Comparative Biochemistry and Physiology, Part A 143 (2006) 361 – 367
www.elsevier.com/locate/cbpa
Changes in sperm motility in response to osmolality/Ca 2+ in three Indonesian
fresh water teleosts: Goby (Oxyeleotris marmorata), Java carp
(Puntius javanicus), and catfish (Clarias batrachus)
Masaya Morita a,e,⁎, Makoto Okuno b , Endang Sri Susilo c , Bambang Pramono Setyo d ,
Diptarina Martarini d , Lilik Harnadi a,e , Akihiro Takemura e
a
Department of Chemistry, Biology and Marine Sciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan
b
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
c
Department of Marine Sciences, Faculty of Fisheries and Marine Sciences Diponegoro University, Indonesia
d
Fresh water Fish Hatchery Center, Ngrajeg, Magelang-Fisheries and Marine Sciences, Diponegoro University, Indonesia
e
Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, 3422 Sesoko, Motobu, Okinawa 907-0227, Japan
Received 27 May 2005; received in revised form 12 December 2005; accepted 18 December 2005
Available online 3 February 2006
Abstract
Sperm of most fresh water teleosts become motile when released into the hypotonic fresh water environment, but the role of osmolality
and Ca2+ on sperm motility is not clear. Osmotic pressure and Ca2+ concentrations increase from fresh water to brackish water. Java carp Puntius
javanicus and catfish Clarias batrachus live and reproduce only in fresh water. On the other hand, goby Oxyeleotris marmorata can acclimate
and reproduce from fresh water to brackish water. In the present study, sperm motility and trajectory were compared among these three
Indonesian endemic species. Sperm of Java carp, goby, and catfish begun to move in the hypotonic condition (b200 mOsm/kg). However, the
response to Ca2+ was different among these teleosts. In the presence of Ca2+, Java carp sperm swam in circular paths and immediately become
quiescent, suggesting that Java carp sperm motility is activated in hypotonic aquatic environment without Ca2+. Goby sperm swam
straightforward in the presence or absence of Ca2+. Percentages of motile sperm increased in 100–200 mOsm/kg but suppressed by removal of
Ca2+. Regarding sperm motility and trajectory, no response was found in catfish sperm. These results suggest that a response to Ca2+ is different
among sperm of the three species and suited to their habitat.
© 2005 Elsevier Inc. All rights reserved.
Keywords: Sperm motility; Ca2+; Osmolality; Flagella; Teleost; Goby; Catfish; Carp
1. Introduction
Sperm motility is necessary for efficient fertilization of eggs. In
teleosts, regulatory mechanisms of sperm motility are known to be
diverse, even though their flagellar axoneme conserves “9 + 2
structure”. Most teleost sperm are quiescent in the testes, because
the osmolality of the testicular fluid is isotonic. Sperm motility is
initiated when they are released to extracorporeal environment
such as hypotonic fresh water or hypertonic seawater. In fresh
⁎ Corresponding author. Department of Chemistry, Biology and Marine
Sciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213,
Japan. Tel.: +81 980 47 6215.
E-mail address: [email protected] (M. Morita).
1095-6433/$ - see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2005.12.020
water teleost sperm, flagellar motility is initiated by the hypoosmotic shock, whereas in marine teleost flagellar motility is
initiated by hyper-osmotic shock (Morisawa and Suzuki, 1980).
Furthermore, in medaka (Inoue and Takei, 2002, 2003;
Kawaguchi et al., in press) and tilapia (Oreochromis mossambicus) (Linhart et al., 1999; Morita et al., 2003, 2004), motility
regulatory mechanisms of sperm flagella are modulated to suit the
spawning environment when they are in fresh water or acclimated
to seawater. In herring sperm, motility initiation required trypsin
inhibitor like sperm-activating peptide from the eggs (HSAPS),
and the sperm exhibited chemotaxis when they are close to eggs
(Yanagimachi and Kanoh, 1953; Yanagimachi, 1957; Yanagimachi et al., 1992; Griffin et al., 1996; Oda et al., 1998). Although
regulation of sperm motility is diverse as described above, it
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M. Morita et al. / Comparative Biochemistry and Physiology, Part A 143 (2006) 361–367
appears that the regulatory mechanisms are suited to the
inhabiting environments.
