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Regular Article |
a Endocrinologie de la Reproduction, Centre de Recherche du CHUQ, Centre de Recherche en Biologie de la Reproduction, Department of OB/GYN, Université Laval, Quebec, Quebec, Canada G1L 3L5
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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calcium, cyclic adenosine monophosphate, gamete biology, signal transduction, sperm capacitation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A large number of membrane and biochemical events occur during sperm capacitation. During this process, a decrease in the membrane cholesterol content [2] resulting in an increased ionic membrane permeability, leading to the hyperpolarization of the membrane [3], has been reported as well as increases in intracellular pH [4, 5], Ca2+ [6–8], and cAMP concentrations [9–11]. In addition, spermatozoa undergoing capacitation show a higher production of superoxide anion [12], and an increase in tyrosine phosphorylation of specific proteins has also been demonstrated in mouse [13–15], bovine [16], and human [17–20] spermatozoa.
The importance of protein tyrosine phosphorylation has been demonstrated in different sperm activities such as motility. Phosphorylation of sperm protein tyrosine residues has been demonstrated in hamster sperm hyperactivation [21], a modification in the motility pattern generally associated in a temporal manner with sperm capacitation. In humans, the increase in phosphotyrosine content of p105 and p81, two fibrous sheath proteins, during capacitation [18–20, 22] is also suggestive of an involvement of protein tyrosine phosphorylation in the control of sperm motility. In fact, an association between the phosphotyrosine content of p105 and p81 and the speed and linearity of sperm motility has been observed [18]. Cyclic AMP-dependent tyrosine phosphorylation of motility-associated proteins has also been described in other species [23, 24].
The mechanisms involved in the control of sperm protein phosphotyrosine content during the process of capacitation result from the interactions between different signaling pathways. Previous studies in spermatozoa from different species have demonstrated that the capacitation-associated increase in protein phosphotyrosine content is regulated by the cAMP-dependent pathway [15, 16, 18, 20, 25, 26]. Sperm capacitation and protein tyrosine phosphorylation are also regulated by oxygen derivatives [1, 19, 27–29]. Capacitating spermatozoa generate the superoxide anion [12] that, along with the hydrogen peroxide, stimulate tyrosine phosphorylation of human sperm proteins [19]. Conflicting results have been shown according to the effects of Ca2+ on sperm protein tyrosine phosphorylation. On one hand, extracellular Ca2+ reduced protein phosphotyrosine content when spermatozoa are incubated under noncapacitating conditions [20, 30, 31]. On the other hand, the presence of Ca2+ is required for the increase in the phosphotyrosine content of human sperm proteins when the cells are incubated under capacitating conditions [20]. Such a result is confirmed by the stimulatory action of the ionophore A23187 [20, 27]. Recently, it has been shown that sperm production of superoxide anion is under cAMP and Ca2+ regulation [20]. Because oxygen derivatives were also shown to increase human sperm cAMP concentrations [11] and because a Ca2+-dependent increase in cAMP concentration is measured during sperm capacitation [9, 10], these results together support the idea that sperm capacitation results from the interactions between Ca2+, cAMP, oxidant, and tyrosine phosphorylation pathways [20, 29]. These signaling reactions possibly occur in a cascade of events triggered by membrane cholesterol efflux [32].
