K+-independent initiation of motility in chum salmon sperm treated with an organic alcohol, glycerol
1 Department of Biology, Graduate School of Arts and Sciences, University of
Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan
2 Department of Physiology, Dokkyo University School of Medicine, Mibu,
Tochigi 321-0293, Japan
Author for correspondence (e-mail:
cokuno{at}mail.ecc.u-tokyo.ac.jp)
Accepted 11 October 2005
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Summary |
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Key words: sperm motility, salmon, organic alcohol, phosphorylation, cAMP, eukaryotic flagella
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Introduction |
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Interestingly, addition of extracellular Ca2+ promotes
initiation of motility, even in the presence of up to 10 mmol
l1 K+
(Tanimoto and Morisawa, 1988).
In addition, motility is suppressed by Ca2+ channel blockers
(Tanimoto and Morisawa, 1988
;
Kho et al., 2001
). These
observations together suggest that the increase in intracellular
[Ca2+], rather than efflux of K+, plays a major role in
the initiation of motility. On the other hand, Boitano and Omoto
(1991
) showed that the presence
of divalent cations other than Ca2+ induces motility initiation,
even in the presence of K+, suggesting that the effect of
Ca2+ is not mainly to initiate motility but a membrane potential
that may be associated with motility initiation. The inconsistency of these
results suggests that membrane hyperpolarization and Ca2+ influx
may act independently in increasing cAMP production. Furthermore, activation
of motility does not require the increase in cAMP in one specific condition.
Demembranated sperm requires the addition of an appropriate concentration of
cAMP and a low concentration of Ca2+ (<108.5
mol l1) to reactivate motility, and high beat frequency can
be achieved in the presence of 200 µmol l1
Mg-ATP2 (Okuno and
Morisawa, 1989
). In the presence of low concentrations of
Mg-ATP2 (<50 µmol l1), however,
addition of cAMP is not required for reactivating demembranated sperm (M.
Okuno, unpublished data). Previous studies have given reliable results that
signal transduction is required for motility initiation in salmonid fish
sperm. However, signal transduction from the plasma membrane to the flagellar
axoneme is still not fully understood. In other words, is the initiation of
motility only regulated by a production of cAMP that is induced by the
membrane hyperpolarization as a result of the decrease in extracellular
K+ and so on?
The aim of this study was to investigate a new membrane-permeabilized sperm model, which conserves membrane structure, rather than the triton-treated demembranated sperm model. The new model gives us clues about the mechanisms of signal transduction. In early cilia and flagella studies, the plasma membranes were permeabilized by treatment with 50% glycerol, and flagella motility was reactivated by an addition of exogenous ATP (Hoffman-Berling, 1955). We treated salmonid sperm with glycerol (CH2OHCHOHCH2OH) under slightly different conditions. The results were unexpected and surprising. The glycerol-treated sperm moved without addition of exogenous ATP and cAMP in the presence of a high concentration of K+, suggesting that the glycerol-treated sperm conserve their plasma membrane and K+ does not inhibit motility. Furthermore, treatment with another organic alcohol, erythritol (CH2OH(CHOH)2CH2OH), also induced motility initiation in the presence of a large amount of K+, although ethylene glycol (CH2OHCH2OH) did not.
The present study reveals another motility initiation mechanism, different from the K+-dependent process, and shows that glycerol treatment alters the regulatory mechanism for inducing motility initiation in a K+-rich solution.
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Materials and methods |
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Collection of sperm
Chum salmon Oncorhynchus keta L. return to the Ohtsuchi River in
Iwate prefecture, Japan, from the end of November to December. Fish were
collected during this period with permission of Ohtsuchi fishermen's union.
Semen was collected directly by inserting a pipette into the sperm duct and
stored on ice until use.
