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First published online November 1, 2006
Journal of Experimental Biology 209, 4574-4579 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02500
Eggs regulate sperm flagellar motility initiation, chemotaxis and inhibition in the coral Acropora digitifera, A. gemmifera and A. tenuis
1 Department of Chemistry, Biology and Marine Sciences, University of the
Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan
2 Sesoko Station, Tropical Biosphere Research Center, University of the
Ryukyus, 3422 Sesoko, Motobu, Okinawa 907-0227, Japan
3 Department of Life Sciences, Graduate School of Arts and Sciences,
University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
4 Comparative Genomics Centre, Molecular Science Building, James Cook
University, Townsville, Queensland 4811, Australia
* Author for correspondence (e-mail: h063107{at}sci.u-ryukyu.ac.jp)
Accepted 17 August 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResults and discussionReferences |
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Key words: coral, Acropora, mass spawning, sperm, chemotaxis, flagellar motility
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResults and discussionReferences |
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). The importance of the mechanism could be an increase of
fertilization efficiency. Corals spawn simultaneously around the night of the
full moon in a phenomenon known as `simultaneous mass spawning'
(Harrison et al., 1984;
Babcock et al., 1986;
Hayashibara et al., 1993). Most
species release gamete bundles, which is a complex of sperm and eggs, into the
seawater (Harrison and Wallece,
1990). The bundles dissipate sperm and eggs to float separately on
the surface of the water where fertilization occurs. There are sperm and eggs
from many species in seawater during mass spawning. Thus, sperm and eggs from
the same species must find each other for efficient fertilization in the
presence of sperm and eggs from many species. We hypothesized that chemotaxis
might be involved. It is possible that sperm/egg have a mechanism(s), which
increases fertilization through regulation of sperm flagellar motility in
order to reach the egg. Within synchronously spawning species of
Acropora, rates of intraspecific fertilization in experimental
crosses are high (often >90%) compared with the rates of fertilization in
many interspecific crosses (Willis et al.,
1997; Hatta et al.,
1999). It has been suggested in Montipora digitata that
interspecific fertilization is reduced by a lack of the sperm attractant in
eggs (Coll et al., 1994).
Acropora species may also have regulatory mechanisms of sperm
flagellar motility to increase fertilization within the same species.
In ascidians, sea urchins and clupeids, eggs secrete a sperm-activating and
-attracting factor to initiate sperm flagellar motility and attract sperm. The
substances, which initiate and attract sperm motility, can range from steroids
to peptides (Coll et al., 1994;
Oda et al., 1998;
Yoshida et al., 2002). In
ascidians, asymmetric flagellar beating that results in a circular trajectory
increases with sperm-activating and attracting factor (SAAF). Sperm swim
towards the eggs, responding to chemoattractant
(Yoshida et al., 1993). In
addition, chemotaxis by SAAF is induced by an increase in
[Ca2+]i, and this involves regulation of the symmetrical
beating of flagella (Yoshida et al.,
2003). Ca2+/calmodulin is associated with the
symmetrical flagellar beating as a result of an increase in
[Ca2+]i in sea urchin sperm
(Brokaw and Nagayama, 1985).
Navigation mechanisms may play an important role in increasing fertilization
success.