Aquatic environment contains several ions such as Na+
and Ca 2+ and these ionic concentrations increase as
osmolality increases from fresh water to seawater. It is likely
that osmolality and ion composition affect sperm motility
regulatory mechanisms. In euryhaline tilapia, sperm motility
of fresh water-acclimated tilapia and those of seawateracclimated tilapia are different (Morita et al., 2003, 2004).
Studies on euryhaline tilapia have suggested that sperm of
fresh water-acclimated tilapia do not require extracellular Ca2+
but sperm of seawater-acclimated tilapia require extracellular
Ca2+. These reports also imply that a response to extracellular
Ca2+ is different between teleost fishes, which live in low and
high osmolality. One clue for the modulation of sperm
flagellar motility regulatory mechanisms is Ca2+ concentration
of spawning ground. Fresh water does not contain large
amount of Ca2+ compared to seawater. Thus, it is speculated
that tilapia creates sperm in response to osmolality and
extracellular Ca2+ concentration. In addition, mode of Ca2+
mobilization via Ca2+ channel for motility activation may also
be associated. It is possible that other teleost fishes inhabit
both in fresh water and brackish water have same features to
Ca2+ and osmolality.
In this study, we focused on fresh water teleost fishes, which
inhabit both in fresh water and brackish water to study the effect
of Ca2+ on sperm motility We observed sperm motility in three
species of Indonesian fresh water teleosts. The goby
(Oxyeleotris marmorata) can acclimate from fresh water to
brackish water. In contrast, the Java carp (Puntius javanicus),
and the catfish (Clarias batrachus), live only in fresh water. As
indicated above, the concentrations of ions, including Ca2+ in
their habitat increase from fresh water to brackish water. How do
sperm of fresh water teleosts acclimate and utilize environmental
differences between fresh water to brackish water? We,
therefore, focused on the effect of extracellular osmolality and
Ca2+ concentration on sperm motility of these fishes. In the
presence of Ca2+, sperm motility of goby increased in the
isotonic condition, however in Java carp and catfish similar
changes were not observed. It is likely that sperm motility
regulatory mechanisms of fresh water teleosts are also suited to
their habitat even though their habitat is hypotonic.
2. Materials and methods
2.1. Fish
Three species of freshwater fishes were used: the goby
(O. marmorata), the Java carp (P. javanicus), and the catfish
(C. batrachus). They were bred in a freshwater hatchery
center of Ngrajek located at Magelang city, central Java,
Indonesia.
2.2. Sperm collection
Sperm of Java carp were collected by inserting disposable
pipette into the sperm duct. Sperm of goby and catfish was not
possible to collect by pressing the abdomen. Hence, these fish
were anesthetized with 0.1% (v/v) 2-phenoxyethanol, and testes
were dissected out from the abdomen. Sperm were collected at
close to sperm duct with a fine disposable transfer pipette from
the isolated testes. Collected sperm were stored at room
temperature and used within 3 h. Sperm motility in the absence
of any treatments was not affected over the course of the
experiments. All experiments were carried out at room
temperature 20–25 °C.
2.3. Motility observation
NaCl and KCl were used as electrolytes and mannitol as a
nonelectrolyte. All solutions contained 10 mM Hepes–NaOH
buffer, pH 8.0. Five-millimolar CaCl2 was added to the NaCl
solutions to examine the effect of Ca2+ on sperm motility.
Semen was diluted into 40 μL of solution on a glass slide and
sperm movements were recorded using a video recorder (GRDVL700, Victor, Japan) and a CCD camera (63W1N,
MINTRON, Taiwan) mounted on a phase contrast microscope
(DAIKO SCIENCE Co. Ltd, Japan). Percentage of motility was
counted from the video recordings. Sperm were counted as
motile when they exhibited either progressive movement or
spontaneous flagellar beatings even when sperm head was
attached to the glass slide. Trajectory was traced using OHP
sheet from video recordings.
2.4. Statistical analysis
The data were subjected to two-way ANOVA followed by
Fisher's PLSD for multiple-group comparisons of motility
within the same osmolality or different osmolalities. Bonferroni's post-hoc test was used whenever any significant differences
were observed between treatments.
