Requirement of Ca2+ on activation of sperm motility in 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
Ryukyu, 3422 Sesoko, Motobu, Okinawa 905-0227, Japan
* Author for correspondence (e-mail: cokuno{at}mail.ecc.u-tokyo.ac.jp)
Accepted 21 November 2002
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Summary |
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Key words: sperm motility, Ca2+, osmolality, phosphorylation, euryhaline tilapia, Oreochromis mossambicus
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Introduction |
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The tilapia Oreochromis mossambicus, a euryhaline teleost, has the
can acclimate to wide range of salinities, from freshwater to seawater, by a
mechanism that includes chloride cells
(Sakamoto et al., 1997;
Balm et al., 1994
;
Borski et al., 1994
). A unique
feature of tilapia is that they can reproduce in both freshwater and seawater,
even though there is a large osmotic difference between those two environments
(Brock, 1954
). Thus, it has
been assumed that tilapia sperm can swim independently of osmolality, or
modulate their regulatory mechanism to suit fertilization in either high or
low salinity. It is possible that the motility regulatory system of tilapia
sperm is quite different from those reported for other teleosts. We have
already reported that tilapia sperm acclimated to seawater were motile in
hypertonic water, i.e. seawater, in the presence of Ca2+, whereas
in tilapia acclimated to freshwater sperm could not swim in hypertonic
conditions even in the presence of Ca2+
(Morita and Okuno, 1998
).
Linhart et al. (1999
) reported
that during acclimation of the fish from freshwater to seawater, tilapia sperm
adapt to conditions of high salinity by changing their motility from being
independent of extracellular Ca2+ to Ca2+ dependent. It
was also reported recently that sperm motility was physically suppressed by
high viscous components in seminal plasma in the Nile tilapia Oreochromis
niloticus, another species that cannot adapt to seawater. This result
suggests that motility activation could be induced simply by mechanical
release from viscosity immobilization
(Mochida et al., 1999
). These
reports led us to question whether the motility regulatory mechanism of
tilapia sperm differs from an osmotic shock related mechanism. In the present
work, we examine the regulatory mechanism of sperm motility in tilapia
acclimated to freshwater and demonstrate that sperm motility is regulated by
osmolality, together with a limited role for extracellular Ca2+.
Furthermore, we demonstrate that on exposure to hypotonic conditions, various
protein phosphorylations and dephosphorylations occur that are associated with
the increase in intracellular Ca2+ levels.
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Materials and methods |
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Fish
All tilapia Oreochromis mossambicus (body mass 500-750 g) used in
this study were collected with a casting net at a blackish water region of Aja
River, in the southern part of Okinawa, Japan. These tilapia were acclimated
to the various conditions in groups of 3 males to 1 female in 1 ton freshwater
tanks for at least 1 month before use.
Sperm collection
Fish were anesthetized with an appropriate amount of 2-phenoxyethanol, and
the 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
transferred to a small Petri dish and stored on ice.
Measurement of sperm motility
In this study, NaCl and KCl were used as electrolytes and mannitol as a
nonelectrolyte. All solutions contained 10 mmol l-1 Hepes-NaOH
buffer, pH 8.0. Approximately 0.05 µl of semen were immediately diluted
into 45 µl of solution on a glass slide with fine glass capillary tube, and
covered with a coverslip. Sperm movements were recorded using a video recorder
(SLV-LF1; Sony, Japan) and a CCD camera (cs 226; Olympus, Japan) mounted on a
phase contrast microscope (Optiphoto, Nikon). Percentage motility was counted
from the video recordings. Sperm were counted as motile if they either
exhibited progressive movement or spontaneous flagellar beating if the sperm
head was attached to the glass slide.
Measurement of components and osmolality of seminal plasma
Dry sperm were transferred to 1.5 ml Eppendorf tubes, and centrifuged for 3
min at 8000 g (4°C). The supernatants were used as seminal
plasma. Osmolality of the seminal plasma and experimental solutions was
measured by vapor-pressure osmometer (VPO5506; Wesco, USA).
