Journal of Andrology, Vol. 25, No. 4, July/August 2004 Copyright q American Society of Andrology
Inhibitors of Phosphoinositide 3-Kinase, LY294002 and Wortmannin, Affect Sperm Capacitation and Associated Phosphorylation of Proteins Differently: Ca21-Dependent Divergences VERONICA NAUC,*† EVE DE LAMIRANDE,* PIERRE LECLERC,† AND CLAUDE GAGNON* From the *Urology Research Laboratory, Royal Victoria Hospital and McGill University, Montre´al, Canada; and the †Endocrinologie de la Reproduction, Pavillon St-Franc¸ois d’Assise, Que´bec, Canada.
Abstract: Sperm capacitation is regulated by multiple pathways that also control sperm motility and tyrosine (Tyr) phosphorylation of several sperm proteins. Among the reported pathways, phosphoinositide 3-kinase (PI3K) signaling and its role in modulating sperm postejaculatory changes and motility remain elusive. It was shown that wortmannin, a selective inhibitor of PI3K, prevents human sperm acrosome reaction. Using LY294002 (2-(4-morphlinyl)-8-phenyl-4H-1-benzopyran-4-one), another chemically different inhibitor of PI3K, it was suggested that this enzyme inhibits human sperm motility. In this study, we used the 2 known inhibitors of PI3K to investigate their effect on sperm capacitation and associated protein phosphorylation events. Our data show that sperm incubated with LY294002 undergo capacitation and increased Tyr phosphorylation of specific sperm proteins in a manner similar to that promoted by the capacitation inducer fetal cord serum ultrafiltrate (FCSu), as well as double phosphorylation of the threonine (Thr)-glutamine (Glu)-Tyr motif. Under similar conditions, wortmannin did not affect these sperm functions on its own, although it did prevent the effect induced
by FCSu. Consistently, wortmannin decreased the phospho (P)-Tyr content of sperm proteins and prevented the phosphorylation of their Thr-Glu-Tyr motif. We also show by means of immunoblotting and cell fractionation experiments the presence of PI3K and its downstream effector Akt (protein kinase B) at the membrane level, as well as sperm heads and flagella. Our data show that human spermatozoa contain a consensus motif usually phosphorylated by Akt and that its P-serine (Ser)/Thr content is increased by both LY294002 and FCSu, while it is decreased by wortmannin. In addition, the 2 inhibitors differently affected the intracellular calcium concentration, [Ca21]i. While LY294002 increased [Ca21]i, wortmannin did not affect its content and did not prevent the LY294002 effect. Thus, we propose that the LY294002-promoted increase in [Ca21]i operates independently of PI3K. In conclusion, we suggest that special care be taken when using LY294002 to investigate the role that PI3K plays in a cellular phenomenon. Key words: Spermatozoa, signal transduction, Akt. J Androl 2004;25:573–585
jaculated mammalian spermatozoa are terminally differentiated cells that are motile but fertilization incompetent. They acquire the ability to fertilize the oocyte only after a finite period of residence in the female reproductive tract. This crucial period conferring fertilization competence to spermatozoa is termed capacitation (Yanagimachi, 1994). During capacitation, spermatozoa undergo a number of physiological, biochemical, and biophysical changes. Although the mechanisms involved in the process of sperm capacitation are still under investigation, the published reports suggest the involvement of an increase in cyclic adenosine monophosphate (cAMP); a modulation of intracellular concentrations of Ca21, K1,
Na1, and Cl2; and an increased generation of reactive oxygen species (Yanagimachi, 1994; de Lamirande et al, 1997; Visconti et al, 1998). Recent studies have clearly established that human sperm capacitation is associated with the phosphorylation of tyrosine (Tyr) residues of a specific subset of proteins (Carrera et al, 1996; Leclerc et al, 1996). This Tyr phosphorylation (P-Tyr) mainly affects proteins from the fibrous sheath; 3 of these, p81, p95, and p105, are related to A-kinase anchoring proteins (AKAP) (Carrera et al, 1996; Leclerc et al, 1997; Mandal et al, 1999). Several factors, such as reactive oxygen species, cAMP, and Ca21, have been shown to regulate the P-Tyr of sperm proteins (Aitken et al, 1995; Visconti et al, 1995; Leclerc et al, 1996, 1997, 1998; Herrero et al, 1999). Since capacitation is a crucial step in the acquisition of sperm fertilizing ability, it is likely that it is controlled by redundant mechanisms and that cross talks between different pathways occur during this process (de Lamirande et al, 1997; Leclerc et al, 1998). Besides the above-mentioned events, several lines of evidence indicate that com-
E
Supported by grants from Canadian Institutes of Health Research (CIHR) and le Fonds Que´be´cois de la Recherche sur la Nature et les Technologies (FQRNT) grants. Correspondence to: Dr Veronica Nauc, Endocrinologie de la Reproduction, D0-708, Pavillon St-Franc¸ois d’Assise, 10, de l’Espinay, Que´bec, Canada G1L 3L5 (e-mail:
[email protected]). Received for publication October 18, 2003; accepted for publication January 23, 2004.
