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From the Centre de Recherche en Biologie de la Reproduction, *
Département des Sciences Animales and
Département d'Obstétrique et
Gynécologie, Université Laval, Sainte-Foy, Québec,
Canada.
| Correspondence to: Dr Janice L. Bailey, Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Pavillon Paul Comtois, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4 (e-mail: janice.bailey{at}crbr.ulaval.ca). |
| Received for publication November 13, 2002; accepted for publication May 13, 2003. |
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Abstract |
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Key words: Tyrosine phosphorylation, chelator, A23187, indo-1
In various mammalian species, protein phosphorylation is known to regulate sperm functions, such as motility (Tash and Means, 1983; Vijayaraghavan et al, 1997) and zona pellucida binding (Naz and Ahmad, 1994). Protein phosphorylation is also implicated in mammalian sperm capacitation (Visconti and Kopf, 1998; Tardif et al, 1999). It appears that several specific signal transduction pathways are involved during sperm capacitation (Visconti et al, 1995b; Baldi et al, 2002). In various species, such as the mouse (Visconti et al, 1995a,1995b), human (Luconi et al, 1996), bovine (Galantino-Homer et al, 1997), and pig (Kalab et al, 1998; Flesch et al, 1999), it has been well documented that capacitation appears to be associated with tyrosine phosphorylation of sperm proteins.
It is well established that calcium plays a crucial role in sperm function (Dragileva et al, 1999; Ho and Suarez, 2001). In 1915, Loeb was the first to demonstrate that extracellular calcium is required for fertilization in invertebrates. A similar requirement has been found for mouse sperm by Iwamatsu and Chang (1971). Since then, many studies have shown the importance of calcium for mammalian sperm capacitation (Handrow et al, 1989; DasGupta et al, 1993; Fraser et al, 1995) and for tyrosine phosphorylation of sperm proteins (Visconti et al, 1995a). We recently identified a tyrosine-phosphorylated protein "p32" in porcine sperm that seems to be closely related to capacitation (Tardif et al, 2001). As calcium is important for sperm capacitation and capacitation is a crucial phenomenon for fertilization, the major goal of this study was to elucidate the importance of calcium in the appearance of the p32 during capacitation in boar sperm. Our hypothesis was that the p32 is a calcium-dependent phosphoprotein related to boar sperm capacitation.
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Materials and Methods |
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Culture Media
The principal culture media used were based on Kreb Ringer Bicarbonate
(Toyoda and Chang, 1974). The
capacitating medium (CM) was composed of 2.7 mM KCl, 1.2 mM
KH2PO4, 8.1 mM Na2HPO4, 95 mM
NaCl, 5.55 mM glucose, 25 mM NaHCO3, 2 mM CaCl2, 0.4%
BSA (type V, free of fatty acids), and 2 mM pyruvate (pH 7.4; 230 mOsm). The
noncapacitating medium (NCM) was similar to the CM but did not contain
calcium, bicarbonate, or BSA (2.7 mM KCL, 1.2 mM KH2PO4,
8.1 mM Na2HPO4, 95 mM NaCl, 5.55 mM glucose, 2 mM
pyruvate, pH 7.4, and 215 mOsm).
Sperm Preparation
Sperm preparation was as previously reported
(Tardif et al, 2001). Briefly,
semen was collected from fertile boars and diluted to 40 x
106 sperm/mL with Beltsville Thawing Solution (BTS) at the Centre
d'Insémination Porcine du Québec (St-Lambert, Québec,
Canada) and transported to the laboratory at 16°C to 18°C within 30
minutes. The diluted semen was divided into 2 equal fractions and centrifuged
once (10 minutes, 270 x g, 25°C). The first fraction was
resuspended in CM (40 x 106 sperm/mL) to induce capacitation,
and the second portion in NCM (40 x 106 sperm/mL) was used as
a noncapacitating negative control. Sperm were then incubated at 38.5°C,
the internal body temperature of the pig, in a humidified 5% CO2
atmosphere.
