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Research Communications |
* Department of Physiology, Wayne State University, Detroit, Michigan 48201 USA; and
Pure and Applied Biology, University of Wales, Cardiff CF1 3TL, U.K.
1Correspondence: Department of Physiology, Gordon H. Scott Hall of Basic Medical Sciences, Wayne State University, 540 E. Canfield Ave., Detroit, MI 48201. E-mail: jeffram{at}med.wayne.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glu-cys inhibited sperm release. Nereithione aroused three
components of the male nuptial behavior: circling, sperm release, and
accelerated swimming. Electrophysiological activity elicited by
nereithione near the sperm release site consisted of initial large
spikes, cyclic bursting activity, and small spikes lasting up to a
minute and was dose dependent, rapid, reversible, and repeatable. This
preparation is an excellent model system for characterizing the
receptors and functions of a marine pheromone.—Ram, J. L.,
Müller, C. T., Beckmann, M., Hardege, J. D. The
spawning pheromone cysteine-glutathione disulfide (`nereithione')
arouses a multicomponent nuptial behavior and electrophysiological
activity in Nereis succinea males.
Key Words: cysteine-glutathione disulfide • pheromone • spawning behavior • sperm release
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the same
cannot be said for comparable water-borne cues. Despite ample
phenomenological evidence from mass spawnings and laboratory
experiments for the existence of pheromones that help synchronize
reproductive behaviors in the marine environment, progress in this area
has been hampered by the lack of identification of the specific
chemicals that mediate these organismal interactions. However, the
recent chemical identification of reproductive pheromones in
polychaetes (2)
, as well as a number of other organisms—e.g., molluscs
(3)
and protozoans (4
5
6)
—now makes such analysis possible.
The polychaete Nereis succinea spawns during a coordinated
`nuptial dance' timed by the phases of the moon, initiated by the
time of the day, and choreographed by the exchange of chemical signals.
Spawning usually takes place according to a lunar cycle, with
populations in different locations spawning primarily at new moon
(Isefjord, Denmark) (7,
8)
, bimodally at new moon and full moon (Woods
Hole, Mass.) (9)
, or at full moon (Cardiff, Wales; J. D. Hardege,
unpublished results). The onset of spawning usually occurs a few hours
after sunset, lasting for an hour or two at most (8,
9)
. To initiate
spawning, mature worms swim to the surface where male and female worms
then swim in small circles around one another (the nuptial dance),
activating both sexes to release gametes in close proximity to one
another. At peak periods in the lunar cycle, the density of animals may
be large enough that the assemblage of animals appearing at the surface
a few hours after sunset has been characterized as a `swarm.'
The behavior of individual animals leading up to and within the nuptial dance suggests that chemical signals may mediate interactions between the males and females. Outside the peak swarming period, when the lower density of animals makes it easier to observe individual interactions, the following behavior can readily be observed. Females emerge and swim slowly at the surface. Male worms usually appear within 10 to 20 s, swimming straight toward the females and at a much faster speed. On close approach to the females, males and females begin to swim in circles, the males release small amounts of sperm, the females release eggs, and then males release massive amounts of sperm. Chemical signals suggested to be involved in this sequence include an attractant or mate recognition pheromone released by the female, an egg release pheromone released by the male, and a sperm release pheromone released by the female.
The sperm release pheromone is best understood. Lillie and Just (9)
noted that the water in which ripe, nonspawned females had been
swimming could trigger ripe males to spawn. Townsend (10)
tested a
number of known biological substances and found that the most effective
chemicals for inducing spawning in male N. succinea were
reduced and oxidized glutathione, of which reduced glutathione was
suggested to be present and released with the coelomic fluid of females
at the time of spawning. However, Townsend (10)
observed that the
sperm-releasing chemical discharged by swimming females appeared to
have much greater potency than could be explained by glutathione
released in the water. Recently, the active sperm release substance
found in coelomic fluid of ripe female N. succinea was
identified as a derivative of glutathione, cysteine-glutathione
disulfide (2)
; this substance, termed `nereithione,', has also been
found to be released into water by swimming ripe females (11)
. Neither
reduced nor oxidized glutathione were found to be released in
measurable quantity with nereithione. Furthermore, male N.
succinea neither release nor have in their coelomic fluid
comparable amounts of substances that elicit spawning in males (11)
.
Therefore, it appears that nereithione is uniquely released by female
N. succinea in requisite amounts and has the appropriate
biological activity to function as the N. succinea sperm
release pheromone.
The receptor for nereithione is so far uncharacterized. Townsend (10)
tested a number of amino acids and also sulfhydryl compounds unrelated
to glutathione; however, her choice of compounds was uninformed by the
knowledge of the active chemical, which has now been identified. In the
present study, efficacy of synthetic nereithione and other substances
related in structure to glutathione has been determined quantitatively.
Nereithione is shown to be the most effective activator of male
spawning tested; one potentially effective inhibitor of the response
has been identified; the efficacy of a glutathione derivative that may
be useful in covalently labeling the receptor has been demonstrated;
nereithione is shown to arouse a multicomponent nuptial behavior
accompanying spawning; and an electrophysiological test system
responsive to nereithione has been developed.