Electrolyte components in seminal plasma were measured after dilution to an appropriate concentration in milliporefiltered water. 10 ml of the diluted seminal plasma solution were subjected to polarized zeeman atomic absorption spectrophotometer (Z-6100; Hitachi, Japan) to measure Na+, K+, Mg2+ and Ca2+ concentrations.
Measurements of intracellular [Ca2+] with fluo-3 and
confocal microscope
Sperm was diluted 1:9 (v/v) with Ca2+-depleted artificial
seminal plasma (CFASP) containing EGTA (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 and 10 mmol l-1
Hepes-NaOH, pH 8.0). The sperm concentration of this suspension was
approximately 4-5x1012 cells ml-1. This sperm
suspension was loaded with fluo-3 AM by incubation with 500 µmol
l-1 fluo-3 AM [from a 20 mmol l-1 stock solution in
anhydrous dimethyl sulphoxide (DMSO)] on ice for 2 h, followed by centrifugion
at 1500 g for 5 min at 4°C. The pelleted sperm were washed
once with CFASP and resuspended in the same volume of CFASP before dilution
1:19 (v/v) into 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, 300 mmol
l-1 NaCl and (iv) 300 mmol l-1 NaCl + 5 mmol
l-1 CaCl2. The diluted sperm were then placed on glass
slides, covered with a coverslip, and sealed with nail varnish to prevent
evaporation. The 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 to examine the effect of
[Ca2+] and osmolality. Glass slides and coverslips were coated with
1% (w/v) bovine serum albumin (BSA) to prevent sperm sticking to the glass
surface. Demembranation and reactivation were carried out according to the
method described previously (Okuno and
Morisawa, 1989). Dry sperm was suspended 1:10 (v/v) in the
demembranation solution [175 mmol l-1 potassium acetate, 1 mmol
l-1 dithiothreithol (DTT), 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
the demembranated sperm was mixed 1:20 (v/v) with the reactivation solution
(175 mmoll-1 potassium acetate, 1 mmoll-1 DTT, 0.5
mmoll-1 EDTA, 0.5 mmoll-1 EGTA, 220
µmoll-1 Mg-ATP2+, 1 mmoll-1 free
Mg2+, 10-9-10-2 moll-1 free
Ca2+, 20 mmoll-1 Hepes-NaOH, pH 8.0).
Fractionation of sperm flagella and sleeve structure
Sperm were activated by suspending the dry sperm in various solutions: (i)
50 mmoll-1 NaCl + 5 mmoll-1 EGTA, (ii) 50
mmoll-1 NaCl + 5 mmoll-1 CaCl2 and (iii) 300
mmoll-1 NaCl, and incubated for 1 min at room temperature.
Movements of sperm in these suspensions were recorded by a video recorder
(SLV-LF1; Sony) and a CCD camera (cs 226; Olympus) mounted on a microscope
(Optiphot; Nikon, Japan) equipped with a dark-field or phase-contrast
condenser. The percentage of motile sperm was counted from the video
recordings. Immotile sperm samples were prepared by suspending them into 300
mmoll-1 NaCl solution (activation solution iii). Sperm were motile
when suspended in 50 mmoll-1 NaCl + 5 mmoll-1 EGTA
(activation solution i) or 50 mmoll-1 NaCl + 5 mmoll-1
CaCl2 (activation solution ii). The immotile and motile sperm
suspensions were centrifuged at 15 000 g for 10 min at
4°C. Sperm pellets were resuspended to a concentration of
1.0x1013 cell ml-1 in urea solution (8
mmoll-1 urea, 2 mmoll-1 thiourea, 1% (w/v) Chaps, 1
mmoll-1 PMSF, 15 µmoll-1 E-64, 1.5
µmoll-1 Pepstatin A and 100 mmoll-1 DTT). Heads were
pelleted by centrifugation at 15 000 g for 10 min at 4°C.
SDS-sample buffer was added to the supernatant containing flagella and sleeve
structures for 1-D polyacrylamide gel electrophoresis (PAGE). Suspensions were
stored at -80°C until used.
Gel electrophoresis and western blotting analysis
Flagella and sleeves (equivalent to approximately 2-3x1011
cells) extracted by urea were subjected to tricine-buffered SDS-PAGE
(Schagger and Jagow, 1987) in
10% polyacrylamide gels containing 0.1% SDS.
