Eggs regulate sperm flagellar motility initiation, chemotaxis and inhibition in the coral Acropora digitifera, A. gemmiferaand A. tenuis

Sperm motility is important for fertilization. Sperm flagellar motility is regulated by several mechanisms through a motility apparatus, the axoneme,which is highly conserved among eukaryotic flagella. Sperm flagellar motility of several animals such as ascidians and clupeids is not initiated before they come in close contact to eggs (Morisawa,1994). 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.

Acropora digitifera L., A. tenuis L. and A. gemmifera L. are widely distributed around Okinawa Island, Japan(26°50′46″N, 128°17′25″E). These three species perform mass spawning and do not hybridize among themselves. Colonies of Acropora digitifera, A. tenuis and A. gemmifera were collected at Oku fishery port in Okinawa Island. These colonies were kept in a running seawater tank under natural light conditions. These colonies were returned to their natural habitat and glued to the substratum.

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 1.0×10^7-9 cells ml^-1. Sperm and eggs were used for experiments within 5 h after collecting.

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.

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.

Thirty eggs were put into 100 ml of filtered seawater. Sperm were diluted with seawater to 1.1×10⁵-1.2×10⁸ 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.

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.

In this study, we observed sperm motility of the coral, A. digitifera,A. gemmifera and A. tenuis. In these species, spawning time is different among the species (Fukami et al.,2003). 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.

As shown above, sperm motility was completely suppressed in ASW containing large amount of Na⁺. However, in the presence of eggs, sperm motility was initiated in ASW when sperm came to about 150-300 μ m from the egg surface. At first, sperm swam in circular paths and then swam in straight paths when they approached the egg (indicated by arrows in Fig. 2A). In the absence of eggs, trajectories of sperm motility were circular(Fig. 2B). It is probable that A. digitifera, A. tenuis and A. gemmifera eggs secret substance(s) to initiate sperm motility via an increase in[pH]_i even in the presence of Na⁺ and modulate symmetrical flagellar beating to swim straight towards the eggs. Sea urchin sperm swim straight in the presence of sperm-activating peptide (SAP) isoform P15 (Shiba et al., 2006). 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.

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⁺]_iinduces 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.

From these results, it is possible to speculate that Ca²⁺ binds to Ca²⁺-binding protein(s) to modulate symmetry of flagellar beating and swimming velocity. Ca²⁺-binding protein, CaM, is known to regulate the asymmetry of flagellar beating in sea urchin sperm(Brokaw and Nagayama, 1985). 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 Acroporasperm.

There appears to be species specificity of initiation/chemotaxis of sperm among the three species. It is reported that the chemoattractants of Montipora digitata are diverse and the ratio of substances are important for chemotaxis of sperm (Coll et al., 1994). 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. digitiferaand 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.

The authors want to express gratitude to Ms Ruth S. Pisingan, Ms S. Kai and Ms M. Nakamura (University of the Ryukyus) and Dr N. Aluru, Department of Biology, University of Waterloo for helpful advice on the manuscript. This study was partially supported by a grant for the 21st Century COE program `The Comprehensive Analyses on Biodiversity in Coral Reef and Island Ecosystems in Asian and Pacific Regions' from the Ministry of Education, Culture, Sports,Science and Technology, Japan (Monbukagakusho) to M.M. and A.N., a Grant-in-Aid for Scientific research (B) (15405029) to A.T. and a Grant-in-Aid for Scientific research (B) (16310158) to K.S.