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First published online March 27, 2009
Journal of Experimental Biology 212, 1092-1100 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.027029
Endogenous signaling pathways and chemical communication between sperm and egg
1 Department of Biological Sciences, California State University, Los Angeles,
CA 90032, USA
2 ARL Division of Neurobiology, University of Arizona, Tucson, AZ 85721,
USA
3 Department of Ecology and Evolutionary Biology, University of California, Los
Angeles, CA 90095, USA
4 Neurosciences Program and Brain Research Institute, University of California,
Los Angeles, CA 90095, USA
* Author for correspondence (e-mail: z{at}biology.ucla.edu)
Accepted 28 January 2009
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Key words: fertilization, sexual reproduction, gamete interactions, sperm, egg, attractant, chemotaxis, tryptophan, amino acid, abalone, Haliotis rufescens
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). Current
theory predicts that membrane-bound proteins promote gamete recognition
(Palumbi, 1999;
Levitan and Ferrell, 2006).
However, soluble egg compounds attracting conspecific sperm could also act as
pre-zygotic factors maintaining species integrity while increasing
fertilization rates (Jantzen et al.,
2001; Riffell et al.,
2004; Eisenbach and Giojalas,
2006; Kaupp et al.,
2008). Fluid-borne sperm attractants would operate upstream of
cell-surface proteins prior to gamete contact. Although cell-surface
recognition between male and female gametes is critical for sexual
reproduction, the roles of soluble egg factors remain largely undescribed.
Gamete passage through a fluid medium is a general phenomenon occurring for
broadcast spawners as well as for internal fertilizers. Principles of sperm
navigation and egg signaling, therefore, may generalize across such distantly
related taxa as marine invertebrates and humans
(Spehr et al., 2003).
In nature, sperm must locate and bind to the egg surface within a fluid
environment. Dissolved signal molecules that cause sperm to orient and
accelerate towards an egg could increase gamete encounter rates.
Species-specific attractants might be evolutionarily important barriers to
hybridization, promoting reproductive isolation among sympatric species
(Harrison et al., 1984;
Miller, 1985;
Miller, 1997). Despite their
importance, few sperm chemoattractants have been chemically identified
(Ward et al., 1985;
Nishigaki et al., 1996;
Olson et al., 2001;
Riffell et al., 2002; Yoshida
et al., 2002; Böhmer et al.,
2005), and none tested for their significance for fertilization
success under natural conditions.
Membrane proteins involved in sperm–egg interactions are better
characterized for abalone (Haliotis spp.) than for any other genus,
and demonstrate strong selection for species-specific gamete recognition as a
pre-zygotic barrier to hybridization
(Swanson and Vacquier, 1995;
Swanson and Vacquier, 1998;
Swanson and Vacquier, 2002a;
Swanson and Vacquier, 2002b).
This system thus represents a logical choice for investigating the
contribution of soluble egg factors and chemosensory-mediated sperm behavior
in fertilization ecology and evolution. We have shown that red abalone
(Haliotis rufescens) sperm detect waterborne signal molecules from
conspecific eggs, and change their swimming behavior to increase the
likelihood of successful contact (Riffell
et al., 2002). As established by bioassay-guided fractionation of
natural egg-conditioned seawater, male gamete attraction is dose-dependent and
stereospecific for the L-isomer of tryptophan (L-Trp).
Closely related metabolites, including serotonin, tyramine and seven other
structural analogs, do not affect sperm motility or orientation towards eggs
(Riffell et al., 2002) (and
R.K.Z., unpublished data). Consequently, red abalone sperm require only a
single, identified compound for gamete attraction.
Improved understanding of how chemical signals interact with the
environment should lead to important insights about the natural selective
forces driving fertilization. Given that eggs release fluid-borne attractants,
investigations are needed to focus on principles of chemical production and
transport (Zimmer and Butman,
2000). Structures, concentrations and fluxes of attractant
molecules must be identified, and rates of advection and diffusion measured to
ascertain chemical distributions over time and in space
(Karp-Boss et al., 1996;
Visser and Jackson, 2004;
Xiang et al., 2005). Knowledge
of these factors would enable analysis of the constraints imposed by natural
physiochemical phenomena on sperm behavioral responses mediating gamete
encounters and determining fertilization success.
