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Gamete Biology |
a Urology Research Laboratory, Royal Victoria Hospital and Faculty of Medicine, McGill University, Montréal, Québec, Canada H3A 1A1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, kinases, nitric oxide, signal transduction, sperm capacitation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The role of O2-·, H2O2, and NO· in human sperm capacitation has been well documented [3, 4]. The observations that capacitation induced by progesterone or biological fluids (e.g., ultrafiltrate from fetal cord serum [FCSu]) is prevented by the cell-impermeant superoxide dismutase (SOD; a scavenger of O2-·) and that spermatozoa generate O2-· from the onset of the capacitation period and over more than 4 h demonstrated the need for extracellular O2-· [5–7]. The O2-· production appears to precede sperm hyperactivation (1–3 h), protein tyrosine (Tyr) phosphorylation (from 1 h), and capacitation (progressive increase from 1–6 h), suggesting that O2-· initiates an early chain of events leading to capacitation [7]. Catalase (a scavenger of H2O2) inhibits, and the direct addition of H2O2 triggers, human sperm capacitation, indicating that H2O2 is also involved in this process [8–10]. Human spermatozoa contain NOS [11, 12], and during capacitation induced by BSA, a 7-fold increase in their NO· production occurs [13]. In addition, exogenous source of NO· causes an increase in intracellular cAMP and sperm capacitation, and L-NAME, a competitive inhibitor of L-Arg for NOS, prevents these events, indicating that NO· plays a role in sperm capacitation [11, 13].
Although the mechanisms by which ROS promote capacitation and the specific role for each ROS in this process are unknown, previous studies demonstrated that they are all involved in signal transduction events leading to time- and cAMP/protein kinase A (PKA)-dependent increase in protein Tyr phosphorylation of two sperm proteins of 80 and 105 kDa (p80/105) [9, 10, 14]. Addition of SOD, catalase, or NOS inhibitors to the incubation medium prevented both human sperm capacitation and the associated Tyr phosphorylation of proteins, whereas exogenous addition of O2-·, H2O2, or NO· promoted these events [3, 4, 8–10, 13]. Therefore, O2-·, H2O2, and NO· appear to be involved in Tyr phosphorylation of p80/105 associated with sperm capacitation.
The presence of the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinase (MAPK) in spermatozoa and the role of this pathway in sperm function have been reported recently [15–18]. The basic assembly of this pathway is an ERK module, which includes Raf as MAPK kinase kinase (for serine/threonine [Ser/Thr]), MEK or MEK-like kinases as MAPK kinase (dual specificity for Ser/Thr and Tyr) [19], and ERK 1 and 2 (p42/44) as MAPK [20, 21]. The MEK and MEK-like kinases phosphorylate Thr and Tyr residues present in a Thr-glutamine (Glu)-Tyr motif, which is present not only in ERK 1 and 2 but also in ERK 5 (big MAPK) [22], ERK 7 [23], and other important signal transduction elements such as MOK [24]. We previously reported early and transient increases in the level of phosphorylation (double phosphorylation) of the Thr-Glu-Tyr motif (P-Thr-Glu-Tyr-P) of sperm proteins of 16–33 kDa and p42/44 (ERK 1 and 2) during the first 5 min of incubation with FCSu; the effect observed on 16- to 33-kDa proteins was prevented by SOD [17]. In addition, a progressive increase in P-Thr-Glu-Tyr-P level of sperm proteins of 80 and 105 kDa (p80/105) was also observed during the course of capacitation induced by FCSu. The MEK or MEK-like kinases, but not PKA, appeared to be involved in this signal transduction event associated with sperm capacitation [17, 18].
