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BOR - Papers in Press, published online ahead of print March 16, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038794
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BIOLOGY OF REPRODUCTION 73, 94–105 (2005)
DOI: 10.1095/biolreprod.104.038794
© 2005 by the Society for the Study of Reproduction, Inc.

Reactive Oxygen Species and Protein Kinases Modulate the Level of Phospho-MEK-Like Proteins During Human Sperm Capacitation1

Cristián O'Flaherty 2 , Eve de Lamirande , and Claude Gagnon 

Urology Research Laboratory, Royal Victoria Hospital and Faculty of Medicine, McGill University, Montréal, Québec, Canada H3A 1A1


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Capacitation is an essential process by which spermatozoa acquire fertilizing ability. Reactive oxygen species (ROS), protein kinase A (PKA), protein kinase C (PKC), protein tyrosine kinases (PTKs), and the extracellular signal-regulated protein kinase (ERK or mitogen-activated protein kinase [MAPK]) pathway regulate sperm capacitation. Our aim was to evaluate the phosphorylation of MEK (MAPK kinase or MAP2K) or MEK-like proteins in human sperm capacitation and its modulation by ROS and kinases. Immunoblotting using an anti-phospho-MEK antibody indicated that the phosphorylation of three protein bands (55, 94, and 115 kDa) increased in spermatozoa treated with fetal cord serum ultrafiltrate (FCSu), BSA, or isobutylmethylxanthine plus dibutyryl cAMP as capacitating agents. These phospho-MEK-like proteins are localized along the sperm flagellum. The MEK-inhibitors PD98059 and U126 prevented this phosphorylation, suggesting that these proteins are MEK-like proteins. The ROS scavengers prevented, and the addition of H2O2 or spermine-NONOate (nitric oxide donor) triggered, the increase of phospho-MEK-like proteins. The capacitation-related increases in phospho-MEK-like proteins induced by FCSu, H2O2, and spermine-NONOate were similarly modulated by PKA, PKC, and PTK, suggesting ROS as mediators in this phenomenon. These results indicate that phospho-MEK-like proteins are modulated by ROS and kinases and probably represent an intermediary step between the early events and the late tyrosine phosphorylation associated with capacitation.

gamete biology, kinases, nitric oxide, signal transduction, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulating evidence suggests that reactive oxygen species (ROS) are not only injurious by-products of cellular metabolism but also fundamental participants in cell signaling and regulation mechanisms [1]. This apparent paradox also is true for spermatozoa, and it appears to be related mostly to the concentration of ROS produced [2]. Motility and other sperm functions are impaired by high amounts of hydrogen peroxide (H2O2) [2]. However, at very low concentration, ROS are involved in the acquisition of fertilizing ability by spermatozoa [3, 4].

Freshly ejaculated spermatozoa are unable to fertilize an oocyte; they need to be capacitated first. The capacitation process occurs over a period of a few hours and is related to metabolic, ionic, and membrane changes as well as to phosphorylation events associated with various kinases and transduction pathways. Proteins from the zona pellucida surrounding the oocyte will trigger the acrosome reaction only in capacitated spermatozoa, and the acrosomal enzymes released during this exocytotic event will digest the zona pellucida, allowing spermatozoa to reach and finally fertilize the oocyte [3, 5].

Although the mechanisms involved in the control of capacitation are not yet well known, strong evidence indicates that this process is associated with or controlled by different signal transduction elements, such as protein kinase A (PKA), its substrates, and the cAMP/PKA-dependent tyrosine (Tyr) phosphorylation of fibrous sheath proteins [6– 12], protein kinase C (PKC) [13], protein tyrosine kinases (PTK) [6, 12], and components of the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinase (MAPK) pathway [13–16].

The observation that the exogenous addition of ROS scavenger, such as superoxide dismutase (SOD) or catalase, inhibits human sperm capacitation and the related protein Tyr phosphorylation was the first indication for the requirement of extracellular superoxide anion (O2·–) and H2O2 in this process. These results indicated that the possible targets of ROS are on the sperm plasma membrane [17–20]. Nitric oxide synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), also prevent human sperm capacitation, suggesting the requirement of nitric oxide (NO·) in this process [21]. Controlled production of O2·–, H2O2, and NO· by spermatozoa and their participation during capacitation have been described in a few species, such as human [7, 18, 21] and bull [22]. The ROS induce capacitation and the related Tyr phosphorylation of several proteins by activating the cAMP/PKA pathway [6, 7, 21] and by modulating the ERK pathway in human spermatozoa [15, 16].

Elements of the ERK pathway are present in human spermatozoa and involved in capacitation [14, 15]. The basic assembly of all MAPK pathways is a module in which three kinases are sequentially activated; the ERK module includes Raf (MAPK kinase kinase, for serine/threonine [Ser/Thr]), MEK (MAPK2K, dual specificity for Ser/Thr and Tyr), as well as ERK 1 (MAPK3) and ERK2 (MAPK1, for Ser/Thr) [23, 24]. The MEK phosphorylates the Thr and Tyr residues within the Thr-Glu-Tyr motif, which is present at the active site not only of ERK1 and ERK2 but also that of ERK 5 (big MAPK) [25], ERK7 [26], and other important signal transduction elements, such as MOK [27]. Inhibitors of MEK and MEK-like kinases (PD98059 and U126) block capacitation and the associated phosphorylation of Thr-Glu-Tyr in sperm proteins, indicating that such kinases are present in spermatozoa and are active during capacitation [13, 15, 16].

The double phosphorylation of the Thr-Glu-Tyr motif associated with human sperm capacitation increases with time, being significant from 1 h of incubation, and it affects two proteins of 80 and 105 kDa. This phosphorylation is regulated by receptor PTK, PKC, and an MEK or MEK-like kinase, and it appears to occur upstream of the known Tyr phosphorylation of fibrous sheath proteins [13, 15]. The ROS modulate transduction, including the ERK pathway [28], and more recent evidence indicates that NO·, but not H2O2 or O2·–, regulate the phosphorylation of the Thr-Glu-Tyr motif during human sperm capacitation [16].