Isolation of Pig Sperm Proteins
Sperm proteins from pig sperm were isolated essentially as described
previously (Tardif et al,
2001). Briefly, aliquots (1-5 x 106 sperm) of
sperm in either NCM or CM were taken before and during incubation at different
times. Sodium orthovanadate (0.2 mM final concentration) was added, and the
samples were centrifuged to obtain a sperm pellet (4 minutes, 16 060 x
g, room temperature), which was resuspended in sample buffer without
ß-mercaptoethanol and heated for 1 minute at 95°C. The sperm solution
was recentrifuged (4 minutes, 16 060 x g), and
ß-mercaptoethanol (5%) was added to the resulting supernatant. At this
point, the samples were stored immediately at -80°C until the day of
electrophoresis. The sperm protein samples were heated for 1 minute at
95°C. Sperm proteins were then ready for sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
SDS-PAGE and Western Blotting
As described in Tardif et al
(2001), the sperm proteins
were loaded on 12% SDS-polyacrylamide gels, and separated proteins were
transferred electrophoretically (overnight, 200 mA) to polyvinylidene fluoride
membranes. Nonspecific protein-binding sites on the membrane were blocked with
5% dry nonfat milk in TRIS-buffered saline (TBS: 25 mM TRIS-HCl and 150 mM
NaCl). Blots were incubated with antiphosphotyrosine antibodies (1:20 000) for
1 hour in TTBS (0.1% Tween 20, 25 mM TRISHCl, and 150 mM NaCl) at room
temperature, washed 3 times for 10 minutes with fresh TTBS, and then incubated
with peroxidase-conjugated goat anti-mouse antibodies (1:20 000 in TTBS, 45
minutes, room temperature). Membranes were again washed 3 times for 10 minutes
with fresh TTBS. Labeled tyrosine phosphoproteins were visualized with a
chemiluminescence detection kit (ECL, Amersham) according to the
manufacturer's instructions.
Measurement of Intracellular Calcium Level
Sperm calcium levels were assessed following a protocol described by Collin
et al (2000) that was modified
to exclude dead or damaged sperm on the basis of propidium iodide
incorporation. Briefly, sperm diluted in BTS were centrifuged (10 minutes, 270
x g, 25°C) and resuspended to 40 x 106
sperm/mL in NCM. For each sample tested, indo-1-AM was diluted in NCM and
added to the sperm solution for a final concentration of 2.5 µM. The sperm
and indo-1-AM suspension was then gently mixed and incubated for 30 minutes at
25°C in the dark. Sperm were washed twice by centrifugation (10 minutes,
350 x g, 25°C) to remove excess indo-1-AM, resuspended in
either CM or NCM at 40 x 106 sperm/mL, and incubated at
39°C in a 5% CO2 humidified atmosphere for up to 3 hours. For
flow cytometric analysis, 20 µL of indo-loaded sperm preparation was
diluted in 980 µL of either CM or NCM, which included 3 µL of propidium
iodide (2.4 mM) to exclude dead or damaged sperm and 20 µL of protamine
sulfate (1 mg/mL) to prevent agglutination. Flow cytometric analysis was
performed using an EPICS ELITE ESP (Beckman Coulter, Miami, Fla). The flow
cytometer detected indo-1 bound to calcium (violet fluorescence) with a 440 DL
filter (381 nm wavelength) and calcium-free indo-1 (blue fluorescence) with a
550 DL filter (525 nm wavelength). The relative intracellular calcium level of
each sperm cell was expressed as the ratio of violet/blue fluorescence.
Analysis was conducted at 37°C, and a total of 10 000 cells were scored
for each aliquot. Debris and cell aggregates were excluded from the analysis
by gating out nonfluorescent cells or particles. Only live sperm were
assessed, as propidium iodide-labeled sperm were excluded from the
analyses.
Statistical Analyses
Differences in calcium levels due to treatment (NCM vs CM) or time were
determined by analysis of variance using General Linear Model procedures
(Statistical Analysis Systems,
1990). The Fisher protected least significant difference test was
conducted when the main effect was found to be significant (P <
.05).
