progesterone or the calcium ionophore A23187 (Luconi et al., 1998a,b). However, the role of ERK1/2 in sperm function is not simple since these kinases are also more tyrosine-phosphorylated and activated during progesterone-induced acrosome reaction, even though PD98059 does not prevent this process (Luconi et al., 1998b).
Rasp21 (Figure 1
) is also present in human spermatozoa and localized on the acrosomal cap (Naz et al., 1992). A monoclonal anti-Rasp21 antibody reduces the levels of hyperactivated motility, acrosome reaction and penetration of zona-free hamster oocyte of human spermatozoa, suggesting that Rasp21 could be involved in the acquisition of fertilizing ability by these cells (Naz et al., 1992). However, a high concentration (100 µg/ml) of antibody was used to obtain these effects (Naz et al., 1992). Another small GTP-binding protein, Rho, is found in the membrane of bovine spermatozoa (Hinsch et al., 1993) whereas rhophilin, a small Rho-binding protein (Nakamura et al., 1999) and ropporin, a sperm-specific binding protein of rhophilin localized exclusively on the fibrous sheath (Fujita et al., 2000), have also been identified in mouse spermatozoa. The adaptor protein Shc (Figure 1) is also localized on the acrosomal region of human spermatozoa (Morte et al., 1998). Only the 52 kDa form, and not the 46 and 66 kDa forms, of Shc has been found by immunoblotting (Morte et al., 1998). The Shc immunoreactive band is more tyrosine-phosphorylated in spermatozoa incubated for 5 h with BSA and then for 15 min with progesterone (Morte et al., 1998); this treatment should induce the acrosome reaction but does not allow differentiation of the effects due to capacitation from those due to the acrosome reaction. The other adaptor protein, Grb2 (Figure 1), has not been detected by immunoblotting or immunoprecipitation in human spermatozoa (Morte et al., 1998).
As summarized above, reports on the presence of elements of the ERK pathway and role of ERK1/2 in sperm function (maturation, motility, capacitation, acrosome reaction) are partial. The presence and involvement of the whole ERK pathway in sperm function has not been firmly ascertained. The first aim of the present study was to confirm the presence and evaluate the role of the ERK pathway in human sperm capacitation induced by fetal cord serum ultrafiltrate (FCSu) (de Lamirande and Gagnon, 1995; de Lamirande et al., 1998a,b). The second aim was to study possible mechanisms of action of the ERK pathway in relation (i) to the O2-· production that initiates capacitation (de Lamirande and Gagnon, 1995; de Lamirande et al., 1998a), (ii) the tyrosine phosphorylation of fibrous sheath proteins that occurs a few hours later (de Lamirande et al., 1997; Leclerc et al., 1997,1998) and (iii) the phosphorylation of specific proteins by MEK and ERK1/2. Finally, since many signal transduction pathways are common for capacitation and the acrosome reaction (de Lamirande et al., 1997), the role of the ERK pathway in the lysophosphatidylcholine-induced acrosome reaction was also studied.
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Materials
Percoll was obtained from Amersham Pharmacia Biotech (Baie d'Urfé, Qué, Canada), the modified Cypridina analogue [MCLA: 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one]from TCI America (Portland, OR, USA) and superoxide dismutase (SOD; from bovine erythrocytes) from Roche Molecular Biochemicals (Laval, Qué, Canada). Lysophosphatidylcholine (LPC), BSA and fluorescein isothiocyanate-conjugated Pisum Sativum agglutinin were purchased from Sigma Chemical Co. (St Louis, MO, USA). CGP85793 was a generous gift of Novartis Pharma Inc. (Basel, Switzerland). 2'-Amino-3'-methoxyflavone (PD98059), 1,4-diamino-2,3-dicyano-1,4bis(methylthio)butadiene (U124), 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U126), N-[5-(3-dimethylaminobenzamido)-2-methylphenyl]-4-hydroxybenzamidine (ZM336372), (Z)-5-fluoro-2-methyl-1-[p-(methylthio)benzylidene]indene-3-acetic acid (sulindac sulphide), FTI-277 and ß-glycerolphosphate were purchased from Calbiochem (La Jolla, CA, USA). Transduction Laboratories (Lexington, KY, USA) was the supplier for antibodies against Shc (polyclonal), Grb2 (monoclonal) and Rasp21 (monoclonal), and Upstate Biotechnology (Lake Placid, NY, USA) for MPM-2 (monoclonal), anti-phosphotyrosine (clone 4G10, monoclonal) and anti-Raf A (polyclonal) antibodies. Antibodies raised against ERK1/2 (polyclonal) and against the motif phospho-threonine-glutamine-phospho-tyrosine (P-Thr-Glu-Tyr-P, found in activated ERK1/2) were bought from New England Biolabs Ltd (Missisauga, Ontario, Canada). Nitrocellulose (0.22 µm pore size; Osmonics Inc., Westborough, MA, USA), goat anti-mouse IgG and goat anti-rabbit IgG, both conjugated to horse-radish peroxidase (Amersham Pharmacia Biotech), an enhanced chemiluminescence kit (Lumi-Light; Roche Molecular Biochemicals) and radiographic films (Fuji, Minami-Ashigara, Japan) were used for immunodetection of blotted proteins. All other chemicals were at least of reagent grade.