The ROS and several kinases, including MEK (or MEK-like proteins), are involved in sperm capacitation, but the mechanisms by which these signaling elements interact are not yet known. As observed in other cells, cross-talk is possible between these kinases [29, 30], as is a modulation of the kinases by ROS [31–33]. The double phosphorylation of the Thr-Glu-Tyr motif increases during sperm capacitation, and MEK or MEK-like proteins are involved in this process and sperm capacitation. Therefore, the first aim of the present study was to determine the presence of phospho-MEK (phosphorylated; active MEK) or phospho-MEK-like proteins in human spermatozoa and their association with capacitation. The phospho-MEK-like proteins were immunolocalized, and then the role of ROS as triggering agent and the modulation of this phosphorylation by protein kinases were evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

The following substances were purchased from Sigma Chemical Company (St. Louis, MO): 3-isobutyl-1-methylxanthine (IBMX), N6,2'-O-dibutyryl cAMP (dbcAMP), H89, L-NAME, NG-nitro-D-arginine methyl ester (D-NAME), Rp-adenosine-3',5'-cyclic monophosphorothionate (Rp-cAMPS), BSA (fraction V, fatty acid-free), lysophosphatidylcholine (LPC), and an anti-phospho-MEK antibody. Diphenyliodonium chloride (DPI) was bought from Aldrich Chemical Company (Milwaukee, WI). Spermine-NONOate (Sp-NONOate; N-(2-aminoethyl)-N-(2-hyrdoxy-3-methrosohydrazino)-1,2-ethylenedeamine) was bought from Cayman Chemical Company (Ann Arbor, MI). The PD98059, U126, U124, chelerythrine, herbimycin A, PP2, PP3, tyrphostin A47, okadaic acid, ß-glycerolphosphate, and bovine liver catalase were purchased from Calbiochem (La Jolla, CA). The SOD from bovine erythrocytes was bought from Roche Molecular Biochemicals (Laval, PQ, Canada). Percoll was purchased from Amersham Pharmacia Biotech (Montréal, PQ, Canada). Nitrocellulose (pore size, 0.22 µm; Osmonics, Inc., Westborough, MA), the antibody raised against the amino acid motif containing a phosphorylated serine (Ser) in an environment similar to that found in human phospho-MEK (Ser217 or Ser221 for MEK1 or MEK2, respectively; Cell Signaling Technology, Beverly, MA), donkey anti-rabbit immunoglobulin (Ig) G conjugated to horseradish peroxidase (Cedarlane Laboratories Ltd., Hornby, ON, Canada), an enhanced chemiluminescence kit (Lumi-Light; Roche Molecular Biochemicals, Laval, PQ, Canada), and radiographic films (Fuji, Minami-Ashigara, Japan) were used for immunodetection of blotted proteins. The blocking agent for the anti-phospho-MEK antibody was provided by Cell Signaling Technology). The biotinylated goat anti-rabbit IgG (H+L) antibody was bought from Cedarlane Laboratories. The Alexa Fluor 555 conjugate of streptavidin and Prolong Antifade kit were purchased from Molecular Probes (Portland, OR). All other chemicals were of at least reagent grade.

Fetal cord blood was collected at the birthing center of the Royal Victoria Hospital (Montréal, PQ, Canada). Informed consent was obtained from the patients, and the ethics board of the Royal Victoria Hospital approved the present study. Fetal cord blood samples were centrifuged (1000 x g, 30 min, 4°C), and supernatants were pooled and frozen at –20°C until used. The ultrafiltrates of fetal cord serum (FCSu) were prepared from three pools of 14–24 different samples using YM3 membranes with an exclusion limit of 3 kDa (Amicon, Oakville, ON, Canada) [18, 34]. Silver stain indicated that no proteins were present in 10% polyacrylamide gels loaded with FCSu alone.

Inhibitors and activators used in the present study were dissolved in water or dimethyl sulfoxide. The concentration of dimethyl sulfoxide 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 at 37°C.

Sperm Preparation and Treatments

Semen samples from 12 healthy volunteers participating in the present study were normal according to World Health Organization criteria [35]. Liquefied semen samples were washed on four-layer (95%-65%-40%-20%) Percoll gradient buffered in Hepes-balanced saline (115 mM NaCl, 4 mM KCl, 0.5 mM MgCl2, 14 mM fructose, 25 mM Hepes, pH 8.0). Samples were centrifuged for 30 min at 2300 x g, and spermatozoa at the 65%-95% Percoll interface and in the 95% Percoll layer were pooled and diluted to 250 x 106 cell/ml with the 95% Percoll solution. Only samples in which progressive motility was greater than 70% were used. Spermatozoa were diluted further to 50 x 106 cells/ml in Biggers, Whitten, and Whittingham (BWW) medium [36] devoid of bicarbonate and BSA and containing 1 mM CaCl2 and 25 mM Hepes (pH 8.0).

The FCSu was shown to trigger capacitation, hyperactivated motility, and cAMP/PKA-dependent Tyr phosphorylation of proteins [3, 6, 19] to similar levels and with kinetics and mechanisms similar to those observed with BSA [3, 17, 37–39]. As observed with other inducers, FCSu-induced capacitation is associated with a cAMP/PKA-dependent Tyr phosphorylation of fibrous sheath proteins [6], generation of ROS [18, 21], activation of PKA [19], PKA-dependent protein phosphorylation [11], and modification of the sulfhydryl content of Triton-soluble proteins [20]. Furthermore, FCSu contributed to low concentration (2 mM) of bicarbonate in the BWW medium, which appeared to be sufficient, because no further increase was observed in the level of capacitation after the addition of bicarbonate to the incubation medium [6]. Sperm capacitation was evaluated after 3.5 h by induction of the acrosome reaction with LPC as previously described [3]. When treated with 10% FCSu (22% ± 5%, values are mean ± SEM throughout), 3 mg/ml of BSA (24% ± 1%), 50 µM H2O2 (18% ± 3%), or 100 µM Sp-NONOate (20% ± 3.5%), spermatozoa had higher levels of capacitation than occurred in control (BWW medium alone, 8.0% ± 0.5%) cells after a 3.5-h incubation.

For the time-course study, sperm samples were incubated in BWW medium at 37°C without (control) or with the capacitation-inducer FCSu (10%, v/v), and aliquots were taken at 0, 2, 5, 15, 30, and 60 min and used for SDS-PAGE and immunoblotting (see below). Because significant increases in the phosphorylation of MEK-like proteins were observed in three protein bands in FCSu-treated spermatozoa at 60 min, the experiments described below were done with this incubation period.

The effects of a 60-min incubation with FCSu and BSA (3 mg/ml) as capacitating agent also were compared to ascertain that these two conditions triggered similar effects on phospho-MEK-like levels. Cells were submitted to a one-step wash over a 20% Percoll layer (2000 x g, 5 min) to remove most of the BSA in the incubation medium; spermatozoa were collected by aspiration of the loose pellet through the Percoll layer as previously described [40].