Organic alcohol treatment of sperm
The semen was diluted 200-fold with ice-cold glycerol solution consisting
of 10% (v/v) glycerol (1.3 mol l1 glycerol), 1.3 mol
l1 ethylene glycol or 1.3 mol l1
erythritol, 150 mmol l1 KCl, 0.5 mmol l1
DTT, 0.5 mmol l1 EDTA and 20 mmol l1
Hepes-NaOH (pH 8.0). After 10 s incubation, 1 µl of the sperm suspension
was suspended in 40 µl of experimental solution on a glass slide coated
with 1% (w/v) BSA for observation. Sperm were completely immotile in the
glycerol solution. The incubation time in the glycerol solution was critical
for motility initiation; incubation for longer than 20 s failed to activate
motility.
Observation of sperm motility
Sperm movements were recorded using a Video recorder (SLV-LF 1; Sony,
Tokyo, Japan) and a CCD camera (CD-5C; RF system, Tokyo, Japan) mounted on a
microscope (Optiphoto; Nikon, Tokyo, Japan) equipped with a dark field or
phase contrast condenser. Sperm trajectories were taken with 0.5 s exposure on
high sensitive film (SUPERIA 1600 or 800, Fuji film, Tokyo, Japan) with
microscope camera (Nikon, UFX-II) mounted on the microscope equipped with dark
field condenser. Then, velocity of sperm was calculated from the length of
trajectories and the beat frequency from the number of waves of
trajectories.
Effect of inhibitors
The semen was diluted tenfold in artificial seminal plasma (ASP) containing
inhibitors as described in Kho et al.
(2001). The composition of ASP
was 130 mmol l1 NaCl, 40 mmol l1 KCl, 2.5
mmol l1 CaCl2, 1.5 mmol l1
MgCl2 and 10 mmol l1 Hepes-NaOH (pH 7.8). ASP
also contained 0.1% (v/v) DMSO, in which inhibitors were resuspended. After an
appropriate incubation time (30 min), the sperm suspension was resuspended in
100 mmol l1 NaCl and 10 mmol l1 Hepes-NaOH
(pH 8.0) in the ratio 1:100. For glycerol treatment, the sperm suspension was
resuspended in glycerol solution for 10 s at a dilution ratio of 1:10. Then, 1
vol of the suspension was resuspended in 40 vol of the activation solution
(100 mmol l1 KCl and 10 mmol l1
Hepes-NaOH, pH 8.0).
Cyclic AMP assay
The cyclic AMP assay was carried out according to the method described
previously (Kho et al., 2001).
In the intact sperm assay, semen was resuspended in ASP at a dilution ratio of
1:10. Then, 6 µl of the sperm suspension were transferred into 600 µl of
100 mmol l1 NaCl or 100 mmol l1 KCl
solution. In the glycerol-treated sperm assay, semen was resuspended in
glycerol solution at a dilution ratio of 1:25. Then, 10 µl of the sperm
suspension were transferred into 400 µl of 100 mmol l1
NaCl, 100 mmol l1 KCl and 300 mmol l1 KCl
solutions. After the appropriate incubation time (15 s), assessment of cAMP
production was carried out using cAMP EIA kit (Biotrak RPN 225; Amersham
Pharmacia Biotech). Briefly, 360 µl of sperm suspension was mixed with 40
µl of Kit buffer (lysis reagent) to stop cAMP synthesis and dissolve the
cells. 100 µl of the mixture was then used for cAMP measurement according
to the manufacture's instructions. The amount of cAMP in each sample was
calculated by measuring absorbance at 450 nm with a micro plate reader (Model
550; Bio-Rad, Richmond, CA, USA).
Phosphorylation assay
In glycerol-treated sperm, extracellular ATP was completely unnecessary.