Fetal cord blood, generously provided by the Blood Bank at the Royal Victoria Hospital (Montréal, Qué, Canada), was centrifuged (1000 g, 10 min, 4°C). Sera were pooled and frozen at –20°C until used. Ultrafiltrates (FCSu) were prepared from three pools of 14–24 different samples using YM3 membranes with an exclusion limit of 3 kDa (Amicon, Oakville, Ont, Canada) (de Lamirande and Gagnon, 1995).
Substances to be tested with spermatozoa 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 or acrosome reaction.
Sperm capacitation and acrosome reaction
Semen samples from healthy volunteers were washed on four-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 cells 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 in which progressive motility was >70% were used. Spermatozoa were further diluted 10-fold in Biggers–Whitten–Whittingham medium (BWW; pH 8.0) (Biggers et al., 1971) devoid of bicarbonate and BSA and containing 1 mmol/l CaCl2.
BWW medium was used without (control) or with FCSu (7.5%, v/v) as a capacitation inducer (de Lamirande and Gagnon, 1995). For capacitation studies, inhibitors of elements of the ERK pathway were added to spermatozoa for 30 min before FCSu was supplemented and the 3.5 h incubation started. Sperm capacitation was then measured by induction of the acrosome reaction with lysophosphatidylcholine (LPC; 100 µmol/l in BWW supplemented with 3 mg BSA/ml) for 30 min as previously described (de Lamirande and Gagnon, 1995; de Lamirande et al., 1997). In experiments in which the effect of PD98059 was tested on the induction of the acrosome reaction, this substance was added to control (BWW alone) or capacitating (FCSu) spermatozoa after 3.25 h of incubation and LPC was added 15 min later. The acrosomal status of ethanol-fixed spermatozoa was evaluated using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (Cross et al., 1986). For each sample, 250–500 spermatozoa were counted. None of the chemicals tested affected the percentage of sperm motility at the concentrations used in this study and over a period of at least 4 h. Unless otherwise stated, all incubations were performed at 37°C.
Measurement of sperm O2-· generation
The production of O2-· by spermatozoa was evaluated by chemiluminescence, which was recorded at 5 min intervals using a computer-driven LKB Wallach 1251 luminometer (Turku, Finland) in the integration mode (5 s), with mixing of the measured sample and at 37°C. Measurements started immediately after the chemiluminescence amplifier MCLA (20 µmol/l) was added to spermatozoa (8x106/ml) exposed to various treatments (± inhibitors, ± FCSu) (de Lamirande and Gagnon, 1995). MCLA is a cell impermeant and very sensitive O2-·-specific probe (Nakano, 1990) and its use requires careful selection of controls and blanks. Every sample, spermatozoa and incubation media, must be run in parallel with a similar one in which SOD is added so that only the SOD-inhibitable signal (the real measure of the presence of O2-·) is considered and so that the contribution of the medium can be subtracted. Calculations were performed as described previously (de Lamirande and Gagnon, 1995).