Fertilization is a complex interaction among the biological and chemical
traits of gametes and physical properties of fluid environments. Red abalone
sperm navigate most effectively and fertilization success is maximized under
experimental conditions simulating the hydrodynamic environment of adults
spawning in nature (Riffell and Zimmer,
2007). Still unresolved, however, are aspects of the dynamics of
gamete attractant production and release. Here, we (1) determine
concentrations of L-Trp, and 18 other free amino acids, from a wide
selection of abalone tissues, (2) measure L-Trp release rates and
link them to egg fertility, and (3) identify whether the ovum, egg jelly, or
both, are source(s) of L-Trp. Establishing vital connections
between gamete physiology and chemical signaling, this study also reveals the
degree to which L-Trp is sequestered by eggs (relative to other
tissues) and, therefore, functions in communication with sperm.
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MATERIALS AND METHODS |
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)], made with ultrapure water and filtered to 0.22 µm
(FSW), and homogenized with a Tissue Tearor (BioSpec Products Inc.,
Bartlesville, OK, USA). Male gonadal tissue (0.5 g) and ovaries (1 g) also
were dissected from specimens (N=3 of each gender) and homogenized in
5 ml FSW. Homogenates were immediately strained, filtered to 0.22 µm, and
kept frozen (at –80°C) prior to analysis. Negative controls,
prepared in parallel with FSW only, yielded no detectable background traces of
DFAA or biogenic amines in high-performance liquid chromatography runs.
Egg and jelly coat analyses
Abalone eggs are surrounded by a jelly coat that potentially could serve as
a reservoir for sperm attractants. Compositional analysis was therefore
performed on (1) freshly spawned eggs with the surrounding jelly coat intact,
(2) eggs from which the jelly coat had been removed, and (3) the egg jelly
itself. Female red abalone (N=11) were spawned using methods
described previously (Riffell et al.,
2002; Riffell et al.,
2004). Eggs were collected within 1 min of spawning and
concentrated over 52 µm Nitex mesh into a suspension of 105 eggs
ml–1, and subsampled for compositional analysis (this
section) and for release rate experiments (see below). For analysis of eggs +
jelly coat, each batch of eggs (0.5 g wet mass of concentrated egg slurry;
N=4 replicates) was immediately homogenized in 1 ml FSW. The egg
jelly was removed from a different lot of eggs (1.0 g; N=4 reps) by
incubation in 15 ml of FSW adjusted to pH 5.0 for 15 min; this treatment
solubilized the jelly coat while leaving the eggs intact
(Glabe and Vacquier, 1977). A
portion of the jelly slurry solution (10 ml) was then removed and filtered.
De-jellied eggs were washed over Nitex mesh with 15 ml FSW, four times, to
remove any residual jelly, and then homogenized in 1 ml FSW. All solutions
were frozen (at –80°C) for later analysis. Jelly slurries were
concentrated tenfold by centrifugation under vacuum prior to compositional
analysis. Total protein was estimated for egg homogenates by comparison with
previously published studies using a BCA colorimetric assay, run according to
manufacturer's recommendations (Bio-Rad Corp., Hercules, CA, USA). The protein
assay was standardized with bovine serum albumin (BSA), and absorbance of
BSA-reacted samples and standards was monitored at 562 nm with a microplate
reader.
High-performance liquid chromatography analysis of amino acid composition
High-performance liquid chromatography (HPLC) was used to quantify
dissolved free amino acids in homogenates of abalone tissues, and from
concentrated, egg-conditioned seawater. The method of Onoue et al.
(Onoue et al., 1983) was
modified to generate fluorescent derivatives of amino acids, prior to
separation by reversed-phase chromatography. Samples were treated with
o-phthalaldehyde (OPA), which reacts with primary amines to generate
fluorescent derivatives (Roth,
1971). A solution of OPA (50 mg) dissolved in absolute methanol
(1.25 ml) was added to 0.4 mol l–1 sodium borate buffer (11.2
ml, pH 9.5) with 2-mercaptoethanol (50 µl). The sample, or a standard
mixture of amino acids, was dissolved in HPLC-grade water mixed with the OPA
reagent in a 4:1 ratio, by a robotic arm designed for automated, pre-column
derivatization (Beckman Coulter Model 508; Fullerton, CA, USA). The reaction
proceeded for 1 min prior to column injection.