Sperm from many species of corals are present during mass spawning. If
chemoattractant(s) is not species-specific, sperm from different species
approach the egg. In addition, polyspermy, which inhibits embryo development
(Oliver and Babcock, 1992),
might occur if eggs do not stop attracting sperm after fertilization or in
response to attachment of many sperm. Therefore, eggs should be able to
regulate sperm motility in a species-specific manner and have a mechanism to
prevent polyspermy. In this study, we identified that sperm of
Acropora have navigation mechanisms and motility inhibition
mechanisms to prevent polyspermy. It is probable that sperm motility
regulatory mechanisms are suited to the spawning phenomenon of
Acropora.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResults and discussionReferences |
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Collection of sperm and eggs
Coral spawning took place in June [3rd, 7th, 8th, 10th, 14th, 22nd (full
moon), 25th and 27th]. Colonies were transferred into individual buckets until
gamete bundles in the polyps were confirmed. Gamete bundles from the colony
were collected after they were released. The following experiments were
conducted at about 28°C. Gamete bundles were washed with filtered seawater
and split into eggs and sperm using a plankton net (diameter 100 µ m). Eggs
were washed 10 times with filtered seawater in order to wash sperm away from
eggs. Detachment of sperm from washed eggs was confirmed by microscopic
observation. Sperm concentration was determined using a Thoma counting chamber
(Kayagaki Iruka Kogyo, Tokyo, Japan). Sperm were stored at room temperature at
1.0x107-9 cells ml-1. Sperm and eggs were used for
experiments within 5 h after collecting.
Solutions
Solutions used to test sperm motility were as follows: artificial seawater
(ASW: 430 mmol l-1 NaCl, 10 mmol l-1 CaCl2, 9
mmol l-1 KCl, 23 mmol l-1 MgCl2, 25 mmol
l-1 MgSO4 and 10 mmol l-1 Hepes-NaOH, pH
8.2), and Na-free ASW (430 mmol l-1 choline chloride, 9 mmol
l-1 KCl, 23 mmol l-1 MgCl2, 25 mmol
l-1 MgSO4, 10 mmol l-1 CaCl2 and
10 mmol l-1 Hepes-KOH, pH 8.2), Na-free ASW containing 10µ mol
l-1 Ca2+ ionophore A23187, or Ca2+-chelated
ASW (430 mmol l-1 choline chloride, 9 mmol l-1 KCl, 23
mmol l-1 MgCl2, 25 mmol l-1 MgSO4,
5 mmol l-1 EGTA and 10 mmol l-1 Hepes-KOH, pH 8.2)
containing 10 mol l-1 Ca2+ ionophore A23187.
NH4Cl (20 mmol l-1) was added to each solution to
increase intracellular pH.
Motility assessment
Sperm flagellar motility was recorded using a video recorder (SLV-LF1;
Victor, Tokyo, Japan/DCR-TRV900; Sony, Tokyo, Japan) and a CCD camera (63W1N,
MINTRON, Taiwan) mounted on a microscope equipped with a phase-contrast or
dark-field condenser (Optiphoto; Nikon, Tokyo, Japan). Motility percentage was
calculated 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. The trajectory of sperm motility
was either recorded under phase-contrast illumination and movement of sperm
heads traced by macros constructed in NIH Image as described by Kihara
(Kihara, 1997), or under
dark-field illumination by modulating the shutter speed of a CCD camera. The
swimming speed was then calculated from the length of trajectory using NIH
image.
Hybridization experiments
Thirty eggs were put into 100 ml of filtered seawater. Sperm were diluted
with seawater to 1.1x105-1.2x108 cells
ml-1 to prevent polyspermy, according to Oliver and Babcock
(Oliver and Babcock, 1992).
Survival rates of eggs were calculated. Unfertilized eggs broke up the next
day. During these experiments, room temperature was kept at 28°C.
Statistical analysis
Data were subjected to one-way ANOVA followed by Fisher's PLSD for
multiple-group comparisons of motility within different solutions.
Bonferroni's post-hoc test was used whenever any significant
differences were observed between treatments.