Western blotting was performed according to the method of Towbin et al.
(1979), with a little
modification. After electrophoresis the tricine SDS-PAGE gels were placed on
polyvinylidene difluoride (PVDF) membranes (Biorad, USA) and electrically
transferred. The membranes were blocked by incubation with 2% (w/v) BSA in
TTBS (137 mmoll-1 NaCl, 0.1% (w/v) Tween 20 and 20
mmoll-1 Tris-HCl, pH 7.4) overnight at 4°C. The membranes were
washed three times with TTBS followed by incubation for 2 h at room
temperature with anti-pS antibody (1:20,000 dilution) or anti-pT antibody
(1:20,000 dilution) as the primary antibodies. Then the membranes were washed
and incubated with extravidine-conjugated with horseradish peroxidase
(1:25,000 dilution) in TTBS for 1 h at room temperature. The membranes were
again washed three times and subjected to the enhanced chemiluminescence (ECL)
reaction, carried out according to the manufacturer's protocol. The membranes
were exposed to X-ray film for 5-30 s.
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Results |
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Sperm retained motility even when suspended in 150 mmoll-1 KCl
solution, which was an almost isotonic condition, suggesting that the system
of motility regulation in this fish is very different from that of salmonid
fish, where decreased [K+] causes increased sperm motility
(Morisawa and Suzuki,
1980).
Addition of Ca2+ to the solutions caused increased sperm motility at the osmolalities less than 500 mosmol kg-1, as shown in Fig. 1B. However, sperm motility ceased above 600 mosmol kg-1. Removal of extracellular Ca2+ by EGTA reduced motility, and sperm were almost quiescent at the isotonic condition. However, even in the presence of EGTA, motility was almost equivalent to that observed in the presence of Ca2+ at osmolalities less than 100 mosmol kg-1. Similar results were observed in solutions containing KCl and mannitol (data not shown). Seminal plasma contains approximately 2 mmoll-1 Ca2+ (Table 1), so it was assumed that Ca2+ contaminated from seminal plasma was responsible for the increased motility in the experiments shown in Fig. 1A. It was also likely that in hypotonic conditions sperm did not require extracellular Ca2+ for motility. Mg2+ had no effect on sperm motility (data not shown).
Measurement of intracellular Ca2+
In freshwater, tilapia sperm exhibited high motility even when the
extracellular [Ca2+] was depleted with EGTA. However, sperm
motility in medium of lower osmolality than isotonic medium was improved by
the addition of Ca2+. Ca2+ might therefore be involved
in the regulatory mechanism of flagellar motility in tilapia.
In order to examine this possibility, the intracellular Ca2+ concentration [Ca2+]i was measured using fluorescent dye. Fig. 2 shows the increase in [Ca2+]i after motility activation. Sperm diluted in solution containing 50 mmoll-1 NaCl + 5 mmoll-1 CaCl2 (Fig. 2A) or 5 mmoll-1 EGTA (Fig. 2B) exhibited high motility for approximately 30 min and then stopped. Sperm did not move in the solution containing high NaCl (300 mmoll-1) regardless of the presence of Ca2+ (Fig. 2C,D). Fluorescence micrographs taken after the flagella motility stopped revealed increased fluorescence in sleeve structures of sperm that had exhibited motility. Even when extracellular Ca2+ was chelated with EGTA, [Ca2+]i were increased (Fig. 2B), suggesting that the increased intracellular Ca2+ was supplied from intracellular Ca2+ stores. No fluorescence was observed in sperm that were immotile under hypertonic conditions.
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The sleeve structures were expanded in solutions containing 50 mmoll-1 NaCl (Fig. 2A,B; low osmotic condition), and contained Ca2+, as indicated by fluo-3 localization, suggesting that this Ca2+ was supplied from Ca2+ stores within the sleeve structures. On the other hand, the sleeve structure was not expanded nor was the [Ca2+]i increased in solutions containing 300 mmoll-1 NaCl with or without 5 mmoll-1 CaCl2 (Fig. 2C,D). It is likely that tilapia sperm do not have the capability to increase [Ca2+]i in a high osmotic environment. In addition, since the sleeve structure is shrunk in the isotonic and hypertonic conditions, supplementation of intracellular Ca2+ stores is stimulated by hypotonic conditions, associated with swelling of the sleeve structure, and high osmotic pressures suppress the function of the Ca2+ store in sleeve structures (Fig. 2E).