The ROS involved in human sperm capacitation are also known for their ability to induce the ERK pathway in other cell types [2]. Therefore, the first objective of the present study was to investigate the role of O2-·, H2O2, and NO· in the regulation of the double phosphorylation of the Thr-Glu-Tyr motif present in sperm proteins p80/105 during capacitation. The second objective was to immunolocalize the P-Thr-Glu-Tyr-P motif in spermatozoa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The following reagents were purchased from Sigma Chemical Company (St Louis, MO): Pisum sativum agglutinin conjugated to fluorescein isothiocyanate (PSA-FITC), 3-isobutyl-1-methylxanthine (IBMX), N6,2'-O-dibutyryl cAMP (dbcAMP), lysophosphatidylcholine (LPC), and H89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide). The NG-nitro-L-arginine methyl ester (L-NAME) and NG-nitro-D-arginine methyl ester (D-NAME) were bought from Research Biochemicals International (Natick, MA). The N-(2-aminoethyl)-N-(2-hydroxy-3-metrosohydrazino)-1,2-ethylenedeamine (spermine NONOate) was bought from Cayman Chemical Company (Ann Arbor, MI). Percoll was obtained from Amersham Pharmacia Biotech (Baie d'Urfé, QC, Canada). The 2'-amino-3'-methoxyflavone (PD98059), chelerythrine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP2), tyrphostin A47, and bovine liver catalase (21 000 IU/mg) were purchased from Calbiochem (La Jolla, CA). The SOD (from bovine erythrocytes) and xanthine oxidase (from cow milk) were purchased from Roche Molecular Biochemicals (Laval, QC, Canada). New England Biolabs (Mississauga, ON, Canada) was the supplier for the polyclonal antibody raised against the P-Thr-Glu-Tyr-P motif and its blocking peptide, P-Thr-Glu-Tyr-P. Nonimmune rabbit immunoglobulins (IgG) were purchased from Cedarlane Laboratories Ltd. (Hornby, ON, Canada). Nitrocellulose (pore size, 0.2 µm; Osmonics, Inc., Westborough, MA), goat anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Pharmacia Biotech), an enhanced chemiluminescence kit (Lumi-Light; Roche Molecular Biochemicals), and radiographic films (Fuji, Minami-Ashigra, Japan) were used for immunodetection of blotted proteins. Alexa Fluor 555 conjugate of streptavidin and Prolong Antifade Kit were purchased from Molecular Probes (Portland, OR). All other chemicals were at least of reagent grade.
Fetal cord blood was collected at the birthing center of the Royal Victoria Hospital (Montréal, QC, Canada). An 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), pooled, and frozen (-20°C) until used. The ultrafiltrate of FCS (FCSu) was prepared from at least 15 individual samples using YM3 membranes (exclusion limit, 3 kDa; Amicon, Oakville, ON, Canada) [5, 6].
Inhibitors to be tested with spermatozoa were dissolved in distilled water or dimethyl sulfoxide (DMSO). The concentration of DMSO in the incubation media never exceeded 1% (v/v), a condition that does not affect sperm capacitation.
Sperm Preparation and Treatments
Semen samples from healthy volunteers were used for the present study and were normal according to World Health Organization criteria [25]. Semen samples were washed on four-layer (95-65-40-20%) Percoll gradients 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 200 x 106 cells/ml with the 95% Percoll solution. Only samples in which progressive motility was greater than 70% were used. Spermatozoa were additionally diluted to 40 x 106 cells/ml in Biggers, Whitten, and Whittingham medium (BWW; pH 8) [26] devoid of bicarbonate and BSA and containing 1 mM CaCl2 and 25 mM Hepes.
In experiment 1, sperm preparations were incubated with capacitation inducers, FCSu (10%, v/v) or a combination of dbcAMP (1 mM) + IBMX (0.1mM) in the absence or in presence of SOD (0.1 mg/ml), catalase (0.1 mg/ml), or NOS inhibitor (L-NAME, 1 mM; or its less active analogue, D-NAME, 1 mM). In experiment 2, the effects of exogenously added ROS were tested by incubating spermatozoa with the combination of xanthine (0.5 mM) + xanthine oxidase (0.05 U/ml) in the presence of catalase (0.1 mg/ml; effect of O2-·), spermine NONOate (0.3 mM; generation of NO·), or H2O2 (50 µM). In experiment 3, reversal of the inhibitory effects of L-NAME (a competitive inhibitor of L-Arg as substrate for NOS) was tested by addition of 5 mM L-Arg to the incubation medium. In experiment 4, sperm preparations were preincubated with H89 (10 µM; PKA inhibitor), PP2 (10 nM; nonreceptor-type protein Tyr kinase [PTK] inhibitor), tyrphostin A47 (100 µM; receptor-type PTK inhibitor), PD98059 (100 µM), or chelerythrine (10 µM) for 30 min and then treated with spermine NONOate (0.3 mM).
In these four series of experiments, the level of P-Thr-Glu-Tyr-P of sperm proteins was evaluated after 2 h of incubation. Sample buffer was added, and samples were used for electrophoresis and immunoblotting as described below. A 2-h incubation period was selected based on our previous results [18] that a progressive increase occurs in the level of P-Thr-Glu-Tyr-P during the course of the capacitation period. The 2-h incubation is optimum for study of the P-Thr-Glu-Tyr-P motif in sperm proteins [18].