Detection of Shc, Grb2, Rasp21, Raf and ERK1/2 by immunoblotting
The detection of ERK pathway components, Shc, Grb2, Rasp21, Raf and ERK1/2, in spermatozoa was first performed. Spermatozoa were treated with Triton X-100 (0.2%, v/v; 10 min, on ice) and then centrifuged (12 000 g for 5 min). The Triton-soluble and -insoluble (resuspended to the original volume with HEPES-balanced saline) fractions were supplemented with electrophoresis sample buffer, boiled, centrifuged, electrophoresed on 10, 12 or 15% polyacrylamide gels and electrotransferred to nitrocellulose as previously described (Leclerc et al., 1996,1997). The membranes were blocked 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) and then incubated overnight at 4°C with the primary antibodies. After washing with TTBS, membranes were incubated with the secondary antibodies for 45 min at 20°C and then washed again with TTBS. Positive immunoreactive bands were detected using the Lumi-Light chemiluminescence kit. Each of the antibodies used was also tested with a cellular extract provided by the supplier as positive control (data not shown). Antibodies raised against Shc (0.5 µg/ml), Grb2 (1 µg/ml), Rasp21 (1 µg/ml) and Raf A (0.5 µg/ml) were diluted in TTBS whereas anti-ERK1/2 (0.02 µg/ml) antibody was diluted with TTBS containing 25 mg BSA/ml. All primary antibody preparations were supplemented with sodium azide (0.1%, w/v). Goat anti-mouse and goat anti-rabbit IgG conjugated with horse-radish peroxidase were diluted to 0.2 µg/ml in TTBS a few minutes before use.
Protein phosphorylation
Protein tyrosine phosphorylation was evaluated after a 2.5 h incubation of spermatozoa under various conditions (see Results) by immunoblotting as described in the preceeding paragraph except that whole spermatozoa were boiled with electrophoresis sample buffer containing 0.1 mmol/l sodium vanadate and that anti-phosphotyrosine antibody (0.1 µg/ml TTBS) was used for 1 h at 20°C.
The presence of the P-Thr-Glu-Tyr-P motif characteristic of activated ERK1/2 and other transduction elements (Lee et al., 1995; Abe et al., 1996,1999; Miyata and Nishida, 1999; Testerink et al., 2000) was determined using the anti-P-Thr-Glu-Tyr-P antibody (0.05 µg/ml in TTBS containing 25 mg BSA/ml) incubated overnight and at 4°C with the blots. This antibody does not react with P-Thr, P-Tyr or P-Thr-X-Tyr-P (X being an amino acid other than Glu) according to a quality control statement by the manufacturer. Because P-Thr-Glu-Tyr-P is not available, no pre-adsorption tests were run. However, we ran experiments to compare immunoblotting with anti-phosphotyrosine (Upstate) and anti-P-Thr-Glu-Tyr-P (New England Biolabs) antibodies. The two antibodies gave very different results (intensity and molecular mass of protein bands) with sperm proteins as well as with the cell extract (A-431 cells stimulated with epidermal growth factor) provided as positive control with the anti-phosphotyrosine antibody. These tests indicated that the two antibodies really measure different protein characteristics.
Potential substrates for ERK1/2 were tested with the MPM-2 antibody, which is specific for the P-Ser/Thr-Pro motif (Westendorf et al., 1994) (2 µg/ml in TTBS containing 25 mg BSA/ml), incubated overnight at 4°C with the blots. In the latter two series of experiments, whole spermatozoa after 5–120 min of incubation under various conditions were boiled with electrophoresis sample buffer containing 0.1 mmol/l vanadate and 0.1 mol/l ß-glycerolphosphate (as phosphoprotein phosphatase inhibitor).
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 proteins loaded in each well was the same.
Statistical analysis
Analysis of variance (two-tailed; unpaired values) was used to evaluate the differences in the levels of capacitation and O2-· generation of spermatozoa submitted to the various treatments. Statistical differences between the effects of various treatments was then determined by the Fisher protected least-significant difference test. A difference was considered statistically significant with P < 0.05.
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Results |
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The ERK1/2 pathway in human spermatozoa: presence and role in capacitation
Immunoblotting experiments confirmed the presence of Shc (Morte et al., 1998
), Rasp21 (Naz et al., 1992) and ERK1/2 (Luconi et al., 1998a,b) and indicated that of Grb2 and Raf in human spermatozoa (Figure 1). Whereas Shc, Rasp21 and Raf appeared to be present exclusively in the Triton-insoluble fraction of spermatozoa, Grb2 and ERK1/2 were found mostly in the Triton-soluble extract of these cells (Figure 2).