The role of ROS on the phosphorylation of MEK-like proteins in human spermatozoa incubated at 37°C for 60 min under capacitating conditions with FCSu (10%, v/v) or the combination of IBMX (0.1 mM) plus dbcAMP (1 mM) was then studied by the addition of SOD (0.1 mg/ml), catalase (0.1 mg/ml), L-NAME (nitric oxide synthase inhibitor, 1 mM), D-NAME (inactive analog of L-NAME; 1 nM), or DPI (NADPH oxidase inhibitor, 100 µM). The effect of the exogenous addition of H2O2 (50 µM) or Sp-NONOate (NO·-releasing agent, 100 µM) on the phosphorylation of MEK-like proteins also was tested.

To study modulation of the phosphorylation of MEK-like proteins by protein kinases, spermatozoa were, or were not, supplemented with a capacitation inducer—FCSu (10%, v/v), H2O2 (50 µM), or Sp-NONOate (100 µM)—in the absence or presence of 10 µM H89 or 200 µM Rp-cAMPS (inhibitors of PKA), 10 µM chelerythrine (inhibitor of PKC), 10 µM tyrphostin A47 (inhibitor of receptor-type PTK), 10 nM PP2 (inhibitor of nonreceptor-type PTK), or 10 nM PP3 (the inactive analog of PP2). All the inhibitors used in the present study were added to spermatozoa 30 min before the capacitation inducers.

SDS-PAGE, Immunoblotting, Densitometry, and Statistical Analysis

At the end of each incubation, treated samples were supplemented with electrophoresis buffer containing vanadate (100 µM), ß-glycerolphosphate (20 mM), sodium fluoride (5 mM), and okadaic acid (10 nM); incubated at 100°C for 5 min; and then centrifuged for 5 min at 21 000 x g. Sperm proteins were electrophoresed on 10% polyacrylamide gels and electrotransferred to nitrocellulose membranes using 10 mM 3-cyclohexylamino-1-propane sulfonic acid (CAPS) buffer (pH 11) containing 10% methanol. The membranes were incubated with a solution of skim milk (5%, w/v) in Tris (20 mM, pH 7.8)-buffered saline containing Tween 20 (0.1%, v/v; TTBS) for 20 min. The anti-phospho-MEK antibody was diluted 1:1000 (v/v) in TTBS supplemented with 25 mg/ml of 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 chemiluminescence kit. At the end of each experiment, blots were rinsed in distilled water and silver stained [41] to ascertain that the amount of protein loaded in each well was the same.

To confirm the specificity of the antibody, the anti-phospho-MEK antibody was preincubated with its blocking agent (the peptide used to raised the antibody) according to the manufacturer's instructions. As another proof that the antibody recognizes MEK and MEK-like proteins and their phosphorylation, spermatozoa were incubated in BWW medium alone or with 10% FCSu in the absence or presence of two MEK inhibitors that act by different mechanisms, PD98059 (100 µM) [42] and U126 (0.3 µM) [43].

After the chemiluminescence detection of the phosphorylation of MEK-like proteins in human spermatozoa, the films were scanned using an Agfa Snapscan 1236 scanner (Agfa-Gevaert, NV). Digital images obtained were analyzed with the Un-Scan-It gel software version 5.1 (Silk Scientific Corporation, Orem, UT). The linearity between the density of the bands and the amount of antigen was verified and confirmed (see Fig. 2D). Different amounts of control and capacitating spermatozoa were blotted with the anti-phospho-MEK antibody, and the intensity of the bands were analyzed. To determine the increase in intensity of the bands in capacitating and noncapacitating spermatozoa for the time-course study and the comparison of various capacitating conditions, the intensities were normalized to 1 with the value obtained at time zero or with the value obtained in spermatozoa incubated in BWW medium alone. This normalization allowed us to determine the relative increases in intensities obtained under various capacitating conditions. In subsequent studies on the triggering effect of ROS and regulation by protein kinases, the intensities were normalized to 1 with the value obtained in capacitating spermatozoa (treated with FCSu, IBMX plus dbcAMP, H2O2, or Sp-NONOate, depending on the respective experiment). This normalization allowed us to determine the percentages of inhibition caused by the presence of the various chemicals. The relative intensities of the three bands of interest were sometimes very different from that of p55, being much lower than for the other two. Because of this, analysis (when needed) was performed on films of different exposure times to allow the best definition of the bands. An experiment was performed in which different amounts of proteins were loaded to ensure that measured densities were proportional to the amounts of proteins on the blots. In all cases, the contribution of the background was subtracted. The normalized intensities were compared using the Student t-test (two tails, paired values) and the different capacitating conditions (FCSu, H2O2, or Sp-NONOate) were analyzed using ANOVA and the Bonferroni test. A difference was considered to be significant at P ≤ 0.05.



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  		FIG. 2. Justification of immunoblotting with the anti-phospho-MEK antibody. A) Antigenic agent. Proteins from capacitating and noncapacitating spermatozoa (60-min incubation) were immunoblotted with the anti-phospho-MEK antibody either preadsorbed or not with the small phosphopeptide used to generate the antibody. Results are from one experiment that is representative of two others (n = 3) performed with semen samples from different donors. B) Effect of PD98059 and U126 on the phospho-MEK-like proteins. Spermatozoa were treated without (–) or with (+) FCSu and in the absence or presence of 100 µM PD98059 or 0.3 µM U126 for 60 min. Sperm proteins were immunoblotted with the anti-phospho-MEK antibody as described in Materials and Methods. The immunoblot presented is representative of six experiments, and relative intensities are presented as the mean ± SEM of six values obtained with semen samples from different donors. *Inhibition compared to that observed in capacitating spermatozoa (treated with FCSu). C) FCSu and BSA cause similar increases of phospho-MEK-like proteins. Spermatozoa were incubated in the absence or presence of 10% FCSu or 3 mg/ml of BSA during 60 min at 37°C (n = 2). §Values are significantly lower than others. D) Relationship between the level of antigen and the intensity of the signal obtained. Different amounts of control and capacitating spermatozoa were electrophoresed and immunoblotted with the anti-phospho-MEK antibody. Intensity is expressed in pixels x 10–3. Different exposure times were used for p55 to have the correct intensity for the analysis. The immunoblot presented is representative of four experiments, and relative intensities are presented as the mean ± SEM of four values obtained with semen samples from different donors

Cellular Fractionation and Immunolocalization of Phospho-MEK-Like Proteins in Spermatozoa

The FCSu-treated spermatozoa (incubated for 60 min) were concentrated to 400 x 106 cells/ml. After the addition of vanadate (100 µM), ß-glycerolphosphate (20 mM), sodium fluoride (5 mM), and okadaic acid (20 nM; these inhibitors were used throughout all procedures described in this section), spermatozoa were quickly frozen at –70°C. After thawing and centrifugation, the supernatant (cytosole-enriched fraction) was collected, and the pellet was incubated with Triton X-100 (0.1%, v/v) for 10 min on ice. The Triton-soluble and -insoluble fractions were collected after centrifugation of these samples (12 000 x g).