Sperm Capacitation
Capacitation was evaluated after a 3.5-h incubation period [3, 6] by induction of the acrosome reaction with LPC (3 mg/ml of BSA and 100 µM LPC) as described elsewhere [3, 6]. The LPC was proven to be effective in inducing the acrosome reaction in capacitated, but not in noncapacitated, human spermatozoa without affecting sperm viability when used in the optimal conditions described above [3]. The acrosomal status of more than 200 spermatozoa per slide was determined using PSA-FITC [27]. The proportion of spermatozoa undergoing acrosome reaction was determined as a measure of capacitation. None of the chemicals and biological agents tested affected the percentage of sperm motility at the concentrations used in the present study and over a period of at least 4 h. Analysis of variance (two-tailed, unpaired values) was used to evaluate the differences in the levels of capacitation. Statistical differences between the effects of various treatments were then determined by the protected least-significant difference test. A difference was considered to be statistically significant with P < 0.05.
SDS-PAGE and Immunoblotting
Sperm proteins were electrophoresed on 12% polyacrylamide gels and electrotransferred using 10 mM CAPS (3-cyclohexylamino-1-propane sulfonic acid) buffer (pH 11) containing 10% methanol to nitrocellulose membranes. 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 1 h. The primary antibody against the P-Thr-Glu-Tyr-P motif 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 goat anti-rabbit IgG conjugated with horseradish peroxidase for 45 min at 20°C and washed again with TTBS. Positive immunoreactive bands were detected using the Lumi-Light chemiluminescence kit. At the end of the experiments, blots were rinsed in distilled water and silver stained [28] to ascertain that the amount of proteins loaded in each well was the same.
We used an antibody specific for the P-Thr-Glu-Tyr-P motif for the immunoblotting experiments. Quality control tests done by the manufacturer (New England Biolabs) indicate that the antibody does not react with P-Thr, P-Tyr, or P-Thr-X-Tyr-P (X being an amino acid other than Glu). The anti-P-Thr-Glu-Tyr-P antibody was incubated with the blocking peptide, P-Thr-Glu-Tyr-P, at a molar ratio of 104 (peptide:antibody). The blocked antibody did not recognize protein bands at 80 and 105 kDa, confirming the specificity of the antibody for the P-Thr-Glu-Tyr-P motif (see Fig. 1B). In addition, we preadsorbed the anti-P-Thr-Glu-Tyr-P antibody with P-Tyr and P-Thr (5 mM each) and observed no reduction of the signal obtained (see Fig. 1B), confirming that the anti-P-Thr-Glu-Tyr-P antibody does not bind to P-Thr or P-Tyr.
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Immunolocalization of P-Thr-Glu-Tyr-P Motif in Spermatozoa
Sperm preparations were incubated without or with FCSu for 3.5 h and prepared for immunocytochemistry. Briefly, smears were prepared on polylysine-coated glass slides and permeabilized by methanol. After rehydration, smears were treated with 5% goat serum in PBS, washed with PBS containing 0.1% Triton X-100, and incubated with anti-P-Thr-Glu-Tyr-P antibody (1:100) for 2 h. Then, smears were washed and incubated with biotinylated goat anti-rabbit antibody (3:1000) and further incubated with an Alexa Fluor 555 conjugate of streptavidin (1:500 w/v) in PBS with 0.1% Triton X-100. Smears were mounted with Prolong Antifade and observed under a Carl Zeiss (Oberkochen, Germany) Axiophot microscope (exciter filter BP 450-490) at 1000x magnification. A control for the primary antibody was performed in which nonimmune rabbit IgG was substituted for the anti-P-Thr-Glu-Tyr-P antibody. A control for the secondary antibody was also performed in which sperm preparations were incubated with the biotinylated goat anti-rabbit antibody followed by Alexa Fluor 555 conjugate of streptavidin as described above. At least 200 spermatozoa were observed, and the proportion of spermatozoa positive for immunostaining was determined. Immunoblotting and capacitation studies were done concurrently using aliquots from same sperm preparations in which capacitation was induced with FCSu and the effects of H89 and PD98059 were tested.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a previous study, we observed a progressive increase in P-Thr-Glu-Tyr-P level only in proteins of 80 and 105 kDa during the course of human sperm capacitation [18]. Therefore, we focused our study on the regulation of this process by ROS. The SOD and catalase did not prevent the increase in P-Thr-Glu-Tyr-P in p80/105 due to the presence of FCSu or IBMX + dbcAMP (Fig. 1). On the other hand, L-NAME (1 mM), a competitive inhibitor of L-Arg for NOS, prevented the increase in the level of P-Thr-Glu-Tyr-P in p80/105 during capacitation (Fig. 1). The less active analogue of L-NAME, D-NAME, had a partial effect on this process. These results paralleled those of capacitation, because L-NAME inhibited capacitation induced by FCSu (Fig. 2A) or dbcAMP + IBMX (Fig. 2B) and because D-NAME had only a partial inhibitory effect.
