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From * Département des Sciences Animales
and Département d'Obstétrique et
Gynécologie, Centre de Recherche en Biologie de la Reproduction,
Université Laval, Sainte-Foy, Québec, Canada; the
Institute of Applied Biochemistry, University
of Tsukuba, Ibaraki, Japan; and the Centre de
Recherche du CHUL, Université Laval, Sainte-Foy, Québec,
Canada.
| Correspondance 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 October 18, 2004; accepted for publication February 26, 2005. |
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Abstract |
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Key words: Fertilization, p32, acrosin, acrosome reaction
Capacitation is regulated by several specific signal transduction pathways (Visconti et al, 1995a; Baldi et al, 2002) involving sperm protein phosphorylation (Visconti and Kopf, 1998; Tardif et al, 1999). In various species, such as the mouse (Visconti et al, 1995b), human (Leclerc et al, 1996), bovine (Galantino-Homer et al, 1997), stallion (Pommer et al, 2003), and pig (Kalab et al, 1998; Flesch et al, 1999; Tardif et al, 2001), capacitation is associated with the tyrosine phosphorylation of sperm proteins. The identification of these tyrosine phosphorylated substrates, such as the calcium-binding tyrosine phosphorylation–regulated fibrous sheath protein CABYR (Naaby-Hansen et al, 2002) and the A-kinase anchoring protein AKAP3 (Ficarro et al, 2003) in human sperm; the pro-A-kinase anchoring protein pro-AKAP83 and the A-kinase anchoring protein AKAP83 (Jha and Shivaji, 2002) in hamster sperm; and the A-kinase anchoring protein AKAP82 (Carrera et al, 1994) in the mouse sperm, allows a better understanding of capacitation. However, the presence of these proteins in the flagellum indicates that they are more involved in motility changes during capacitation than sperm-zona binding or the acrosome reaction.
Recently, we reported that a tyrosine phosphorylated protein in porcine sperm, named "p32," appears only during incubation under capacitating conditions (Tardif et al, 2001, 2003). We also described a tyrosine kinase, termed TK-32, in pig sperm (Tardif et al, 2001), which was then found to more likely be a dual-specificity kinase such as a MAPK (Tardif et al, 2003). The activity of this enzyme increased during sperm capacitation, and immunoprecipitation experiments clearly showed that "p32" and "TK-32" are likely to be two different proteins (Tardif et al, 2003). Therefore, the identity of the p32 tyrosine phosphorylated protein remains unknown. Moreover, if and how p32 is involved during the capacitation process at the molecular level is not understood. Our general hypothesis is that the appearance of the p32 tyrosine phosphorylated protein is implicated in boar sperm capacitation. The objective of the present study was to identify p32 by proteomic techniques. In this report, we establish that "p32" is, in fact, "sp32," a binding protein implicated in (pro)acrosin maturation (Baba et al, 1989).
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Materials and Methods |
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Culture Media
The principal culture media used were based on Krebs Ringer Bicarbonate
(Toyoda and Chang, 1974).
Capacitating medium (CM) was composed of 4.8 mM KCl, 1.2 mM
KH2PO4, 95 mM NaCl, 5.56 mM glucose, 25 mM
NaHCO3, 2 mM CaCl2, 0.4% bovine serum albumin (BSA; type
V, fatty acid-free), and 2 mM pyruvate, pH 7.4. The noncapacitating medium
(NCM) was similar to CM but was without calcium, bicarbonate, and BSA (4.8 mM
KCL, 1.2 mM KH2PO4, 95 mM NaCl, and 5.56 mM glucose, pH
7.4). The CM has been previously shown to support capacitation, whereas NCM
acts as a negative control (Tardif et al,
2001).