Nevertheless, the enzymes playing the key role in the regulation of sperm protein phosphotyrosine content, the tyrosine kinases (PTK) and/or the phosphotyrosyl-protein phosphatases (PTPase), remain to be identified and characterized. Whether the increase in protein phosphotyrosine content occurs through the activation of PTK or the inhibition of PTPase remains to be elucidated. The involvement of PTK is strongly suggested by the inhibitory action of different PTK inhibitors on sperm protein tyrosine phosphorylation and acrosomal exocytosis [13, 19, 22, 31, 33–36]. The basal and capacitation-associated increase in sperm tyrosine phosphorylation is abolished by herbimycin A [19] and erbstatin [22, 31], two inhibitors of cytosolic src-related PTK, which suggests the involvement of this family of tyrosine kinases in sperm protein phosphorylation. This family of tyrosine kinase is involved in many different cellular processes such as proliferation, differentiation, adhesion, migration, and cytoskeletal alteration [37]. In addition, the stimulatory effect of oxygen derivatives on sperm capacitation [19, 38] and protein phosphotyrosine content [1, 19] support the hypothesis that src-related tyrosine kinases are involved in these processes, with members of this family of tyrosine kinases being activated by an oxidative stress [39–41]. This is emphasized by the fact that c-src is expressed during rat spermatogenesis [42] and by the presence of c-yes in rat spermatozoa [43]. Because a Ca2+-dependent inhibition in protein tyrosine kinase activity has been reported in human spermatozoa [31] and the src-related tyrosine kinase c-yes is known to be inhibited by Ca2+ [44], the goal of the present study was to determine whether c-yes is present in human spermatozoa and whether it is regulated by Ca2+.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Percoll used for washing spermatozoa was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden) and Protein G sepharose was bought from Pharmacia (Baie d'Urfée, PQ, Canada). Ham F10 medium used for the incubation of spermatozoa was from Gibco/BRL/Life Technologies (Grand Island, NY). Rabbit muscle enolase, IBMX (3-isobutyl-1-methylxanthine), BAPTA (1,2-bis-[o-aminophenoxy]-ethane-N,N,N',N'-tetraacetic acid), and Tween-20 were purchased from Sigma Chemical Co. (St. Louis, MO). Monoclonal anti-yes antibody was obtained from Transduction Laboratories (Lexington, KY) and a polyclonal anti-yes antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-mouse IgG conjugated to horseradish peroxidase or to fluorescein isothiocyanate (FITC) were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). The enhanced chemiluminescence detection kit (ECL) was from Amersham Life Science Inc. (Oakville, ON, Canada), nitrocellulose (0.22-µm pore size) was from Micron Separations Inc. (Westboro, MA), and x-ray films were from Fuji (Tokyo, Japan).
Preparation of Spermatozoa
Ejaculates were obtained by masturbation from healthy volunteers after 3 days of sexual abstinence. After liquefaction, the semen was layered on top of a gradient composed of 2-ml fractions each of 20%, 40%, and 65% and 0.1 ml of 95% Percoll made isoosmotic in Hepes-buffered saline (HBS; 25 mM Hepes, 130 mM NaCl, 4 mM KCl, 0.5 mM MgCl2, 14 mM fructose, pH 7.6) and was centrifuged (30 min, 1000 x g) to wash the spermatozoa. Sperm cells at the 65%–95% interface and within the 95% Percoll fraction were collected and washed once by centrifugation (5 min, 500 x g) in HBS.
Immunodetection of the Tyrosine Kinase c-yes in Human Spermatozoa
Sperm proteins were solubilized by the addition of 5x concentrated solubilization buffer (1x final concentrations: 2% SDS, 10% glycerol, 5% ß-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8) and heated at 100°C for 5 min. Particulate material was removed by centrifugation (10 000 x g, 5 min, 4°C). The proteins were separated by electrophoresis through a 7.5% SDS-polyacrylamide gel [45] and were electrotransferred [46] to nitrocellulose. Nonspecific sites on the membrane were blocked with 5% (w/v) skimmed dry milk in Tris buffered saline (TTBS; 0.9% NaCl, 20 mM Tris-HCl, pH 7.8, 0.1% Tween-20). The nitrocellulose membrane was incubated for 1 h at room temperature with the anti-yes monoclonal antibody. The membrane was extensively washed in TTBS, and goat anti-mouse IgG conjugated with horseradish peroxidase was added. After a 1-h incubation period at room temperature, the membrane was extensively washed, and positive immunoreactive bands were detected by chemiluminescence using ECL according to the manufacturer's instructions.
