Acclimation of sperm motility apparatus in seawater-acclimated euryhaline tilapia Oreochromis mossambicus
1 Department of Life Sciences, Graduate School of Arts and Sciences,
University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
2 Sesoko Station, Tropical Biosphere Research Center, University of the
Ryukyus, 3422 Sesoko, Motobu, Okinawa 905-0227, Japan
* Author for correspondence (e-mail: cokuno{at}mail.ecc.u-tokyo.ac.jp)
Accepted 10 October 2003
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Summary |
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Key words: sperm motility, protein phosphorylation, Ca2+, osmolality, tilapia, Oreochromis mossambicus
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Introduction |
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Euryhaline tilapia Oreochromis mossambicus can habituate and
reproduce in both freshwater and seawater, so the question arises as to how
the sperm of tilapia overcome the difference in osmolality to activate
motility in both habitats. We previously reported that sperm of
freshwater-acclimated tilapia (FWT) adjust to low osmotic pressure
corresponding to freshwater (Morita et
al., 2003). FWT sperm exhibit motility only in lower osmolality
solutions (<500 mOsm kg-1). Thus, the motility regulatory
mechanism of FWT sperm suits a low osmolality environment such as freshwater.
However, euryhaline tilapia also reproduce in seawater, where the osmolality
is approximately 1000 mOsm kg-1. In addition, physiological studies
have shown that seawater-acclimated tilapia (SWT) sperm should suit a high
osmolality (Morita and Okuma,
1998
; Linhart et al., 2000). SWT must therefore modulate their
motility apparatus to suit high osmolality.
In FWT sperm, an increase in intracellular Ca2+
([Ca2+]i) is associated with motility activation
(Morita et al., 2003). In
hypotonic conditions, both motility activation and increased
[Ca2+]i occur even when extracellular Ca2+ is chelated.
However, the increase in Ca2+ and motility activation do not occur
in conditions more hypertonic than 500 mOsm kg-1, even in the
presence of extracellular Ca2+. On the other hand, SWT sperm must
move in hypertonic conditions in order to reproduce in seawater of osmolality
>500 mOsm kg-1. If SWT sperm require increased
[Ca2+]i for motility activation, the method of increasing
[Ca2+]i must be modulated to supply Ca2+ in a hypertonic
environment.
Protein phosphorylation is also involved in sperm motility activation in
many animals. Protein phosphorylation in FWT sperm is observed only in
motility-feasible hypotonic conditions associated with the increase in
[Ca2+]i (Morita et al.,
2003). Therefore, it is also important to investigate if the
protein phosphorylation cascades exist in SWT sperm for motility activation in
hypertonic conditions.
The aim of the present study was to further our understanding of the sperm motility regulatory mechanism in SWT. We report here that increased [Ca2+]i is also tightly linked with sperm motility activation; however, the flow of Ca2+ was modulated. It is likely that influx of extracellular Ca2+ also plays a significant role and that subsequent protein phosphorylation cascades are also modulated.
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Materials and methods |
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Fish
All tilapia Oreochromis mossambicus L. (body mass 500-750 g) used
in this study were collected using a casting net at a brackish water region of
Aja River, south of Okinawa, Japan. These tilapia were acclimated to
freshwater or seawater. For freshwater-acclimated tilapia (FWT), fishes were
directly transferred to freshwater. For seawater-acclimated tilapia (SWT),
fishes were maintained for 3 days in 30% seawater, then the salinity was
increased up to 100% seawater within 5 days by gradual addition of 100%
seawater. All FWT and SWT were acclimated in the ratio three males to one
female in 1 ton freshwater tanks or in 1 ton running seawater tanks for at
least 1 month before use.
Sperm collection
Fishes were anesthetized with 0.1% (v/v) 2-phenoxyethanol and testes
dissected out from the abdomen. Sperm were collected by inserting a fine
disposable transfer pipette (Iuchiseieido, Japan) into the sperm duct, taking
great care not to contaminate the blood. Collected sperm were put onto a small
Petri dish and stored on ice during the experiments, which lasted less than 7
h.