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Six inhibitors of the ERK pathway were tested for their effect on capacitation: CGP85793 blocks the SH2 domain of Grb2 (Gay et al., 1999a
,b); FTI-277 inhibits farnesylation reactions, therefore activation of Rasp21 (Suzuki et al., 1998); sulindac sulphide prevents the Rasp21/Raf interaction (Rice et al., 2001); ZM336372 inhibits Raf activity (Hall-Jackson et al., 1999; Kolch, 2000); PD98059 (Alessi et al., 1995; Dudley et al., 1995) and U126 (Favata et al., 1998) inhibit MEK (Figure 1). All these substances prevented human sperm capacitation induced by FCSu in a dose-dependent fashion (Figure 3). The inactive analogue of U126, U124, had only a mild effect on capacitation. The concentrations of inhibitors needed to prevent sperm capacitation were of the same order of magnitude as those that are effective to block the ERK pathway in other cell types (Alessi et al., 1995; Dudley et al., 1995; Favata et al., 1998; Suzuki et al., 1998; Gay et al., 1999a,b; Hall-Jackson et al., 1999; Rice et al., 2001). The O2-· generated by human spermatozoa during capacitation is essential for this process (de Lamirande and Gagnon, 1995; de Lamirande et al., 1997). However, none of the inhibitors of the ERK pathway tested (CGP85793, FTI-277, U126 and PD98059; Figure 1) modified the sperm generation of O2-· related to FCSu-induced capacitation (Table I), suggesting that the ERK pathway plays a role downstream of, or in parallel to, O2-· production.
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Value different from zero but not from that obtained with the inhibitor at the same concentration and in the absence of FCSu.
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On the other hand, CGP85793 (30 µmol/l), FTI-277 (1 µmol/l), PD98059 (30 µmol/l) and U126 (0.3 µmol/l), but not the inactive analogue U124 (0.3 µmol/l), prevented the tyrosine phosphorylation of two fibrous sheath proteins, p81/p105 (according to their molecular masses) related to FCSu-induced sperm capacitation (Figure 4
), suggesting that the ERK pathway plays a role upstream of this event.
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Phosphorylation of the Thr-Glu-Tyr motif of sperm proteins during FCSu-induced capacitation
ERK1/2 contain a Thr-Glu-Tyr motif, that is phosphorylated by MEK on both Thr and Tyr (position is 202 and 204, respectively) at the time of activation (Windmann et al., 1999
) and that will be referred to as P-Thr-Glu-Tyr-P. The P-Thr-Glu-Tyr-P motif is present not only in activated ERK1/2 but also in other proteins that are associated with signal transduction pathways (Lee et al., 1995; Abe et al., 1996,1999; Miyata and Nishida, 1999; Testerink et al., 2000). A time course study was run to determine whether ERK1/2 are activated during capacitation. Two proteins of 42 and 44 kDa had a higher level of P-Thr-Glu-Tyr-P in FCSu-treated spermatozoa than in control (BWW medium alone) spermatozoa 5 min after the beginning of the incubation and a progressive and important decrease in activity was noted over the next 2 h (Figure 5). The same two proteins were recognized by an anti-ERK1/2 antibody when stripping and reblotting of the membrane was performed (data not shown), strongly suggesting an activation of ERK1/2 at the beginning of sperm capacitation. The addition of superoxide dismutase (SOD), which prevents capacitation, to FCSu did not change this pattern, indicating that extracellular O2-· is not involved in this event (Figure 5).
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The P-Thr-Glu-Tyr-P motif was also found in sperm proteins of 16, 20, 27–33 and 80 kDa (Figure 5
). The intensity of the 16–33 kDa bands increased more in spermatozoa treated with FCSu than in control spermatozoa 5 min after the beginning of the incubation and then also progressively decreased. In contrast to what was observed with ERK1/2, SOD prevented the early increases due to FCSu, and later caused an even faster decrease, in the intensity of the bands (Figure 5), suggesting a role for O2-· in this phenomenon. There was also an increase in the P-Thr-Glu-Tyr-P level of a protein band of 80 kDa at 2 h of incubation and this was higher in spermatozoa treated with FCSu than with FCSu + SOD (Figure 5, both panels). This 80 kDa protein was found in the Triton-insoluble fraction of spermatozoa and corresponded to one of the two proteins recognized by the anti-phosphotyrosine antibody (as evidenced by stripping the anti-P-Thr-Glu-Tyr-P antibody and reprobing with the anti-phosphotyrosine antibody; data not shown).