However, [-32P]ATP was added in glycerol solution,
activation solutions and inactivation solution to investigate phosphorylation
of proteins in glycerol-treated sperm. 20 µl of semen were resuspended and
incubated for 1 min in the ice-cold 200 µl glycerol solution containing 4
MBq ml1 [
-32P]ATP (222 TBq
mmol1) and centrifuged at 10 000 g for 5 min
at 4°C. The sperm pellet was diluted into 50 µl activation solution
(100 mmol l1 KCl or NaCl and 10 mmol l1
Hepes-NaOH, pH 8.0) or inactivation solution (300 mmol l1
KCl and 10 mmol l1 Hepes-NaOH, pH 8.0). Both of them
contained 8 MBq ml1 [
-32P]ATP (222 TBq
mmol1). After 5 min incubation at room temperature, the
suspensions were centrifuged at 10 000 g for 5 min at 4°C.
The supernatant was then decanted and the sperm pellet eluted for 1 min on ice
with 30 µl of elution solution (8 mol l1 urea and 10%
2-mercaptoethanol). Residuals were removed by centrifugation at 12 000
g for 5 min at 4°C. Then, 2x sample buffer [100 mmol
l1 Tris-HCl, pH 6.8, 4% (w/v) SDS, 12% (v/v)
2-mercaptoethanol, 20% (v/v) glycerol and0.02% (w/v) Bromophenol Blue] was
added to the supernatants and boiled for 2 min.
In the Triton model (demembranated sperm), sperm were treated with
demembranation solution [0.4% (w/v) Triton X-100, 150 mmol
l1 potassium acetate, 1 mmol l1 EDTA, 1
mmol l1 DTT and 20 mmol l1 Hepes-NaOH, pH
8.0] for 10 min on ice and centrifuged at 10 000 g for 1 min
at 4°C. The demembranated sperm were resuspended with reactivation
solution [150 mmol l1 potassium acetate, 1 mmol
l1 EGTA, 1 mmol l1 DTT, 2 mmol
l1 MgCl2, 2 MBq ml1
[-32 P]ATP (6000 Ci mmol) and 20 mmol l1
Hepes-NaOH, pH 8.0] with or without 10 µmol l1 cAMP.
Phosphorylation reactions were carried out for 5 min at room temperature and
suspensions were centrifuged at 10 000 g for 5 min at 4°C.
The supernatant was then decanted, the demembranated sperm pellet eluted and
sample prepared for SDS-PAGE as described above.
Samples were subjected to 12.5% SDS-PAGE
(Laemmli, 1970), followed by
staining with Coomassie Brilliant Blue. 32P-labeled proteins were
examined by exposing the gel to the X-ray film for 72 h at
80°C.
Statistical analysis
The data were subjected by two-way analysis of variance (ANOVA) followed by
Fisher's PLSD for multiple-group comparisons. If there was a significant
difference, the Bonferroni post-hoc test was conducted. Beat
frequency and velocity data were subjected to analysis of covariance (ANCOVA;
StatView J-5).
All experiments were carried out at room temperature (1520°C), and the results are given as means ± S.D.
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Results |
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Additions of ATP and cAMP were not required to initiate the motility of glycerol-treated sperm, whereas the demembranated sperm required ATP and cAMP to be reactivated. This observation suggests that the properties of the glycerol-treated sperm were quite different from both the intact sperm and demembranated sperm, since fluctuations in extracellular [K+] are not closely related to the initiation of motility.
Other organic alcohols, ethylene glycol (CH2OHCH2OH) and erythritol (CH2OH(CHOH)2CH2OH) were also tested (Fig. 2A). Ethylene glycol treatment did not induce motility initiation in 100 mmol l1 KCl solution (Fig. 2B), whereas erythritol treatment induced motility initiation in KCl solution (Fig. 2B).
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Sperm velocity increased significantly with beat frequency (ANCOVA, F1,80=282, P<0.0001, Fig. 4A). The slopes for glycerol-treated and intact sperm were significantly different (F1,80=77.4, P<0.0001). Dark field microscopy revealed that the trajectory pitch of glycerol-treated sperm was smaller than that of intact ones (Fig. 4B,C), indicating that swimming velocity achieved with one stroke of the glycerol-treated sperm was lower than that of intact sperm.