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574 ponents of the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases (MAPK) are present in spermatozoa and are involved in capacitation (Naz et al, 1992; Luconi et al, 1998; de Lamirande and Gagnon, 2002). The key players in this cascade (Shc, Grb2, Ras, Raf-1, mitogen-activated protein kinase [MEK], ERK1, and ERK2) are present in ejaculated spermatozoa (de Lamirande and Gagnon, 2002). ERK1 and ERK2 are activated by MEK, a dual-specificity kinase that phosphorylates the Thr-Glu-Tyr motif present at its active center (Widmann et al, 1999). However, the ThrGlu-Tyr motif is also present in other key signaling elements such as ERK5 (Yan et al, 2001), ERK8 (Abe et al, 2002), MOK (Miyata and Nishida, 1999), and MAP1B (Lien et al, 1994). Our recent studies have shown that an increase in the P-Thr-Glu-Tyr-P motif of several sperm proteins other than the ERK1 and ERK2 is associated with sperm capacitation (de Lamirande and Gagnon, 2002; Thundathil et al, 2002). Although protein phosphorylation appears indispensable during sperm capacitation, the phosphorylation-specific cell signaling is complex and not completely clear. The effects that chemical inhibitors have on key regulators of cellular signaling have provided insights into specific signaling events that regulate sperm functions. Thus, a role for phosphoinositide 3-kinase (PI3K) has been suggested in sperm functions (Fisher et al, 1998; Luconi et al, 2001). PI3K is a heterodimeric protein consisting of a p85 regulatory (adaptor) subunit and a p110 catalytic subunit (Cantrell, 2001). PI3K is implicated in many biological processes, including cell survival and chemotaxis, membrane ruffling and DNA synthesis, receptor internalization, and vesicular trafficking (Wymann and Pirola, 1998; Cantrell, 2001). In somatic cells, PI3K also phosphorylates a large spectrum of protein substrates (Wymann and Pirola, 1998). One of the known effectors of the PI3K, Akt (also named protein kinase B [PKB]), was identified as a serine/threonine (Ser/Thr) protein kinase with a catalytic domain closely related to those of both protein kinase A (PKA) and protein kinase C (PKC) and also as a cellular homolog of the viral oncogene v-akt (Datta et al, 1999). The activated Akt phosphorylates and inactivates a series of proteins implicated in cell survival and metabolism and affects the MAPK (ERK1 and ERK2) signaling cascade (Datta et al, 1999; Perkinton et al, 2002). Two unrelated products, wortmannin and LY294002, are extensively used as pharmacological agents for characterizing the role of PI3K in cellular signaling (Cantrell, 2001). Wortmannin is a cell-permeable, potent, and selective inhibitor of PI3K (IC50 5 50 nmol/L) and pleckstrin phosphorylation (Barker et al, 1995). The quercetin derivative LY294002 (2-(4-morphlinyl)-8-phenyl-4H-1benzopyran-4-one) is a specific and cell-permeable inhib-
Journal of Andrology · July/August 2004 itor of PI3K but is less potent (IC50 5 50 mmol/L) than wortmannin (Vlahos et al, 1994). Although there are numerous reports that these 2 agents are being used for the inhibition of PI3K and its signaling pathway and that they induce similar cell responses, in spermatozoa, the published data suggest that wortmannin and LY294002 have distinct and opposed effects. On the one hand, wortmannin inhibited the acrosome reaction induced by either mannose-bovine serum albumin (BSA) or the antibody raised against the sperm zona receptor kinase (Fisher et al, 1998), suggesting that PI3K activity is needed for sperm function. On the other hand, LY294002 promoted sperm motility and hyperactivation (Luconi et al, 2001), an event that is usually associated with sperm capacitation (Yanagimachi, 1994), suggesting that the inhibition of PI3K is required for this process. Because there are only 2 independent studies that examine the possible role of PI3K in human spermatozoa, because each of those were performed using only 1 of the above 2 inhibitors, and because of the apparent contradiction in the published results, we decided to investigate how these 2 inhibitors affect human sperm capacitation and associated events. We investigated the effect of both LY294002 and wortmannin on human sperm capacitation by measuring their effect on the lysophosphatidylcholine (LPC)-induced acrosome reaction and the associated phosphorylation of Tyr residues and the Thr-GluTyr motif of sperm proteins. We also investigated whether PI3K, Akt, and proteins recognized by the antibody raised against the consensus motif phosphorylated by Akt are present in human spermatozoa and involved in sperm capacitation. As a possible explanation for the differences observed between the 2 inhibitors, the effect of LY294002 and wortmannin on the intracellular free Ca21 concentration of spermatozoa was also measured.
Materials and Methods Percoll, along with goat anti-mouse immunoglobulin (IgG) and goat anti-rabbit IgG, both of which were conjugated to horseradish peroxidase, were all obtained from Amersham Pharmacia Biotech (Montre´al, Canada). LPC, BSA, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin, and LY294002 were purchased from Sigma Chemical Co (St Louis, Mo). Wortmannin and b-glycerophosphate were purchased from Calbiochem (La Jolla, Calif). Upstate Biotechnology (Lake Placid, NY) was the supplier for the anti-PI3K (85 kd) and anti–phospho-Tyr (PTyr, clone 4G10, monoclonal) antibodies. Antibodies raised against the P-Thr-glutamine (Glu)-Tyr-P motif, against Akt, and against the anti–arginine (Arg)-X-Arg-X-X-P-Ser/Thr motif (where X represents any amino acid) were bought from Cell Signalling Technology (Beverly, Mass). INDO-1/AM, Pluronic F-127, and propidium iodide (PI) were purchased from Molecular Probes (Eugene, Ore). Nitrocellulose (0.22-mm pore size; Osmonics Inc, Westborough, Mass), an enhanceds chemilumi-
Nauc et al · Different Effects of PI3K Inhibitors nescence kit (Lumi-Light; Roche Molecular Biochemicals, Laval, Canada), and radiographic films (Fuji, Minami-Ashigara, Japan) were used for the immunodetection of blotted proteins. BioRad Laboratories Inc (Hercules, Calif) was the supplier for the Quantity One software. All other chemicals were at least of reagent grade. Fetal cord blood was collected at the birthing center of the Royal Victoria Hospital (Montre´al, Canada). For this purpose, informed consent was obtained from the patients, and the ethics board of the hospital approved the present study. Fetal cord blood was centrifuged (1000 3 g, 30 minutes at 48C), and sera were pooled and frozen (2208C) until use. FCSu was prepared by ultrafiltration of the fetal cord sera from at least 15 individual samples using YM3 membranes (exclusion limit, 3 kd; Amicon, Oakville, Canada) (de Lamirande and Gagnon, 1995). Reagents to be tested with spermatozoa were dissolved in distilled water or dimethylsulfoxide (DMSO). The concentration of DMSO in the incubation media never exceeded 1% (vol/vol), a condition that does not affect sperm capacitation and protein phosphorylation.