The increased level of P-Thr-Glu-Tyr-P on 16-33 kDa proteins from FCSu-treated spermatozoa, 5 min after the beginning of incubation, appeared to be related not only to the presence of O2-· but also to activation of MEK since it was prevented by PD98059, CGP85793 and ZM336372 (Figure 6). Treatment of spermatozoa with dibutyryl cAMP, a cell-permeant analogue of cAMP, plus isobutylmethylxanthine (IBMX), a cyclic nucleotide phosphodiesterase inhibitor, also prevented the increase in P-Thr-Glu-Tyr-P (Figure 6), suggesting a down-regulation of this phenomenon by cAMP or the cAMP/PKA system.
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Possible substrates for ERK1/2 in human spermatozoa
Substrates for ERK1/2 are known to be phosphorylated on Ser or Thr when proline is at the position P+1 (P-Ser/Thr-Pro motif) (Windmann et al., 1999
). The monoclonal antibody, MPM-2, which is selective for such a P-Ser/Thr-Pro motif (Westendorf et al., 1994), recognized mainly a doublet of Triton-insoluble proteins (75 and 80 kDa) (Figure 7). The P-Ser/Thr-Pro content of this protein doublet was increased in FCSu-treated spermatozoa and reduced to control levels when SOD or PD98059 were added to FCSu to prevent capacitation (Figure 7), suggesting a regulation by both O2-· and the ERK pathway.
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Role of the ERK pathway in human sperm acrosome reaction
Capacitation can be considered as a regulatory event that primes spermatozoa for the acrosome reaction, and these two physiological processes appear to be regulated, even though differently, by many common mechanisms (cAMP, Ca2+, ROS, protein tyrosine phosphorylation, etc.) (Yanagimachi, 1994
; de Lamirande et al., 1997,1998a, b). Therefore, the involvement of the ERK pathway in the acrosome reaction was also tested. Addition of PD98059 to capacitated spermatozoa reduced the LPC-induced acrosome reaction (Figure 8) after incubation in FCSu. As previously reported (de Lamirande et al., 1998b), LPC-induced acrosome reaction caused a mild increase in tyrosine phosphorylation of sperm proteins as compared to that observed after capacitation (Figure 8B). PD98059 not only prevented this increase but also reduced the protein tyrosine phosphorylation to low levels (Figure 8).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Data presented in this article demonstrate the presence and the role of key signalling elements of the ERK cascade in human sperm capacitation induced by FCSu. The ERK pathway appeared to play a role in capacitation downstream of sperm O2-· generation but upstream of tyrosine phosphorylation of p81/p105. Interestingly, the P-Thr-Glu-Tyr-P motif, characteristic of MEK substrates, was found not only on p42/p44 (ERK1/2) but also on other sperm proteins of low (16–33 kDa) and high (80 kDa) molecular masses. The increased level of P-Thr-Glu-Tyr-P in 16–33 kDa proteins observed after treatment of spermatozoa with FCSu was regulated by the ERK pathway and O2-·. Furthermore, two Triton-insoluble proteins could be substrates for ERK1/2 during sperm capacitation and the regulation of their P-Ser/Thr-Pro content would involve O2-· and the ERK pathway. Finally, the ERK pathway also appeared to be involved in the acrosome reaction.