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NO ·, but not O2-· or H2O2, Increases the Double Phosphorylation of Thr-Glu-Tyr in p80/105
The exogenous addition of O2-· (a combination of xanthine + xanthine oxidase in the presence of catalase) [5] or H2O2 did not affect the level of P-Thr-Glu-Tyr-P in p80/105 (Fig. 3) even though these conditions trigger capacitation [5, 10]. However, NO· generated by spermine NONOate induced a significant increase in the level of P-Thr-Glu-Tyr-P in p80/105. The percentage of spermatozoa undergoing capacitation was higher for cells that received spermine NONOate (11.5% ± 0.8%) as compared to that of spermatozoa incubated with BWW alone (4.5% ± 0.3%) (n = 6).
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L-Arg Reverses the Inhibition of the Double Phosphorylation of Thr-Glu-Tyr due to L-NAME
The D-NAME caused a partial inhibition of capacitation and of the associated increase in P-Thr-Glu-Tyr-P phosphorylation, level (Figs. 1 and 2), which could indicate a nonspecific effect of L-NAME on some sperm proteins other than NOS. Therefore, an experiment was performed in which L-Arg (5 mM) was used in an attempt to reverse the effect of the competitive inhibitor L-NAME during capacitation. The inhibitory effect of L-NAME on the increase of P-Thr-Glu-Tyr-P during capacitation induced by FCSu or a combination of dbcAMP + IBMX was reversed by L-Arg (Fig. 4A). When used alone, L-Arg caused an increase in P-Thr-Glu-Tyr-P that was prevented by L-NAME (Fig. 4A). The results of capacitation experiments paralleled the results of immunoblotting (Fig. 4B).
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Regulation of the Double Phosphorylation of P-Thr-Glu-Tyr-P During Capacitation Induced by Spermine NONOate
To determine the possible mechanism by which NO· regulates the P-Thr-Glu-Tyr-P phosphorylation, the effect of various inhibitors of signal transduction elements were tested on capacitation induced by spermine NONOate. The increase in P-Thr-Glu-Tyr-P induced by spermine NONOate was prevented by tyrphostin A47 (for receptor-type PTK), PD98059 (for MEK or MEK-like kinases), and chelerythrine (for protein kinase C) and, to a lesser extent, by PP2 (for nonreceptor-type PTK) but was not affected by H89 (for PKA) (Fig. 5A). Capacitation results were consistent with those of immunoblotting: Preincubation of sperm samples with PP2, tyrphostin A47, chelerythrine, and PD98059 totally prevented capacitation induced by spermine NONOate, whereas H89 caused a partial inhibition (Fig. 5B).
