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From the Department of Life Science, Graduate School of Science and Technology, Kobe University, Kobe, Japan.
| Correspondence to: Dr Hiroshi Harayama, Department of Life Science, Graduate School of Science and Technology, Kobe University, 1 Rokkodai, Nada, Kobe 657-8501, Japan. |
| Received for publication May 1, 2003; accepted for publication July 11, 2003. |
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Abstract |
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Key words: Serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation
We are making a unique approach for the disclosure of the details of the signaling cascades that are activated by the intracellular cAMP in boar sperm head, and our approach is assessment by assay for the sperm head-to-head agglutination. Briefly, it has been observed that boar spermatozoa usually become agglutinated at the head during incubation in the capacitation-supporting medium (Harayama et al, 1999). This agglutination is apparently promoted by an adenylyl cyclase stimulator (bicarbonate) and a cell-permeable cAMP analog (dibutyryl cAMP), but is not affected by a cell-permeable cyclic guanosine 3',5'-monophosphate (cGMP) analog (dibutyryl cGMP). Moreover, the signaling cascades leading to the agglutination are disturbed by the exposure of spermatozoa to the cell-permeable cAMP antagonist diasteromer of adenosine (Rp-adenosine) 3',5'-cyclic monophosphorothioate (Harayama et al, 1998, 2000). Based on these facts, we believe it acceptable that the head-to-head agglutination is valid as an indicator showing the activity of cAMP-signaling cascade in boar sperm head. In addition, we have recently suggested that this cAMP-signaling cascade is connected to mobilization of calcium from the putative acrosomal store to the cytoplasm by the sperm head-to-head agglutination assay (Harayama et al, 2003). However, characteristics of the cAMP-dependently agglutinated spermatozoa remain unclear. The aim of the present study is to examine the viability and the protein phosphorylation patterns of boar spermatozoa agglutinated by the treatment with a cell-permeable cAMP analog.
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Materials and Methods |
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Assessment for Agglutination
Spermatozoa were gently smeared on glass slides, dried, and stained in a
phosphate-buffered solution of Giemsa (Merck, Darmstadt, Germany) for 90
minutes. More than 300 spermatozoa on each preparation were counted at random
by light microscopy (400x) to determine percentages of head-to-head
agglutinated cells, as described previously
(Harayama et al, 2003).
Assessment for Viability
Sperm viability was assessed by using a Live/Dead Sperm Viability kit
(Molecular Probes, Inc, Eugene, Oreg). Briefly, each sperm suspension (1 mL)
was stained with 0.3 µM SYBR14 at 25°C for 10 minutes and subsequently
with 24 µM propidium iodide (PI) at 25°C for 10 minutes. After
staining, spermatozoa were washed in mKRH (5 mL) by centrifugation at 700
x g for 10 minutes, resuspended in mKRH (1 mL), and observed
under a differential interference microscope equipped with epifluorescence
(mirror unit B2 set filter: excitation filter EX450-490, dichroic mirror
DM510, and emission filter BA520, Nikon Company, Tokyo, Japan). One hundred
agglutinated spermatozoa and 100 free spermatozoa on each preparation were
counted separately to determine the percentage of PI-positive spermatozoa
(dead spermatozoa).
Sperm viability also was assessed by the subjective observation of sperm motility. Briefly, the motility was observed in a 5-µL drop of sperm suspension on a heated stage (38.5°C) under the bright-field microscope. In each sample, agglutinated spermatozoa and free spermatozoa were separately evaluated. Cells showing any movement were considered as motile spermatozoa, irrespective of their progressive motility. The percentages of motile spermatozoa were converted into the 5 grades of motility scores (score 1: <20% motile spermatozoa, score 2: 21%-40% motile spermatozoa, score 3: 41%-60% motile spermatozoa, score 4: 61%-80% motile spermatozoa, and score 5: >81% motile spermatozoa).