3. Results
3.1. Effect of osmolality on sperm motility of goby, Java carp,
and catfish
In Java carp and catfish, sperm began to move after dilution
in 10 mM Hepes buffer and stopped moving after 22.7 ± 2.5 and
28.5 ± 2.1 s (means ± S.D.), respectively. Goby sperm showed
motility for more than 20 min.
Sperm of these fishes were diluted with various concentrations of electrolytes (NaCl and KCl) and a nonelectrolyte
(mannitol). As shown in Fig. 1, Java carp sperm showed about
80% motility when they were diluted into hypotonic solutions
below 100 mOsm/kg. In KCl solution, sperm were motile up to
100 mM (200 mOsm/kg), however, the motility decreased to
about 20% at 125 mM (250 mOsm/kg). In NaCl solution, the
motility decreased to 60% at 100 mM (200 mOsm/kg) and
almost to 0 at 125 mM (250 mOsm/kg). In mannitol solution, the
motility decreased to 60% at 150 mM (150 mOsm/kg),
30% at 200 mM (200 mOsm/kg) and almost 0 at 250 mM
(250 mOsm/kg). Effect of NaCl, KCl and mannitol above
150 mOsm/kg on sperm motility was different in Java carp sperm.
M. Morita et al. / Comparative Biochemistry and Physiology, Part A 143 (2006) 361–367
100
100
80
NaCl
KCl
Mannitol
60
40
Motility (%)
Motility (%)
80
NaCl
60
KCl
Mannitol
40
20
20
0
0
10
50
100
150
200
Osmoality (mOsm/kg)
250
50
100
150
200
250
300
Osmolality (mOsm/kg)
0
25
50
75
100
NaCl or KCl (mM)
125
150
0
50
100
150
Mannitol (mM)
250
300
200
10
300
Fig. 1. Effect of osmotic pressure of electrolytes and a nonelectrolyte on sperm
motility of Java carp Puntius javanicus. Sperm were suspended in 10 mM
Hepes–NaOH, pH 8.0, containing different concentrations of electrolytes (NaCl
and KCl) and a nonelectrolyte (mannitol). Motility was shown in NaCl (blank
bar) and KCl (grey bar) and in mannitol (black bar). Values were means ± S.D.;
N = 103–138 sperm from 3 fish for each point.
100
0
25
50
75
100
NaCl or KCl (mM)
125
150
0
50
100
150
Mannitol (mM)
250
300
200
Fig. 3. Effect of osmotic pressure of electrolytes and a nonelectrolyte on sperm
motility of goby Oxyeleotris marmorata. Sperm were suspended in 10 mM
Hepes–NaOH, pH 8.0, containing different concentrations of electrolytes (NaCl
and KCl) and a nonelectrolyte (mannitol). Motility was shown in NaCl (blank
bar) and KCl (grey bar) and in mannitol (black bar). Values were means ± S.D.;
N = 108–137 sperm from 3 fish for each point.
A)
100
NaCl
*
NaCl+5 mM CaCl2
80
Motility (%)
In catfish, like as in Java carp sperm, sperm was motile under
hypotonic conditions (Fig. 2). Approximately 80% sperm
showed motility from 10 to 100 mOsm/kg in NaCl, KCl and
mannitol solutions. However, motility gradually decreased at
150 mOsm/kg and was completely suppressed at 250 mOsm/kg
solutions. Sperm motility was not significantly different at the
same osmotic pressure solutions of electrolytes and a nonelectrolyte. The motility of catfish sperm was longer duration at
75 mOsm/kg than at osmolality below 50 mOsm/kg (Fig. 6B).
80
NaCl+5 mM EGTA
60
40
20
NaCl
Motility (%)
363
60
0
KCl
10
50
100
150
200
250
300
Osmolality (mOsm/kg)
Mannitol
B)
40
20
0
10
50
100
150
200
Osmolality (mOsm/kg)
250
300
0
25
50
75
100
NaCl or KCl (mM)
125
150
0
50
100
150
Mannitol (mM)
250
300
200
Fig. 2. Effect of osmotic pressure of electrolytes and a nonelectrolyte on sperm
motility of catfish Clarias batrachus. Sperm were suspended in 10 mM Hepes–
NaOH, pH 8.0, containing different concentrations of electrolytes (NaCl and
KCl) and a nonelectrolyte (mannitol). Motility was shown in NaCl (blank bar)
and KCl (grey bar) and in mannitol (black bar). Values were means ± S.D.;
N = 104–256 sperm from 3 fish for each point.