Fluorescent derivatives were separated by reversed-phase HPLC on an Ultrasphere ODS column (4.6 mmx25 cm, 5 µm particle size; Beckman Coulter), eluting at 1 ml min–1. Before each run, the column was equilibrated in Solvent A for 15 min. One minute after injection, a linear gradient from 0 to 20% of Solvent B in Solvent A was run for 15 min, increasing to 50% Solvent B in 9 min, and finally to 100% Solvent B in 15 min (Solvent A, 1:19:80 tetrahydrofuran:methanol:0.05 mol l–1 sodium acetate; Solvent B, 80:20 methanol:0.05 mol l–1 sodium acetate; both solvent mixtures were pH 6.8). Fluorescent derivatives were detected after elution from the column with a fluorometer (Jasco Instruments, Easton, MD, USA), monitoring at 453 nm with an excitation wavelength of 332 nm. Measurements were sensitive and reproducible to picomole levels.
Commercial standards (Sigma-Aldrich, St Louis, MO, USA) were used to calibrate retention times, and response factors for integrating peak areas, in Gold Nouveau software (Beckman Coulter). Samples were analyzed with separate HPLC runs for standard amino acids plus taurine, and biogenic amines. One standard included 18 of the 20 coding amino acids (all but cysteine and proline) plus taurine, a sulfonic amino acid that is an osmolyte in the tissues of many invertebrates. A second set of standards for biogenic amines included dopamine, L-DOPA, tyramine and serotonin. Standards were run at four concentrations: 2.5x10–9, 5x10–10, 1x10–10 and 2.5x10–11 moles of each analyte per injection. Replicate standard injections were done at the beginning, middle and end of sample runs (i.e. three replicates per concentration level) to control for changes in retention times over the course of a run. Two calibrations were done to maximize accuracy. Calibrations with standards ranging from 5x10–10 to 2.5x10–11 moles accurately measured analytes present at <5x10–10 moles per injection, but over-estimated more abundant compounds; calibrations using all four standard concentrations were therefore only used to measure compounds present at >5x10–10 moles.
All samples were run at two concentrations because taurine was up to 20 times more abundant than the next most common amino acid, and thus was quantified separately. Prior to derivatization and injection, an aliquot was diluted either 1:10 for quantifying the 18 coding amino acids or biogenic amines, or 1:100 for measuring taurine. The average of two replicate injections for each sample at each concentration was used as the value for that replicate tissue preparation. Co-injection with standards confirmed the identity of critical peaks.
For statistical analyses, the DFAA total was determined for each tissue by summing the 18 coding amino acids. The proportion of Trp in each replicate sample of each tissue was then determined, and the means and standard deviations calculated. A one-way ANOVA was used to test the null hypothesis of no difference in the percentage of Trp in the DFAA pool among abalone tissues. A post-hoc Dunnett's t-test was then applied to compare the proportion of Trp in egg homogenate against all other tissues.
Taurine and tyramine were quantified for each tissue but were not initially included in total DFAAs used to compute the percentage composition of Trp. Subsequently, tyramine was included in the DFAA total and the proportion of tyramine was computed for each tissue. A one-way ANOVA tested the null hypothesis of no difference in the proportion of tyramine across tissues. A post-hoc Dunnett's t-test compared the proportion of tyramine in ovarian tissue against all other tissues. We followed the same procedures for taurine, including an ANOVA test but no post-hoc comparisons.
Release rate of sperm attractant
To quantify release rates for the abalone sperm attractant, secretion and
accumulation of tryptophan from eggs into seawater were measured at
ecologically relevant time scales. Female abalone were spawned and eggs
concentrated as described above to yield a slurry of 105 eggs
ml–1. Eggs were immediately transferred to experimental
vessels, sterile glass Ehrlenmeyer flasks containing FSW that were held in a
water bath at 15°C (a typical field temperature for subtidal abalone
populations in southern California) for the duration of the experiment. Based
on pilot studies, 3x105 eggs (
3 g) were incubated in 25
ml FSW, a sufficient concentration for HPLC measurements of released
tryptophan. Five replicate vessels were used for each of the following time
points: 1, 15, 30, 45, 60 and 120 min. These intervals were chosen based on
preliminary results indicating abalone eggs only remain fertile for an hour
after spawning. Egg-conditioned seawater (ESW) samples were immediately
filtered at the end of the incubation time and frozen at –80°C prior
to HPLC analysis.