Assessment of sperm motility
Sperm motility was observed as described previously
(Morita et al., 2003). Sperm
were suspended in various solutions: electrolyte solution (0-500 mmol
l-1 NaCl or KCl, 10 mmol l-1 Hepes-NaOH, pH 8.0),
nonelectrolyte solution (0-900 mmol l-1 mannitol, 10 mmol
l-1 Hepes-NaOH, pH 8.0), NaCl+Ca2+ solution (0-750 mmol
l-1 NaCl, 0, 2 or 10 mmol l-1 CaCl2, 10 mmol
l-1 Hepes-NaOH, pH 8.0), Ca2+-chelated NaCl solution
(0-200 mmol l-1 NaCl, 5 mmol l-1 EGTA, 10 mmol
l-1 Hepes-NaOH, pH 8.0), artificial seawater (ASW; 420 mmol
l-1 NaCl, 9.0 mmol l-1 KCl, 10 mmol l-1
CaCl2, 24.5 mmol l-1 MgCl2, 25.5 mmol
l-1 MgSO4, 2.15 mmol l-1 NaHCO3,
10 mmol l-1 Hepes-NaOH, pH 8.0). Velocities and beat frequency of
sperm were determined from photographic records. Briefly, photographs (0.5 s
exposure time) were taken using a Nikon camera system mounted on a microscope
(Optiphoto; Nikon, Japan) with dark field condenser and 10x objective.
The waveform was traced from video recordings of dark field images using
20x objective lens.
Measurements of [Ca2+]i using fluo-3 and confocal
microscopy
One volume of sperm was diluted into nine volumes of
Ca2+-depleted artificial seminal plasma (CFASP) containing EGTA
(FWT: 143 mmol l-1 NaCl, 50.7 mmol l-1 KCl, 0.18 mmol
l-1 MgSO4, 0.15 mmol l-1 glucose, 5 mmol
l-1 EGTA, 10 mmol l-1 Hepes-NaOH, pH 8.0; SWT: 132.4
mmol l-1 NaCl, 52.5 mmol l-1 KCl, 1.14 mmol
l-1 MgSO4, 0.15 mmol l-1 glucose, 5 mmol
l-1 EGTA, 10 mmol l-1 Hepes-NaOH, pH 8.0). The sperm
concentration of this suspension was about 4-5x1012 cells
ml-1. This sperm suspension was loaded with fluo-3 AM by incubating
with 500 µmol l-1 fluo-3 AM (from a 20 mmol l-1 stock
solution in anhydrous dimethylsulphoxide) on ice for 2 h. Then, the sperm
suspension was centrifuged at 1500 g for 5 min at 4°C. The
pelleted sperm were washed once with CFASP and resuspended into the same
volume of CFASP. The sperm suspension was diluted to 19 volumes of various
experimental solutions: (i) 50 mmol l-1 NaCl, (ii) 50 mmol
l-1 NaCl + 5 mmol l-1 EGTA, (iii) 50 mmol l-1
NaCl + 5 mmol l-1 CaCl2, (iv) 300 mmol l-1
NaCl and (v) 300 mmol l-1 NaCl + 10 mmol l-1
CaCl2. Suspended sperm were then placed on the slide glass, covered
with the coverslip, and sealed with nail varnish to prevent evaporation. These
preparations were observed with a confocal microscope (40x objective
lens) (Fluoview FV 500; Olympus, Japan).
Reactivation of the demembranated sperm
Demembranated tilapia sperm were reactivated in reactivation solutions to
examine the effect of Ca2+ and osmolality. Slides and coverslips
were coated with 1% (w/v) bovine serum albumin (BSA) to prevent sperm sticking
to the glass surface. 1 volume of dry sperm was suspended into 10 volumes of
the demembranation solution (175 mmol l-1 potassium acetate, 1 mmol
l-1 dithiothreitol, 1 mmol l-1 EDTA, 0.04% w/v Triton
X-100, 20 mmol l-1 Hepes-NaOH, pH 8.0) for 30 s on ice. Then, 1
volume of the demembranated sperm suspension was mixed with 20 volumes of the
reactivation solution (containing 75, 175, 350, 500 or 650 mmol l-1
potassium acetate, 1 mmol l-1 dithiothreitol, 0.5 mmol
l-1 EDTA, 0.5 mmol l-1 EGTA, 220 µmol l-1
Mg-ATP2-, 1 mmol l-1 free Mg2+,
10-9-10-2 mol l-1 free Ca2+, 20
mmol l-1 Hepes-NaOH, pH 8.0).
Fractionation of sperm flagella and sleeve structure
Sperm were activated by suspending dry sperm, prepared by centrifuging at
8000 g for 10 min at 4°C to remove seminal plasma, into
solutions containing (i) 50 mmol l-1 NaCl + 5 mmol l-1
CaCl2 and (ii) 300 mmol l-1 NaCl + 10 mmol
l-1 CaCl2, and incubating for 1 min at room temperature.