Capacitated spermatozoa supplemented with Triton X-100 were sonicated (5 sec at 30% output), and the dissociation of heads and tails were monitored by phase-contrast microscopy. Heads and flagellar fragments were then separated by a 5-min centrifugation (2000 x g, 4°C) over a discontinuous 45%-90% Percoll gradient in Hepes-balanced saline. The fractions enriched with flagellar fragments (45%-90% Percoll interface) or heads (pellet) were solubilized in electrophoresis sample buffer.

For the immunolocalization of phospho-MEK-like proteins in human spermatozoa, cell suspensions were incubated without or with FCSu for 60 min before smears were prepared on Superfrost Plus slides (Fischer Scientific, Montréal, PQ, Canada). After permeabilization with 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-phospho-MEK antibody (dilution 1:100) for 2 h at 20°C. Then, smears were 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 (Oberkochen, Germany) Axiophot microscope (exciter filter BP450-490) at 1000x magnification. As controls, smears were incubated with the anti-phospho-MEK antibody preadsorbed with the blocking agent for the antibody (prepared as described above) or with the biotinylated goat anti-rabbit antibody alone.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of MEK-Like Proteins Increases in Capacitating Spermatozoa

The antibody used in the present study was raised against a small amino acid motif containing a phosphorylated Ser in an environment similar to that found in human phospho-MEK (Ser217 and Ser221 for MEK1 and MEK2, respectively) and recognized protein bands of 55, 94, and 115 kDa in human spermatozoa (Fig. 1). An increased intensity of the protein bands recognized by the antibody was observed only for p55, p94, and p115 when spermatozoa were incubated under capacitating (FCSu) as compared to control (BWW medium alone) conditions. The level of phosphorylation of these proteins in spermatozoa incubated in BWW medium alone remained unchanged over the course of the incubation. Phosphorylation of p115 increased as early as 2 min after the beginning of capacitation and plateaued 30–60 min later (Fig. 1). Phosphorylation of p55 and p94 started to increase at 2 min, but the difference in the phosphorylation level between noncapacitating and capacitating spermatozoa was significant only at 60 min (Fig. 1). Although variations were found in the intensity of phosphorylation among the different samples used, all of them followed the same general pattern of activation, and statistical analysis confirmed the differences. Because the phosphorylation appeared to be maximal at 60 min, spermatozoa were incubated for this period of time in subsequent experiments.



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  		FIG. 1. Time course for the phosphorylation of MEK-like proteins in human spermatozoa during capacitation induced by FCSu. Percoll-washed spermatozoa resuspended in BWW medium were incubated in the absence (–) or presence (+) of FCSu (10%, v/v). Proteins from 0.5 x 106 cells were loaded in each well, electrophoresed, electrotransferred, and immunoblotted using the anti-phospho-MEK antibody as described in Materials and Methods. The position of the molecular mass markers is indicated on the left. Results are from one experiment that is representative of six others (n = 7) performed with semen samples from different donors. The level of phosphorylation was estimated by densitometric analysis as described in Materials and Methods. The density value of bands obtained in spermatozoa incubated in BWW medium alone at time zero was used to normalize the values obtained with the other samples. Relative intensity of bands is presented as the mean ± SEM of seven determinations performed on sperm samples from different donors. #Value obtained in FCSu-treated spermatozoa (black dots) is significantly different from that obtained in control spermatozoa (white dots) at the same time.

The specificity of the antibody was confirmed by using the anti-phospho-MEK antibody preincubated with the small phosphopeptide that was used to generate the antibody; the protein bands of p55, p94, and p115 were not detected under these conditions (Fig. 2A). Another way to demonstrate that the anti-phospho-MEK antibody recognized MEK or MEK-like proteins was tested by using two inhibitors of MEK and MEK-like proteins that act by different mechanisms, PD98059 [42] and U126 [43]. As expected, the inhibitors prevented the phosphorylation of p55, p94, and p115, induced by FCSu, providing functional evidence that phospho-MEK-like proteins are recognized by the antibody (Fig. 2B). The inactive analog of U126, U124, did not have any effect (data not shown). The same bands of proteins were detected when another anti-phospho-MEK antibody, generated by a different manufacturer (Sigma Chemical Company), was used (data not shown).

The effect of BSA (3 mg/ml) as a capacitation inducer was the same, both for the phosphorylated proteins bands and for the intensity of the bands as observed with FCSu (Fig. 2C). Furthermore, the levels of capacitation of spermatozoa incubated with FCSu or BSA for 3.5 h were similar (22% ± 5% and 24% ± 1%, respectively) and close to those already reported by others [3, 37–39]. These data indicate that the results obtained for phospho-MEK-like proteins really correspond to capacitating conditions. The level of spontaneous acrosome reaction (no inducer added) was similar (6.5% ± 1.2%) in all sperm samples irrespective of the incubation conditions.

Localization of Phospho-MEK-Like Proteins in Human Spermatozoa

The supernatant recovered after the disruption of sperm membranes by a freezing-thawing treatment (cytosole-enriched fraction) did not present any phospho-MEK-like protein. The p55 was found mainly in the Triton-insoluble fraction, whereas p94 and p115 were found in the Triton-insoluble fraction (Fig. 3). Subsequent fractionation of the Triton-insoluble portion by sonication indicated that phospho-MEK-like proteins are present only in the flagella-enriched fraction.