Ca2+ requirement for reactivation of the demembranated
sperm
Soluble components of the cytoplasm, including ions, soluble proteins,
nucleotides etc, diffuse away from the axoneme when sperm are demembranated.
It is then possible to examine the effect of intracellular factors, such as
Ca2+, directly by changing the composition of the solution. It is
very difficult to control the [Ca2+]i because the cell
membrane is impermeable to Ca2+. We used the demembranated sperm
model to apply Ca2+ directly to the flagellar axoneme. As shown in
Fig. 3A, demembranated sperm
were not reactivated in the presence of ATP only. An appropriate concentration
of Ca2+ (10-4 moll-1) in the reactivating
solution was necessary for motility to occur. Addition of either 10
µmoll-1 cAMP or 10 µmoll-1 cGMP failed to
reactivate the sperm movement.
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More detailed experiments using the demembranated sperm were performed to investigate the effect of Ca2+ on reactivation. Motility was very low at 10-9 moll-1 Ca2+, began to increase at 10-7 moll-1 and was maximal at 10-4 moll-1 Ca2+. Reactivated sperm motility was suppressed at Ca2+ levels of 10-2 moll-1 or higher (Fig. 3B). It seems that increased [Ca2+]i rather than increased cAMP or cGMP levels was necessary to activate sperm motility. We found no difference in motility when Ca2+ was either present or absent in the demembranation solution.
Protein phosphorylation during activation of sperm motility
Tilapia sperm exhibited vigorous motility under conditions of low osmotic
pressure associated with increased [Ca2+]i. It is known
that many kinds of kinases are activated by Ca2+, including protein
kinase C. Therefore, we examined the effect of protein phosphorylation on the
activation of motility introduced by transferring sperm from a hypertonic (300
mmoll-1 NaCl) to a hypotonic (50 mmoll-1 NaCl) medium.
The effect of Ca2+ in the hypotonic solution was also examined.
Western blotting using phosphoserine (pS) and phosphothreonine (pT) antibodies
were carried out on PVDF membranes transferred from tricine SDSPAGE. We
found no evidence of tyrosine phosphorylation using phosphotyrosine (pY)
antibody in this study.
Western blotting using anti-phosphoserine antibodies revealed that two proteins were dephosphorylated when sperm shifted from the immotile to the motile phase (Fig. 4). The changes in phosphorylation patterns differed in the presence and the absence of extracellular Ca2+ (Fig. 4B,C). Two protein bands of 18 kDa and 15 kDa were dephosphorylated after dilution into both hypotonic solutions (50 mmoll-1 NaCl + 5 mmoll-1 EGTA and 50 mmoll-1 NaCl + 5 mmoll-1 CaCl2). Therefore, protein dephosphorylation accompanied the motility change whether or not extracellular Ca2+ was present.
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In order to investigate further the effect of protein phosphorylation on motility activation, phosphorylation of threonine residue(s) was investigated. Changes in phosphorylation were detected in three proteins after motility activation was attained in hypotonic conditions (Fig. 5). The 41 kDa protein was phosphorylated and the 18 kDa and 15 kDa proteins were both dephosphorylated in 50 mmoll-1 NaCl solution with or without Ca2+. Protein phosphorylation and dephosphorylation of threonine residue(s) thus occurred in conditions where sperm motility was activated.
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Discussion |
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Increased [Ca2+]i on initiation of motility was
reported in the marine teleost, pufferfish
(Oda and Morisawa, 1993) and
salmonid fish (Cosson et al.,
1989
; Boitano and Omoto,
1992
). Increased [Ca2+]i seems to play an
important role in the initiation process of sperm motility. In tilapia sperm,
in hypotonic conditions, extracellular Ca2+ is not necessary to
initiate motility (Fig. 1), although extracellular Ca2+ increases motility in certain extent.
By contrast, sperm from seawater-reared tilapia require Ca2+ for
motility (Linhart et al.,
1999
; Morita and Okuno,
1998
).