-Ca2+
+Ca2+
Fig. 4. Effect of extracellular Ca2+ on (A) sperm motility and (B) trajectory of
goby Oxyeleotris marmorata. Sperm were suspended in NaCl solution (blank
bar), NaCl solution containing 5 mM CaCl2 (grey bar) or 5 mM EGTA (black
bar). There were significant differences between motility in the presence of
5 mM CaCl2 and that in the absence or chelated of Ca2+. ⁎P b 0.01 Motility in
solutions more than 100 mOsm/kg. Values were means ± S.D.; N = 115–
263 sperm from 3 fish for each point.
364
M. Morita et al. / Comparative Biochemistry and Physiology, Part A 143 (2006) 361–367
In goby, about 80% sperm showed motility from 10 to
100 mOsm/kg in mannitol solutions (Fig. 3: black bars), but
the motility gradually decreased at 150 mOsm/kg and was
completely suppressed at 250 mOsm/kg. In NaCl solutions,
motility decreased above 150 mOsm/kg and was almost
completely suppressed at 200 mOsm/kg (Fig. 3: blank bars),
whereas, in KCl solutions, sperm motility decreased above
50 mOsm/kg and was suppressed at 150 mOsm/kg (Fig. 3:
grey bar). In goby sperm, large amount of K+ was inhibitory
on sperm motility. By contrast, in mannitol, sperm retained
motility even at high osmolality. The effect of K+ and mannitol
on motility in the goby sperm was quite different from those of
Java carp sperm.
sperm motility was suppressed at osmolalities greater than
100 mOsm/kg (Fig. 4A: black bar), suggesting that Ca2+
increases the motility in goby sperm as observed in tilapia
(Morita et al., 2003). Trajectory of swimming sperm showed
straightforward even in the presence or absence of Ca2+ (Fig.
4B). Motility duration also did not change with or without
Ca2+ (data not shown).
The sperm motility response to extracellular Ca2+ in the Java
carp sperm was quite different compare to goby sperm. In the
presence of Ca2+, the percentage of motile sperm in Java carp
decreased at all osmolalities (Fig. 5A). Sperm swam in a circular
path and became quiescent in the presence of Ca2+ within 12 s
(Fig. 5B and C). Sperm flagella bent at proximal region like a
cane. By contrast, sperm motility increased when extracellular
Ca2+ was chelated with EGTA (Fig. 5A) and motility duration
was longer than in the presence of Ca2+ (Fig. 5B). Trajectory of
swimming sperm was also in the straightforward direction (Fig.
5C), suggesting that the extracellular calcium induces an
asymmetrical beating of flagellum. In Java carp, extracellular
Ca2+ did not regulate motility initiation but affects the
regulatory mechanism controlling symmetry of the waveform.
In catfish sperm, extracellular Ca2+ did not affect sperm
motility both in motility activation and waveform as shown in
Fig. 6A and C. Sperm motility either in the presence or
absence of Ca2+ did not change significantly. On the other
hand, the duration of sperm motility increased around 100–150
mOsm/kg from that at 0–50 mOsm/kg (Fig. 6B). Motility
3.2. Effect of extracellular Ca2+ on sperm motility
Extracellular Ca2+ concentration increases with increase in
extracellular salinity. Fresh water environment contains very
little Ca2+. Brackish water environment close to isotonic
osmolality, contains several millimolar Ca2+. In euryhaline
tilapia, O. mossambicus, acclimated to fresh water, sperm
motility increases with millimolar concentration of extracellular
Ca2+ (Morita et al., 2003).
Goby sperm motility at 150–200 mOsm/kg increased
drastically in the presence of Ca2+ (Fig. 4A). In addition, the
motility was also observed at 250 and 300 mOsm/kg osmolality.