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  		FIG. 3. Presence of the phospho-MEK-like proteins in sperm extracts. Proteins from whole spermatozoa (1 x 106 cells/well), cytosole-enriched fraction (equivalent to 5 x 106 cells/well), Triton-soluble (equivalent to 5 x 106 cells/well), and Triton-insoluble (equivalent to 1 x 106 cells/well) extracts as well as from flagella-enriched and heads-enriched (both equivalent to 6 x 106 cells/well) fractions were immunoblotted as described previously. Results are from one experiment that is representative of two others (n = 3) performed with spermatozoa from different donors

Immunocytochemistry indicated that sperm proteins recognized by the anti-phospho-MEK antibody are located all along the flagellum (Fig. 4). The intensity of the fluorescence was higher in capacitating than in noncapacitating spermatozoa, and the former showed a bright spot at the end of the mitochondrial sheath. The percentage of spermatozoa carrying this bright spot (Fig. 4C) significantly increased from 22% ± 4% in noncapacitating spermatozoa to 70% ± 1% in capacitating spermatozoa (mean ± SEM, n = 4, P < 0.05). As observed with immunoblots (Fig. 2), preadsorption of the antibody with the small phospho-peptide used to generate the antibody reduced the signal (Fig. 4D) to that observed with the second antibody alone (a weak labeling on the equatorial segment) and in the postacrosomal region, but not on the flagellum (Fig. 4E).



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  		FIG. 4. Immunolocalization of phospho-MEK-like proteins in human spermatozoa. Fluorescence (A–E) and phase-contrast (F–J) images of the same microscopic field are presented. Same time of exposure was used for all fluorescence and phase-contrast microphotographs. Spermatozoa were incubated with BWW medium alone or with FCSu (10%, v/v) for 60 min. Immunocytochemistry was done using the anti-phospho-MEK antibody alone (A and C) or preadsorbed with the small phosphopeptide that was used to generate the antibody (B and D) or with the second antibody alone (E). Note the spermatozoa carrying a bright spot localized at the end of the mitochondrial sheath (white arrows). Results are from one experiment that is representative of three others (n = 4) performed on spermatozoa from different donors. Original magnification x1000

ROS Modulate Phosphorylation of MEK-Like Proteins in Capacitating Spermatozoa

Because ROS and the ERK pathway are involved and interact during human sperm capacitation [15, 16], modulation of the phospho-MEK-like proteins by ROS was studied. In capacitating spermatozoa, SOD (0.1 mg/ml), catalase (0.1 mg/ml), L-NAME (1 mM), the combination of these three compounds, or DPI (100 µM) prevented the phosphorylation of p55, p94, and p115 (Fig. 5). The inactive analog of L-NAME, D-NAME, did not affect the phosphorylation of these proteins (data not shown).



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  		FIG. 5. The ROS are involved in the phosphorylation of MEK-like proteins during capacitation induced by FCSu. Percoll-washed spermatozoa were treated with SOD (0.1 mg/ml); catalase (CAT; 0.1 mg/ml); L-NAME (1 mM); the combination of SOD, CAT, and L-NAME; or DPI (100 µM) and then incubated for 60 min in the absence (–) or the presence (+) of FCSu. Proteins from 0.5 x 106 cells were loaded in each well. The immunoblots presented over the histograms for the three protein bands are from the same gel and are representative of results obtained in six experiments performed with semen samples from different donors. As explained in Materials and Methods, the relative intensities of the three bands of interest were sometimes very different, with that of p55 being much lower than those of p94 and p115. Because of this, the densitometric analysis, when needed, was performed on films of different exposure times to allow the best definition of the bands. The images presented over the histograms are those that were used for the densitometry. In addition, the band intensities evaluated by densitometry were normalized to 1 with the value obtained in capacitating spermatozoa (treated with FCSu). This normalization allowed easier evaluation of the inhibition because of the presence of the various chemicals. The immunoblots presented are representative of 4 experiments, and relative intensities are presented as the mean ± SEM of four values obtained with semen samples from different donors. *Value is significantly lower than that obtained in capacitating spermatozoa (treated with FCSu, normalization standard)

Human sperm capacitation and Tyr phosphorylation induced by the combination of IBMX (phosphodiesterase inhibitor) and dbcAMP (cell-permeant analog of cAMP) is not prevented by SOD, indicating that the O2·– generated by spermatozoa acts upstream of cAMP and PKA in this process [3]. Therefore, the effects of IBMX plus dbcAMP in the absence or presence of SOD, catalase, and L-NAME on the phosphorylation of p115, p94, and p55 were tested. Catalase, SOD, and L-NAME, either alone or in combination, prevented the phosphorylation of p55, p94, and p115, suggesting that an action of ROS downstream from that of cAMP and PKA (Fig. 6).



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  		FIG. 6. The ROS are involved in the increase in phospho-MEK-like proteins during capacitation induced by IBMX plus dbcAMP. Percoll-washed spermatozoa were treated with SOD, catalase (CAT), L-NAME, or the combination of SOD, CAT, and L-NAME as described in Figure 5 and then incubated for 60 min in the absence (–) or the presence (+) of IBMX (0.1 mM; phosphodiesterase inhibitor) plus dbcAMP (1 mM; cell-permeant cAMP analog). Proteins from 0.5 x 106 cells were loaded in each well. The immunoblots presented over the histograms for the three protein bands are from the same gel and are representative of results obtained in four experiments. Relative intensities are presented as the mean ± SEM of four values performed with semen samples from different donors. The images presented over the histograms are those that were used for the densitometry. *Value is significantly lower than that obtained in capacitating spermatozoa (treated with IBMX plus dbcAMP; normalization standard)

PKA, PKC, and PTK Are Involved in FCSu-Dependent Phosphorylation of MEK-Like Sperm Proteins

The cAMP/PKA pathway is essential for sperm capacitation and the associated Tyr phosphorylation of proteins [6, 12, 21]. Because of this and of the results presented above (Fig. 6), the effect of two inhibitors of PKA (H89 and Rp-cAMPS) that act by different mechanisms were tested on the levels of phospho-MEK-like proteins. Both inhibitors prevented the phosphorylation of p55, p94, and p115 (Fig. 7A).



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  		FIG. 7. PKA, PKC, and PTK are involved in the modulation of phospho-MEK-like proteins during FCSu-induced sperm capacitation. Percoll-washed spermatozoa were pretreated with (A) H89 (10 µM) or Rp-cAMPS (200 µM) or with (B) chelerythrine (10 µM), tyrphostin A47 (10 µM), or PP2 (10 nM) and then incubated for 60 min in the absence (–) or the presence (+) of FCSu. Proteins from 0.5 x 106 cells were loaded in each well. The immunoblots presented and the histograms showing the relative intensities (mean ± SEM) for the three protein bands are from the same gel and are representative of results obtained in seven (A) and eight (B) experiments performed with semen samples from different donors. *Value is significantly lower than that obtained with capacitating spermatozoa (treated with FCSu; normalization standard)

Both PKC and PTK also are involved in phosphorylation events associated with sperm capacitation [11–13, 19]. Therefore, the effects of PP2 and herbimycin A (both of which are nonreceptor-type PTK inhibitors) as well as of tyrphostin A47 (receptor-type PTK inhibitor) and chelerythrine (PKC inhibitor) were tested. Both PP2 (Fig. 7B) and herbimycin A (data not shown) prevented the increase in phosphorylation of the three MEK-like proteins. The inactive analog of PP2, PP3, did not prevent the phosphorylation on any of the three bands (data not shown). Chelerythrine and tyrphostin A47 prevented the phosphorylation of p55, but not those of p94 and p115, indicating a different regulation for the three proteins (Fig. 7B).