Osmotic shock induces initiation of motility in sperm of teleosts
fertilizing in external spawning grounds
(Morisawa and Suzuki, 1980).
However, the environment controlling the regulatory mechanism seems to be
different, since osmosis between outside and inside the cell is reversed.
Hypotonic shock triggers initiation of sperm motility in freshwater teleosts
such as cyprinids. By contrast, hyper-osmolality triggers sperm motility in
seawater teleosts such as pufferfish. Tilapia mossambicus can spawn
from freshwater to seawater, as their sperm adapt to the external spawning
ground by changing their sensitivity to extracellular [Ca2+]
(Morita and Okuno, 1998
;
Linhart et al., 1999
).
However, the process by which the motility regulatory mechanism adapts remains
obscure. In the present study, we examined the motility of sperm from
freshwater-acclimated tilapia, and demonstrate that
[Ca2+]i plays a significant role in motility activation,
although sperm can move without extracellular Ca2+.
It is likely that [Ca2+]i is necessary for motility activation, for the following reasons. Firstly, confocal microscopy using fluo-3 loaded sperm revealed that the [Ca2+]i was increased independently of the extracellular [Ca2+] when sperm were suspended into a hypotonic solution (Fig. 2A,B). Increase in [Ca2+]i occurred even when external Ca2+ was chelated (Fig. 2B). Therefore, it could be assumed that hypotonic shock triggered the supplementation of Ca2+ in the cytoplasm from some intracellular Ca2+ store, not from the outside the cell. Secondly, demembranated sperm were reactivated only in the presence of Ca2+ concentrations greater than 10-6 moll-1. cAMP and cGMP failed to reactivate the motility.
How is [Ca2+]i increased in tilapia sperm? In
salmonid fish sperm, decreased extracellular [K+] on ejaculation of
sperm into freshwater is thought to induce hyperpolarization of the plasma
membrane followed by a transient influx of Ca2+ via
Ca2+ channels (Tanimoto and
Morisawa, 1988). Thus, extracellular Ca2+ is necessary
for sperm motility in this species. By contrast, extracellular Ca2+
is not necessary for motility of tilapia sperm, and high concentrations of
KCl, approximately 100 mmoll-1, had no effect on motility
activation in the present study. Therefore, Ca2+ supplementation in
tilapia must occur intracellularly. Since hypotonic shock induces swelling of
the sleeve structure together with increased [Ca2+] in this area
(Fig. 2A,B), sleeve structure
and some other organelle may be candidates for supplying the Ca2+,
and regulated by hypotonic shock.
In salmonid fish sperm, cAMP plays an important role as a second messenger.
Both adenylate cyclase activity and the cAMP concentration increase on
motility initiation in intact sperm
(Morisawa and Ishida, 1987),
and cAMP is necessary for reactivating the demembranated sperm
(Morisawa and Okuno, 1982
). It
has been suggested that a cAMP dependent phosphorylation cascade is the main
cause of motility activation (Hayashi et
al., 1987
; Jin et al.,
1994
). In tilapia, however, cAMP failed to activate the motility
of demembranated sperm, which was activated by Ca2+. In cyprinid
sperm, cAMP is not required and only increased [Ca2+] is necessary
to reactivate demembranated sperm
(Krasznai et al., 2000
). Thus,
an increase in [Ca2+]i could be sufficient to cause
activation of motility. Increased [Ca2+]i was obtained
from extracellular Ca2+ via Ca2+ channels in
Cyprinid sperm; however, the present study suggested that extracellular
Ca2+ influx was not the major source of increased
[Ca2+]i in sperm of tilapia. Ca2+ stores are
assumed to be involved in sperm of tilapia, operating only in hypotonic
conditions to increase [Ca2+]i in sleeve structures in
the neck region of sperm (Fig.
2A,B,E). the sleeve was only expanded in hypotonic solutions,
whereas in hypertonic conditions this structure was shrunk; this cell swelling
is probably linked with the Ca2+ releasing mechanism in hypotonic
conditions.