Assessment for Protein Phosphorylation Patterns
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western
Blotting—
Each sperm suspension (100 µL, sperm concentration: 1.0 x
108 cells/mL) was mixed with an equal volume of a double-strength
sample buffer (pH 6.8) composed of 125 mM Tris-HCl, 4% sodium dodecyl sulfate
(SDS), 10% 2-mercaptoethanol, 20% glycerol, and 0.02% bromophenol blue
(Laemmli, 1970), and then
heated in a boiling water bath for 5 minutes. After this procedure, the
samples were clarified by centrifugation at 12 500 x g for 15
minutes at 4°C. Twenty microliters of each sperm extract (the extract from
1.0 x 106 sperm) was loaded on each lane of the acrylamide
gel. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed by using a 10% acrylamide gel with Laemmli's buffer system
(Laemmli, 1970). Prestained
SDS-PAGE standards (Amersham) were used as molecular mass standards. The
separated proteins were transferred to the polyvinylidene difluoride (PVDF)
membrane (Immobilon P, Millipore, Bedford, Mass) in a semidry transfer cell
for 60 minutes at 2.0 mA/cm2 in a transfer buffer composed of 48 mM
tris(hydroxymethyl)aminomethane, 39 mM glycine, and 20% (vol/vol) methanol
(Bjerrum and Schafer-Nielsen,
1986) supplemented with 0.13 mM SDS.
The blotted PVDF membrane was blocked with 10% calf serum (CS, Invitrogen
Corp, Carlsbad, Calif) in PBS containing 0.1% Tween 20 (Wako Pure Chemical
Industries, Ltd, Osaka, Japan) (PBS-Tween) for 60 minutes. Either rabbit
anti-phosphoserine/phosphothreonine PKA substrate polyclonal antibody (Cell
Signaling Technology, Inc, Beverly, Mass, 1:2000) or mouse
anti-phosphotyrosine monoclonal antibody (clone 4G10, Upstate Cell Signaling
Solutions, Charlottesville, Va, 1:10 000) was diluted with PBS-Tween
containing 5% CS, and incubated with the membrane for 180 minutes. After
washing three times for 10 minutes each in PBS-Tween, the membrane was blocked
in PBS-Tween containing 10% CS for 60 minutes and then treated with
horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulins
(1:1000) (Amersham) or HRP-conjugated goat anti-mouse immunoglobulins (1:2000)
(Dako Cytomation Denmark A/S, Glostrup, Denmark) in the blocking buffer for 60
minutes. After washing three times, peroxidase activity was visualized with
the Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Inc, Santa
Cruz, Calif). The specificities of the primary antibodies were described
below. The anti-phosphoserine/phosphothreonine PKA substrate polyclonal
antibody was produced by immunizing rabbit with synthetic phospho-PKA
substrate peptide and then was sequentially purified by Protein A and peptide
affinity chromatography. This antibody is specific for proteins containing
phosphothreonine with arginine at the -3 position and proteins containing
phosphoserine with arginine at the -2 or -3 positions. The phosphorylation at
these amino acid motifs is catalyzed by PKA and other Arg-directed kinases.
However, this antibody does not recognize the nonphosphorylated PKA substrate
motif (see catalog from Cell Signaling;
Bruce et al, 2002). The
anti-phosphotyrosine monoclonal antibody (IgG2b
) was
produced in vitro by mouse-mouse hybridoma 4G10 (FOX-NY [NS-1 derivative]
myeloma x spleen cells) and then purified by Protein A-Sepharose
chromatography. Immunogen was phosphotyramine-KLH. Many reports
(Druker et al, 1989;
Cohen et al, 1990;
Kanakura et al, 1991) have
demonstrated that this antibody recognizes the phosphotyrosine-containing
proteins (see catalog from Upstate Cell Signaling).
Indirect Immunofluorescence— All procedures were undertaken at room temperature. Each of sperm suspensions (10 µL) was gently smeared on a glass slide and fixed in methanol for 10 minutes. The slides were gently rinsed with PBS twice, blocked with 5% bovine serum albumin (BSA, Serologicals Corporation, Norcross, Ga) in PBS (PBS-BSA) for 60 minutes, and then treated with either rabbit anti-phosphoserine/phosphothreonine PKA substrate polyclonal antibody (1:25) for 180 minutes or mouse anti-phosphotyrosine monoclonal antibody (clone 4G10, 1:1000) for 30 minutes in PBS-BSA. After being rinsed twice with PBS, the slides were treated with PBS-BSA for 60 minutes and then with fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit immunoglobulins (Dako, 1:25) for 60 minutes or FITC-conjugated rabbit anti-mouse immunoglobulins (Dako, 1:100) for 30 minutes. After being rinsed twice with PBS, the slides were covered with 0.22 M 1,4-diazabicyclo [2,2,2] octane (Sigma) dissolved in glycerol : PBS (9:1) and coverslips. The sperm preparations were examined under a differential interference microscope equipped with epifluorescence (mirror unit U-MNIBA2: excitation filter BP470-490, dichroic mirror DM505, and emission filter BA510-550, Olympus Optical Company Ltd, Tokyo, Japan).