Sperm Preparation
The research was conducted with the approval of the university committee
for animal care. The sperm-rich semen fraction was collected from fertile
boars at the Centre d'Insémination Porcine du Québec (St
Lambert, Canada) and was transported to the laboratory at
16°C–18°C within 30 minutes, as previously reported
(Tardif et al, 2001). The
sperm-rich semen was diluted to 40 x 106 sperm/mL directly in
either NCM, as a noncapacitating negative control, or in CM to induce
capacitation. 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 (Dubé et al,
2003). Briefly, aliquots (5 x 106 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
2-mercaptoethanol and heated for 1 minutes at 95°C. The sperm solution was
recentrifuged (4 minutes, 16 060 x g) and 2-mercaptoethanol
(5%, final concentration) was added to the resulting supernatant. For
nonreduced samples, the addition of 2-mercaptoethanol was omitted. At this
point, 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).
Immunoprecipitation
Sperm were solubilized by vortexing every 15 minutes (1 hour, 4°C) in
lysis buffer (4% CHAPS; 50 mM Tris-HCl, pH 7.4; 0.2 mM
Na3VO4; and protease inhibitor cocktail). Lysates were
centrifuged, and 1–5 µg of either anti-sp32 antibody or
anti-phosphotyrosine antibody were added to the supernatant. The sperm protein
solution was incubated at 4°C overnight with gentle agitation. The
immunocomplexes were sequestered by adding 100 µL protein G-coupled
sepharose (Amersham Biosciences AB, Uppsala, Sweden) for 2 hours at 4°C.
The immune complexes linked to the beads were collected by a quick spin (8
seconds, 16 060 x g) and the supernatant was kept for acetone
precipitation. The beads were washed 3 times with cold phosphate-buffered
saline, resuspended in 2x sample buffer, heated for 2 minutes at
90°C, and centrifuged (5 minutes, 16 060 x g). The
supernatant, with or without 2-mercaptoethanol, was submitted to SDS-PAGE and
immunoblotting.
SDS-PAGE and Western Blotting
As described in Dubé et al
(2003), sperm proteins were
subjected to SDS-PAGE, and separated proteins were electrophoretically
transferred (overnight, 200 mA) to polyvinyldienne fluoride (PVDF) membranes
(PerkinElmer Life Sciences, Boston, Mass). Nonspecific binding sites on the
membrane were blocked with 5% dry nonfat milk in Tris-buffered saline (TBS: 25
mM Tris-HCl, 150 mM NaCl). Blots were incubated with anti-phosphotyrosine
(1:20 000) or anti-sp32 antibodies (1:10 000) for 1 hour in Tween TBS (TTBS;
0.1% Tween 20, 25 mM Tris-HCl, 150 mM NaCl) at room temperature, washed 3
times for 10 minutes each time with fresh TTBS, and then incubated with
peroxidase-conjugated goat anti-mouse antibody (1:3000) or
peroxidase-conjugated goat anti-rabbit antibody (1:3000) in TTBS for 45
minutes at room temperature. Membranes were washed again, 3 times for 10
minutes each time, with fresh TTBS. Labeled proteins were visualized using a
chemiluminescence detection kit (ECL, Amersham) according to the
manufacturer's instructions.
For some experiments, membranes were reprobed with another antibody. Therefore, the membranes were first stripped at 50°C for 20 minutes in a solution of 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM 2-mercaptoethanol to remove the original antibodies. The membranes were then washed in TBS 1x, blocked with 5% milk, and reprobed as described above.
Consecutive Nonreducing/Reducing SDS-PAGE
Sperm proteins were extracted in nonreducing conditions (without
2-mercaptoethanol) and separated by 1-dimensional 12% SDS-PAGE. At the end of
the migration, the lane of interest on the gel was cut out using a scalpel and
straight edge to slice from the well comb to the bottom (the adjacent wells
were left empty to prevent contamination from neighboring proteins), then
incubated under reducing conditions (sample buffer with 5% 2-mercaptoethanol)
for 2 hours at room temperature with gentle agitation. The gel strip was
layered in the application well made in the stacker of another 12% SDS gel for
a second consecutive round of 1-dimensional SDS-PAGE under reducing
conditions. Before and after placing the strip, 200 µL of reducing sample
buffer were poured into the well. Electrophoresis was carried out at 200 V for
approximately 45 minutes. Proteins were then transferred on PVDF membranes and
probed with antibodies as described previously.