Induction of Phosphorylation of MEK-Like Protein in Human Spermatozoa by O2·–, H2O2, and NO·: Role of Protein Kinases

The results shown in Figures 5 and 6 suggest that ROS are involved in the increase in phospho-MEK-like proteins associated with sperm capacitation. Therefore, the effect of the direct addition of H2O2 and NO· at concentrations that induce sperm capacitation [3, 21, 34] as well as the regulation of the phosphorylation observed by kinases were studied (Fig. 8). The exogenous addition of 50 µM H2O2 (Fig. 8A) or 100 µM Sp-NONOate (Fig. 8B) caused an increase in the level of the same three proteins (p55, p94, and p115) recognized by the anti-phospho-MEK antibody. Furthermore, the increases in phosphorylation (noncapacitating vs. capacitating spermatozoa) were of the same order as those observed when FCSu was used as capacitation inducer (Fig. 9).



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  		FIG. 8. PKA, PKC, and PTK are involved in the modulation of phospho-MEK-like proteins during H2O2 and Sp-NONOate-induced sperm capacitation. Percoll-washed spermatozoa were pretreated with H89 (10 µM), Rp-cAMPS (200 µM), chelerythrine (10 µM), tyrphostin A47 (10 µM), or PP2 (10 nM) and then incubated for 60 min in the absence (–) or the presence (+) of (A) H2O2 (50 µM) or (B) Sp-NONOate (100 µM). Proteins from 0.5 x 106 cells were loaded in each well. The immunoblots presented over the histograms for the three protein bands are from the same gel and are representative of the results obtained in four experiments. Relative intensities are presented as the mean ± SEM of four values obtained with semen samples from different donors. *Value is significantly lower than that obtained in capacitating spermatozoa (treated with H2O2 [A] or Sp-NONOate [B]; normalization standard)



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  		FIG. 9. Capacitation induced by FCSu, H2O2, or Sp-NONOate trigger similar increases in the levels of phospho-MEK-like proteins in human spermatozoa. Percoll-washed spermatozoa were incubated for 60 min in BWW medium alone or with FCSu (10%, v/v), H2O2 (50 µM), or Sp-NONOate (100 µM). Proteins from 0.5 x 106 cells were loaded in each well. The intensities of bands were normalized to those obtained from control spermatozoa (BWW medium alone), because the aim of this experiment was to compare the effects of various capacitating agents. Values are presented as the mean ± SEM of four experiments performed with semen samples from different donors. Similar levels of phospho-MEK-like proteins were reached with the three capacitating conditions. §Value is significantly lower than all the others

As observed in FCSu-treated spermatozoa (Fig. 7), H89 prevented phosphorylation of the three protein bands and tyrphostin A47 blocked only that of p55 in spermatozoa treated with H2O2 or Sp-NONOate (Fig. 8). Chelerythrine caused a decrease in the phosphorylation of p55 in spermatozoa treated with H2O2 (as seen with FCSu) (Fig. 7B) but an increase in those treated with Sp-NONOate. The inhibitory effect of PP2 affected p55 and p94 in spermatozoa treated with H2O2 but only p55 when Sp-NONOate was used as capacitation agent. Therefore, the effects of inhibitors of PKA, PKC, and PTK generally were the same on spermatozoa treated with FCSu, H2O2, or Sp-NONOate.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we report, to our knowledge for the first time, an increase in the levels of phospho-MEK-like proteins during human sperm capacitation induced by FCSu, BSA, the combination of IBMX plus dbcAMP, or the exogenous addition of ROS (H2O2 and NO·). Furthermore, FCSu and the exogenous addition of ROS similarly increased the level of phospho-MEK-like proteins, and the modulation of this phenomenon by kinases appeared to be similar.

Previous studies from our laboratory demonstrated that PD98059 and U126, both of which are inhibitors of MEK, not only inhibited human sperm capacitation and the associated Tyr phosphorylation but also prevented the phosphorylation of the Thr-Glu-Tyr motif, which is characteristic of the MEK substrates in sperm proteins. These data suggest that a dual-specificity kinase similar to MEK is responsible for this phosphorylation [13, 15]. The anti-phospho-MEK antibody used in the present study recognized sperm proteins with molecular masses of 55, 94, and 115 kDa (Fig. 1), which differ from those of MEK1 and MEK2 (~45 kDa). Another anti-phospho-MEK antibody, provided from a different supplier (Sigma Chemical Company), recognized the same three protein bands (data not shown) supporting the previous data. The level of phospho-MEK-like proteins of spermatozoa increased during capacitation as evidenced by immunoblotting (Fig. 1) and immunocytochemistry (Fig. 4). Moreover, PD98059 and U126 prevented the phosphorylation of these proteins induced by FCSu (Fig. 2), indicating that the proteins recognized shared functional similarities with MEK1 and MEK2. Considering that a small amino acid motif containing the phospho-Ser in an environment similar to that found in human phospho-MEK (Ser217 and Ser221 for MEK1 and MEK2, respectively) was used to generate the antibody, it may not be surprising that this small motif recognized by the anti-phospho-MEK antibody could be present in other MEK-like proteins. This could explain the differences in molecular masses between the proteins recognized in spermatozoa and MEK1 and MEK2. These findings could be compared to those of a previous report in which two proteins of 45 and ~70 kDa were detected with an anti-{alpha}-MEK1 antibody, suggesting the presence of MEK1 and a MEK-like protein in human spermatozoa [14]. Taken together, these results (Figs. 1, 2, and 4) strongly suggest that p55, p94, and p115 are MEK-like proteins and involved in human sperm capacitation.