Preparation of Sperm Samples Semen samples from healthy volunteers were washed on 4-layer (95%-65%-40%-20%) Percoll gradients buffered in HEPES-balanced saline (HBS; 115 mmol of NaCl per liter, 4 mmol of KCl per liter, 0.5 mmol of MgCl2 per liter, 14 mmol of fructose per liter, and 25 mmol of HEPES per liter, pH 8.0). Samples were centrifuged for 30 minutes at 2300 3 g, and sperm cells at the 65%–95% Percoll interface and in the 95% Percoll layer were pooled and diluted to 200 3 106 cells/mL with the 95% Percoll solution. Sperm concentration and motility were determined using the CellSoft computer-assisted sperm analyzer (Montgomery, NY). Only samples in which motility was greater than 70% were used for experiments.
Treatments of Spermatozoa and Evaluation of Capacitation For capacitation studies, Percoll-separated spermatozoa were diluted to 40 3 106 cells/mL with Biggers-Whitten-Whittingham medium (BWW, pH 8) (Biggers et al, 1971) devoid of bicarbonate and BSA and containing 1 mmol of CaCl2 per liter. Inhibitors of PI3K, LY294002 and wortmannin, were added to spermatozoa for 30 minutes (378C) prior to supplementation or not (control, BWW alone) with FCSu (10% [vol/vol]) as the capacitation inducer. This capacitation inducer has been shown to promote sperm capacitation (as measured by the LPC- or A23187-induced acrosome reaction) and protein Tyr phosphorylation (de Lamirande and Gagnon, 1995; Leclerc et al, 1996, 1997, 1998; de Lamirande et al, 1997) to levels (and with kinetics) similar to those observed with BSA (Griveau et al, 1994; Aitken et al, 1995; Luconi et al, 1996; de Lamirande et al, 1997; Herrero et al, 1999). Neither LY294002 nor wortmannin affected the percentages of sperm motility, at the concentrations used in this study, for a period of at least 4 hours at 378C. Sperm capacitation was evaluated after 3.5 hours of incubation by the induction of the acrosome reaction with LPC as previously described (de Lamirande and Gagnon, 1995). In brief, spermatozoa were washed with HBS, resuspended in BWW medium contain-
575 ing 3 mg of BSA per milliliter, and 100 mmol/L of LPC, and incubated for another 30 minutes at 378C to induce the acrosome reaction. Then, spermatozoa were washed with HBS and fixed in ethanol. The acrosomal status of sperm cells was evaluated using fluorescein isothiocyanate-conjugated P sativum agglutinin (Cross et al, 1986). On each slide, the acrosomal status of at least 200 cells was evaluated. Differences in the level of capacitation obtained after various treatments of spermatozoa were analyzed for significant differences by the Tukey multiple comparison test following a 1-way analysis of variance. A difference was considered significant at P , .05.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Immunoblotting Sperm proteins (extracted after 2.5 hours of sperm incubation under conditions described above) were reduced and denatured in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer that was supplemented with 1 mmol of sodium vanadate per liter, 10 mmol of b-glycerophosphate per liter, and 100 mmol of NaF per liter and subsequently electrophoresed on 10% polyacrylamide gels. The separated proteins were transferred (using 10 mmol of CAPS per liter [3-(cyclohexylamino) 1-propane sulfonic acid] buffer, pH 11, and 10% methanol) onto nitrocellulose membranes and then incubated with 5% skim milk in Tris-buffered saline with Tween-20 (TTBS; 20 mmol of Tris-HCl per liter, pH 7.8, 0.9% NaCl, and 0.1% Tween-20). The incubation with the primary antibodies was performed for either 1 hour at room temperature (the anti– P-Tyr was diluted 10 000-fold with TTBS, and the anti-PI3K was diluted 2500-fold with TTBS containing 2.5% BSA) or overnight at 48C (the anti–P-Thr-Glu-Tyr-P, anti-Akt, and anti–ArgX-Arg-X-X-P-Ser/Thr were diluted 1000-fold with TTBS containing 2.5% BSA). After incubation with the primary antibodies, the membranes were washed with TTBS and then incubated with respective secondary antibodies for 1 hour. The immunoreactive bands were detected using the Lumi-Light chemiluminescence kit. At the end of the experiments, blots were silver stained (Jacobson and Karsnas, 1990) to ascertain that the amount of protein loaded in each well was the same. Protein bands were scanned, and their density was evaluated using Quantity One software. Values of separate experiments were analyzed by the Tukey multiple comparison test following a 1-way analysis of variance. A difference was considered significant at P , .05.