Movements of sperm in these suspensions were recorded as described above. The
percentage of motile sperm was determined from video recordings. SWT sperm
were motile when suspended in hypotonic (50 mmol l-1 NaCl + 5 mmol
l-1 CaCl2) and hypertonic (300 mmol l-1 NaCl
+ 10 mmol l-1 CaCl2) solutions. Immotile and motile
sperm suspensions were centrifuged at 15 000 g for 10 min at
4°C. Then 1 volume of flagella and sleeve structures in each sperm pellet
were eluted with 7 volumes of urea solution (8 mol l-1 urea, 2 mol
l-1 thiourea, 1% w/v Chaps, 1 mmol l-1 EDTA, 100 mmol
l-1 dithiothreitol, 1 mmol l-1 phenylmethylsulphonyl
fluoride (PMSF), 15 µmol l-1 E-64, 1.5 µmol l-1
Pepstatin A) to a concentration of 1.0x1013 cells
ml-1. Heads were pelleted by centrifugation at 15 000
g for 10 min at 4°C. SDS sample buffer was added to these
flagella and sleeve suspensions, which were stored at -80°C before
analysis by 1-D polyacrylamide gel electrophoresis (PAGE) and western
blotting.
An equal volume of SDS-PAGE sample buffer was added to the urea-extracted
flagella and sleeve. The samples (equivalent to 1.5x1011
cells) were subjected to SDS-PAGE in tricine buffer, according to the method
of Schagger and Jagow
(1987).
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Western blotting was performed according to Towbin et al.
(1979), with a little
modification. Gels obtained from tricine-SDS-PAGE were placed on
polyvinylidene difluoride membranes (PVDF; Biorad, USA) and electrically
transferred. The membranes were blocked by incubation with 5% (w/v) bovine
serum albumin in TTBS (137 mmol l-1 NaCl, 0.1% w/v Tween 20, 20
mmol l-1 Tris-HCl, pH 7.4) overnight at 4°C for detection of
phosphoproteins. The membranes were washed three times for 10 min with TTBS
followed by incubation for 2 h at room temperature with anti-phosphoserine
antibody (diluted 1:20 000) and anti-phosphothreonine antibody (diluted 1:20
000). Then, the membranes were washed and incubated with horseradish
peroxidase-conjugated extravidin (diluted 1:25 000) in TTBS. The membranes
were again washed three times and subjected to an enhanced-chemiluminescence
(ECL) reaction, performed according to the manufacturer's instructions
(Amersham Pharmacia Biotech). The resulting membranes were exposed to X-ray
film for 5-30 s.
Statistical analysis
Group comparisons were performed using a one-way or two-way analysis of
variance (ANOVA) followed by the least-significant test, where quality of
variance criteria were met. Otherwise Mann-Whitney and Kruskal-Wallis
procedures were used for the swimming velocity and beat frequency results.
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Results |
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To further examine the effect of Ca2+, EGTA was added to remove
Ca2+ completely, since considerable amounts of Ca2+ are
derived from seminal plasma, which contains approx. 2 mmol l-1
Ca2+ in the experimental conditions shown in
Fig. 1A. Under these
conditions, sperm motility was drastically suppressed
(Fig. 1B). The response of SWT
sperm to changes in osmolality was different from that of FWT sperm. The
motility of FWT sperm (Fig. 2A)
in 200 mmol l-1 NaCl solution (approximately 400 mOsm
kg-1) increased from 0% to approximately 20% on addition of
Ca2+ (2 mmol l-1). However, no motility was observed
at 350 mmol l-1 NaCl even in the presence of 10 mmol l-1
Ca2+. In contrast, the effect of Ca2+on SWT sperm was
more marked, as shown in Fig.
2B, and rose dose-dependently in the presence of extracellular
Ca2+. More than 90% of SWT sperm were motile in the presence of 5
mmol l-1 Ca2+ at 250 mmol l-1 NaCl. The
higher the osmolality, the larger the extracellular [Ca2+]
necessary to activate motility. It is likely that SWT sperm require
extracellular Ca2+ to acquire motility in the hypertonic
environment.