To determine whether c-yes was present in the head or flagellum, spermatozoa were sonicated 3 times (30 sec followed by a 30-sec rest period each) on ice, and heads and flagellar fragments were then separated by a 15-min centrifugation (700 x g) at 4°C through a 75% Percoll layer in HBS. Flagellar fragments were recovered at the surface of the Percoll layer while the heads were found in the pellet. The supernatant was centrifuged for 10 min (10 000 x g, 4°C) and the resulting supernatant was further centrifuged (100 000 x g) to separate the membrane from the cytosolic fraction. Both the flagellar and head fractions were diluted in HBS and centrifuged again individually through Percoll to ensure minimal contamination between the two sperm cellular fractions. With this procedure, the flagella were broken in small fragments and minimal cross-contamination between the two cellular fractions was observed. An aliquot of each fraction was diluted in electrophoresis solubilization buffer, sonicated, and heated to 100°C to extract proteins, and the amount of solubilized proteins was measured using the micro-BCA protein assay kit (Pierce, Rockford, IL) upon precipitation with trichloroacetic acid (TCA) to get rid of the detergents and reducing agents. The presence of c-yes was investigated by Western blot on proteins from each fraction.
Indirect Immunofluorescence
Percoll-washed spermatozoa were placed on a poly-L-lysine-coated coverslip, fixed for 15 min in 3.7% formaldehyde in phosphate buffered saline (PBS; 1.5 mM KH2PO4, 8.1 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4), rinsed with PBS, permeabilized for 10 min in 0.2% Triton X-100 in PBS, and rinsed again with PBS. Nonspecific sites were blocked with PBS supplemented with 1% BSA. Samples were then incubated for 2 h at 37°C with the polyclonal c-yes antibody diluted in the blocking solution, rinsed with PBS, and incubated with a FITC-labeled secondary antibody. Following rinses with PBS, the coverslips were mounted on slides with 90% glycerol containing an antibleaching agent (1.5% DABCO) and sealed with nail polish. Immunofluorescence was detected by epifluorescence microscopy with a UV light. On one occasion, the washed sperm cells were extracted first with Triton X-100 (1% in PBS), washed, deposited on poly-L-lysine-coated coverslips, then fixed with formaldehyde and processed as described above to determine whether yes is associated with the cytoskeleton.
Immunokinase Assay of Human Sperm c-yes
Tyrosine kinase activity was assayed in the immune complex using a c-yes monoclonal antibody. Washed spermatozoa were resuspended (100 x 106 sp/ml) in immunoprecipitation buffer (IP buffer; 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, 1 mM EDTA, 1 mM EGTA, 0.2 mM Na orthovanadate, 250 µM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin A) and kept on ice for 30 min and frequently vortexed to facilitate kinase extraction. The homogenate was centrifuged (10 000 x g, 4°C, 20 min), and the soluble fraction was next incubated on ice for 2 h in the presence of 1 µg immunoglobulins from the antibody. The immune complex was precipitated with Protein G sepharose after a 2-h incubation on ice with frequent inversions. The beads were washed 3 times by centrifugation with IP buffer, then resuspended in the kinase buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 2.5 mM MnCl2, 0.5 mM DTT, and 0.1 mM Na orthovanadate). The kinase assay was performed at 30°C for 10 min in the presence of 25 µM ATP and 5 µCi [32P]
ATP in the absence or presence of 2.5 µg acid-treated enolase to discriminate between autophosphorylation and kinase activity, respectively. The phosphorylation reaction was stopped by the addition of concentrated electrophoresis solubilization buffer, and the proteins were heated for 5 min at 100°C and separated by SDS-PAGE. The gels were stained with Coomassie Brilliant Blue and then dried, and the phosphorylated proteins were visualized by autoradiography at -80°C using intensifying screens. Using this procedure, the kinase activity of c-yes was also measured on spermatozoa incubated for 4 h in either Ham F10 supplemented with 500 µM IBMX to induce capacitation and to increase protein phosphotyrosine content [18] or in Ham F10 added with 1 mM BAPTA as a treatment to increase protein tyrosine phosphorylation without inducing sperm capacitation [20].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When spermatozoa are incubated under noncapacitating conditions, it has been shown that Ca2+ inhibits the tyrosine kinase activity in sperm homogenates [20, 31]. Since a Ca2+-dependent inactivation of the src-related tyrosine kinase c-yes has been demonstrated in keratinocytes [44], we first looked at the expression of this kinase in spermatozoa using a commercial monoclonal antibody. As shown in Figure 1, a 60-kDa protein is detected by the antibody, which is in agreement with the reported mass of that tyrosine kinase. In experiments where the sperm cells were dislocated and both the heads and tails were isolated, Western blot analyses revealed that c-yes is mostly found in the membrane fraction (Fig. 1). The weak signal detected in the flagellar fraction might be due to contamination by outer acrosomal membrane fragments as detected by peanut agglutinin binding (data not shown). Similar results were obtained when the membranes were isolated following nitrogen cavitation of the washed sperm cells (data not shown).