By contrast, when extracellular Ca2+ was chelated by EGTA,
A)
B)
30
100
NaCl
25
NaCl+5 mM CaCl2
NaCl+5 mM EGTA
60
40
Motility duration (sec)
Motility (%)
80
20
0
20
-Ca2+
+Ca2+
15
10
5
10
50
100
150
200
Osmolality (mOsm/kg)
250
300
0
0
50
100
150
200
Osmolality (mOsm/kg)
C)
12 seconds later
-Ca2+
+Ca2+
Fig. 5. Effect of extracellular Ca2+ on (A) sperm motility, (B) motility duration, and (C) trajectory of Java carp Puntius javanicus. Sperm were suspended in NaCl
solution (blank bar), NaCl solution containing 5 mM CaCl2 (grey bar) or 5 mM EGTA (black bar). Sperm motility either in the presence or absence and chelated of
Ca2+ was significantly different (P b 0.005). While, motility in 200 mOsm/kg containing 5 mM CaCl2 and NaCl alone was not significantly different. (B) Motility
duration was measured in NaCl solutions containing 5 mM CaCl2 (grey bar) or 5 mM EGTA (white bar). Motility duration in the presence or absence of Ca2+ was
significantly different (P b 0.01). Values were means ± S.D.; N = 110–121 sperm from 3 fish for each point.
M. Morita et al. / Comparative Biochemistry and Physiology, Part A 143 (2006) 361–367
B)100
100
80
NaCl
Motility (%)
NaCl+5 mM CaCl2
60
NaCl+5 mM EGTA
40
80
60
-Ca2+
+Ca2+
40
20
20
0
Motility duration (sec)
A)
365
10
50
100
150
200
250
Osmolality (mOsm/kg)
300
0
0
50
100
150
200
Osmolality (mOsm/kg)
C)
-Ca2+
+Ca2+
Fig. 6. Effect of extracellular Ca2+ on (A) sperm motility, (B) motility duration, and (C) trajectory of catfish Clarias batrachus. Sperm were suspended in NaCl solution
(blank bar), NaCl solution containing 5 mM CaCl2 (grey bar) or 5 mM EGTA (black bar). Motility duration up to 1500 mOsm/kg was significantly different (P b 0.05)
between in the conditions in the presence and absence of Ca2+. Values were means ± S.D.; N = 109–164 sperm from 3 fish for each point.
duration was not altered either in the presence or absence of
Ca2+ (Fig. 6B). Trajectory of motile sperm also did not change,
and the sperm swam in a straight path either in the presence
or absence of Ca2+ (Fig. 6C).
4. Discussion
The periods of motility were different among three species.
Sperm of Java carp and catfish stopped flagellar movement
within 10 to 90 s after the initiation. On the contrary, that of
goby was more than 20 min. In the present study, we did not
demonstrate the differences in mechanism. It is likely that ways
of energy supplemented for flagellar beating of sperm in these
fishes are diverse.
Most of the teleosts sperm are quiescent in the isotonic
testicular fluid. Sperm motility is initiated when they are
released to outer environment such as hypotonic fresh water or
hypertonic seawater (Morisawa and Suzuki, 1980). Motility
regulatory mechanisms of fresh water teleosts, therefore, are
assumed to suit only hypotonic condition (fresh water). In all of
three species, sperm motility was initiated when sperm were
suspended to hypotonic solutions (Figs. 1, 2, and 3). However,
effects of electrolytes and nonelectrolytes on sperm motility
initiation were different among species. Electrolytes are known
to affect membrane potential. In common carp, sperm plasma
membrane is in depolarized state in seminal plasma, and plasma
membrane is hyperpolarized when sperm are released into
hypotonic condition (Krasznai et al., 2003). If sperm motility
initiation was not different among electrolytes and a nonelectrolyte, it is likely that osmotic pressure is only associated.
In Nernst equation, ratio of [K+]o and [K+]in denotes membrane
potential. Membrane potential is hyperpolarized when [K+]in
decreases as a result of K+ outward current through K+ channel.
It is speculated that K+ efflux does not occur when large amount
of K+ is present compare to intracellular region containing
about 60.5 mM K+ (Balkay et al., 1997; Emri et al., 1998). In
Java carp and goby, sperm motility initiation was different
among electrolytes and nonelectrolyte solutions (Figs. 1 and 3).