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We then assessed sperm motility by determining swimming velocity, flagellar beat frequency and waveform, as shown in Fig. 3. The swimming velocities of FWT and SWT sperm were different in the hypotonic Ca2+-containing condition (50 mmol l-1 NaCl + 5 mmol l-1 CaCl2). In this hypotonic solution, the swimming velocity of FWT sperm was greater that that of SWT sperm (Fig. 3A.). By contrast, SWT sperm in hypertonic Ca2+-containing solution (300 mmol l-1 NaCl + 10 mmol l-1 CaCl2) swam as fast as FWT sperm in hypotonic solution (Fig. 3A). In this Ca2+-containing hypertonic solution, SWT sperm showed vigorous motility, and the swimming velocity of SWT sperm was faster than that of sperm in the Ca2+-containing hypotonic condition (Fig. 3A).
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On the other hand, the beat frequency of FWT and SWT in Ca2+-containing hypertonic and hypotonic conditions was the same (Fig. 3B). Hence, the pitch of one stroke by SWT sperm in the hypertonic condition was larger than that of sperm in the hypotonic condition. As shown in Fig. 3C, the waveform of SWT sperm in the hypertonic condition was different from that in the hypotonic condition but similar to the waveform of FWT sperm in the hypotonic condition. Therefore, it is plausible that FWT and SWT sperm are suited to hypotonic or hypertonic conditions, corresponding to their habitat, suggesting that the mechanism for regulating the sperm motility is modulated during acclimation of these fish.
Demembranated sperm of FWT and SWT sperm
The present study revealed that demembranated SWT sperm, like demembranated
FWT sperm, required Ca2+ but not cAMP or cGMP to reactivate the
motility (Fig. 4). It is likely
that increased [Ca2+]i is necessary to activate both FWT and SWT
sperm motility, even though motility-feasible conditions were different for
FWT and SWT sperm.
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The effect of [K+] on motility reactivation in both FWT and SWT
sperm was studied. Takai and Morisawa
(1995) reported that
[K+] is important in marine and freshwater teleosts. In marine
teleosts, an increase in [K+] is necessary to reactivate the
demembranated sperm. In freshwater teleosts such as zebra fish, on the other
hand, a decrease in [K+] is necessary. We examined the effect of
[K+] in demembranated FWT and SWT sperm. Both FWT and SWT
demembranated sperm exhibited a high ratio of reactivation in hypotonic and
isotonic [K+] solutions (Fig.
5A,B) in the presence of 0.1 mmol l-1 Ca2+.
In hypertonic solutions, motility was decreased gradually, suggesting that
[K+] did not affect the activation cascades in tilapia sperm in the
isotonic and hypotonic range of solutions tested.
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Increase in intracellular [Ca2+]
SWT sperm showed motility in hypotonic and hypertonic conditions in the
presence of extracellular [Ca2+] (Figs
1B,
2B). In SWT sperm, increased
[Ca2+]i was observed in motility-feasible hypotonic and hypertonic
conditions with Ca2+ present
(Fig. 6A,D). As shown in our
previous study (Morita et al.,
2003), in hypertonic conditions, [Ca2+]i in FWT sperm
did not increase even on addition of extracellular Ca2+. By
contrast, in SWT sperm, [Ca2+]i only increased when extracellular
Ca2+ was added, suggesting that the increased [Ca2+]i is
extracellular [Ca2+]-dependent. The increase in [Ca2+]i
of FWT and SWT sperm occurred in each different motility-feasible condition,
suggesting that ways of mobilising Ca2+ also differ between FWT and
SWT sperm. It is possible that the mechanism of raising [Ca2+]i in
FWT sperm is driven hypo-osmotically and that of SWT sperm depends on
Ca2+ influx from extracellular regions.
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Increased [Ca2+]i was mainly observed in the head area,
including the sleeve structure (arrows in
Fig. 6A,D). A weak fluorescence
signal was also observed in flagella (arrowheads in
Fig. 6A,D). As shown in our
previous study, swelling of the sleeve structure in FWT sperm occurred in
hypotonic conditions corresponding to the motility practicable condition in
which the increase in [Ca2+]i was observed
(Morita et al., 2003). The
sleeve structure in SWT sperm was swollen in both hypotonic and hypertonic
conditions independently of [Ca2+], as shown in
Fig. 6E.
Protein phosphorylation in SWT sperm during motility activation
Activation of SWT sperm was dependent on extracellular [Ca2+].