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Results |
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To confirm that calcium is necessary for the appearance of the p32, different chelators were added to the sperm incubation media, and the results are shown in Figure 2. Since EDTA chelates both Mg2+ and Ca2+, we also tested ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid potassium salt (BAPTA-K+), which predominantly chelate Ca2+. As expected, in NCM with or without chelators at 0 and 3 hours of incubation, the p32 is not visible. As seen previously, when porcine sperm are incubated in CM, the tyrosine phosphoprotein p32 appears after 3 hours of incubation (Tardif et al, 2001). The addition of 6 mM EGTA, 6 mM EDTA, and 6 mM BAPTA-K+ (determined by dose response, data not shown) almost totally reduces the appearance of the tyrosine-phosphorylated protein, confirming the hypothesis that calcium is essential for p32 appearance.
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To further investigate the role of calcium in the appearance of the p32, we used the nonfluorescent calcium ionophore Br-A23187, which promotes calcium influx into cells. We added 10 µM Br-A23187 after 1 hour of incubation, and sperm proteins were extracted at 0, 0.5, and 1.5 hours post-A23187 of incubation. Results are shown in Figure 3. The effects of the addition of the calcium ionophore are evident at 0.5 hours post-A23187, as the p32 is clearly enhanced by incubation with Br-A23187 in CM and, to a lesser degree, in NCM. The enhancement is even more obvious at 1.5 hours post-A23187. A tyrosine phosphoprotein also appears at Mr 21 000 in a time-dependent manner and with the addition of calcium ionophore; this is likely the same protein (Mr 23 000) that was observed in Figure 1. As well, the tyrosine phosphoproteins present at approximately Mr 45 000 and Mr 48 000 appear to be slightly down-regulated in the presence of A23187, but only in CM after 1.5 and 3 hours.
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Sperm Calcium Increases During Capacitation
To investigate the relationship between extracellular calcium,
intracellular calcium, and p32 appearance, indo-1-loaded sperm were suspended
in either NCM or CM (Figure 4A)
and in NCM plus or minus Ca2+ (2 mM) or CM plus or minus
Ca2+ (2 mM) before incubation
(Figure 4B). Calcium levels
were detected by flow cytometry, and the level of calcium was expressed as the
ratio of violet/blue fluorescence (RU indicates relative units). At 0 hours,
there was no significant difference in intracellular calcium levels between
sperm incubated in NCM or CM (9.92 RU ± 1.4 vs 13.10 RU ± 2.0,
respectively; P > .05), which represent approximately 28 and 34 nM
calcium, respectively (according to the calculation of
Grynkiewicz et al, 1985).
After 3 hours of incubation, the level of intracellular calcium increased
markedly in sperm incubated in CM (33.58 RU ± 6.3; approximately 128
nM). The level of intracellular calcium in NCM at this time of incubation was
not significantly different from that at 0 hours of incubation (15.04 RU
± 4.3; P > .05).
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At 0 hours, the addition of calcium to the media (Figure 4B) showed no significant effect in media without calcium (NCM without calcium: 11.65 RU ± 3.9, CM without calcium: 11.35 RU ± 1.2; P > .05) or with calcium (NCM with calcium: 15.75 RU ± 8.2, CM without calcium: 12.35 RU ± 1.5; P > .05). In contrast, after 3 hours of incubation, there was a significant difference between NCM without and with calcium (12.67 RU ± 3.5 vs 21.32 RU ± 3.8, respectively; P < .05) and between CM without and with calcium (13.45 RU ± 4.9 vs 25.22 RU ± 2.7, respectively; P < .05).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionConclusionReferences |
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When calcium was added to the negative control, NCM, the p32 appeared after 3 hours of incubation (Figure 1), as in capacitating conditions (CM), supporting the hypothesis that the p32 (and thereby capacitation) requires calcium for its appearance. The presence of a tyrosine-phosphorylated protein of Mr 32 000 in NCM supplemented with calcium could also indicate that the p32 does not require BSA and bicarbonate to appear. These components have been reported to be essential for protein tyrosine phosphorylation and capacitation observed in mouse sperm (Visconti et al, 1995a). Occasionally, an Mr 32 000-tyrosine phosphoprotein was observed at 0 hours in NCM supplemented with calcium, but this seems to depend on the boar. Our calcium-free treatments confirmed the importance of calcium for p32 appearance: the p32 does not appear in NCM or CM without calcium at any time. Taken together, these results clearly demonstrate that calcium is essential for the appearance of the p32.