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Localization of c-yes within the spermatozoa was performed by indirect immunofluorescence using a polyclonal c-yes antibody. A strong signal is detected at the acrosomal portion of the head, and some staining was also observed at the midpiece region (Fig. 2A). However, the midpiece labeling does not appear to be specific because it is also observed when the antibody is preadsorbed with the immunizing peptide (data not shown). The localization of the yes kinase was also investigated on spermatozoa extracted with 1% Triton X-100 prior to the fixation with formaldehyde on coverslips. In these extracted cells, yes was still present at the acrosomal level (Fig. 2B). On coverslips run in parallel to determine whether the outer acrosomal membrane or content was still present, the Triton X-100-extracted cells show no binding to the Pisum sativum agglutinin in the acrosomal or equatorial segment regions (data not shown). Taken together with the results presented in Figure 1, it appears that c-yes is associated both with the cytoskeletal elements present mostly at the subacrosomal region and with the plasma membrane surrounding the acrosome and/or the outer acrosomal membrane.
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Immunoprecipitation of Sperm yes Kinase Activity
Investigations were next attempted to determine whether sperm yes is an active kinase. This was done by a kinase assay in the absence or presence of enolase as a substrate protein using proteins immunoprecipitated by the c-yes monoclonal antibody. Immunodetection of yes on sperm proteins collected after the extraction by the immunoprecipitation buffer, which contains nonionic detergents, shows that the kinase is not completely solubilized (Fig. 3). This result is in agreement with the strong yes signal still observed by indirect immunofluorescence when sperm are Triton X-100 extracted prior to fixation (Fig. 2B). Densitometric analyses of the films after immunodetection reveal that 55 ± 4% (n = 33) of the sperm kinase is solubilized in the immunoprecipitation buffer. Nonetheless, the soluble yes kinase is active since it phosphorylated the protein substrate enolase following immunoprecipitation (Fig. 4A). In addition to enolase, another phosphorylated protein migrating at 60 kDa is detected. This latter protein might be the kinase itself, yes, that is autophosphorylated since it was also detected when the kinase assay was performed in the absence of enolase. This statement is emphasized by the detection of a positive signal when the procedure is run in parallel, in the absence of radiolabeled ATP, and probed by Western blot using the anti-yes monoclonal antibody (Fig. 4B).
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Regulation of Sperm yes Kinase Activity
In order to determine whether yes kinase activity is modulated during sperm capacitation, the immunoprecipitation-kinase assay was performed on extracts obtained from spermatozoa incubated under conditions known to increase their protein phosphotyrosine content. The immune-kinase activity was assayed using spermatozoa treated for 4 h with the phosphodiesterase inhibitor IBMX, which has been shown to induce an increase in protein phosphotyrosine content and to induce sperm capacitation [1, 18]. As for sperm protein phosphotyrosine content, an increase in yes kinase activity was observed in 4 out of 6 independent experiments when spermatozoa were subjected to an IBMX treatment (Fig. 5). Since Western blot analyses revealed no increase in the yes kinase content in the soluble extract or in the immunoprecipitation complex (data not shown), the increase in enolase phosphorylation most likely results from a higher yes activity.