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Results and discussion |
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TOPSummaryIntroductionMaterials and methodsResults and discussionReferences |
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). As described elsewhere
(Fukami et al., 2003), A.
digitifera and A. gemmifera spawned from 21:00 h to 22:00 h, and
A. tenuis spawned from 19:00 h to 20:00 h. Similar to starfish sperm,
sperm of these three species were completely immotile in seawater
(Fig. 1A). Sperm of most of
marine invertebrates are quiescent in seawater and begin to move in response
to several signals, some of which are steroids or peptides from eggs
(Yoshida et al., 2002;
Morisawa, 1994). In starfish
sperm, an increase in intracellular pH ([pH]i) induces motility
activation through axonemal protein phosphorylation
(Nakajima et al., 2005). When
sperm were suspended in artificial seawater (ASW) containing NH4Cl,
which increases [pH]i, sperm flagella bent in the proximal region
but the bending motion did not propagate distally (to the tip of the
flagellum), to create the cyclical sinusoidal bending motion. Instead, sperm
showed vigorous motility when NaCl was replaced by choline chloride (Na-free
ASW containing NH4Cl) (Fig.
1B). Addition of a membrane-permeable cAMP, dbcAMP (50 µ mol
l-1), did not initiate motility (data not shown), suggesting that
an increase in [pH]i, not an increase in [cAMP]i,
induces initiation of flagellar motility, and Na+ antagonizes
elevation of [pH]i even though NH4Cl was added. It is
likely that sperm flagellar motility of Acropora species is
suppressed by Na+ in seawater.
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).
These reactions were almost similar among three Acropora species. It
is possible that each species should have species-specific mechanism(s) to
initiate and attract sperm to choose appropriate eggs, which are not
fertilized. Acropora eggs may secret species-specific substance(s) to
activate and attract sperm.
Sperm flagellar motility was initiated by NH4Cl treatment and
sperm became quiescent within 1 min when they came close to eggs after many
sperm had attached to the egg surface (Fig.
3A,B). In the absence of eggs, sperm showed motility for a longer
period (Fig. 3B), suggesting
that a secretion of motility inhibition substance(s) from eggs occurs when
many sperm attach to the egg. In addition, quiescent sperm reinitiate their
flagellar motility by a re-addition of NH4Cl, suggesting that a
decrease in [pH]i causes a suppression of sperm motility. It is
possible that an inhibition of flagellar motility by eggs is reversible. Sperm
motility and swimming velocity decreased quickly unlike those in the absence
of an egg (Fig. 3B,C). However,
swimming speed was faster in the first 10 s after dilution
(Fig. 3C), implying that
fertilization occurs within 10 s and the egg begins to secret motility
suppressor(s). In ascidians, sperm-activating and -attracting activities of
the egg disappear when fertilization is completed
(Yoshida et al., 1993).
Therefore, swimming velocity could increase in the presence of eggs that are
not fertilized. However, swimming velocity of sea urchin sperm is not changed
in the presence of asterosap isoform p15
(Shiba et al., 2006). It is
reasonable to assume that the egg secretes motility suppressor(s) to prevent
polyspermy, which inhibits embryo development
(Oliver and Babcock, 1992).
One clue about the evolution of a sperm motility inhibition mechanisms is that
eggs of coral do not have a fertilization membrane, which develops after
fertilization to prevent sperm entry
(Babcock and Heyward, 1986;
Hayashibara et al., 1997). In
corals, many different species spawn at once, and many eggs and sperm from
other species are present during mass spawning. Mechanism(s) to restrict sperm
motility may be useful to increase the fertilization success.
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This study suggests that an elevation of [pH]i induces motility
initiation and a decrease in [pH]i suppresses motility. However,
chemotactic behaviour of sperm, swimming in a straight path, did not occur
with the addition of NH4Cl. In the ascidians Ciona
intestinalis and C. savignyi, an increase in intracellular
Ca2+ concentration ([Ca2+]i) induces an
increase in asymmetrical flagellar beating
(Yoshida et al., 1994;
Yoshida et al., 2003). In
contrast to ascidians, Acropora sperm swam straight when they
approach the egg (Fig. 2A,
Fig. 3A). It is possible that
Ca2+ affects chemotactic behaviour of sperm. To elucidate the
role(s) of Ca2+ in chemotaxis, the effect of Ca2+ on
trajectory and swimming velocity was considered. In A. digitifera,
sperm swam straight in a solution containing EGTA and the Ca2+
ionophore A23108 (which decreases [Ca2+]i)
(Fig. 4A). By contrast, sperm
swam in circles when extracellular Ca2+ and Ca2+
ionophore A23187 was present (Fig.