Statistical Analysis
Percentages of head-to-head agglutinated spermatozoa, percentages of
PI-positive spermatozoa and motility scores were subjected to 1-way analysis
of variance (ANOVA). When F test results were significant in ANOVA,
individual means were further tested by Tukey multiple range test
(Motulsky, 1995).
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Results |
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Figure 2 shows time-related changes of the percentages of head-to-head agglutinated spermatozoa in the samples incubated with or without cBiMPS (0.1 mM). In the control samples incubated without cBiMPS, the percentages of spontaneously agglutinated spermatozoa slightly increased to 22% during the first 10-minute incubation, and were followed by only minor changes thereafter. However, in the samples incubated with cBiMPS, the percentages of head-to-head agglutinated spermatozoa increased to 61% during 30 minutes of incubation. These results revealed that cBiMPS could activate the cAMP-signaling cascades in the sperm head during the first 30-minute incubation.
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Table 1 shows changes in sperm motility during the incubation with or without cBiMPS (0.1 mM). In the control samples incubated without cBiMPS, motility scores of the agglutinated and free spermatozoa showed no significant changes throughout the 180-minute incubation. However, in the samples incubated with cBiMPS, motility scores of the agglutinated and free spermatozoa were significantly changed during the first 30-minute incubation, namely coincidentally with the increase of the head-to-head agglutinated spermatozoa. These results confirmed the above-mentioned suggestion that the cAMP analog-induced agglutination occurs mainly in live spermatozoa. Additionally, in the samples incubated with cBiMPS for more than 120 minutes, motility scores were much higher in the agglutinated spermatozoa than in the free spermatozoa, and many of motile spermatozoa exhibited hyperactivationlike motility.
Protein Phosphorylation Patterns of Spermatozoa
Figure 3 shows incubation
time-related changes in the SDS-PAGE patterns of sperm suspensions
immunodetected with the anti-phosphoserine/phosphothreonine PKA substrate
antibody. In the control samples without cBiMPS, 2 bands with molecular masses
of 63 kd and 54 kd were detected before incubation but were gradually
eliminated during incubation for 60 minutes. In the samples incubated with
cBiMPS (0.1 mM), at least 8 bands (>220 kd, 220 kd, 180 kd, 84 kd, 56 kd,
54 kd, 37 kd, and 34 kd) increased during the first 30-minute incubation, and
some of them diminished thereafter. Moreover, 2 major bands (93 kd and 59 kd)
apparently increased later than 30 minutes. As shown in
Figure 4, protein tyrosine
phosphorylation patterns of the control samples incubated without cBiMPS were
varied among 5 replicates. However, 3 major bands (42 kd, 40 kd, and 37 kd)
were usually detected throughout the incubation period for 180 minutes, and a
32-kd band increased in the all samples after a 5-minute incubation. In the
all samples incubated with cBiMPS (0.1 mM), at least 6 bands (>220 kd, 190
kd, 93 kd, 59 kd, 54 kd, and 32 kd) appeared or increased later than 30
minutes, although no cBiMPS-induced change was found during the first
15-minute incubation. To examine whether the cBiMPS-phosphorylated bands were
sperm-associated or sperm-internal proteins, the following experiment was
undertaken. Briefly, aliquots of the sperm suspensions after incubation for 15
and 180 minutes were centrifuged at 12 500 x g for 5 minutes at
4°C. The resultant sperm pellets and supernatants were recovered and then
used separately for SDS-PAGE and Western blotting
(Figure 5). The
anti-phosphoserine/phosphothreonine PKA substrate antibody detected the bands
with molecular masses of >220 kd, 220 kd, 180 kd, 93 kd, 84 kd, 59 kd and
54 kd only in the extracts from sperm pellets. Likewise, the
anti-phosphotyrosine antibody reacted with the bands with molecular masses of
>220 kd, 190 kd, 93 kd, 59 kd, 54 kd, and 32 kd only in the extracts from
sperm pellets, but no bands in the supernatants. These results confirmed that
most of the cBiMPS-phosphorylated bands shown in Figures
3 and
4 were sperm-associated or
sperm-internal proteins.