Osmotic regulation of [Ca2+]i have also been reported
in cyprinids, where Ca2+ influx for motility initiation is
triggered by membrane hyperpolalization induced by the decreasing ion
concentration resulting from the decrease in osmolality
(Krasznai et al., 2000). In
salmonid fish, a transient increase in [Ca2+]i occurs by
the release of Ca2+ from intracellular stores
(Boitano and Omoto, 1992
). In
the marine teleost, pufferfish, it is reported that increased
[Ca2+]i initiates sperm motility in hypertonic
conditions (Oda and Morisawa,
1993
). Changes in osmotic pressure triggered an increase in
[Ca2+]i, initiating motility in both freshwater and
marine teleosts. It is supposed that osmolality-regulated changes in cell
membranes trigger intracellular Ca2+ releasing pathways
via some unknown mechanism.
Electron microscopic analysis (Don and Avtalion, 1993) showed that large
amounts of cytoplasm surrounded by plasma membrane similar to endoplasmic
reticulum (ER) exist in sleeve structures, and this ER-like structure could be
a Ca2+ store operated by cell swelling in hypotonic conditions. It
is well known that inositol triphosphate and ryanodine receptors attached to
the ER, a Ca2+ store, and mobilize Ca2+ from this store
into cytoplasm (Berridge,
1993). Hypotonic shock could then induce mobilization of
Ca2+ via the inositol triphosphate and ryanodine
receptors. Osmolality regulation of these two receptors has not, however, yet
been reported, and it is possible that the increase in
[Ca2+]i is the result of rupture of ER-like structures
in the sleeve caused by cell swelling. Low osmolality has an inhibitory effect
on sperm motility in cyprinid fish, however, and tip of flagellum of carp
sperm became folded and swollen within 90-120 s of initiation of motility in
freshwater, resulting in serious damage to sperm function
(Perchec et al., 1996
). Sperm
of tilapia must therefore have a means of overcoming potential damage in
conditions of low osmolality.
Protein phosphorylation occurs during initiation and activation of sperm
motility in salmonid fish, echinoderms and mammals
(Morisawa, 1994;
Bracho et al., 1998
;
Nomura et al., 2000
). The
present study shows that serine and threonine residues of various proteins are
phosphorylated and dephosphorylated in solutions that support motility. Since
phosphorylation and dephosphorylation occur in many proteins, our criterion
for detecting motility-associated phosphophorylation was that since the
motility activation occurred within several seconds, any phosphorylation or
dephosphorylation events associated with motility activation must occur within
a time equal to or quicker than that of the motility activation. In the
present study, we therefore prepared protein samples from sperm for analysis 1
min after the activation.
Phosphorylation at threonine residues occurred in a 41 kDa protein independently of extracellular [Ca2+] (Fig. 5) and dephosphorylation at serine and threonine residues occurred in 18 kDa and 15 kDa proteins (Figs 4 and 5). It is likely that these proteins phosphorylation and dephosphorylation of these proteins was probably linked with increased [Ca2+]i (Fig. 2), and possibly with the Ca2+ uptake mechanisms. Whether Ca2+ uptake also involved activation of kinases such as protein kinase C remains for to be investigated. It is likely that the phosphorylation/dephosphorylation of these proteins in hypotonic solutions is associated with initiation of motility and flagellar activation via dynein activation.
18 kDa and 15 kDa proteins are similar in molecular mass to the 15 kDa
protein that plays an important role in initiation process of sperm motility
in salmonids (Morisawa and Hayashi, 1986;
Hayashi et al., 1987;
Jin et al., 1994
), although
phosphorylation in the latter protein occurred at tyrosine residues. A 15 kDa
protein was phosphorylated during initiation of motility of chum salmon sperm,
in contrast to the dephosphorylation of 15 kDa and 18 kDa proteins seen in
tilapia sperm under hypo-osmotic conditions. Therefore, it is possible that
these proteins are not the same as the 15 kDa proteins in chum salmon sperm,
where increased [Ca2+] is required to stimulate cAMP synthesis
needed to initiate sperm motility (Jin et al., 2000), via protein
phosphorylation cascades. It is thus likely that not only protein
phosphorylation but also protein dephosphorylation play important roles in the
sperm motility activation process under hypotonic conditions in sperm of
freshwater-acclimated tilapia.
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Acknowledgments |
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