For tryptophan measurements, the majority of each ESW sample was desalted and concentrated prior to analysis using Sep-Pak reversed-phase C-18 cartridges. Sep-Paks were initially washed with HPLC-grade methanol (10 ml) to wet the resin, then equilibrated with ultra-pure water (15 ml). Each ESW sample was loaded onto a column by gravity feed, and the column washed with ultrapure water (15 ml) to desalt. Tryptophan was then eluted with 30% methanol (10 ml), which was concentrated under vacuum to 100 µl for HPLC analysis. Replicate standards of tryptophan (2.5 nmol l–1 and 0.25 nmol l–1; N=3 per concentration) were added to FSW and concentrated on Sep-Pak cartridges in parallel for calibrating HPLC runs of ESW. This procedure controlled for any Trp loss during the concentration steps.
For the entire dataset, release of Trp over time was modeled by polynomial
regression, using a second-order model. Release over the first 45 min (the
period of egg fertility) was separately modeled using linear regression. Based
on inspection of residual plots, a log-transformation was applied to the
response variable (Trp concentration, expressed as fmol
egg–1) to improve homogeneity of variances
(Quinn and Keough, 2002).
Attenuation of sperm attractant signal
Based on results of the preceding experiment, we investigated quenching of
the tryptophan signal produced by freshly spawned eggs at later time points
(see Egg fertility and sperm attractant signal in the Results
section). Three alternative hypotheses were tested: Trp initially accumulates
around eggs but is gradually eliminated by (1) oxidation; (2) uptake by
bacteria in seawater; or (3) a Trp-degrading factor released by eggs, in a
negative feedback loop. To test the first two hypotheses, three sets of
standard solutions (N=4 replicates each) were made of 1 µmol
l–1 Trp in seawater (25 ml). In the first treatment, Trp
solutions were placed in conical tubes and floated in 15°C seawater for 3
h. To test for rapid oxidation, controls were prepared in parallel but
immediately frozen. In the second treatment, solutions were prepared and
incubated for 3 h using seawater to which the following antibiotics had been
added: streptomycin sulfate (150 mg l–1), gentamycin (100 mg
l–1), and penicillin G (150 mg l–1). A
decline in Trp concentrations in the seawater-only incubation, but not in the
antibiotic-seawater treatment, would suggest microbial uptake. A decrease in
both 3 h incubations, relative to controls, would support rapid oxidation of
Trp (a highly reduced molecule) within hours of spawning.
To test the third hypothesis, freshly spawned eggs were concentrated as before, added to replicate containers (N=4) of seawater at 15°C, and incubated for 60 min to produce ESW. This treatment presumably would contain any soluble egg factor that degrades Trp. An aliquot (1 ml) of each ESW preparation was filtered to 0.22 µm, and a Trp standard added to yield a final concentration of 1 µmol l–1 Trp in filtered ESW. The control was Trp standard added to FSW only (N=4 reps). Samples were incubated at 15°C for 2 h, then frozen prior to analysis.
Egg fertility and sperm attractant signal
Bioassays established the relationship between egg fertility and tryptophan
concentration (in solution) as a function of time. These tests were conducted
simultaneously with experiments on tryptophan release and accumulation. Here,
100 freshly spawned eggs from a single female were placed into each of 52 (13
treatments x four replicates) sterile Petri dishes (32 mm diameter)
containing FSW. Sperm from a single male were added (105 cells
ml–1, final concentration) to four replicate dishes at each
of 13 time treatments (1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120
min post-spawn). All dishes were incubated for 3 h (at 15°C) after sperm
were added, eggs were fixed in 5% formalin, and percentage fertilized
(cleaved) were counted under a compound microscope (x100; Olympus Model
IX70). Because both male and female gametes were the same age when combined, a
separate control isolated an egg from a sperm effect. In parallel with the
main experiment, four additional dishes contained 120-min-old sperm, from the
original male, added to a new batch of fresh eggs from a second female (1 min
post-spawn). All other methods were identical. If percentage fertilization in
the control is equal to that at 1 min in the main experiment, sperm fertility
would be eliminated as a causative factor, and any change over time in
reproductive success attributable only to eggs. This entire experiment was
repeated four times, using gametes from different males and females in each
run.
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0.8 pmol egg–1,
whereas total soluble protein was estimated at 125 ng
egg–1.