In goby, sperm motility was inhibited when more than 50 mM
(100 mOsm/kg) KCl was present (Fig. 3). On the other hand, in
Java carp, sperm motility initiation in the presence of more than
100 mM (200 mOsm/kg) KCl was higher than motility initiation
in a nonelectrolyte, mannitol (Fig. 1). In goby and Java carp
sperm, K+ current between intracellular region and extracellular
region may affect motility initiation. Therefore, it is possible
that membrane potential is associated with motility initiation in
Java carp sperm and goby sperm. It is also possible that an effect
of membrane hyperpolarization induced by K+ efflux is positive
on motility of goby sperm but negative on motility of Java carp
sperm. On the other hand, there was no significant difference
between the electrolytes and a nonelectrolyte in catfish sperm
(Fig. 2), suggesting that sperm motility of catfish is initiated
only by osmotic pressure.
Membrane potential is associated with the regulation of
several ion channels. It is reported in common carp sperm that a
change in membrane potential is associated with a regulation of
Ca2+ channel. In common carp sperm, change in the membrane
potential opens voltage-gated Ca2+ channels (Krasznai et al.,
1995, 2000) and as a consequence, an increase in intracellular
Ca2+ concentration is assumed to initiate sperm flagellar
motility (Cosson et al., 1989; Krasznai et al., 2000; Takai and
Morisawa, 1995). In goby, sperm motility was inhibited when
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M. Morita et al. / Comparative Biochemistry and Physiology, Part A 143 (2006) 361–367
more than 75 mM (150 mOsm/kg) NaCl was present (Fig. 3),
and sperm motility increased when extracellular Ca2+ was
present (Fig. 4A). It is possible in above 150 mOsm/kg that
membrane hyperpolarization as a result of K+ efflux induces an
increase in intracellular Ca2+ and an increase in Ca2+ is
necessary for motility initiation. On the other hand, as shown in
Fig. 5A, sperm motility in Java carp was inhibited in the
presence of Ca2+, suggesting that an increase in Ca2+ as a result
of membrane hyperpolarization seems to be harmful to motility.
In catfish sperm, Ca2+ had no effect on their sperm motility (Fig.
6A). Euryhaline tilapia sperm, like as catfish, do not require
membrane potential-dependent Ca2+ uptake from extracellular
region. However, an increase in Ca2+ is required for motility
initiation (Morita et al., 2003).
Tilapia sperm have endoplasmic-like structure at the neck
region of the head (Don and Avitalion, 1993). In catfish, there
are also some endoplasmic reticulum-like structures in the neck
region of the head (Mansour et al., 2002). If an increase in
intracellular Ca2+ is required to initiate sperm motility of
catfish, the endoplasmic reticulum-like structure is possible to
work as a Ca2+ store, supplying Ca2+ for initiation. However,
further studies are required to verify the role of Ca2+. In this
study, it is likely that an effect of an increase in Ca2+ on motility
initiation is diverse among species and membrane potential may
be associated with a regulation of Ca2+ influx. Furthermore, in
goby sperm, it is possible that an increase in Ca2+ is required for
motility initiation in high osmotic pressure.
Ca2+ concentration in aquatic environment is likely to
increase from fresh water to brackish water. Goby can acclimate
from fresh water to brackish water. Whereas Java carp and
catfish can acclimate only in fresh water. It is possible that role
(s) of Ca2+ is reflected in the Ca2+ concentration in the habitat
of these fish. In goby sperm, sperm motility increased from
150 mOsm/kg (Fig. 4A) but sperm motility of other two species
did not increase with Ca2+ (Fig. 5A and B). It is likely that sperm
of teleost fish which can acclimate to brackish water require
extracellular Ca2+ to move into high osmolality. One support to
this idea is that sperm of fresh water-acclimated tilapia do not
require extracellular Ca2+ to be motile in low osmolality
whereas Ca2+ enhances the motility around 200–600 mOsm/kg
(Morita et al., 2003). It is possible that teleost fish which can
acclimate to brackish water evolved Ca2+-dependent motility
regulatory mechanisms.