Similar modifications in phospho-MEK-like proteins were observed in spermatozoa incubated with FCSu or BSA (Fig. 2C). Furthermore, the percentages of capacitation in spermatozoa treated with these inducers for 3.5 h were similar and close to those reported by others [3, 37– 39]. Although most of capacitation media contain BSA, the presence of protein is not an essential requirement to support capacitation. Spermatozoa from different species undergo capacitation, hyperactivation, and in vitro fertilization in a protein-free medium [44, 45]. Moreover, human sperm capacitation and in vitro fertilization have been performed in a protein-free medium [46]. Results obtained with FCSu and BSA were similar, strongly suggesting the involvement of these phospho-MEK-like proteins in sperm capacitation.

Cellular fractionation indicated that MEK-like proteins are mostly Triton-insoluble and present in the flagella-enriched fraction but not in the heads-enriched fraction (Fig. 3). This location was confirmed by immunocytochemistry data indicating that phospho-MEK-like proteins are present all along the sperm flagellum (Fig. 4). The intensity of phosphorylation of these MEK-like proteins and the percentage of cells carrying a bright spot at the end of the mitochondrial sheath (Fig. 4H) increased in capacitating spermatozoa, providing additional indications that this phosphorylation is related to capacitation.

The level of phosphosphorylation of the three phospho-MEK-like proteins increased as early as 2 min after the beginning of capacitation and plateaued 30–60 min later (Fig. 1), an effect that was inhibited by PD98059 and U126 (Fig. 2). This time course suggests that ERK1 and ERK2 (p42 and p44, respectively), two common substrates for MEK, probably are not targets for the MEK-like proteins detected by the antibody. During human sperm capacitation, the phosphorylation of ERK1 and ERK2 occurs 5 min after the beginning of the incubation and is back to the control level 10 min later [15]. Another hypothesis was that the phospho-MEK-like proteins are responsible for the dual phosphorylation of the Thr-Glu-Tyr motif that occurs from 60 min and increases over the course of capacitation [13]. Both MEK1 and MEK2 as well as similar MEK-like kinases are responsible for the phosphorylation of the Thr-Glu-Tyr motif in cells [23]. Therefore, one of our aims was to study the modulation of the phosphorylation of MEK-like proteins during FCSu-induced capacitation. Here, we report that H89, Rp-cAMPS, and PP2 prevented the phosphorylation of p55, p94, and p115 (Fig. 7), suggesting a role for PKA and a nonreceptor-type PTK. Chelerythrine and tyrphostin A47 inhibited the phosphorylation of p55 (Fig. 7), indicating the involvement of PKC and a receptor-type PTK. On the other hand, the double phosphorylation of the Thr-Glu-Tyr motif is regulated by MEK and PTK but not by PKA or PKC [13, 16]. Furthermore, in the present study, the three ROS (O2·–, H2O2, and NO·) appear to be involved in the phosphorylation of MEK-like proteins (Figs. 5, 6, and 8) but that of the Thr-Glu-Tyr motif was triggered only by NO· [16]. Therefore, the differences in the modulation of phosphorylation for the Thr-Glu-Tyr motif reported previously [12, 15] and three MEK-like proteins observed in the present study are such that we have to conclude these two phosphorylation systems probably are not directly related. The MEK-like protein responsible for the Thr-Glu-Tyr phosphorylation previously reported [13, 16] may be different enough from MEK not to be detected by the antibodies used here.

The determination of MEK activity could give us more information about the actual activation of MEK-like proteins during human sperm capacitation. However, the specific substrates for these phospho-MEK-like proteins are presently unknown and are probably not ERK1 and ERK2. Both ERK1 and ERK2 are phosphorylated as early as 5 min after the beginning of capacitation [15], and then phosphorylation decreases to basal levels within the next 10 min. The maximum level of phospho-MEK-like proteins occurred at 1 h under the same capacitating conditions (Fig. 1). Moreover, PKA seems to play a role in the regulation of these phospho-MEK-like proteins (Figs. 7A and 8) but not of phospho-ERK1 and phospho-ERK2 [15]. Therefore, the measurement of MEK-like activity is not possible at present, because the substrates for these kinases are still unknown. However, the phosphorylation of MEK-like proteins of 55, 94, and 115 kDa increased during capacitation, and PD98059 and U126 blocked both of these events, indicating the participation of the phospho-MEK-like proteins. Therefore, we pursued our study on the regulation of MEK-like protein phosphorylation by ROS and protein kinases, because these elements are recognized to play an important role in human sperm capacitation.

The role of ROS as signal elements to induce the cAMP/ PKA pathway and the associated protein Tyr phosphorylation during human sperm capacitation is well known [3, 7, 21]. Superoxide anion, H2O2, and NO· [7, 18, 21, 22] are produced on the surface of capacitating spermatozoa. Furthermore, SOD and catalase, which are nonpermeable scavengers of O2·– and H2O2, respectively, prevent human sperm capacitation and the related protein Tyr phosphorylation induced by various agents, such as FCSu, follicular fluid ultrafiltrate, and progesterone [34]. Here, SOD, catalase, L-NAME, and DPI prevented, and H2O2 and Sp-NONOate triggered, phosphorylation of the MEK-like proteins (Figs. 5 and 8). Furthermore, the increase in phosphorylation of MEK-like proteins caused by H2O2 and Sp-NONOate was prevented by H89, suggesting that participation of ROS in the phosphorylation of MEK-like proteins is upstream of PKA. Intracellular levels of cAMP are increased in spermatozoa exposed to NO· [21], H2O2 [7], and O2·– [47] at concentrations that trigger human sperm capacitation. It could be hypothesized that the increase in cAMP levels related to ROS produced by spermatozoa activates PKA and triggers the phosphorylation of MEK-like proteins during capacitation. In other cell types, PKA regulates the ERK1 and ERK2 pathway by various mechanisms. For example, in PC12 cells [48], and in ß cells from pancreatic islets [49], PKA activates Raf-B, which subsequently will phosphorylate MEK. The action of ROS in their effects upstream of PKA also could be directed toward PTK, which participates in the protein Tyr phosphorylation associated with sperm capacitation [6, 7, 21], and a nonreceptor-type PTK seems to regulate the activation of PKA [11]. The phosphorylation of MEK-like proteins also appeared to be modulated by PTK, although probably in a different fashion for the three MEK-like proteins (Fig. 7B). A nonreceptor-type PTK (inhibited by PP2) would be involved in the phosphorylation of the three phospho-MEK-like proteins and a receptor-type PTK (inhibited by tyrphostin A47) only in p55 (Fig. 7B). The ROS, such as H2O2, were shown to activate PTK directly [50] or to increase indirectly the level of protein Tyr phosphorylation by inhibition of protein Tyr phosphatases [51]. Subsequently, the activated PTK can trigger, in cascade, the action of other kinases, such as PKC{delta} [32, 33] and then Raf-1 [29].