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Arginine, alanine, glycine and glutamic acid were the four most prevalent coding amino acids in all tissues except eggs, which had very low levels of alanine and were enriched in aspartic acid and serine relative to all other tissues (Fig. 2). In eggs, no single amino acid was particularly common, with arginine, aspartic acid and glutamic acid each constituting 14% of the DFAA pool, followed by serine and glycine at 9% apiece. The most abundant amino acid differed between tissues (Fig. 2). Glycine was 35.4±5.4% (±s.e.m.) of the DFAAs in testes, but only about 10% of DFAAs in other tissues. Arginine represented almost half the DFAA pool in muscle (44.4±8.5%) and was elevated in hemolymph (29.4±3.7%), but constituted less than 20% of the DFAAs in all other tissues. Glutamic acid was the most abundant amino acid in ovarian tissue (25.1±0.1%), nearly twice that found in eggs (13.7±0.8%). Taurine was more abundant in each tissue than the total pool of 18 coding amino acids (Table 2, Fig. 3). Out of all tissues studied, the ratio of taurine to the 18 coding amino acids was greater than 5:1 for eggs and gills and about 4:1 for all other tissues, except testis.
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The biogenic amine tyramine was detected in eggs, ovary, gill and stomach tissue (Table 2, Fig. 1). When included in the total DFAA pool, tyramine made up a significantly greater proportion in ovaries, compared with all other tissues (Fig. 1, and results of a one-way ANOVA: F7,23=236.71, P<0.0001; post-hoc Dunnett's t-tests comparing ovary against each tissue, P<0.001). Ovaries and eggs contained a trace of DOPA, but no serotonin was detected. Conversely, muscle tissue contained serotonin and a trace of DOPA, but no detectable tyramine.
There was no difference in the concentration of any DFAA between eggs with an intact jelly layer, versus eggs from which jelly was experimentally removed (Table 1, Fig. 2). The small amount of Trp detected in the jelly slurry did not exceed the amount that would be released by eggs over 15 min, the period of incubation for jelly removal (see below). Thus, the jelly layer surrounding fresh eggs did not serve as a reservoir of Trp, or any other amino acid.
Egg fertility and sperm attractant signal
Natural rates of Trp release from eggs were quantified and linked with
fertility. Concentration surrounding eggs increased linearly over the first 45
min post-spawn (Fig. 4A, and
linear regression of time on log-concentration Trp:
F1,18=36.77, R2=0.67,
P<0.0001). The rate of release was 0.2 fmol
egg–1 min–1 during this period. After 45
min, however, Trp dwindled rapidly from solution. Release and accumulation
kinetics, therefore, were best modeled overall by a quadratic function,
because of the decay of Trp in the second hour (polynomial regression of time
on log-transformed Trp concentration: F2,25=20.76,
R2=0.62, P<0.0001). Closely paralleling these
events, fertilization success peaked during an initial 30 min period
(post-spawn), but decreased to nil over the next 50 min
(Fig. 4B). Whereas egg
fertility diminished over time, sperm fertility did not. Thus, older eggs
stopped releasing Trp as they became infertile.
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; Riffell and Zimmer,
2007). Here, we quantified levels of this ecologically important
signaling molecule in various abalone tissues, and in freshly spawned eggs. As
a percentage of the total DFAA pool, Trp was substantially more concentrated
in eggs than in any other tissue. Tryptophan levels were even higher in eggs
than in ovarian tissue. Thus, red abalone selectively concentrate a
sperm-attracting amino acid in their ova. A higher initial Trp concentration
would intensify the odorant plume released by an egg, and thereby, create a
stronger signal for sperm in surrounding seawater
(Karp-Boss et al., 1996;
Visser and Jackson, 2004).
When male gametes of red abalone approach a conspecific egg, they swim
faster and navigate directly towards the egg surface
(Riffell et al., 2002;
Riffell and Zimmer, 2007).
Sperm tracks around single conspecific eggs were computer imaged in the
presence or absence of the enzyme tryptophanase, which selectively digests
free tryptophan (Riffell et al.,
2004). In the enzyme treatment, sperm did not orient towards
fertile eggs; yet, male and female gametes otherwise were not adversely
affected. The overall impacts of eliminating tryptophan were to extinguish
sperm chemotaxis, reduce gamete encounter rates, and therefore, lower
fertilization success. In the present study, older eggs stopped producing Trp
at the point when their fertility dropped below 50%, a key link between gamete
physiology and chemical signaling. This observed covariation between signal
production and egg fertility was consistent with the ecological role of Trp in
promoting fertilization. From combined results, egg fertility does not depend
on the presence of Trp in free solution, but Trp release rate is highly
dependent on egg age, and hence, fertility
(Riffell et al., 2002;
Riffell et al., 2004) (and
present study).