Ca2+ also affects waveform of flagellar motility. Sperm swim
circular when symmetry of flagellar beating increases. In Java
carp, sperm swam with circular path and became quiescent
quickly in the presence of Ca2+ (Fig. 5B and C), whereas sperm
swam straightforward when extracellular Ca2+ was removed
(Fig. 5C). In other species, sperm swam straightforward with or
without extracellular Ca2+ (Figs. 4B and 6B). It is likely that
only in Java carp that asymmetry of flagellar beating increased
in the presence of Ca2+. Flagellum also bent at the proximal
region near the head, referred to as the “cane shape”. In
demembranated sea urchin sperm model, asymmetry of flagella
beating increases with elevated Ca2+ concentrations (Brokaw,
1979). Furthermore, in the presence of high concentrations of
Ca2+ (10− 4 M), an increase in asymmetry of flagella leads to
“cane shape” quiescent state (Gibbons and Gibbons, 1980).
Brokaw and Nagayama (1985) reported that calmodulin
modulates the asymmetry of sea urchin sperm flagella beating.
Together these studies suggest that asymmetry of flagellar
beating is induced by Ca2+. It is plausible in Java carp that
sperm became quiescent as a result of an increase in Ca2+,
which induces an increase in the asymmetry of flagella beating.
As described above, membrane potential and Ca2+ play an
important role in regulation of flagellar motility. However,
catfish sperm did not match. In catfish sperm, Ca2+ had no effect
on sperm motility, but motility duration in 100–150 mOsm/kg
was longer than at concentrations below 100 mOsm/kg (Fig.
6B). Percentage of motile sperm in 150 mOsm/kg was lower
than 100 mOsm/kg (Fig. 2), suggesting that catfish sperm
have ability to increase fertilization success in high osmolality
(N 100 mOsm/kg) without Ca2+. The response to Ca2+ was
different between Java carp and catfish. In catfish sperm, Ca2+
had no effect on sperm motility but motility duration of catfish
increased in higher osmolality (Fig. 6A and B). On the other
hand, in Java carp sperm, Ca2+ modulates asymmetry of
flagellar beating and they became a quiescent state quickly (Fig.
5B and C). It is likely that Java carp sperm suited to low Ca2+
environments but catfish sperm may be suited to higher
osmolality environment, which contains higher Ca2+ concentrations, compare to Java carp sperm.
In conclusion, sperm motility of three Indonesian teleosts,
Java carp, goby, and catfish were initiated when they are
released in hypotonic environment (below 200 mOsm/kg).
However, the response against extracellular Ca2+ was quite
Java carp
Osmolality that
allows sperm motility
catfish
Goby
0
100
Freshwater
Osmolality (mOsm/kg)
250
Brackishwater
Ca2+ concentration
Fig. 7. Schema of possible spawning ground of Java carp, catfish and goby. As shown in Figs. 1–6, motility-feasible range was different among three species. Sperm
motility features such as percentages of motile sperm and motility duration are likely to be suited to the fresh water to brackish water in aspect of extracellular Ca2+
concentrations. Arrows indicates the osmolality that could allow sperm motility of each teleosts.
M. Morita et al. / Comparative Biochemistry and Physiology, Part A 143 (2006) 361–367
different among these three species. The Ca2+ response seen
here among the three species appear to have evolved to adapt to
either fresh water containing low Ca2+ content or brackish water
containing reliably higher Ca2+ (Fig. 7).
Acknowledgement
The authors wish to express their deepest gratitude to staff
and students of Department of Marine Sciences, Diponegoro
University. Thanks are respectfully due to head of Fisheries and
Marine Services Government of Central Java, Prof. Dr. Ir.
Slamet Budi Prayitno, MSc and staff of his office for their kind
support to carry out experiments in east Java of Indonesia.
Thanks are sincerely due to Dr. M.M. Vijayan and Dr. N. Aluru,
Department of Biology, University of Waterloo, for critically
reading the manuscript. This study is supported in part by a
Grant-in-Aid for Scientific research (B) (15405029) to AT and
21st Century COE program of the University of the Ryukyus
from the Ministry of Education, Culture, Science and
Technology, Japan to MM.
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