Immunoblotting experiments confirmed the presence of Shc (Morte et al., 1998), Rasp21 (Naz et al., 1992) and ERK1/2 (Luconi et al., 1998a,b) and indicated for the first time that of Grb2 and Raf in human spermatozoa (Figure 2). Whereas, in most cells, ERK pathway elements are present in the Triton-soluble fraction (Windmann et al., 1999; Kolch, 2000), Shc, Rasp21 and Raf|and to a lesser extent Grb2 and ERK1/2|were found in the Triton-insoluble sperm fraction. Furthermore, this binding to the sperm particulate fraction was sometimes very strong; in the case of Shc, the use of sodium dodecyl sulphate (1%, w/v) and boiling for 10 min was needed to solubilize the protein (our unpublished data) (Morte et al., 1998). The localization of some elements of the ERK pathway to the Triton-insoluble fraction could suggest that scaffolding proteins (Kolch, 2000) of this system are located on the sperm cytoskeleton. In mouse spermatozoa, rhophilin, a small Rho-binding protein (Nakamura et al., 1999) and ropporin, a sperm-specific binding protein of rhophilin, have been found exclusively on the fibrous sheath (Fujita et al., 2000). It could be hypothesized that the fibrous sheath of human spermatozoa similarly binds Rasp21 and that this small GTP binding protein recruits Raf, and indirectly MEK and ERK1/2, to the cytoskeleton instead of recruiting them to the plasma membrane. Consistent with this is the observation that some of the potential targets for MEK (as measured by the level of P-Thr-Glu-Tyr-P) and ERK1/2 (as measured by the level of P-Ser/Thr-Pro) are present in the Triton-insoluble fraction of spermatozoa and more phosphorylated during capacitation (Figures 5 and 7). In both cases, the 80 kDa band corresponds also to one of those that is more tyrosine-phosphorylated during capacitation (data not shown). It is therefore possible that the 80 kDa band contains other proteins than the known sperm AKAP (Carrera et al., 1996; Mandal et al., 1999) or that this AKAP contains a Thr-Glu-Tyr motif phosphorylated by MEK or a similar kinase at the time of capacitation.
Substances acting at the level of Grb2, Rasp21, Raf and MEK inhibited human sperm capacitation (Figure 3) and the associated tyrosine phosphorylation of p81/p105 (Figure 4), confirming the role of the ERK pathway in this process. It was important to test inhibitors that act on many elements of this cascade because cross-talking occurs between different pathways, bypassing the need for some elements. For example, Raf activation can occur independently of Rasp21 via direct phosphorylation by PKC (Van Der Hoeven et al., 2000), a kinase involved in sperm capacitation (de Lamirande et al., 1997). The cAMP/PKA system can also up-regulate or down-regulate ERK1/2 by specific phosphorylations on Raf (Bornfeldt and Krebs, 1999; Rice et al., 2001). In addition, kinases other than Raf (mos, tp1/2 and MEKK1/2) can stimulate MEK (Windmann et al., 1999). Furthermore, the recruitment of Raf can be mediated by direct interaction with phosphatidic acid and be independent of association with Rasp21 (Rizzo et al., 2000). Finally, Rasp21 has effector proteins other than Raf, such as phospholipase C
, phosphatidyl-inositol 3-kinase and the Ra1GSD family of activators of Ra1 GTPases (White et al., 1995; Kelley et al., 2001). Even though it is not possible at the present time to evaluate the cross-talks with other transduction pathways (described above), our results indicate that the whole cascade, from Grb2 to ERK1/2, is involved in sperm capacitation.
The activation of ERK1/2 (as measured by the increase in P-Thr-Glu-Tyr-P content) occurred very early after the beginning of treatment with FCSu (capacitating conditions) and was followed by a progressive but major decrease in activity (Figure 5). This result is different from that obtained previously (Luconi et al., 1998a) where a progressive activation of p42/p44 kinases (ERK1/2) over the course of a 24 h incubation was observed. This discrepancy could in part be due to the use of a different inducer as BSA promotes capacitation but at a much slower rate than FCSu. Similarly to Luconi et al. (1998a), we previously observed an increased tyrosine phosphorylation of two protein bands at ~42 and ~44 kDa after a 3.5 h capacitation period (de Lamirande et al., 1998b). However, these protein bands probably contain other tyrosine-phosphorylated proteins because immunoblotting with anti-P-Thr-Glu-Tyr-P antibody clearly indicated different kinetics for ERK1/2 activation (Figure 5). Immunoblotting with an anti-P-Thr-Glu-Tyr-P antibody was preferred to in-gel kinase assay to evaluate ERK1/2 activation because renaturation of proteins after electrophoresis is a process of low yield for most enzymes and because myelin basic protein can be a substrate for many kinases.