As shown in Figure 2, extracellular calcium seems to be crucial for the appearance of the p32 in capacitating conditions. We used calcium chelators (EGTA, EDTA, and BAPTA-K+) to investigate whether the p32 depends on calcium by inhibiting its appearance. As expected, chelation of extracellular calcium by EDTA and BAPTA-K+ eliminated the p32. BAPTA-K+ is more selective for calcium than are EGTA and EDTA, although EDTA and BAPTA-K+ also chelate magnesium. EGTA is predominantly a calcium chelator. A faint p32 band appeared in the presence of EGTA, but we attribute this observation to the possible implication of magnesium in p32 appearance; however, this hypothesis remains to be tested. Conversely, by using calcium ionophore Br-A23187 (Figure 3), we demonstrated that increased calcium entry into the sperm promotes the appearance of the p32. We added calcium ionophore after 1 hour of incubation because we presumed that the elevated membrane fluidity due to the onset of capacitation facilitated the action of calcium ionophore. In fact, it is obvious on the Western blot at 1.5 and 3 hours that the inclusion of Br-A23187 in the incubation medium enhances the appearance of the p32. These results imply that an artificial entry of calcium into the sperm cell by Br-A23187 accelerates p32 appearance by increasing the calcium influx or bypassing the membrane mechanism necessary for calcium influx. Therefore, we could suggest that extracellular calcium is essential for the signaling events leading to tyrosine phosphorylation of the p32. The signaling pathway implicated in the appearance of the p32, however, remains unknown. As discussed in Tardif et al (2001), the p32 may be comparable to the Mr 33 000 protein reported by Flesch et al (1999). Indeed, the p32 is likely the same as that reported by Green and Watson (2001). The minor differences in the reported molecular weights could be due to the protocol. However, it should also be mentioned that a preliminary investigation by our laboratory indicates that a cohort of various tyrosine-phosphorylated proteins appears around Mr 32 000, although the identities of the capacitation-associated proteins remain to be confirmed (Dubé et al, 2002).
Increased Sperm Intracellular Calcium During Capacitation Coincides
With the Appearance of the p32
The results obtained by flow cytometry
(Figure 4A) indicate that the
level of cytosolic calcium increases markedly in sperm incubated in conditions
known to support capacitation (3 hours of incubation in CM). These results are
similar to observations by Zhou et al
(1990), who reported that
intracellular calcium increases during capacitation in boar sperm. The
intracellular calcium levels increased in CM supplemented with calcium after
an incubation of 3 hours (Figure
4B), coinciding with the appearance of the p32 tyrosine
phosphoprotein visualized by Western blotting
(Figure 1). In the absence of
calcium in the medium, the level of intracellular calcium did not rise in the
sperm. Flow cytometry analysis shows that extracellular calcium in the medium
is essential to increase intracellular levels and that this corresponds to p32
appearance. So, the appearance of the p32 phosphoprotein and the elevation of
the intracellular calcium level in boar sperm both occur during capacitation
and could be linked. Our negative control NCM, which contains no calcium,
bicarbonate, or BSA, does not support capacitation
(Tardif et al, 1999). In
noncapacitating conditions, there is no increase in the intracellular calcium
level between 0 and 3 hours, and the p32 does not appear. These results
suggest a strong correlation between the increase in intracellular calcium
level and the appearance of the p32.
It is notable that in human sperm, increased extracellular calcium and the addition of calcium ionophore to the medium resulted in the dephosphorylation of phosphotyrosine-containing human sperm proteins (Carrera et al, 1996; Luconi et al, 1996; Leclerc and Goupil, 2002). Similarly, we observed that the boar sperm tyrosine phosphoproteins of Mr 45 000 and Mr 48 000 also seem to be down-regulated in the presence of calcium (Figures 1 and 3). In contrast, however, calcium seems to act differently in tyrosine phosphorylation in boar sperm for the appearance of the tyrosine phosphoprotein p32.
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Conclusion |
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TopAbstractMaterials and MethodsResultsDiscussionConclusionReferences |
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Acknowledgments |
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Footnotes |
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References |
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