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Figure 6 shows incubation time-related changes in indirect immunofluorescence patterns of sperm antigens detected by the anti-phosphoserine/phosphothreonine PKA substrate antibody. In the spermatozoa before incubation, fluorescence was observed in the postacrosomal region and central spot of the head, but fluorescent intensity was varied among the cells (panels 0(-) and 0). During the first 30-minute incubation with 0.1 mM cBiMPS, many spermatozoa became agglutinated with one another and greatly increased fluorescence in the connecting and principal pieces (panels 5, 10, 15, and 30). Furthermore, fluorescence was eliminated in the sperm head after incubation for 30 minutes (panel 30), but was intensified in the middle piece in addition to the connecting and principal pieces by the prolonged incubation (panel 120). In contrast, no change in the immunofluorescence pattern of the sperm flagellum was observed in the control samples incubated without cBiMPS throughout the incubation for 180 minutes (data not shown). As shown in Figure 7, the anti-phosphotyrosine antibody recognized the antigens that were located in the apical region of the acrosome and central spot of the head in the samples before incubation (panels 0(-) and 0) or after incubation for 15 minutes (panels 15(-) and 15). However, prolonged incubation with cBiMPS (0.1 mM) changed fluorescence patterns in sperm flagellum (panels 180, S180, and 180(-), and Figure 8). Specifically, the connecting and principal pieces exhibited intense fluorescence preferentially in the head-to-head agglutinated spermatozoa. Moreover, moderate fluorescence also was detected in the middle pieces of the agglutinated spermatozoa. However, these changes of the fluorescence patterns were rarely found in the flagellum of free spermatozoa. In addition, when the treatment with the primary antibodies (anti-phosphoserine/phosphothreonine PKA substrate antibody and anti-phosphotyrosine monoclonal antibody) was skipped to examine the specificity of secondary antibodies, no fluorescence was observed in any sample (representative results are shown in Figure 6 panel C 0 and Figure 7 panel C 180).
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Discussion |
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In the samples before incubation, 63-kd and 54-kd proteins from boar spermatozoa reacted with anti-phosphoserine/phosphothreonine PKA substrate antibody (Figure 3). Indirect immunofluorescence showed that this antibody recognized antigens of postacrosomal region and central spot of the head but scarcely recognized antigens of the flagellum (Figure 6). This anti-phosphoserine/phosphothreonine PKA substrate antibody also cross-reacts with proteins containing serine/threonine with arginine at the -2 or -3 position that is phosphorylated by other Arg-directed kinases (see "Materials and Methods"). These suggest relatively higher activity of the Arg-directed kinase, including PKA, or lower activity of protein serine/threonine phosphatase in the head than in the flagellum of boar spermatozoa before incubation. However, the activity of these enzymes in the sperm head seems to be changed during incubation without the intracellular cAMP-increasing reagents such as bicarbonate and cAMP analog, because the 63-kd and 54-kd sperm proteins were gradually eliminated in the control samples (Figure 3). In contrast, incubation with 0.1 mM cBiMPS rapidly (within 30 minutes) raised or increased serine/threonine-phosphorylated proteins (>220 kd, 220 kd, 180 kd, 84 kd, and 54 kd; Figures 3 and 5). As revealed by indirect immunofluorescence, protein serine/threonine phosphorylation state was enhanced mainly in the connecting and principal pieces during the same incubation period (Figure 6). These results are interpreted as showing that incubation with the cAMP analog rapidly (within 30 minutes) activates PKA, other Arg-directed kinases, or both in the connecting and principal pieces of boar spermatozoa. Additionally in the samples incubated with 0.1 mM cBiMPS, the percentages of head-to-head agglutinated spermatozoa rose coincidentally with the protein serine/threonine phosphorylation in the connecting and principal pieces during the first 30-minute incubation (Figures 2 and 6). This enhanced state of sperm agglutination is a sign of the activation of the cAMP-signaling cascades in the sperm head, because the agglutination is apparently a cAMP-dependent event (as described in "Introduction"). Thus, these indicate that the incubation of spermatozoa with the cAMP analog also activates the cAMP-signaling cascades in the head as rapidly as in the connecting and principal pieces.
As shown in Figures 3 and 5, 2 bands (93 kd and 59 kd) that were recognized by the anti-phosphoserine/phosphothreonine PKA substrate antibody increased in the samples containing 0.1 mM cBiMPS during incubation prolonged up to 180 minutes. Interestingly, the 2 bands with the same molecular masses (93 kd and 59 kd) also were detected in the samples incubated with 0.1 mM cBiMPS by the anti-phosphotyrosine antibody (Figures 4 and 5). These findings suggest that the 93-kd and 59-kd bands are phosphorylated at both serine/threonine and tyrosine residues, and that tyrosine kinase activation or tyrosine phosphatase inactivation occurs in response to the action of the cAMP analog during prolonged incubation. In addition, the 93-kd tyrosine-phosphorylated band has been already identified as a valosine-containing protein in boar spermatozoa (Geussova et al, 2002).