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The immune kinase assay was also performed on proteins extracted from spermatozoa previously incubated for 4 h in the presence of the Ca2+ chelator BAPTA, a treatment known to increase the phosphotyrosine content of sperm proteins without inducing capacitation [20]. In 5 out of 7 independent experiments, an increase in yes kinase activity was observed when the sperm cells were incubated under these conditions (Fig. 6A). The BAPTA treatment did not affect the solubilization of yes nor its content in the immunoprecipitated complex (data not shown), which supports the idea that sperm yes kinase activity is modulated by extracellular Ca2+. Similar results were obtained when spermatozoa were incubated with EGTA instead of BAPTA to chelate extracellular Ca2+ (data not shown). On the other hand, when proteins were solubilized from spermatozoa incubated under control conditions, in Ham F10 medium, and immunoprecipitated in the absence of the Ca2+ chelators EDTA and EGTA, yes kinase activity markedly decreased (Fig. 6B). These results clearly demonstrate that sperm c-yes kinase activity is inhibited by Ca2+.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, it is clearly shown that sperm tyrosine kinase yes is an active kinase since it could phosphorylate acid-treated enolase upon immunoprecipitation. Moreover, when spermatozoa were first incubated under conditions known to induce an increase in protein tyrosine phosphorylation such as in the presence of the phosphodiesterase inhibitor IBMX or the Ca2+ chelator BAPTA (data not shown and [18, 20]), a higher level of phosphorylated enolase was observed in the kinase assay upon c-yes immunoprecipitation. This effect appears to be related to an increase in c-yes tyrosine kinase activity since the cellular localization, the amount of the kinase in the solubilized material, and the c-yes level in the immunoprecipitated material were unaffected by the sperm treatment (not shown). A Ca2+-dependent decrease in human sperm total tyrosine kinase activity has been previously reported [31], although no specific tyrosine kinase was identified. The results presented here showing an increase in the activity of c-yes in spermatozoa incubated in the absence of extracellular Ca2+ support such findings. Even though the increase in c-yes kinase activity agrees with the increase in sperm protein phosphotyrosine content, more investigations have to be done in order to determine whether this latter effect results exclusively from an increase in c-yes activity. The increase in yes tyrosine kinase activity when spermatozoa are incubated in the presence of the Ca2+ chelator BAPTA is in agreement with the report of Zhao et al. [44], where it was demonstrated that agents causing an increase in intracellular Ca2+ inhibited c-yes kinase activity. The mechanisms involved in the Ca2+-mediated kinase inhibition are not elucidated. However, it does not appear to occur through phosphorylation of the regulatory tyrosine-535 in the C-terminal domain of the kinase since it is rapidly dephosphorylated upon increase in intracellular Ca2+ concentration [44]. Such dephosphorylation of the C-terminal regulatory tyrosine is an event normally required to activate the kinase. In the present study, we could not demonstrate any tyrosine phosphorylation of sperm c-yes, suggesting that the regulatory C-terminal tyrosine was either already dephosphorylated or the level of the kinase in the immunoprecipitated complex was too low to detect any tyrosine phosphorylation by Western blot technique (data not shown). On the other hand, the Ca2+-dependent inhibition of c-yes might be mediated through the binding of an inhibitory protein to the kinase [44] forming a complex that would resist the solubilization in the presence of ionic detergents. In our study, because sperm protein solubilization was performed using nonionic detergents, this would favor the coimmunoprecipitation of proteins associated with or forming a complex with the kinase. Moreover, the increase in yes kinase activity observed when sperm proteins were solubilized and immunoprecipitated in the absence of Ca2+ (in the presence of EDTA and EGTA), as compared with the activity detected in its presence (Fig. 6B), supports the hypothesis of a Ca2+-dependent inhibitory protein. Nonetheless, the c-yes kinase activity in sperm incubated for 4 h with BAPTA was measured in the protein complex immunoprecipitated and solubilized in the presence of the Ca2+ chelators EDTA and EGTA. Under those conditions, the extracellular BAPTA would have caused a decrease in intracellular Ca2+, reverting the inhibitory action of Ca2+ on c-yes activity. In addition, it appears conceivable that a higher increase in yes kinase activity is observed in spermatozoa incubated in the presence of BAPTA when the solubilization/immunoprecipitation is performed in the absence of EGTA/EDTA, such as in the present report (data not shown). The presence of these 2 chelators in the solubilization/immunoprecipitation buffers most likely explain why, in 2 out of 7 experiments, no increase in c-yes activity was observed when sperm were incubated with BAPTA.