Several mechanisms might explain the apparent negative feedback loop
quenching the sperm signal after 1–2 h. Tryptophan is typically one of
the least abundant amino acids in seawater, probably because the aromatic ring
system is readily oxidized (Fuhrman and
Ferguson, 1986; Coffin,
1989; Keil and Kirchman,
1991; Suttle et al.,
1991). We tested whether rapid oxidation caused the observed
decline in Trp from ESW by spiking FSW with micromolar levels of a Trp
standard. After several hours of incubation, there was no decrease in Trp
concentration. Consequently, oxidation is not the cause of signal decay in ESW
preparations. Our seawater was filtered to 0.22 µm which removes most
bacteria, and no difference was found in Trp levels after incubation with or
without antibiotics. It is therefore unlikely that Trp was taken up by
suspended microbes. Feasibly, eggs could have secreted a Trp-metabolizing
enzyme when they ceased to be fertile. This negative feedback loop could be
adaptive if it prevented sperm from being `distracted' by infertile eggs,
decreasing contact rates with longer-lived eggs produced by a given female.
When seawater previously conditioned by eggs was spiked with a Trp standard,
no digestion of the attractant signal occurred, ruling out a secreted egg
factor. We thus hypothesize that microbes on the surface of abalone eggs
scavenge amino acids from the surrounding seawater. Amino acid efflux would
greatly exceed microbial uptake, but only while eggs are fertile. Although
speculative, there are precedents for this phenomenon in other systems
(Decho et al., 1998;
Hidalgo-Grass et al.,
2004).
Among free-spawning invertebrates, gamete signaling has been especially
well studied in deuterostomes (echinoderms and ascidians)
(Ward et al., 1985; Yoshida et
al., 2002; Yoshida et al., 2003; Kaupp et
al., 2003). Sea urchin and sea star eggs, for example, release
short peptides that bind receptors on the sperm flagellum, activating sperm
and altering patterns of motility in a concentration-dependent manner
(Ward and Kopf, 1993;
Nishigaki and Darszon, 2000;
Nishigaki et al., 1996;
Böhmer et al., 2005). The
cascade of internal signal transduction events includes activation of guanylyl
cyclase and production of cGMP, followed by an influx of calcium ions that
triggers asymmetric flagellar beating
(Matsumoto et al., 2003;
Neill and Vacquier, 2004;
Kaupp et al., 2006;
Hirohashi et al., 2008). To
date, only a few peptidic egg factors have been shown to induce chemotaxis,
and the mechanism by which ionic fluxes lead to directional sperm navigation
remains unclear (Ward et al.,
1985; Nishigaki et al.,
1996; Kaupp et al.,
2003; Böhmer et al.,
2005; Kaupp et al.,
2008). Comparative studies with abalone could reveal if homologous
transduction pathways regulate sperm motility and egg recognition among
protostomes.
Amino acid profiles of tissues
The profile of soluble amines in ovaries did not mirror that in spawned
eggs. Tyramine, for example, was more abundant in ovaries than in any other
tissue type. Thus, different biologically active amines can be selectively
enriched in various abalone tissues. Red abalone ovary contained 140 mg
tyramine kg–1 wet mass, a much higher level than reported for
ovaries of the scallop Pecten maximus (10 mg
kg–1). Yields from testes were approximately the same for red
abalone (25 mg kg–1 tissue) and scallop (28 mg
kg–1 tissue) (Mackie et
al., 1997). Future studies should examine whether molecules
biosynthetically related to aromatic amino acids are concentrated in eggs of
other Haliotis species, and if so, whether they function as
species-specific sperm signals.
The proportional composition of red abalone eggs differed somewhat from
that reported for the blacklip abalone Haliotis rubra; levels of Glu
and Asp were lower in eggs of H. rubra, compared with H.
rufescens (King et al.,
1996; Litaay et al.,
2001). Tryptophan was not quantified in H. rubra eggs.
Total concentrations of DFAAs and soluble protein were 1.5–2.0
times higher in eggs of H. rufescens than H. rubra. Per-egg
content of protein (125 ng) and taurine (500 pmoles), however,
showed excellent agreement between the present and a previous study on H.
rufescens (Wellborn and Manahan,
1995).
