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From the * Urology Research Laboratory, Royal
Victoria Hospital and McGill University, Montréal, Canada; and the
Endocrinologie de la Reproduction, Pavillon
St-François d'Assise, Québec, Canada.
| Correspondence to: Dr Veronica Nauc, Endocrinologie de la Reproduction, D0-708, Pavillon St-François d'Assise, 10, de l'Espinay, Québec, Canada G1L 3L5 (e-mail: vero_na{at}hotmail.com). |
| Received for publication October 18, 2003; accepted for publication January 23, 2004. |
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Abstract |
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Key words: Spermatozoa, signal transduction, Akt
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 Ca2+, 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 components 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 Thr-Glu-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 = 50 nmol/L) and pleckstrin phosphorylation (Barker et al, 1995). The quercetin derivative LY294002 (2-(4-morphlinyl)-8-phenyl-4H-1-benzopyran-4-one) is a specific and cell-permeable inhibitor of PI3K but is less potent (IC50 = 50 µmol/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-Glu-Tyr 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 Ca2+ concentration of spermatozoa was also measured.
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Materials and Methods |
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Fetal cord blood was collected at the birthing center of the Royal Victoria Hospital (Montréal, 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 x g, 30 minutes at 4°C), and sera were pooled and frozen (-20°C) 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
x g, and sperm cells 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. 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
x 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 (37°C) 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
37°C. 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 containing 3 mg of BSA per milliliter, and 100 µmol/L of LPC,
and incubated for another 30 minutes at 37°C 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
ß-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 4°C (the
anti–P-Thr-Glu-Tyr-P, anti-Akt, and anti–Arg-X-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
µg of aprotinin per milliliter, 10 µg of leupeptin per milliliter, 10
µg of pepstatin per milliliter, 250 µmol of phenylmethylsulphonyl
fluoride, and 1 mmol of sodium vanadate per liter) at 600 x g
for 5 minutes. Sperm pellets (20 x 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 x
g for 15 minutes). The Triton-soluble and Triton-insoluble fractions
(resuspended to the original 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 4°C. The Percoll-separated 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 x g for 10 minutes. The supernatant was decanted and then subjected to ultracentrifugation at 100 000 x 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 x 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 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 x 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 100°C. 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 µg of total protein was loaded per well onto the 7.5% SDS-polyacrylamide gels.
Evaluation of the Intracellular Free Ca2+ Concentration
Percoll-washed spermatozoa were diluted to 25 x 106
cells/mL in a calcium-free BWW medium and incubated for 30 minutes at room
temperature in the presence of 2.5 µmol of INDO-1/AM and 0.00625% Pluronic
F-127 as previously described (Collin et
al, 2000). The sperm suspension was washed with calcium-free BWW
medium to remove the noninternalized Ca2+ probe and was then
resuspended in complete BWW medium. For the evaluation of the intracellular
free Ca2+ concentration, spermatozoa were diluted to 1 x
106 cells/mL. As an indicator of sperm viability, 5 µg of PI per
milliliter was added to each sample. LY294002 µmol/L (3, 10, or 30
µmol/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,
Ca2+-bound)/blue (525 nm, Ca2+-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.
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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 Capacitation-Associated 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 proteins of 81 and 105 kd
(Figure 2). Similar to what has
been observed for sperm capacitation, LY294002 (30 µmol/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).
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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 µmol/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 µmol/L) (Figure 5). On the other hand, wortmannin alone did not affect the P-Thr-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).
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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 PI3K-independent 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 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.
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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
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).
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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 µmol/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 µmol/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).
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The Effect of LY294002 and Wortmannin on Sperm Intracellular Ca2+ 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
Ca2+ (Ethier and Madison,
2002). When tested in human spermatozoa, LY294002 induced a very
rapid increase in [Ca2+]i
(Figure 10A). The noted effect
occurred in freshly diluted spermatozoa in a concentration-dependent manner
(Figure 7B). Higher LY294002
concentrations (100 µmol/L) resulted in a further increase in
[Ca2+]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 [Ca2+]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 µmol/L), this
agent did not affect the LY294002-mediated increase in
[Ca2+]i (data not shown). Using PI incorporation as a
tool to evaluate sperm viability throughout the intracellular Ca2+
measurements, no difference was observed in any of the treatments with the
PI3K inhibitors (data not shown).
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Discussion |
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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 µmol/L) increased the content of P-Thr-Glu-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 findings, 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 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 X-100
(Figure 6B), whereas in most
cells, Akt is in the Triton-soluble fraction
(Cantrell, 2001). Once
activated, 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, I
B
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-X-Arg-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 FCSu-induced 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 µmol/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 Ca2+ in the presence of either LY294002 or wortmannin. Intracellular free Ca2+ rapidly increased in sperm cells after the addition of LY294002 at 30 µmol/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 [Ca2+]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 [Ca2+]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 [Ca2+]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 Ca2+ from intracellular stores, thus promoting an increase in [Ca2+]i (Ethier and Madison, 2002). We must also consider the effect of wortmannin, which triggered a minor decrease in [Ca2+]i (Figure 10B). This effect may be similar to that found in human platelets, where 2 µmol of wortmannin inhibited a store-depletion–evoked Ca2+ influx and reduced the rise of [Ca2+]i (Jenner et al, 1996). However, at the concentrations used for PI3K inhibition (10–100 nmol/L), wortmannin did not greatly affect [Ca2+]i (present study and Agell et al, 2002); thus, we did not further investigate the noted tendency.
Evidence supports that an increase in intracellular Ca2+ 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 Ca2+ 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 [Ca2+]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 [Ca2+]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.
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Acknowledgments |
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Footnotes |
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References |
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Agell N, Bachs O, Rocamora N, Villalonga P. Modulation of the Ras/Raf/MEK/ERK pathway by Ca2+, and calmodulin. Cell Signalling. 2002;14: 649 -654.[Medline]
Aitken RJ, Paterson M, Fisher H, Buckingham DW, van Duin M. Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J Cell Sci. 1995; 108: 2017 -2025.[Abstract]
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