Cell Fractionation Triton Extraction—Percoll-separated spermatozoa were subsequently washed with cold HBS supplemented with protease inhibitors (10 mg of aprotinin per milliliter, 10 mg of leupeptin per milliliter, 10 mg of pepstatin per milliliter, 250 mmol of phenylmethylsulphonyl fluoride, and 1 mmol of sodium vanadate per liter) at 600 3 g for 5 minutes. Sperm pellets (20 3 106 cells) were treated with 1% Triton X-100 buffered in HBS supplemented with protease inhibitors (see above) for 30 minutes on ice and then centrifuged (12 000 3 g for 15 minutes). The Tritonsoluble and Triton-insoluble fractions (resuspended to the orig-
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assessed by light microscopy. When about 90% of the spermatozoa were properly separated, the cell suspension was layered on the top of a 75% Percoll solution. After a 15-minute centrifugation (700 3 g), the flagella fraction was collected at the interface between the suspension and Percoll layer, and the sperm head fraction was recovered at the tube bottom. Proteins from each fraction were denatured and reduced using SDS-PAGE sample buffer and were then heated for 5 minutes at 1008C. Protein aliquots corresponding to each fraction were precipitated (trichloroacetic acid, 25%) and then resuspended with NaOH 0.1 N to initial volume. The obtained preparations were assessed for protein concentration (Micro BCA Protein Assay Reagent Kit; Pierce, Rockford, Ill). All sperm fractions were normalized by the total protein concentrations, and 5 mg of total protein was loaded per well onto the 7.5% SDS-polyacrylamide gels.
Evaluation of the Intracellular Free Ca21 Concentration
Figure 1. The effect that LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one) and wortmannin have on the capacitation of human spermatozoa. Spermatozoa resuspended in Biggers-Whitten-Whittingham (BWW) medium were incubated with (A) LY294002 or (B) wortmannin at the indicated concentrations for 30 minutes and then without (white bars) or with fetal cord serum ultrafiltrate (FCSu) (black bars) for 3.5 hours. Sperm capacitation was evaluated as described in ‘‘Materials and Methods.’’ Results are mean 6 SEM of 5 determinations performed on sperm samples from different donors. * indicates that a value is different from that of control spermatozoa (BWW alone), and # indicates that a value is different from that of FCSu-treated spermatozoa.
inal volume with HBS) were supplemented with SDS-PAGE sample buffer, heated, and centrifuged. Equal volumes of resulting fractions (corresponding to 106 cells) were loaded on 7.5% polyacrylamide gel for protein separation and were then processed for immunoblotting with either anti-PI3K (85 kd) or anti-Akt antibodies. Cavitation—All steps were carried out at 48C. The Percollseparated sperm cells were diluted with cold HBS (pH 7.4), placed in ice, and then subjected to nitrogen cavitation according to the procedure proposed by Noland et al (1983) with several modifications. Briefly, the cavitation was performed at 700 psi for 10 minutes in a Parr bomb. Cavitated cells were centrifuged at 10 000 3 g for 10 minutes. The supernatant was decanted and then subjected to ultracentrifugation at 100 000 3 g for 1 hour. The ultracentrifugation supernatant was concentrated four- to fivefold using centrifugal filters (Microcon YM-10; Millipore, Bedford, Mass). Membranes were collected at the bottom of the ultracentrifugation tubes. The 10 000 3 g pellets representing cells devoid of plasma membrane were subjected to sonication to separate sperm heads from flagella (3 times, 10 seconds). The separation results were
Percoll-washed spermatozoa were diluted to 25 3 106 cells/mL in a calcium-free BWW medium and incubated for 30 minutes at room temperature in the presence of 2.5 mmol of INDO-1/ AM and 0.00625% Pluronic F-127 as previously described (Collin et al, 2000). The sperm suspension was washed with calciumfree BWW medium to remove the noninternalized Ca21 probe and was then resuspended in complete BWW medium. For the evaluation of the intracellular free Ca21 concentration, spermatozoa were diluted to 1 3 106 cells/mL. As an indicator of sperm viability, 5 mg of PI per milliliter was added to each sample. LY294002 mmol/L (3, 10, or 30 mmol/L) and wortmannin (100 nmol/L) were injected to samples prior to readings. Measurements were performed by flow cytometry (Epics Elite ESP; Beckman Coulter, Miami, Fla) with a flow cytometer that was equipped with a helium cadmium laser (Model 100; Omnichrome, Chino, Calif) having an excitation wavelength of 325 nm. The violet (381 nm, Ca21-bound)/blue (525 nm, Ca21-unbound) INDO-1 emission ratios were plotted against time, as indicated in the Current Protocols in Cytometry (June et al, 1997). More than 5000 cells were analyzed for each treatment in different experiments. The kinetic analysis was performed using the shareware WinMDI 2.8 (http://facs.scripps.edu). Values of separate experiments were analyzed by the Tukey multiple comparisons test following a 1-way analysis of variance. A difference was considered significant at P , .05.