The 32-kd tyrosine-phosphorylated boar sperm protein has been well characterized as p32. Briefly, this p32 can be detected more intensely in the samples incubated in the capacitation-supporting medium than in the less capacitation-supporting medium lacking bicarbonate and calcium (Tardif et al, 2001; Kaneto et al, 2002). Recently, Bailey and her collaborators (Tardif et al, 2003) have reported that tyrosine phosphorylation in p32 is not dependent on bicarbonate (an intracellular cAMP-increasing reagent) but on extracellular calcium, although the 32-kd tyrosine kinase (TK32, which is distinct from p32) activation and sperm capacitation require both bicarbonate and calcium. These results are supported by our observation that the 32-kd tyrosine-phosphorylated protein was apparently detected in the control samples incubated with calcium but without bicarbonate and cAMP analog. Moreover, we found that the 32-kd tyrosine-phosphorylated protein further increased only in the samples containing both calcium and cBiMPS later than 30 minutes (Figures 4 and 5). Probably, this additional tyrosine phosphorylation results from the cAMP analog-induced activation of the tyrosine kinase including TK32 or inactivation of the tyrosine phosphatase.
Indirect immunofluorescence showed protein serine/threonine phosphorylation in the middle piece of spermatozoa during incubation with 0.1 mM cBiMPS prolonged up to 180 minutes (Figure 6). This result reveals that protein serine/threonine phosphorylation in response to the cAMP analog is slower in the middle piece than in the connecting and principal pieces, suggesting the existence of different cAMP-signaling systems among pieces of sperm flagellum. On the other hand, protein tyrosine phosphorylation state was enhanced intensely in the connecting and principal pieces and moderately in the middle piece of the agglutinated spermatozoa during this prolonged incubation with 0.1 mM cBiMPS (Figure 7). This cAMP analog-induced protein tyrosine phosphorylation in the sperm flagellum likely is hardly associated with the head-to-head agglutination. However, it is very interesting that the protein tyrosine phosphorylation of the flagellum was limited almost entirely to the head-to-head agglutinated spermatozoa (Figures 7 and 8). This suggests that the agglutinated spermatozoa may possess higher activity of the tyrosine kinase or lower activity of the tyrosine phosphatase in the flagellum than free spermatozoa.
In humans, incubation of spermatozoa with intracellular cAMP-increasing reagents enhances both hyperactivated motility and the percentages of spermatozoa exhibiting protein tyrosine phosphorylation in the flagellum. The A-kinase anchoring proteins (AKAPs) localized to the fibrous sheath of the flagellum, such as AKAP82, pro-AKAP82, and FSP95, are the major tyrosine-phosphorylated proteins during capacitation (Urner and Sakkas, 2003). In this study, prolonged incubation of spermatozoa with cBiMPS increased the tyrosine-phosphorylated proteins in the whole flagellum preferentially in the agglutinated spermatozoa (Figures 7 and 8). In the samples incubated with cBiMPS for more than 120 minutes, motility scores were much higher in agglutinated spermatozoa than in free spermatozoa, and many of motile agglutinated spermatozoa exhibited hyperactivationlike motility (Table 1). These results suggest a possible relationship between hyperactivated motility and increase of tyrosine-phosphorylated proteins in the whole flagellum in head-to-head agglutinated boar spermatozoa.
Table 2 summarizes events in boar spermatozoa induced by the activation of cAMP-signaling cascades. Our present data are consistent with the following conclusions: activation of the cAMP-signaling cascades leads to rapid (within 30 minutes) head-to-head agglutination in live spermatozoa; rapid (within 30 minutes) protein serine/threonine phosphorylation in the connecting and principal pieces of both cAMP-dependently agglutinated and free spermatozoa and subsequent (later than 30 minutes) phosphorylation in the middle piece of them; slow (later than 30 minutes) protein tyrosine phosphorylation preferentially in the connecting, middle, and principal pieces of the cAMP-dependently agglutinated spermatozoa; and slow shift of motility pattern to hyperactivationlike motility in many of agglutinated spermatozoa. Based on these conclusions, we indicate that many of cAMP-dependently agglutinated spermatozoa are live cells in which cAMP-signaling cascades leading to protein serine/threonine and tyrosine phosphorylation and to hyperactivationlike motility are activated in the whole flagellum. We expect these conclusions could contribute to disclosure of the mechanism of the unique cAMP-protein phosphorylation signaling system in boar spermatozoa.
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Acknowledgments |
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