The participation of ROS in MEK-like protein phosphorylation also could be downstream of PKA action, because SOD, catalase, and L-NAME prevented the phosphorylation of p55, p94, and p115 induced by IBMX plus dbcAMP (Fig. 6). It is possible that exogenously added, or endogenously produced, H2O2 and NO· (diffusible molecules) by spermatozoa pass through the plasma membrane and act directly on elements of the ERK pathway and/or modulate the action of still-unknown PKA substrates as intermediates participating in the phosphorylation of MEK-like proteins. The observation that phospho-MEK-like proteins (Fig. 4) and phospho-PKA substrates [11] have similar localization (as evaluated by immunocytochemistry) and similar response to ROS could substantiate this hypothesis. The results presented here strongly suggest that during human sperm capacitation, ROS are involved not only in the modulation of the early activation of the cAMP/PKA pathway, as suggested before [9, 11], but also in subsequent steps related to the phosphorylation of MEK-like proteins.

The level of phospho-MEK-like proteins increased to similar levels (Fig. 9) in spermatozoa capacitated with H2O2 and NO·, as with FCSu. The mechanism by which ROS trigger this effect is not presently known. It is possible that ROS could induce the phosphorylation of MEK-like proteins in spermatozoa by activating Rasp21, as seen in a human T-cell line [52], that in turn will activate Raf (the activator of MEK). In addition, H2O2 at low concentrations can activate PKC [31], and this kinase can stimulate Raf-1, which will then phosphorylate MEK, as observed in bovine tracheal smooth muscle cells [29]. Therefore, it could be hypothesized that one mechanism by which phosphorylation of MEK-like proteins occurs is through modulation of the ERK pathway or PKC activation by ROS during capacitation of human spermatozoa.

The role of PKA in human sperm capacitation is well documented [3, 7–11]. One of the mechanisms by which ROS induce the Tyr phosphorylation of several proteins appears to be by activating the cAMP/PKA pathway in human spermatozoa, at least in part by triggering the increase in cAMP levels [6, 7, 19, 21, 47]. Recently, we found that the phosphorylation of sperm proteins of 80 and 105 kDa and carrying the arginine-X-X-phospho-(Ser/Thr) motif characteristic of PKA substrates is increased during capacitation and that this effect is modulated by ROS [11]. The phosphorylation of these PKA substrates reaches a maximum 30 min after the beginning of capacitation and, therefore, appears to be related in time with the stimulation of PKA activity [9]. In the present study, we observed that the phosphorylation of the MEK-like proteins p55, p94, and p115 plateaued after 60 min of capacitation (Fig. 1) and is dependent on PKA (Fig. 7A) and stimulated by ROS (Fig. 8). This phosphorylation of MEK-like proteins occurs after the early activation of PKA (30 min) and the increase of the phospho-PKA substrates [9, 11] but before the capacitation-related Tyr phosphorylation [6]. From these data, we could hypothesize that the phospho-PKA substrates of 80 and 105 kDa are potential intermediates involved in the phosphorylation of the MEK-like proteins p55, p94, and p115. Because the capacitation-related Tyr phosphorylation is cAMP/PKA dependent [6, 7, 12] and prevented by the two inhibitors of MEK, PD98059 and U126 [15], we also could hypothesize that these phospho-MEK-like proteins are involved in the regulation of the later Tyr phosphorylation during human sperm capacitation. Further studies are needed to confirm this hypothesis. On the basis of our results, we present a schema of the hypothetical signaling pathways involving the different elements studied here and their possible role in human sperm capacitation (Fig. 10).



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  		FIG. 10. Hypothetical mechanisms regulating phospho-MEK-like proteins during human sperm capacitation. The ROS could activate the cAMP/PKA pathway that phosphorylates several yet-unknown substrates that could, either directly or indirectly, promote the phosphorylation of MEK-like proteins. The SOD (scavenger of O2·–), catalase (CAT; scavenger of H2O2), and inhibitors of NOS synthase (L-NAME) or NADPH oxidase (DPI) could prevent the action of PKA and the subsequent phosphorylation of substrates [11]. The PKA is involved in the phosphorylation of MEK-like proteins (Fig. 7A) and protein Tyr residues [6], because H89 and Rp-cAMPS (PKA inhibitors) prevent these events. The MEK-like proteins are needed for protein Tyr phosphorylation and capacitation, because PD98059 blocks these processes [15] and prevented the phosphorylation of MEK-like proteins (Fig. 2B). The ROS participate upstream and downstream of PKA action, possibly by inducing PKC and/or PTK. These kinases also could be activated by ROS or through other mechanisms. The phosphorylation of MEK-like proteins appears to link some of the early events (e.g., ROS generation and PKA activation) [9, 11, 18] and late events (e.g., protein Tyr phosphorylation) [6, 12] of sperm capacitation. Because of the complexity of the capacitation process, only mechanisms that appear to be related directly to the results presented in our study are shown

In summary, this report presents, to our knowledge for the first time, the role and regulation of phosphorylation of MEK-like proteins during human sperm capacitation triggered by FCSu, IBMX plus dbcAMP, as well as H2O2 and NO·. These phospho-MEK-like proteins are immunolocalized all along the sperm flagellum, and their level increased in capacitating spermatozoa. The phosphorylation of MEK-like proteins is modulated by PKA, PKC, and PTK, and ROS generated by spermatozoa themselves during capacitation could be responsible, both upstream and downstream of PKA action, for this effect. The phosphorylation of MEK-like proteins probably represents an intermediary step between the early events and the late tyrosine phosphorylation associated with sperm capacitation.


   ACKNOWLEDGMENTS
 
We are grateful to Daniel White for reviewing the article. We thank Dr. Lucie Morin, Royal Victoria Hospital, for collecting fetal cord blood. We also wish to acknowledge the participation of the volunteers in the present study.


   FOOTNOTES
 
1 Supported by a grant from the Canadian Institutes for Health Research (CIRH), Canada, to C.G. Back

2 Correspondence: Cristian O'Flaherty, Urology Research Laboratory, H6.47, Royal Victoria Hospital, 687 avenue des Pins ouest, Montréal, Québec H3A 1A1, Canada. FAX: 514 843 1457; coflaher{at}yahoo.com Back

Received: 3 December 2004.

First decision: 22 December 2004.

Accepted: 14 March 2005.


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 INTRODUCTION
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 DISCUSSION
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