Identification by MS/MS
Proteins were visualized by Coomassie Blue staining of the gels after
protein electrophoresis under nonreducing followed by reducing conditions.
Selected spots of interest were excised from the gel and trypsin-digested for
peptide mass spectrometric analysis using the mass spectrometry/mass
spectrometry (MS/MS) technique (Zhu et al,
2003) at the Institute for Biomolecular Design, University of
Alberta (Edmonton, Canada). Identification was performed by "mass
fingerprinting" in a peptide mass computer data bank
(http://www.matrixscience.com).
Indirect Immunofluorescence
Pig sperm were incubated in appropriate media at 40 x 106
sperm/mL, centrifuged (10 minutes, 270 x g), fixed with 2%
formaldehyde for 1 hour, centrifuged again (10 minutes, 270 x
g), and then blocked with 2% BSA overnight to eliminate nonspecific
binding. Approximately 8 x 106 sperm were air dried onto
PolysineTM microscope slides (Esco, Portsmouth, NH) and then
permeabilized in anhydrous ethanol or not. Acrosome-reacted sperm were
obtained by incubation with 10 µM of the nonfluorescent calcium ionophore
Br-A23187 (Molecular Probes, Eugene, Oreg) for 60 minutes
(Moos et al, 1993a). For
double labeling, samples on slides were incubated for 1 hour with a mixture of
anti-sp32 antibody and anti-phosphotyrosine antibody with 5% goat serum,
washed in TBS, then coincubated for 1 hour with a mixture of the second
antibody conjugated to FITC (green) and CYTM3 (orange) diluted in TBS
with 5% goat serum. One drop of 90% glycerol was added on the slide to limit
photobleaching, and slides were mounted with coverslips. The sperm were
examined by epifluorescence using a Nikon Eclipse E600 microscope equipped
with fluorescence (FITC: maximal excitation: 495 nm, DM 510 filter;
CYTM3: maximal excitation: 550 nm, DM 400 filter; 40 x 0.75).
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Results |
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Immunoprecipitation Corroborates That sp32 Is Tyrosine Phosphorylated in Capacitated Sperm
To confirm the results obtained in Figures
1,
2,
3,
4, proteins from capacitated
sperm were immunoprecipitated with anti-phosphotyrosine or with anti-sp32
antibodies. Figure 5A shows the
Western blot of anti-phosphotyrosine and anti-sp32 immunoprecipitated proteins
labeled with anti-phosphotyrosine antibody. The total extract in
Figure 5A (lane 1) demonstrates
again that p32 is tyrosine phosphorylated. Tyrosine phosphorylated proteins
were immunoprecipitated as well by anti-phosphotyrosine antibody in the pellet
(Figure 5A, lane 2), and some
remained in the supernatant (Figure
5A, lane 3). When proteins are subjected to immunoprecipitation
with anti-sp32 antibody, tyrosine phosphorylated proteins are detected in the
pellet (Figure 5A, lane 4), but
mostly in the supernatant (Figure
5A, lane 5), indicating that sp32 was immunoprecipitated as a
tyrosine phosphorylated protein.
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Sperm labeled with anti-phosphotyrosine antibody exhibited an intense spot of fluorescence on the equatorial segment in noncapacitating conditions (Panel 3); however, the fluorescence was displaced to the acrosomal cap after incubation in capacitating conditions (Panel 6), coincident with the sp32 labeling. When sperm were acro-some reacted (Panel 9), only the spot of anti-phosphotyrosine antibody fluorescence on the equatorial segment remained, confirming the anti-sp32 antibody results. Controls incubated only with secondary antibodies validate our treatments (Panels 11 and 12).
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Discussion |
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Sequencing Reveals p32 to Be sp32
The p32 tyrosine phosphoprotein was isolated as described by Kalab et al
(1994) using two consecutive
series of SDS-PAGE, under nonreducing and reducing conditions, respectively
(Figure 1). Amino acid
sequencing of the peptides from p32 by MS/MS revealed 100% identity with the
sp32 precursor (Figure 2), an
acrosomal binding protein implicated in acrosin maturation
(Baba et al, 1989). The
identity of the putative sp32 was confirmed by rehybridizing the original
membranes (Figure 1) with an
anti-sp32 antibody (Figure 3).