Results The Effect of LY294002 and Wortmannin on Sperm Capacitation It has been proposed that PI3K negatively modulates human sperm hyperactivated motility (LY294002-induced effect) (Luconi et al, 2001) while promoting the sperm acrosome reaction (wortmannin-induced prevention of acrosome reaction) (Fisher et al, 1998). Since hyperactivated motility is associated with sperm capacitation and these events prime spermatozoa to undergo the acrosomal exocytosis, we analyzed the effects of these 2 agents
Nauc et al · Different Effects of PI3K Inhibitors (LY294002 and wortmannin), which have been recognized as selective inhibitors of PI3K, for the role they play in human sperm capacitation. LY294002 alone enhanced sperm capacitation in a concentration-dependent manner (Figure 1A). A further increase in LY294002 concentration up to 100 mmol/L did not affect this stimulatory effect (data not shown). LY294002 had no effect on FCSu-induced capacitation (Figure 1A). When incubated for 3.5 hours with spermatozoa resuspended in BWW, this agent did not trigger a spontaneous acrosome reaction without further stimulation with LPC (n 5 3; data not shown). Under similar conditions, wortmannin did not change the capacitation status of spermatozoa incubated with BWW but prevented the FCSu-induced capacitation at 10 nmol/L (Figure 1B), the lowest concentration tested in the present study.
LY294002, Wortmannin, and Their Role in CapacitationAssociated Phosphorylations Since an increase in protein Tyr phosphorylation is commonly associated with capacitation, we examined this phenomenon in sperm incubated in the presence of LY294002 and wortmannin. As reported previously, FCSu increased the P-Tyr content in human sperm pro-
577 teins of 81 and 105 kd (Figure 2). Similar to what has been observed for sperm capacitation, LY294002 (30 mmol/L) induced an increase in Tyr phosphorylation of proteins of 81 kd (Figure 3A) and 105 kd (Figure 3B). Phosphorylation was further increased in spermatozoa incubated in the presence of FCSu after a 30-minute pretreatment with LY294002 (Figures 2 and 3). On the other hand, wortmannin alone slightly decreased the level of Tyr phosphorylation of p81 and p105 (Figure 3), while it did not modify the P-Tyr content of these proteins in spermatozoa that were incubated with FCSu (Figure 3). Recent evidence (Thundathil et al, 2002) indicates that capacitation is associated with an increase in the level of P-Thr-Glu-Tyr-P in sperm proteins during the course of capacitation. Therefore, the effects of LY294002 and wortmannin on this process were evaluated in either the presence or absence of FCSu. The addition of 30 mmol/ L of LY294002 to sperm cells induced an increase in the double phosphorylation of the Thr-Glu-Tyr motif of proteins at 80, 105, and greater than 200 kd (Figures 4 and 5). This phosphorylation was further increased when the cells were stimulated with FCSu, and there was an additive effect when FCSu was added to spermatozoa pretreated with LY294002 (30 mmol/L) (Figure 5). On the other hand, wortmannin alone did not affect the P-Thr-
Figure 2. Phosphorylation of tyrosine residues in spermatozoa treated with LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and wortmannin. Spermatozoa pretreated with LY294002 or wortmannin at the indicated concentrations were incubated without (lanes 2–6) or with (lanes 7– 11) fetal cord serum ultrafiltrate (FCSu) for 2 hours. Sperm proteins (0.3 3 106 cells per well) were immunoblotted with anti–phospho-tyrosine (P-Tyr) antibody. Results are representative of 4 independent experiments performed using spermatozoa from different donors.
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Journal of Andrology · July/August 2004 with the T-leukemic cell line Jurkat control (Figure 7, lane 6), strongly suggest that the regulatory subunit of PI3K is present in human spermatozoa and that its molecular mass is 85 kd. Since Akt is one of the best-characterized downstream effectors of the PI3K signaling pathway and its presence in spermatozoa has not been reported, similar localization experiments were performed with an anti-Akt antibody. No immunoreactive protein band was extracted with Triton X-100 (Figure 6B). Similar to the localization of the 85-kd band, a 60-kd band was present in all cell fractions except for cytosol (Figure 7B). These data strongly suggest that Akt is present in human spermatozoa and that its molecular mass is similar to that of proteins found in other cell types (Datta et al, 1999).
Phosphorylation of the Arg-X-Arg-X-X-Ser/Thr Motif
Figure 3. Phospho-tyrosine (P-Tyr) content of 81- and 105-kd proteins in spermatozoa treated with LY294002 (2-(4-morpholinyl)-8-phenyl-4H1-benzopyran-4-one) and wortmannin. Spermatozoa were incubated in either Biggers-Whitten-Whittingham (BWW) medium (white bars) or fetal cord serum ultrafiltrate (FCSu) (black bars) after cell pretreatment with either LY294002 (LY, at 3 and 30 mmol/L) or wortmannin (W, at 10 and 100 nmol/L). Following immunoblotting, major proteins recognized by the anti–P-Tyr antibody were further analyzed by densitometry as stated in ‘‘Material and Methods.’’ (A) The densitometric assessment of the 81-kd protein and (B) of the 105-kd protein is shown. Results are mean 6 SEM of 4 determinations performed on sperm samples from different donors. * indicates that a value is different from that of control spermatozoa (To).
Glu-Tyr-P content of the 80- and 105-kd proteins, whereas it prevented the FCSu-induced phosphorylation of the Thr-Glu-Tyr motif of these proteins (Figure 5).