The increased level of P-Thr-Glu-Tyr-P of two sperm proteins of 42 and 44 kDa that are also recognized by an anti-ERK1/2 antibody strongly suggest that activation of ERK1/2 occurs during capacitation. This activation may appear very rapid (Figure 5) but such an effect has been observed in other cell types. For example, the activation of ERK1/2 during integrin-dependent adhesion of human neutrophils to endothelial cells occurs 5 min after stimulation with peroxynitrite (Zouki et al., 2001). In many systems, the activation of ERK1/2 is transient and closely regulated by specific phosphatases and negative feedback by phosphorylation of the guanidine exchange factor Sos and/or increase in cAMP (Windmann et al., 1999). The progressive and important decrease in ERK1/2 activity observed after the early activation and over 2 h of capacitation could be related to the intracellular increase in cAMP known to occur at the beginning of this process (Parinaud and Milhet, 1996) and the resulting activation of PKA (Visconti et al., 1997). As stated above, cAMP/PKA-dependent phosphorylation can down-regulate Raf activity (Bornfeldt and Krebs, 1999).
The anti-P-Thr-Glu-Tyr-P antibody recognized p42/p44 (ERK1/2) but also other sperm proteins (16–33 and 80 kDa). This did not appear to be due to non-specific binding of the antibody since the P-Thr-Glu-Tyr-P content of sperm proteins was modified with time and incubation conditions (Figure 5). This finding is interesting since other proteins beside ERK1/2, such as ERK5, ERK7, MOK and RISK-1 (Lee et al., 1995; Abe et al., 1999; Miyata and Nishida, 1999; Testerink et al., 2000; Sung et al., 2001), have been shown to contain the Thr-Glu-Tyr motif and be doubly phosphorylated at the time of their activation. The changes in P-Thr-Glu-Tyr-P content of low molecular mass (16–33 kDa) sperm proteins followed the same kinetics than that of p42/p44 (ERK1/2), i.e. an early increase followed by a progressive decrease over the next 2 h of capacitation (Figure 5). However, in contrast to what was observed for p42/p44 (ERK1/2), SOD prevented the early increase, and later caused an even faster decrease, in the P-Thr-Glu-Tyr-P level of proteins, suggesting a role for O2-· in this effect. This result is consistent with that indicating that elements of the ERK cascade do not influence the generation of O2-· by capacitating spermatozoa (Table I) but rather play a role downstream of the O2-· production. In other cell types, ROS have also been shown to modulate the activity of MAPK (including ERK) pathways (Abe et al., 1996,1999; Zhang et al., 1998; Adler et al., 1999). The FCSu-induced increase in P-Thr-Glu-Tyr-P content of proteins of 16–33 kDa was also prevented by CGP85793, ZM336372, PD98059 and the combination of dibutylyl cAMP plus IBMX (Figure 6), indicating the participation of elements of the cascade leading to MEK and of the cAMP/PKA system in this effect. Together, these results strongly suggest that spermatozoa may contain some substrates for MEK or a similar kinase, that would be directly or indirectly regulated by O2-·. The nature of the proteins containing the P-Thr-Glu-Tyr-P motif remains to be elucidated.
These results again emphasize that multiple pathways regulate sperm capacitation and that a fine regulation is needed for its completion. Both the ERK pathway (Luconi et al., 1998a,b; this article) and the cAMP/PKA system (Leclerc et al., 1996; de Lamirande et al., 1997; Visconti et al., 1997) are needed for capacitation. However, an increase in cAMP or cGMP and the resulting activation of PKA or protein kinase G (PKG), respectively, have been shown to prevent and/or reverse ERK1/2 activation (Bornfeldt and Krebs, 1999; Rice et al., 2001). We could hypothesize that the action of the ERK pathway and the cAMP/PKA system are needed sequentially during capacitation. The early activation of the ERK pathway would trigger some event of capacitation and be stopped by action of cAMP/PKA, which would then take over and act on other effector(s). Targets for the ERK1/2 pathway and the cAMP/PKA system would be different but eventually affect a common system, possibly the tyrosine phosphorylation of fibrous sheath proteins, leading to capacitation. However, results presented in Figure 6 seem to contradict this hypothesis. The combination dbcAMP plus IBMX triggers capacitation (Leclerc et al., 1996; this article) even though it prevents the FCSu-associated increase in P-Thr-Glu-Tyr-P of sperm proteins (Leclerc et al., 1996). This effect could perhaps be explained by the high amount of dbcAMP, 1 mmol/l (100–200-fold higher than sperm intracellular cAMP concentration), used for such experiments (Leclerc et al., 1996), that could possibly bypass the need for activation of the ERK pathway.