The presence of 3 tyrosine phosphorylation sites on the active form of sp32 is
consistent with the results obtained with the anti-phosphotyrosine antibody.
However, the molecular mass of the protein on the gel (Mr 32 000) indicates
that the active sp32 is observed, not the precursor (Mr approximately 61 000).
Moreover, the sp32 precursor is processed into the mature form sp32 in the
testis during spermatogenesis (Baba et al,
1994). A protein of approximately Mr 35 000, detected by anti-sp32
antibody on Figure 4, is not
present on Figure 3A and B. Presently we have no firm explanation about this protein detected with
anti-sp32 antibody, but the difference between Figures
3 and
4 could be explained by
different epitope conformations detected by anti-sp32 antibody due to the
techniques. We are currently conducting 2-dimensional gel electrophoresis and
Western blotting followed by sequencing to identify all proteins recognized by
the anti-sp32 antibody.
The first report of a proacrosin binding protein was in 1977, when Polakoski and Parrish revealed that the proacrosin samples in their study were contaminated with a Mr 29 000 protein in boar sperm. Shortly thereafter, in 1978, Parrish and Polakoski (1978) demonstrated that this protein forms a complex with proacrosin. In 1989, Baba et al (1989) were the first to describe the function of the 32-kd protein as a facilitator of proacrosin conversion. In 1992, Yi et al (1992) detected a proacrosin binding protein between Mr 28 000 and Mr 32 000 in the boar sperm using a proacrosin binding protein antibody. A similar protein of Mr 28 000–29 000 was reported by Moos et al (1993b). As well, a protein of Mr 28 000–29 000 was detected in guinea pig sperm by Hardy et al (1991) and Noland et al (1994). Finally, in 1994, Baba et al reported the amino acid sequence of the sp32 (and its precursor), and a few years later, Huh and Yi (1999, 2001) determined the partial sequence of a Mr 28 000–29 000 protein similar to sp32. The importance of this sp32 protein was of greater interest when Howes et al (2001) and Howes and Jones (2002) presented evidence that proacrosin/acrosin are complementary binding proteins on sperm for ZP2, the secondary receptor that retains acrosome reacted sperm on the egg's zona pellucida. The importance of sp32 in reproduction is also underscored by the discovery that the sp32 precursor is a potential human cancer/testis antigen (Ono et al, 2001). Whether tyrosine phosphorylation of sp32 is involved in these putative roles has never been discussed.
A Tyrosine Phosphorylated Form of sp32 Is p32
Immunoprecipitation experiments confirm that the tyrosine phosphorylated
sp32 is p32. Figure 5A shows
that most of the tyrosine phosphorylated proteins are immunoprecipitated
(pellet, lane 2), but some remain in the supernatant (lane 3). The anti-sp32
pellet (the immunoprecipitate) confirms that sp32 is tyrosine phosphorylated
in capacitated sperm (Figure
5A, lane 4). The strong band appearing in the anti-sp32
supernatant (Figure 5A, lane 5)
indicates that other proteins are tyrosine phosphorylated, and some of these
are unlikely to be sp32. Moreover, the presence of a sp32 band in the
anti-phosphotyrosine immunoprecipitate
(Figure 5B, lane 2) indicates
that some of the sp32 content is tyrosine phosphorylated, while the strong
sp32 band remaining in the anti-phosphotyrosine supernatant
(Figure 5B, lane 3) shows a
major portion of the sp32 to be non–tyrosine phosphorylated. Taken
together with our previous research
(Tardif et al, 2001), we
conclude that in pig sperm, some of the sp32 molecules are tyrosine
phosphorylated during capacitation. Ficarro et al
(2003) showed that sp32 is also
tyrosine phosphorylated in capacitated human sperm, lending support to our
findings. To the best of our knowledge, it is not known if sp32 is one of the
tyrosine phosphorylated substrates in the sperm of other species (such as the
guinea pig, which contains sp32; Noland et
al, 1994).