Localization of PI3K and Akt in Human Sperm The divergent effects of LY294002 and wortmannin may suggest that at least one of these inhibitors acts in a PI3Kindependent manner. Therefore, the presence of this latter enzyme in human spermatozoa was investigated. PI3K comprises a 110-kd catalytic subunit and an 85-kd regulatory adapter subunit (Cantrell, 2001). The antibody raised against the PI3K regulatory subunit recognized a 85-kd protein band in Triton-soluble and Triton-insoluble fractions (Figure 6). Further fractionation of spermatozoa provided sperm fractions enriched in heads, flagella, cytosol, and plasma membranes. The anti-PI3K regulatory subunit antibody recognized a protein band of 85 kd in all sperm fractions except for the cytosol, with a major portion being present in the plasma membrane fraction (Figure 7A). These data, combined with the data obtained
The PI3K/Akt pathway is essential for many cellular functions, and further signal transduction occurs through its downstream effectors. Akt phosphorylates substrates only at Ser/Thr in a conserved motif Arg-X-Arg-X-X-Ser/ Thr (Allesi et al, 1996). Our preliminary experiments indicated that an antibody raised against the phosphorylated Arg-X-Arg-X-X-Ser/Thr motif recognized the sperm proteins of 70, 80, 97, 110, and 140 kd. There was an increase in the phosphorylation of this motif within 5 minutes after the beginning of the incubation with the human sperm capacitation inducer FCSu (Figure 8), which was followed by a decrease in P-Ser/Thr contents 30 minutes later (data not shown). FCSu stimulation induced an increase in the phosphorylation of proteins at 70, 80, 97, 110, and 140 kd (Figures 8 and 9). The treatment of spermatozoa with 30 mmol/L of LY294002 resulted in an increased phosphorylation of the same protein bands, with greater phosphorylation (when compared with the FCSu effect) occurring for bands at 97 and 110 kd. The addition of FCSu to spermatozoa pretreated with LY294002 (30 mmol/L) induced the highest phosphorylation of proteins recognized by the anti–Arg-X-Arg-X-X-P-Ser/Thr antibody. Contrary to the effect observed with LY294002, wortmannin prevented the phosphorylation of the Arg-X-Arg-X-X-Ser/Thr motif in spermatozoa incubated with FCSu (Figure 9).
The Effect of LY294002 and Wortmannin on Sperm Intracellular Ca21 Concentration Although both LY294002 and wortmannin are extensively used for investigating roles that PI3K and Akt may play in a variety of cellular processes (Allesi et al, 1996; Datta et al, 1999), these 2 agents also have effects unrelated to PI3K. LY294002 has a PI3K-independent effect in smooth muscle cells that results in an increase in intracellular Ca21 (Ethier and Madison, 2002). When tested in human spermatozoa, LY294002 induced a very rapid
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Figure 4. Phosphorylation of the threonine-glutamine-tyrosine (Thr-Glu-Tyr) motif in spermatozoa treated with LY294002 (2-(4-morpholinyl)-8-phenyl4H-1-benzopyran-4-one) and wortmannin. Spermatozoa pretreated with these inhibitors at the indicated concentrations were incubated without (lanes 2–6) or with (lanes 7–11) fetal cord serum ultrafiltrate (FCSu) for 2 hours. Sperm proteins (1.5 3 106 cells per well) were immunoblotted with the anti– P-Thr-Glu-Tyr-phospho (P) antibody. Results are representative of 4 independent experiments performed using spermatozoa from different donors.
increase in [Ca21]i (Figure 10A). The noted effect occurred in freshly diluted spermatozoa in a concentrationdependent manner (Figure 7B). Higher LY294002 concentrations (100 mmol/L) resulted in a further increase in [Ca21]i (data not shown), suggesting that the noted effect is independent of PI3K. In contrast to LY294002, wortmannin (100 nmol/L) induced a minor decrease in [Ca21]i in human spermatozoa (Figure 10B). Moreover, when spermatozoa were preincubated with wortmannin (for 30 minutes or 4 hours) and were further stimulated with LY294002 (30 mmol/L), this agent did not affect the LY294002-mediated increase in [Ca21]i (data not shown). Using PI incorporation as a tool to evaluate sperm viability throughout the intracellular Ca21 measurements, no difference was observed in any of the treatments with the PI3K inhibitors (data not shown).
Discussion Data presented in the current study bring new insights regarding the controversial effects of 2 commonly used inhibitors of PI3K, LY294002 and wortmannin, in human spermatozoa. Discrepancies between the effects of the 2 inhibitors were observed while assessing sperm capacitation. LY294002 alone promoted sperm capacitation (Figure 1A), which is consistent with the previous observation regarding LY294002-induced sperm hyperactiva-
tion (Luconi et al, 2001), an event that is commonly associated with capacitation (Yanagimachi, 1994). On the other hand, wortmannin did not affect sperm capacitation on its own but prevented the sperm capacitation induced by FCSu (Figure 1B). This finding is in agreement with the observation of a wortmannin inhibitory role on the human sperm acrosome reaction induced either by mannose-BSA or the antibody against the sperm zona receptor kinase (Fisher et al, 1998). Consistent with our data on sperm capacitation, LY294002 alone triggered an increase in the P-Tyr content of sperm proteins of 81 and 105 kd, which is similar to the effect observed when FCSu was used as a capacitating agent (Figures 2 and 3). The proteins of 81 and 105 kd were extensively characterized as proteins from the sperm fibrous sheath and as being related to AKAPs (Carrera et al, 1996; Leclerc et al, 1997). Furthermore, although mostly characterized as playing roles during PKA anchoring, some AKAPs may simultaneously bind other signal transduction molecules such as PKC (Faux et al, 1999), ropporin (Carr et al, 2001), and G proteins (Niu et al, 2001), which suggests that they also function in cells as a substrate and effector for the anchored kinases and phosphatases. Thus, an LY294002-induced increase in AKAP P-Tyr content suggests that this agent promotes signaling pathways independent of those activated by PI3K. Consistent with this prediction, LY294002 alone (30 mmol/L) increased the content of P-Thr-Glu-
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Figure 5. Evaluation of the content of the phospho-threonine-glutaminetyrosine-phospho (P-Thr-Glu-Tyr-P) motif of 80- and 105-kd proteins in spermatozoa treated with LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one) and wortmannin. Spermatozoa were incubated in either Biggers-Whitten-Whittingham (BWW) medium (white bars) or fetal cord serum ultrafiltrate (FCSu) (black bars) after cell pretreatment with either LY294002 (LY, at 3 and 30 mmol/L) or wortmannin (W, at 10 and 100 nmol/L). Following immunoblotting, major proteins recognized by the anti–P-Thr-Glu-Tyr-P antibody were further analyzed by densitometry as stated in ‘‘Materials and Methods.’’ (A) The densitometric assessment of the 80-kd protein and (B) of the 105-kd protein is shown. Results are mean 6 SEM of 4 determinations performed on sperm samples from different donors. * indicates that a value is different from that of control spermatozoa (To).