4B,C). Sperm swam slower in the presence of Ca2+ or
Ca2+ and A23187 (Fig.
4D). Therefore, a reduction of [Ca2+]i
induces an increase in swimming velocity and symmetrical flagellar beating. It
is likely that chemotaxis does not depend on a rise of [pH]i but
regulation of [Ca2+]i.
|
). In
higher Ca2+ concentrations, sperm swim circular paths as a result
of an increase in asymmetry of flagellar beating through
Ca2+/CaM-dependent regulation. It is possible that this type of
regulation exists in Acropora. However, further studies are required
to identify the role of CaM on chemotactic behaviour in Acropora
sperm.
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). Acropora species may have species-specific
substance(s), which induces sperm flagellar motility initiation, implying that
fertilization among A. digitifera, A. gemmifera and A.
tenuis could not occur as a result of the lack of sperm flagellar
motility initiation by eggs from different species. Sperm of each
Acropora species did not respond to eggs of the other two species,
suggesting that a substance(s) from eggs that induces flagellar motility
initiation is species specific. In mass spawning, eggs of different species
should be present in the seawater. It is likely that sperm begin to move when
they come in close to eggs of the same species. Therefore, hybridization among
different species could be avoided and sperm and eggs of same species could
undergo fertilization.
As shown above, sperm did not respond to eggs from other species in the
present study, because motility initiation occurred in a species-specific
manner. However, hybridization does occur among some Acropora species
(Hatta et al., 1999). Hatta et
al. (Hatta et al., 1999)
described that 71.5±28.5% fertilization success was observed when sperm
of A. nobilis and eggs of A. florida were mixed. It seems
that eggs of A. florida activates A. nobilis sperm. On the
other hand, fertilization did not occur when sperm of A. digitifera
and eggs of A. florida were mixed
(Hatta et al., 1999). Thus, it
is possible that specificity of activation factor(s) from eggs is different
among species. One possible reason why hybridization within the genus
Acropoa occurs is that Acropora have possibly evolved in
reticulate ways (Veron,
1995a). Some species could have the same activating substrate(s)
and fertilization mechanism(s). Further studies are necessary to elucidate the
importance of substances that initiate sperm motility and attract sperm during
fertilization among different species.
To gain insight into this relationship, hybridization experiments among
A. digitifera, A. gemmifera and A. tenuis were carried out.
Motility initiation did not occur among these species. Hybridization among
A. digitifera, A. tenuis and A. gemmifera did not completely
occur. Therefore, it is possible that initiation of sperm flagellar motility
in a species-specific manner plays an important role in fertilization.
However, A. nobilis hybridizes with A. florida
(Hatta et al., 1999), but it is
unknown whether sperm of A. nobilis show
initiation/chemotaxis toward eggs of A. florida. Further studies are
necessary to identify the role of motility initiation and chemotaxis on
hybridization among Acropora species.
In summary, in the corals, A. digitifera, A. tenuis and A.
gemmifera, eggs suppressed sperm motility when many sperm approached
(Fig. 5). This study is the
first report to show that eggs inhibit sperm motility in response to sperm
attachment. Eggs initiated sperm motility via an increase in
[pH]i (Fig. 5). In
addition, sperm approached eggs by modulating bending motion to swim straight,
in a [Ca2+]i-dependent manner
(Fig. 5). These phenomena could
be induced by a secretion of substance(s) from eggs. Mass spawning occurs
synchronously in many coral species. It has been reported that there are more
than 150 Acropora species (Veron,
1995b). Since sperm must fertilize eggs in the presence of many
different Acropora species, it is possible that sperm motility
regulatory mechanisms could have been selected to increase fertilization
success.
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Acknowledgments |
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References |
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