Tardif et al (2001) observed, in nonpermeabilized boar sperm, that tyrosine phosphoproteins relocalized from the equatorial segment of the sperm to the acrosome during capacitation, whereas permeabilized sperm always displayed labeling over the acrosome. By double labeling, we compared those patterns with those obtained using anti-sp32 antibody (Figure 6). The anti-sp32 antibody labeling was uniformly observed over the acrosomal cap in capacitated and noncapacitated sperm, regardless of permeabilization. We suggest that sp32, a (pro)acrosin binding protein, is constitutively present in the acrosome. Inducing the acrosome reaction confirmed that sp32 is present, because loss of the acrosome leads to the loss of the fluorescent anti-sp32 signal. We previously used Triton X-114 to extract sperm proteins, which revealed that p32 is present in the cytosolic fraction in uncapacitated pig sperm and in both the cytosolic and membrane (micellar) fractions after capacitation (Tardif et al, 2001). Given our current hypothesis that p32 is a tyrosine phosphorylated form of sp32, it is tempting to speculate that the partitioning of sp32 in the acrosomal compartments (soluble, the matrix, acrosomal membrane) is regulated by the phosphorylation status of the sp32. We will test this hypothesis in future experiments.
Hypothetical Roles of Tyrosine Phosphorylated sp32
The role of sp32 in acrosin maturation/capacitation in porcine sperm leads
to different theories. First, Baba et al
(1989) speculated that the
proacrosin binding protein accelerates proacrosin activation. Hardy et al
(1991) then co-localized
proacrosin and its binding protein in the acrosomal matrix. Finally, Yi et al
(1992) hypothesized that
proacrosin binding protein might be involved in proacrosin processing. In
2001, Kim et al demonstrated that AM67, a proacrosin binding protein, was
localized in the acrosomal matrix, a finding that was in agreement with those
of other reports (Hardy et al,
1991; Yi et al,
1992; Baba et al,
1994). Those studies suggest that proacrosin binding proteins
immobilize and stabilize proacrosin until the acrosome reaction. In addition,
proacrosin might play a role in the secondary sperm binding to the zona
pellucida via ZP2 (Howes et al,
2001,
2002), and sp32 could anchor
proacrosin in the acrosomal matrix until a later phase of capacitation or
until early in the acrosome reaction. Here our indirect immunofluorescence
results on acrosome-reacted sperm showed no sp32 signal after the acrosome
reaction; the sp32 could be liberated or proteolytically degraded
(Hardy et al, 1991;
Moos et al, 1993b) during this
process and thereby might no longer be detected by the anti-sp32. The tyrosine
phosphorylated portion of the degraded sp32 could remain in the sperm, as
reflected by the persistent anti-phosphotyrosine labeling following the
acrosome reaction (Figure
6).
During capacitation, maturation of some proacrosin to acrosin could be facilitated by tyrosine phosphorylation of sp32. Since sp32 does not bind acrosin (Baba et al, 1989), the tyrosine phosphorylated sp32 would be released to the acrosomal cap as acrosin is transferred to the soluble compartment of the acrosome. Binding to the ZP leads to the acrosome reaction and release of the acrosomal contents, including acrosin. The secondary binding of the sperm to the ZP could thereby be accomplished through the proacrosin still anchored in the acrosomal matrix.
In conclusion, this study demonstrates that p32, a tyrosine phosphorylated protein related to capacitation of porcine sperm, is a tyrosine phosphorylated form of sp32, a proacrosin binding protein. Our interest will now focus on the actual implication of the tyrosine phosphorylation of sp32 during capacitation and acrosin maturation.
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Acknowledgments |
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Footnotes |
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 M. L. Vadnais and K. P. Roberts Effects of Seminal Plasma on Cooling-Induced Capacitative Changes in Boar Sperm J Androl, May 1, 2007; 28(3): 416 - 422. [Abstract] [Full Text] [PDF]
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