ERK1/2 selectively phosphorylate Ser or Thr but only when a Pro is present at position P+1 (Ser/Thr-Pro motif) (Windmann et al., 1999). Results obtained with the MPM-2 antibody suggest that two Triton-insoluble sperm proteins (75 and 80 kDa) could be the main substrates for ERK1/2 or a similar kinase in spermatozoa (Figure 7). The P-Ser/Thr-Pro content of this protein doublet was increased in FCSu-treated spermatozoa and reduced to control level when SOD or PD98059 were added to FCSu to prevent capacitation (Figure 7), suggesting a regulation both by O2-· and the ERK pathway. This result may seem contradictory to that presented in Figure 5 since activation of ERK1/2 did not appear to be modulated by O2-·. We could hypothesize that, beside ERK1/2, other factors are needed for the phosphorylation of the protein doublet, and that these factors are modulated by O2-·. Alternatively, we may propose that another kinase with an ERK1/2-like activity and triggered by the same signalling cascade as ERK1/2, may be present in spermatozoa and regulated by O2-·. Support for this proposition rests on the finding that spermatozoa contain other proteins with the P-Thr-Glu-Tyr-P motif, characteristic of activated ERK1/2 and of an increasing number of signalling elements and kinases (Lee et al., 1995; Abe et al., 1999; Miyata and Nishida, 1999; Testerink et al., 2000; Sung et al., 2001) and that the P-Thr-Glu-Tyr-P content of some of these sperm proteins was regulated by O2-· (Figures 5 and 6).
The ERK pathway may be activated by the triggering of many different receptor types, such as the epidermal growth receptor, the insulin receptor, the platelet-derived growth factor, T-cell receptor and specific cytokine receptors (Windmann et al., 1999). At the present time, there is no indication of the sperm receptor responsible for activation of the ERK pathway during capacitation. However, we may hypothesize that O2-·, which is produced by spermatozoa as one of the initiating events of capacitation (de Lamirande and Gagnon, 1995) and/or its dismutation product, H2O2, may activate a plasma membrane receptor and as a consequence trigger the ERK cascade as observed in other cellular systems (Zhang et al., 1998; Adler et al., 1999). It is also possible that O2-· or other ROS, such as H2O2 and NO·, which are both cell permeant and generated by spermatozoa during capacitation (de Lamirande et al., 1997; Herrero et al., 2000), directly affect elements of this transduction pathway. Rasp21 is a known target for O2-· and NO·; these free radicals modify the Rasp21 structure so that the GTP-bound (active) form is maintained (Lander et al., 1997; Adler et al., 1999).
In contrast to what was observed by one study (Luconi et al., 1998b), data presented in Figure 8 suggest that the ERK pathway may also be involved in the induction of the acrosome reaction and the associated tyrosine phosphorylation of sperm proteins. This discrepancy could again be explained by the use of inducers (progesterone and A23187 versus LPC) that have very different mechanisms of action. However, our result may not be that surprising since capacitation and the acrosome reaction appear to be regulated, even though differently, by many common mechanisms (cAMP, Ca2+, ROS, protein tyrosine phosphorylation, etc.) (Yanagimachi, 1994; de Lamirande et al., 1997,1998a,1998b).
In conclusion, our data demonstrate the presence and role of the ERK signalling cascade in human sperm capacitation induced by FCSu. The activation of the ERK pathway is rapid and transient. We identified some few sperm proteins beside p42/p44 (ERK1/2) as potential substrates for MEK and a doublet of proteins as potential ERK1/2 substrates. The phosphorylation of these proteins appears to be regulated, at least in part, by O2-· and/or cAMP. These results emphasize again the role of multiple transduction pathways and the need of a tight regulation of these during capacitation.
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We thank Miss Grace Ho for her skilful assistance in the study on tyrosine phosphorylation of sperm proteins and Ms Linda Lefièvre and Mr Daniel White for the review of the manuscript. We also thank the staff of the Blood Bank at the Royal Victoria Hospital for generously providing fetal cord blood and all the volunteers who participated in this study. This work was supported by a grant from the Canadian Institute of Health Research to C.G.
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1 To whom correspondence should be addressed at: Urology Research Laboratory, H6.47, Royal Victoria Hospital,687 ave des Pins ouest, Montréal, Qué Canada H3A 1A1. E-mail: edelamirande{at}hotmail.com
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