Tyr-P in sperm proteins of 80 and 105 kd (Figures 4 and 5), which was similar to the increase promoted by FCSu (present study and Thundathil et al, 2002). Although these later proteins remain to be identified, our group showed recently that their phosphorylation is prevented by PD98059 and U126 (inhibitors of MEK), suggesting a role for a dual-specificity (Ser/Thr and Tyr) kinase similar to MEK in the phosphorylation of their Thr-Glu-Tyr motif (de Lamirande and Gagnon, 2002; Thundathil et al, 2002). The noted discrepancies regarding cellular responses toward the 2 inhibitors of PI3K triggered some doubts concerning the presence of PI3K in human spermatozoa. Immunoblotting experiments strongly suggested the presence of the 85-kd regulatory subunit of PI3K in human spermatozoa (Figures 6A and 7A). The results indicate that the major proportion of the regulatory unit of PI3K is located in the sperm membrane and, to a lesser extent, in the sperm fractions composed mainly of sperm flagella and internal organelles (Figure 7A). Similar to our find-
Figure 6. Presence of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B) in human spermatozoa. Spermatozoa were treated with Triton X-100. Proteins corresponding to 106 cells (total, Triton-soluble, and insoluble fractions) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then immunoblotted with (A) anti-PI3K and (B) anti-Akt antibodies. Results are representative of 2 independent experiments performed using spermatozoa from different donors.
ings, Cantrell (2001) reported that the regulatory subunit of PI3K (85 kd) is usually found at the cell membrane site in complex with the receptor Tyr kinase. We further verified the presence of the PI3K main downstream effector, Akt, in human spermatozoa. While Akt is known to be present during spermatogenesis (Feng et al, 2000), its presence in spermatozoa has not, to our
Nauc et al · Different Effects of PI3K Inhibitors
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Figure 7. Cellular localization of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B) in human spermatozoa. Spermatozoa were fractionated as described in ‘‘Materials and Methods.’’ Proteins from each fraction (5 mg) were immunoblotted with (A) anti-PI3K and (B) anti-Akt antibodies. Jurkat cells (lane 6) were used as a protein control. Results are representative of 3 independent experiments performed using spermatozoa from different donors.
Figure 8. Phosphorylation of the motif implicated in signal transduction downstream of Akt (protein kinase B). Spermatozoa pretreated with LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and wortmannin at the indicated concentrations were incubated without (lanes 2–6) or with (lanes 7–11) fetal cord serum ultrafiltrate (FCSu) for 5 minutes. Sperm proteins (1.5 3 106 cells per well) were immunoblotted with anti–arginine (Arg)-X-ArgX-X-phospho (P)-serine (Ser)/threonine (Thr) motif (where X represents any amino acid) antibody. Results are representative of 4 independent experiments performed using spermatozoa from different donors.
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Figure 9. Evaluation of the content of the arginine (Arg)-X-Arg-X-X-phospho (P)-serine (Ser)/threonine (Thr) motif of proteins recognized by the respective antibody. Spermatozoa were incubated in either Biggers-Whitten-Whittingham (BWW) medium (white bars) or fetal cord serum ultrafiltrate (FCSu) (black bars) after cell pretreatment with either LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) (LY, at 3 and 30 mmol/L) or wortmannin (W, at 10 and 100 nmol/L). Following immunoblotting, major proteins recognized by the anti–Arg-X-Arg-X-X-P-Ser/Thr antibody were further analyzed by densitometry as stated in ‘‘Materials and Methods.’’ (A) The densitometric assessments of the 140-kd protein, (B) the 110-kd protein, (C) the 97-kd protein, (D) the 80-kd protein, and (E) the 70-kd protein are shown. Results are mean 6 SEM of 4 determinations performed on sperm samples from different donors. * indicates that a value is different from that of control spermatozoa (To).