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Immunolocalization of P-Thr-Glu-Tyr-P Motif in Spermatozoa
Immunostaining with the anti-P-Thr-Glu-Tyr-P antibody indicated that sperm proteins containing this motif are present in the principal piece region of spermatozoa (Fig. 6A). Furthermore, the proportion of cells with positive immunostaining increased when spermatozoa were incubated with FCSu (Fig. 6D); PD98059, but not H89, prevented this increase. Results of immunoblotting studies (Fig. 6E) done concurrently with immunocytochemistry (Fig. 6D) using aliquots from the same sperm preparations gave comparable results. The proportion of cells immunostained for P-Thr-Glu-Tyr-P was significantly lower for the sperm sample pretreated with PD98059; however, H89 did not have such an effect.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously reported that O2-· modulates the ERK pathway in spermatozoa. A decrease because of SOD was observed in the phosphorylation of Thr-Glu-Tyr, mainly in proteins of 16–33 kDa, and of the Ser/Thr-Pro motif (characteristic of ERK substrates) in proteins of 75 and 80 kDa [17]. Because a progressive increase in the level of P-Thr-Glu-Tyr-P motif of p80/105 was observed during the course of capacitation and that capacitation is blocked when the rise of P-Thr-Glu-Tyr-P of p80/105 is inhibited [18], a study was done to evaluate the role of ROS in the regulation of this process. The removal of O2-· or H2O2 by scavengers (Fig. 1) or their presence, through exogenous sources (Fig. 3), during sperm incubation did not affect the level of P-Thr-Glu-Tyr-P. These results indicate that although O2-· and H2O2 are involved in protein Tyr phosphorylation, sperm hyperactivation, and sperm capacitation, these ROS did not seem to be involved in regulation of the level of P-Thr-Glu-Tyr-P. Taken together, these results suggest that in capacitating spermatozoa, the level of P-Thr-Glu-Tyr-P of p80/105 is regulated through a mechanism that is independent of O2-· and H2O2. Previous results suggested that SOD prevents the P-Thr-Glu-Tyr-P in p80 of human spermatozoa [17]. We do not have an explanation for the discrepancy between these data and those presented here. However, the present data are more reliable, because SOD was shown to have no effect on P-Thr-Glu-Tyr-P in p80/105 in spermatozoa treated with two capacitation inducers (FCSu and dbcAMP + IBMX) and because exogenous addition of O2-· did not induce any increase in P-Thr-Glu-Tyr-P in p80/105 (Fig. 3). Therefore, the present results do not support a role for O2-· in regulation of the level of P-Thr-Glu-Tyr-P in p80/105 during sperm capacitation.
The NO·-releasing compounds prime spermatozoa to respond earlier to human follicular fluid, whereas the NOS inhibitors prevent this process [18]. In addition, a positive association between capacitation and Tyr phosphorylation involving NO· was observed. Our present findings that incubation of spermatozoa with NOS inhibitors prevents (Fig. 1), and that addition of spermine NONOate triggers (Fig. 3), the increase of P-Thr-Glu-Tyr-P suggest that NO· is involved in the regulation of this process. In addition, NO· is also required for capacitation, because incubation of spermatozoa with L-NAME inhibited capacitation induced not only by albumin [13] but also by FCSu (Fig. 2A) or dbcAMP + IBMX (Fig. 2B). The partial inhibitory effect of D-NAME (a less active analogue of L-NAME) on the increase of P-Thr-Glu-Tyr-P as well as capacitation could suggest a nonspecific effect of L-NAME on sperm proteins other than NOS. However, our findings that L-Arg (5 mM) reverses the inhibitory effect of L-NAME on the level of P-Thr-Glu-Tyr-P (Fig. 4A) and on capacitation (Fig. 4B) is indicative of its specific action on NOS. In addition, L-Arg alone caused an increase in the level of P-Thr-Glu-Tyr-P and capacitation (Fig. 4, A and B). It can be hypothesized that this exogenous substrate for NOS causes an increased synthesis of NO·, which is responsible for these effects. The unique characteristics or mechanism of action of NO· that enables this ROS to specifically trigger the P-Thr-Glu-Tyr-P remains unknown. The effects of ROS on molecules depend on the quantity and species of ROS involved [1]. The longer half-life (1–7 sec) and the cell permeability of NO· may allow this ROS to interact with different cell-signaling mechanisms than O2-· and H2O2.
The exact mechanism by which NO· would regulate the double phosphorylation of Thr-Glu-Tyr during sperm capacitation is presently unknown. However, the observation that tyrphostin A47, chelerythrine, PD98059, and, to a lesser extent, PP2 prevent the increase in P-Thr-Glu-Tyr-P suggests the involvement of PTK, PKC, and MEK or MEK-like kinases in the NO·-mediated regulation of this process. However, the mechanisms by which NO· acts on these enzymes remain unknown. The NO· could directly activate PTK or PKC, as observed in other cell types [29, 30]. These kinases could then activate elements of the ERK pathway. Such an effect was observed for PKC, which can activate Raf [20, 21], but is also associated with a prolonged activation of the ERK pathway through a MEK-independent mechanism [31]. Studies on Ras, an upstream element in the ERK pathway, have identified a single cysteine residue, Cys 118, which is S-nitrosylated by NO·. This S-nitrosylation triggers downstream activation of Ras and then of MEK or MEK-like kinases [32]. The NO· could also inactivate phosphoprotein phosphatases [33], which would cause an indirect and/or prolonged activation of some kinases or other enzymes upstream of MEK or MEK-like kinases responsible for the phosphorylation of Thr-Glu-Tyr.