Protein phosphorylation and dephosphorylation occur in FWT sperm during
activation of motility (Morita et al.,
2003). Therefore, we examined protein phosphorylation in relation
to the [Ca2+] increase and motility activation. In hypotonic
Ca2+-containing conditions (50 mmol l-1 NaCl + 5 mmol
l-1 CaCl2), serine and threonine residues of 15 kDa and
18 kDa proteins were dephosphorylated (Fig.
7A,B, lanes b) compared to those of the dry sperm
(Fig. 7A,B, lanes a). These
dephosphorylations did not occur in the absence of extracellular
Ca2+ even in hypotonic conditions (data not shown). By contrast, in
hypertonic conditions, serine and threonine residues of 15 kDa and 18 kDa
proteins were phosphorylated (Fig.
7A,B, lanes c). As previously shown
(Morita et al., 2003
),
threonine residue(s) of a 41 kDa protein in FWT sperm were also phosphorylated
in motility-feasible hypotonic conditions. This 41 kDa protein was also
phosphorylated in threonine residues in dry sperm
(Fig. 7B; lane a) and was
retained under motile conditions (Fig.
7B, lanes b,c) in SWT sperm.
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Discussion |
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In sperm of puffer fish (Oda and
Morisawa, 1993), lancelet
(Tanaka et al., 2002
),
ascidians (Nomura et al.,
2000
; Yoshida et al.,
1994
,
2003
), carp
(Krasznai et al., 2000
) and
salmonidae (Boitano and Omoto,
1992
; Tanimoto and Morisawa, 1998;
Kho et al., 2001
), increased
[Ca2+]i is known to play an important role in activation of the
sperm motility. Demembranated sperm is assumed to simulate a regulatory
mechanism of motility in intact sperm. In the presence of >10-4
mol l-1 Ca2+, demembranated FWT sperm were reactivated
(Morita et al., 2003
). In the
present study we have shown that demembranated sperm of SWT is also required
Ca2+ for reactivation (Fig.
4), suggesting that Ca2+ activates a second messenger
for motility activation. In salmonid fishes, increased [Ca2+]i was
also observed on motility initiation/activation
(Cosson et al., 1989
). These
results suggest that Ca2+ is tightly coupled to the initial phase
of motility regulation. However, a series of experiments have demonstrated
that Ca2+ sometimes works as an inhibitor. [Ca2+] of
more than 10-8.5 mol l-1 inhibits reactivation of
demembranated salmonid sperm (Okuno and
Morisawa, 1989
). In demembranated sea urchin sperm, sperm flagella
become quiescent and `cane'-shaped in high [Ca2+] (10-4
mol l-1; Gibbons and Gibbons,
1980
; Okuno and Brokaw,
1981
). In these cases, Ca2+ seems to suppress some wave
parameters directly. Therefore, it is likely that Ca2+ has a
biphasic role. Ca2+ in tilapia works predominantly in motility
initiation/activation.
As shown above, increased [Ca2+]i is necessary for activation of
both demembranated FWT and SWT sperm motility. The levels of Ca2+
in habitats of FWT and SWT are quite different. Freshwater does not contain
large amounts of Ca2+, but seawater does, although both FWT and SWT
sperm require increased [Ca2+]i as a second messenger for motility
activation. FWT sperm have difficulties in getting sufficiently high levels of
extracellular Ca2+ for activation in freshwater, because there are
not high enough amounts of Ca2+ present. By contrast, it would be
easier for SWT sperm to utilize extracellular Ca2+ in seawater for
motility activation. Therefore, it is reasonable to suggest that one major
component in acclimation of motility regulatory mechanisms in tilapia is
modulation of the flow of Ca2+ supply. The Ca2+
indicator, fluo 3, revealed that levels of [Ca2+]i increased in SWT
sperm in the presence of extracellular Ca2+ even at high osmolality
(Fig. 6A,D). Increase in
[Ca2+]i in SWT sperm, however, did not occur in the absence of
extracellular Ca2+ (Fig.
6B,D), suggesting that the Ca2+ influx is required to
raise the intracellular Ca2+ level. We previously reported that FWT
sperm do not exhibit motility at high osmotic pressure even in the presence of
extracellular Ca2+, if the increase in [Ca2+]i does not
also occur (Morita et al.,
2003). It is plausible that FWT sperm have Ca2+ stores
that can be released by osmotic shock to increase [Ca2+]i, whereas
SWT sperm do not. However, it is possible that SWT sperm may also have
Ca2+ stores controlled by a ryanodine receptor, which is activated
by attaching Ca2+ (Berridge,
1993
). Thus, in SWT sperm, it is possible that the increase in
[Ca2+]i occurs via Ca2+-induced Ca2+
release. Preliminary experiments show that the ryanodine receptor inhibitor
did inhibit SWT sperm motility in hypotonic conditions (M. Morita and M.