Journal of Andrology · July/August 2004
Figure 10. LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4one)–induced effect in intracellular Ca21. (A) INDO-1/AM was incorporated into spermatozoa as described in ‘‘Materials and Methods.’’ Spermatozoa were next diluted with Biggers-Whitten-Whittingham (BWW) medium (1 mmol of Ca21 per liter). The evaluation of the intracellular free [Ca21] was performed by flow cytometry. (A) The relative [Ca21]i represents the ratio between the intensity of fluorescence emitted by the Ca21bound (violet)/Ca21-unbound (blue) INDO-1 probe according to the method described in Current Protocols in Cytometry (June et al, 1997). An experiment representative of 4 in which similar results were obtained is shown. The arrow indicates the time of LY294002 (30 mmol/L) injection. (B) INDO-1/AM–loaded spermatozoa were diluted with BWW medium (1 mmol of Ca21 per liter) and then supplemented with a vehicle dimethylsulfoxide (DMSO), LY294002 (LY, 3 and 30 mmol/L), or wortmannin (W, 100 nmol/L). Results are expressed as the percentage of increase in relative [Ca21]i after subtraction of control (BWW medium) values of the same parameter. Values are mean 6 SEM of 4 determinations performed independently on spermatozoa from different donors. *: Data are significantly different from those obtained in control (DMSO) spermatozoa.
knowledge, been reported previously. Our data show that a band (60 kd) recognized by the anti-Akt antibody is present in sperm membranes, flagella, and heads while absent from the cytosolic fraction (Figure 7B). This protein kinase was resistant to extraction with 1% Triton X100 (Figure 6B), whereas in most cells, Akt is in the Triton-soluble fraction (Cantrell, 2001). Once activated,
Nauc et al · Different Effects of PI3K Inhibitors Akt phosphorylates proteins that contain the consensus phosphorylation sites Arg-X-Arg-X-X-Ser/Thr (Allesi et al, 1996). Several protein targets of Akt are well characterized for their role in modulating cell functions (BAD, caspase-9, folkhead transcription factors, IkB kinases, GSK-3, and Raf-1) (Datta et al, 1999). In human spermatozoa, as Allesi et al (1996) have observed in other cell types, Akt activation and phosphorylation of characteristic substrates occurred within 1–5 minutes of the cells being stimulated and decreased rapidly afterward (after 30 minutes of incubation; data not shown). A series of sperm proteins were recognized by the anti–Arg-XArg-X-X-P-Ser/Thr antibody (Figure 8), and the level of phosphorylation was increased in cells stimulated with FCSu (Figures 8 and 9), suggesting that the PI3K/Akt pathway is involved in capacitation events. The observation that wortmannin effectively prevented the FCSuinduced phosphorylation of these proteins (Figures 8 and 9) suggests that the phosphorylation occurs via a wortmannin-sensitive PI3K/Akt pathway. Under similar conditions, LY294002 promoted, in a concentration-dependent manner (up to 100 mmol/L; data not shown), increases in the P-Ser/Thr content of proteins of 80, 97, and 110 kd, confirming the previously noted divergences between these 2 agents. An explanation for the LY294002 effect was found when analyzing the amount of intracellular Ca21 in the presence of either LY294002 or wortmannin. Intracellular free Ca21 rapidly increased in sperm cells after the addition of LY294002 at 30 mmol/L (Figure 10A), a concentration that inhibits PI3K in many cell types (Rameh et al, 1998; Pasquet et al, 1999; Siddiqui and English, 2000). However, it is unlikely that the increases in [Ca21]i observed in response to LY294002 were due to PI3K inhibition for 2 reasons. First, a different and irreversible inhibitor of the PI3K, wortmannin, at a concentration that effectively inhibits PI3K (Barker et al, 1995), had a minor effect on [Ca21]i (Figure 10B). Second, pretreating cells with wortmannin did not inhibit subsequent responses to LY294002 (data not shown). Therefore, our findings suggest that LY294002 increases [Ca21]i in a PI3K-independent manner, although the mechanism involved remains to be established. These data are in agreement with recent evidence showing that, in smooth muscle cells, LY294002 releases Ca21 from intracellular stores, thus promoting an increase in [Ca21]i (Ethier and Madison, 2002). We must also consider the effect of wortmannin, which triggered a minor decrease in [Ca21]i (Figure 10B). This effect may be similar to that found in human platelets, where 2 mmol of wortmannin inhibited a store-depletion–evoked Ca21 influx and reduced the rise of [Ca21]i (Jenner et al, 1996). However, at the concentrations used for PI3K inhibition (10–100 nmol/L), wortmannin did not greatly affect
583 [Ca21]i (present study and Agell et al, 2002); thus, we did not further investigate the noted tendency. Evidence supports that an increase in intracellular Ca21 concentration allows a positive regulation of the Ras/Raf/ MEK/ERK pathway (Agell et al, 2002) and PKC signaling (Lenz et al, 2002). Furthermore, the rise of the intracellular free Ca21 concentration is essential for spermatozoa to complete capacitation and to undergo the acrosome reaction (Stock and Fraser, 1989; Yanagimachi, 1994) as well as to promote Tyr phosphorylation of specific sperm proteins (Dorval et al, 2002). Similarly, an increase in the [Ca21]i is one of the main factors triggering sperm hyperactivation (Ho et al, 2002), an event reported to occur in the presence of LY294002 (Luconi et al, 2001). Therefore, we propose that the reported LY294002 effects on human sperm capacitation and protein phosphorylation are due to its effect on increasing [Ca21]i rather than the inhibition of PI3K. In conclusion, the present study indicates that special care must be taken when using LY294002 to investigate the PI3K signaling pathway and the role that PI3K plays in cellular phenomena.
Acknowledgments We would like to address a special thanks to Dr E. Asselin for providing antibodies against Akt and Dr M. Dufour for help with [Ca21]i assessment. We would also like to thank Dr Lucie Morin (Royal Victoria Hospital) for collecting and providing us with fetal cord blood samples, as well as all volunteers who took part in the study.
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