Previous studies indicated that NO·- as well as O2-·- and H2O2-induced capacitation and associated protein Tyr phosphorylation are mediated through a cAMP/PKA pathway [7, 10, 13, 14, 34]. The observations that capacitation induced by dbcAMP + IBMX is inhibited by L-NAME (Fig. 2B) and that H89 partially inhibited capacitation induced by spermine NONOate (Fig. 5B) also confirm that NO· regulates human sperm capacitation through a cAMP/PKA-dependent pathway. On the other hand, we recently observed that the increase in P-Thr-Glu-Tyr-P level associated with capacitation appears to be regulated by PTK and MEK or MEK-like kinases but not by cAMP/PKA [18]. The present report indicates that among the ROS, only NO· would be involved in the regulation of P-Thr-Glu-Tyr-P of p80/105 (Figs. 1 and 3), which appears again to be independent of the cAMP/PKA pathway because it was not affected by H89 (Fig. 5A). Therefore, NO· appears to be involved in capacitation through two mechanisms, one dependent on cAMP/PKA and one dependent on the ERK pathway. These two pathways are essential for capacitation, because inhibition of PKA or elements of the ERK pathway prevent capacitation and Tyr phosphorylation [17, 35, 36]. The PKA and ERK pathways would act in parallel but, ultimately, would lead to Tyr phosphorylation of proteins of 80 and 105 kDa during capacitation.
It is interesting to note that NO· mediated the regulation of P-Thr-Glu-Tyr-P through similar mechanisms when capacitation was induced by FCSu or spermine NONOate [18; present study]. Results of our previous study indicated that during capacitation induced by biological agents such as FCSu, the level of P-Thr-Glu-Tyr-P is regulated through the action of PTK and MEK or MEK-like kinases [18]. Similarly, PTK, PKC, and MEK or MEK-like kinases were involved in the regulation of P-Thr-Glu-Tyr-P when capacitation was induced by spermine NONOate (Fig. 5A).
The anti-P-Thr-Glu-Tyr-P antibody recognized proteins located throughout the principal piece region of spermatozoa (Fig. 6, A–C) as well as Triton X-100-insoluble proteins of 80 and 105 kDa (Fig. 6E). The molecular masses of proteins containing the P-Thr-Glu-Tyr-P motif and their location in the principal piece region of sperm could suggest that these proteins are part of the fibrous sheath. From these data, we could hypothesize that fibrous sheath proteins contain the P-Thr-Glu-Tyr-P motif or that this motif is present in proteins other than those from fibrous sheath but having similar molecular masses and cellular location. Both ERK 1 and 2 have been localized in the sperm head at the postacrosomal/equatorial regions during capacitation [15]. The anti-P-Thr-Glu-Tyr-P antibody used in the present study did not localize to the sperm head even if the P-Thr-Glu-Tyr-P motif is present in activated ERK. This is probably because Thr-Glu-Tyr phosphorylation of ERK 1 and 2 occurs very early (5 min) after the beginning of treatment with FCSu and then dramatically decreases over the next 2 h of incubation [17]. The main two proteins recognized by the anti-P-Thr-Glu-Tyr-P antibody in immunoblotting experiments are p80/105 and not p42 and p44. Because the phosphorylation of Thr-Glu-Tyr during capacitation induced by FCSu is independent of the cAMP/PKA pathway and involves the ERK pathway [18], we used H89 (an inhibitor of PKA) and PD98059 (an inhibitor of MEK or MEK-like kinases) in immunocytochemistry to demonstrate the effect of these inhibitors on the double phosphorylation of Thr-Glu-Tyr. These results (Fig. 6D) were consistent with that of immunoblotting (Fig. 6E) using anti-P-Thr-Glu-Tyr-P antibody and support our finding that regulation of P-Thr-Glu-Tyr-P during capacitation does not involve a cAMP/PKA pathway.
In summary, our data show, to our knowledge for the first time, an effect of ROS that appears to be specific for NO· but not for O2-· or H2O2 in the transduction mechanisms leading to human sperm capacitation. The NO· appears to regulate the double phosphorylation of the Thr-Glu-Tyr motif present in sperm proteins of 80 and 105 kDa during capacitation through a pathway dependent on PTK, PKC, and MEK or MEK-like kinases but independent of cAMP/PKA.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jacob Thundathil, Urology Research Laboratory, H6.44, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, QC, Canada H3A 1A1. FAX: 514 843 1457; jc.thundathil{at}umontreal.ca
Received: 27 June 2002.
First decision: 29 July 2002.
Accepted: 23 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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