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Change in intracellular content of cyclic AMP on the process of motility initiation
Intracellular cyclic AMP (cAMP) concentration ([cAMP]i) was
increased in intact sperm when they were exposed to K+-free
solution (100 mmol l1 NaCl), but not to K+-rich
solution (100 mmol l1 KCl;
Fig. 6). Glycerol-treated sperm
showed vigorous motility even in K+-rich solutions.
[cAMP]i in glycerol-treated sperm, however, was not increased in
K+-rich solution even though 90% of sperm showed motility, and no
increase in [cAMP]i was observed with 300 mmol l1
KCl, although sperm did not move because of the high osmolality
(Fig. 6). It is unlikely that
[cAMP]i is important in the motility initiation process in
glycerol-treated sperm. In other words, [cAMP]i increases in the
initiation process of intact sperm motility.
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In demembranated sperm, a 18 kDa protein corresponding to the 15 kDa
protein described in Morisawa and Hayashi
(1985), dynein light chain, 29
kDa protein, PKA regulatory subunit, 80, 125 and 300 kDa proteins were
phosphorylated in a cAMP-dependent manner
(Fig. 7A). Glycerol-treated
sperm presented a very low incorporation of 32P, suggesting that
activity of protein kinase is very low. Among the phosphoproteins the PKA
regulatory subunit and 300 kDa protein were only weakly phosphorylated
(Fig. 7B; lane, glycerol
treatment). Most proteins were phosphorylated when sperm were diluted in
activating solutions. Dynein light chain, 125 and 300 kDa proteins were
phosphorylated in 100 mmol l1 NaCl and KCl solutions
(Fig. 7B). These proteins were
also phosphorylated in the demembranated sperm in a cAMP-dependent manner
(Fig. 7A). A 70 kDa protein was
phosphorylated in 100 mmol l1 NaCl and KCl solutions. This
protein was phosphorylated only in the glycerol-treated sperm, and not the
demembranated sperm (Fig. 7A).
The 18 and 29 kDa proteins in the glycerol-treated sperm were not as strongly
phosphorylated as those of the demembranated sperm
(Fig. 7A,B).
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Discussions |
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Glycerol-treated sperm motility was initiated even when a large amount of extracellular K+ was present (Fig. 1). Not only glycerol but also erythritol (Fig. 2A) induced motility initiation in the presence of a large amount of K+ (Fig. 2B), but ethylene glycol treatment did not induce motility activation (Fig. 2B). The molecular size of alcohol is important for induction of motility initiation, and must be equal to or larger than glycerol.
In intact sperm, the decrease in extracellular [K+] is
identified as a trigger for the initiation of flagellar motility.
K+ efflux via K+ channels induces
hyperpolarization of the plasma membrane
(Tanimoto and Morisawa, 1988;
Kho et al., 2001
), followed by
Ca2+ influx via the dihydropyridine-sensitive
Ca2+ channel (Kho et al.,
2001
). Kho et al.
(2001
) then postulated that
the membrane hyperpolarization activates adenylyl cyclase for the production
of cAMP. It is known that activation of adenylyl cyclase is associated with
membrane hyperpolarization in ascidian (Izumi et al., 1999) and sea urchin
sperm (Beltran et al., 1996
).
However, in the present study, hyperpolarization did not occur with a high
concentration of extracellular K+ in glycerol and
erythritol-treated sperm. Moreover, cAMP production was not observed in the
K+-rich condition. Motility of glycerol-treated sperm was not
strongly inhibited by an adenylyl cyclase inhibitor, SQ22536
(Fig. 5B). Furthermore, the
present study shows that an increase in [cAMP]i did not occur in
glycerol-treated sperm (Fig.
6). By contrast, [cAMP]i of intact sperm increased in
the motility-permissive 100 mmol l1 NaCl solution
(Fig. 6). Therefore, in
glycerol-treated sperm, it is likely that initiation of flagellar motility is
not associated with the cAMP synthesis pathway, which is induced by
hyperpolarization as a result of K+ efflux and Ca2+
influx in intact sperm. It is possible that the motility regulatory mechanism
of glycerol-treated sperm is different from that in intact sperm.