Okuno, unpublished data).
SWT sperm also showed swelling of the sleeve structure in hypertonic
conditions, whereas the sleeve of FWT sperm was shrunk
(Fig. 6E). Volume regulation of
the sleeve structure might possibly act as an important candidate to increase
[Ca2+]i or another function of motility in conditions of high
osmolality. It is reported that cell swelling induces increased
[Ca2+]i in Necturus erythrocytes
(Light et al., 2003).
Therefore, it is likely that volume regulation of the sleeve structure is
important for the motility regulatory mechanism by increasing
[Ca2+]i even if the Ca2+ was influxed from the
extracellular region.
The ionic environment is also an important factor responsible for motility
activation. High concentrations of electrolytes, such as KCl and NaCl, are
necessary for activating the motility of demembranated sperm of marine
teleosts such as puffer fish. On the other hand, a decrease in electrolytes is
required for motility activation of freshwater teleosts such as zebra fish
sperm (Takai and Morisawa,
1995), where reactivation of the demembranated sperm failed to
occur when the electrolytes were substituted for mannitol, suggesting that the
presence of appropriate concentrations of ions is necessary for attaining
motility. In comparison with puffer fish and zebra fish, the properties of
demembranated tilapia sperm appeared similar to those of freshwater teleosts,
even when acclimated to seawater. Furthermore, it is likely that the
regulatory mechanism of tilapia sperm motility is not controlled by
[K+], since the demembranated tilapia sperm were reactivated in
isotonic K+ solution (Fig.
5A,B) and the decrease and increase in [K+] had no
effect on the reactivation ratios of either FWT or SWT demembranated sperm
(Fig. 5A,B).
In sperm of salmonid fishes, it has been accepted that motility initiation
is induced by phosphorylation of a 15 kDa protein via the increase in
cAMP (Morisawa and Hayashi,
1985; Hayashi et al.,
1987
). We failed to reactivate demembranated tilapia sperm with
cAMP as shown in Fig. 4.
Furthermore, we added both cAMP and the catalytic subunit of A-kinase to
eliminate the possibility that the latter was depleted on demembranation.
Again, we failed to reactivate the demembranated sperm (data not shown). Thus,
we conclude that a cAMP-dependent system is not involved in tilapia sperm
motility activation. We previously reported that protein phosphorylation
occurred during activation of FWT sperm motility accompanied by an increase in
[Ca2+] (Morita et al.,
2003
). In the present study, protein phosphorylation of serine and
threonine residues of various proteins was closely related to the activation
of SWT sperm motility (Fig. 7).
Taken together with our previous results, it is apparent that a protein
phosphorylation cascade is involved in motility activation mechanism in
tilapia sperm although the protein phosphorylation cascades of FWT and SWT
sperm seem to be different.
Considering the relationship between motility activation and protein
phosphorylation, three phosphoproteins, of 15 kDa, 18 kDa and 41 kDa, were
detected. The 15 kDa and 18 kDa proteins were dephosphorylated in
motility-feasible hypotonic conditions in both FWT and SWT sperm. By contrast,
the 15 kDa and 18 kDa proteins were strongly phosphorylated in hypertonic
conditions (Fig. 7). A
threonine residue(s) of a 41 kDa protein in FWT sperm was phosphorylated in
the motility-feasible hypotonic condition
(Morita et al., 2003). The 41
kDa protein in SWT sperm was also phosphorylated in the dry sperm condition
(Fig. 7, lane a), suggesting
that the protein phosphorylation cascades related to motility activation are
different in FWT and SWT sperm. It is possible that phosphorylation of the 41
kDa protein in the dry sperm condition is required for motility in the
hypertonic condition. It is also likely that modulation of protein
phosphorylation cascades is related to acclimation of the motility regulatory
mechanism in FWT and SWT sperm. It is therefore suggested that acclimation of
sperm is caused by modulation of spermatogenesis, involving modulation of
protein phosphorylation cascades and mechanism of Ca2+ supply.
However, it is still not certain what kinds of signal transduction are related
to motility activation with respect to the supply of Ca2+ and
protein phosphorylation.
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Acknowledgments |
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