An increase in c-yes activity was also noted when spermatozoa were previously incubated in the presence of the phosphodiesterase inhibitor IBMX to maintain high intracellular cAMP concentrations (Fig. 5). As for spermatozoa incubated in Ca2+-depleted medium, this increase in yes activity is in agreement with the increase in sperm protein phosphotyrosine content (data not shown and [18]). At the present time, it is unlikely that c-yes kinase activity is directly affected by cAMP because, unlike v- or c-src [47–49], yes does not possess a cAMP-dependent phosphorylation site. Using inhibitors of phosphoseryl/threonyl-protein phosphatase inhibitors, it has been demonstrated that cAMP increases sperm tyrosine phosphorylation indirectly through phosphorylation on Ser/Thr residues of an undetermined protein [18]. Again, it is not known whether c-yes is responsible for all sperm protein tyrosine phosphorylation detected in those reports. At this time, the mechanisms by which cAMP affects c-yes kinase activity are still elusive. It has been demonstrated that csk, the tyrosine kinase that phosphorylates the C-terminal regulatory tyrosine on src-related kinase, can be phosphorylated through cAMP-dependent protein kinase [50]. However, the effect of such phosphorylation appears to be controversial because both an increase [51] and a decrease [50] in csk activity have been reported.
The respective stimulatory and inhibitory effects of cAMP and Ca2+ on c-yes activity were measured on the kinase extracted with nonionic detergents, which resulted in an incomplete extraction of the yes kinase. Nonetheless, sperm treatment with the ionic detergent SDS also resulted in an incomplete yes kinase extraction (data not shown). In the context of sperm capacitation, both cAMP and intracellular Ca2+ increase [10], which might suggest that the stimulatory effect of cAMP on yes kinase activity overcomes the inhibitory effect of Ca2+. At the present time, it is not known whether the activity of the nonextracted kinase would be similarly affected by these agents. Moreover, during sperm capacitation, c-yes might not be the only tyrosine kinase involved in the increase in protein tyrosine phosphorylation since the majority of human sperm phosphotyrosine-containing proteins remain with the extracted cells (not shown), associated with the fibrous sheath [19, 30], whereas the results obtained by indirect immunofluorescence strongly suggest that this specific src-related tyrosine kinase associates with sperm head cytoskeletal elements. The presence of a 32-kDa protein (PT32) sharing homologies with a c-yes interacting protein, WBP2, within bull sperm perinuclear theca has been recently reported [52]. Such a finding is in perfect agreement with the presence of c-yes associated with head Triton X-100-insoluble cytoskeleton. Because the perinuclear theca is not extracted by nonionic detergents [53], this would explain why c-yes is present in spermatozoa extracted by nonionic detergents in both Western blots and indirect immunofluorescence experiments presented here (Figs. 2B and 3). In addition, c-yes was also present over the anterior portion of the head when spermatozoa were sonicated to isolate heads and flagella (data not shown). Sperm perinuclear theca is resistant to the sonication treatment, which is an important preparative step in the isolation of that cytoskeletal element [53]. However, it is not known whether human sperm possesses a protein responsible for the anchoring of c-yes, a cytosolic/membrane tyrosine kinase, to the head perinuclear theca at the subacrosomal area.
The results presented in this paper demonstrate that the src-related tyrosine kinase c-yes is present in human spermatozoa in head membranes as well as in the head Triton X-100-insoluble fractions. An increase in sperm yes kinase activity is measured when the sperm cells are incubated under conditions known to increase protein phosphotyrosine content.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Canadian Institutes in Health Research to P.L. P.L. is supported by a scholarship from the Fonds de la Recherche en Santé du Québec. 
2 Correspondence: Pierre Leclerc, Endocrinologie de la Reproduction, D0-708, Pavillon Saint-François d'Assise 10, de l'Espinay, Québec, PQ, Canada G1L 3L5. FAX: 418 525 4195; pierre.leclerc{at}crsfa.ulaval.ca
Accepted: February 6, 2002.
Received: November 20, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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