The high levels of Arg, Ala and Gly measured in abalone foot are
characteristic of muscle from a variety of mollusks
(Carr et al., 1996). The same
four amino acids (Arg, Gly, Glu and Ala) were proportionally the most abundant
in H. rufescens and H. diversicolor muscle
(Chiou et al., 2001). In both
species, Arg represented the majority of the DFAA pool in muscle but not in
viscera. Taurine is also present in abalone muscle at exceedingly high levels,
where it acts as a substrate for a dehydrogenase enzyme that recycles NADH
during hypoxia (Gäde,
1988). The high concentration of Arg in H. rufescens
hemolymph is in agreement with similarly high levels in hemolymph of the sea
hare Aplysia californica. In both species, the top three DFAAs were
Arg, Ala and Gly in that order (Derby et
al., 2007). Major patterns of DFAA profiles for select tissues are
therefore shared by red abalone and other marine gastropods.
Why tryptophan?
Identifying signal molecules is essential to linking behavior, chemosensory
physiology and ecological function. Studies are needed to distinguish unique
natural products from more common metabolites that serve a variety of roles.
One strategy for producing chemical signals is to biosynthesize a novel
substrate for each required cue, as with anthopleurine, a sea anemone alarm
pheromone (Howe and Sheikh,
1975), or adrenaline, the mammalian `fight-or-flight' hormone
(Baulieu and Kelly, 1990).
Alternatively, ordinary molecules can have extraordinary effects. Cellular
mechanisms of chemosensory reception are highly converged
(Ache, 1994;
Hildebrand and Shepherd, 1997;
Bargmann, 2006). The same
molecules, therefore, may function in chemical communication by
phylogenetically diverse species and among different cell types. Histamine,
for example, initiates local immune responses
(Jutel et al., 2006;
Sugata et al., 2007),
regulates receptor-mediated physiological processes in the gut (Filippova and
Nozdrachev, 2007), functions as a neurotransmitter
(Jones, 2005;
Stuart et al., 2007), and acts
as an environmental cue of habitat suitability for invertebrate larvae
(Swanson et al., 2004;
Swanson et al., 2006).
Similarly, the ubiquitous amino acid L-Trp has multiple and
varied functions in diverse tissues and organisms. It is an essential amino
acid required for protein formation
(Yanofsky, 2003;
Ladner et al., 2007), acts as
a sperm attractant for red abalone
(Riffell et al., 2002;
Riffell et al., 2004), and
serves as a precursor for the biosynthesis of many biogenic signals including
plant hormones and brain neuromodulators
(Schaechter and Wurtman, 1990;
Sarwar and Frankenberger,
1994). One tryptophan metabolite, serotonin, plays an especially
critical role in nervous system development, including the apical sense organ
(Byrne et al., 2006;
Croll, 2006). Larvae of red
abalone and other marine taxa use this sense organ to select a suitable site
for irreversible metamorphosis into the juvenile form
(Hadfield et al., 2000); a
critical period in marine life histories
(Palmer and Strathmann, 1981;
Young, 1990).
Given the importance of serotonin during the larval stage, elevated levels of Trp in red abalone eggs may reflect a redirection of resources to biosynthetic pathways involved in nervous system functions. If high Trp concentrations are correlated with enhanced serotonin production, neural function, and settlement success, Trp release could be an honest indicator of egg fitness. Likewise, if the intensity of this signal predicts egg quality, sexual selection may favor sperm that respond to Trp. Alternatively, the paucity of Trp in natural seawater may have selected for enrichment of this common metabolite in eggs, producing a sperm cue with no appreciable `background noise' under ambient conditions. Future work should test whether chemoattractants reflect selection for sperm that recognize fitness cues from potential eggs, or physico-chemical constraints on effective signaling in the fluid medium through which gametes communicate.
Once intractable to manipulation, remote communication between sperm and
eggs is now experimentally accessible using abalone as a model system. Recent
results have shown species specificity in sperm chemoattraction among
congeneric abalone, presenting novel opportunities for comparative study
(Riffell et al., 2004).
Further research should reveal if Trp and related metabolites function as
different dialects of an evolutionarily conserved language that communicate
gamete location and fitness, as well as species identity.
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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  K. Knight ABALONE SPERM FOLLOW Trp TRAIL TO FIND EGGS J. Exp. Biol., April 15, 2009; 212(8): i - ii. [Full Text] [PDF]
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