As described above, cAMP production was not necessary for motility
initiation in glycerol-treated sperm. However, a PKA inhibitor, H-89, weakly
inhibited motility initiation of glycerol-treated sperm
(Fig. 5C). PKA exists as an
inactive tetramer, consisting of two regulatory and two catalytic subunits, at
low cAMP concentrations (Robinson,
1970). Binding of cAMP to PKA regulatory subunits causes the
release of PKA catalytic subunits, inducing PKA activation and resulting in
protein phosphorylation (Taylor et al.,
1990
). Therefore, activation of PKA is tightly related to the
increase in cAMP. The present study shows that the cAMP concentration
increased on motility activation without K+, as shown in
Fig. 6, in agreement with
previous studies that demonstrated an increase in cAMP followed by protein
phosphorylation (Morisawa and Hayashi,
1985
; Hayashi et al.,
1987
; Inaba et al., 1989). This means that protein phosphorylation
could not occur unless cAMP was produced. However, the present experiments
show that dynein light chain and PKA regulatory subunit of the
glycerol-treated sperm were phosphorylated in both 100 mmol
l1 NaCl and KCl solutions
(Fig. 7B). These proteins are
known to be phosphorylated in a cAMP-dependent manner
(Fig. 7A;
Inaba, 2002
;
Inaba et al., 1998
). Therefore,
it is plausible that PKA is activated without the synthesis of cAMP. It is
possible that glycerol- or erythritol treatment can release the regulatory
subunits from an inactive PKA tetramer in the absence of cAMP. Then, the
following question arises. Does organic alcohol itself affect the PKA state?
If this is the case, proteins under the regulation of PKA could be
phosphorylated during the glycerol treatment. However, 32P-uptake
in sperm was very low in the glycerol solution, except that the PKA regulatory
subunit was weakly phosphorylated (Fig.
7B). Large amounts of 32P-uptake occurred after sperm
were treated with 100 or 300 mmol l1 KCl. It is speculated
that the high osmolality of glycerol solution (about 1300 mOsm
kg1) inhibited the kinase activity in the presence of
glycerol. Then, kinase activity is likely to rise as osmolality decreases.
As shown in Figs 6 and
7B, synthesis of cAMP is not
required to induce protein phosphorylation of glycerol-treated sperm, although
the 18 and 29 kDa proteins were not as strongly phosphorylated as those in
demembranated sperm (Fig.
7A,B). In Paramecium cilia, phosphorylation of a 29 kDa
protein regulates the sliding velocity of microtubules and swimming speed
(Hamasaki et al., 1991). The
swimming speed of glycerol-treated sperm at the same beat frequency was
significantly lower than that of non-treated intact sperm
(Fig. 4A). It is possible that
phosphorylation of the 29 kDa protein regulates the swimming speed of salmonid
fish sperm by changing the wave form via the sliding velocity of
microtubules. But further studies are necessary to elucidate the
mechanisms.
Glycerol- and erythritol-treated sperm exhibited surprising features, not only in sperm flagellar motility but also in protein phosphorylation, which is believed to be important in cell signaling. Further studies could provide more interesting and valuable information.
We express thanks to the staff of Otsuchi Kaiyo Center of the Ocean Research Institute of University of Tokyo, Fishery Institutes of Nakagawa, Tochigi prefecture and Otsuchi, Iwate prefecture, Japan. We also wish to thank Dr S. Awata, COE postdoctoral fellow of University of the Ryukyus, for teaching the statistical required for this study. This study was supported in part by a Grant-in-Aid for Scientific Research(B) (15405029) to M.O. and 21st century COE program of the University of the Ryukyus from the ministry of Education, Culture, Sports, Science and Technology, Japan, to M.M.
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Footnotes |
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References |
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