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MATERIALS AND METHODS |
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-glu-cys (also known as des-gly-glutathione; Sigma), and
S-(p-azidophenacyl)-glutathione (APG;2
Sigma). All but the last compound were dissolved directly in water;
APG was first dissolved at 1 mg/21 µl dimethyl sulfoxide, and then
diluted 100-fold to 10-3 M in 15 ppt diluted sea
water (DSW), from which further dilutions were made.
Animals
N. succinea were collected by netting from the water
surface in the Roath Basin and adjacent lock, Cardiff, Wales, between
11:00 PM and 1:00 AM on
several evenings between 27 July and 25 August, 1998. All animals were
kept in standard laboratory conditions for these animals in 15 ppt DSW.
Animals were stored individually for 1–7 days in ~50 ml DSW each at
8–10°C until use in assays. For use in assays, individual male worms
were allowed to warm up gradually to ambient temperature (21–25°C)
in 65 mm diameter Pyrex crystallizing dishes, in which they began
swimming actively around the circumference. Animals that showed
evidence of having spawned in their storage containers (cloudy water)
or that did not swim actively were not used for spawning assays;
however, some additional experiments on activation of swimming or
identification of anatomical regions sensitive to the chemicals used
animals that had not begun to swim spontaneously.
Spawning assay
For spawning bioassay, individual male worms were gently
transferred into 10 ml of the test solution in a 65 mm diameter
crystallizing dish. Test solutions consisted of DSW or various
concentrations of chemicals diluted in DSW. Animals were allowed to
swim in the test solution for 30 s, releasing sperm if an
appropriate stimulus were present, after which the medium was removed
for quantitation, and the animal was put into DSW to rinse off the test
chemical. Animals were usually serially exposed to DSW and increasing
concentrations (10-fold increasing concentrations starting with
10-8 or 10-7 M, depending
on the chemical and the responses obtained), with rinses in DSW between
each chemical test. On any particular experimental day, animals that
had not responded to any of the various test chemicals were tested for
their response to either 10-5 M oxidized
glutathione or 10-4 M reduced glutathione (known
to be effective at producing spawning); if spawning was still not
obtained, then prior data from that animal were discarded. Animals that
spawned could be used for many tests, even with successively increasing
concentrations of effective spawning stimulators, as it is well known
(and also observed by us) that males can respond multiple times (2,
9,
10)
. Townsend (10)
reported spawning animals up to 40 times each.
The amount of sperm released was estimated visually on a 5 point scale (0 = no spawning to 4 = intense response, producing very cloudy water), but for a more quantitative measurement, 200 µl of the water into which sperm had been released was added to 40 µl of Bio-Rad protein assay reagent (Bio-Rad, Hercules, Calif.) in a 96-well microtiter plate, and absorbance at 590 nm was read after 1–2 h. Blanks for DSW alone were subtracted. None of the test chemicals produced absorbance increases on their own. To determine the relationship of sperm density to absorbance, sperm densities for several samples were determined with a hemacytometer and compared with absorbance measured by this assay. Linearity of the response with amount of sperm was examined by measuring absorbances of sperm samples diluted to 25%, 50%, and 75% of their released densities. The significance of sperm density measurements over the range of concentrations tested for each chemical was determined by one-way analysis of variance (ANOVA). Regression analyses and t tests for other comparisons were used as appropriate.
Video observations
Behavioral responses to nereithione were further characterized
by video recording. Behavior of individual swimming worms (and in one
case, a nonswimming but responsive worm) in 20 ml DSW in 65 mm diameter
crystallizing dishes was recorded by video camera placed directly above
the dish. Test solutions were applied by Hamilton syringe or volumetric
pipette, injecting 5–100 µl of 10-5 M
nereithione or DSW into the dish near the animal.
Electrophysiology
Worms were pinned to a 35 mm Sylgard dish, ventral side up. A
suction electrode was applied externally to parapodia at various points
along the animal from head to tail. Spike activity was amplified by a
differential amplifier (Campden 900 Biological Amplifier, 4000x gain,
HF cutoff at 10K Hz, LF cutoff at 100 Hz), monitored by oscilloscope,
and recorded on pen recorder and a Racal FM tape recorder for later
playback. Measurements of spike durations were the width of spikes at
half their peak height. Small volumes (10 or 20 µl) of DSW,
10-6 M nereithione, or
10-5 M nereithione were applied to the animal
while continuously recording.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, using sperm released in response to APG. The relationship is nearly
linear (for a linear regression, r=0.99,
P<0.01). For four different sperm samples, the relationship
of sperm density to absorbance averaged 1.5 ± 0.4 x
106 sperm/0.1 absorbance units. Absorbance
measurements also correlated well with visual estimates of sperm
release (r=0.83, P<0.01, n=51
points).
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Responses to nereithione and related chemicals
Nereithione was the most effective activator of male spawning,
producing the most consistently large spawning responses and at the
lowest concentration. Two of five animals spawned a moderate amount of
sperm (visual scale = +1 and +2) in response to
10-7 M nereithione, and four of five produced
the most intense response (visual scale = +4) in response to
10-6 M nereithione. The fifth animal had a +3
response. These responses are reflected in the absorbance measurements
illustrated in Fig. 2
(ANOVA, P<0.001). In video-recorded experiments (see
below), three additional animals produced +4 responses to applications
of 5–100 µl of 10-5 M nereithione.
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Oxidized and reduced glutathione and a phenacyl glutathione derivative
all produced spawning (ANOVA, P<0.05), but the responses
were usually less intense and required significantly higher
concentrations than nereithione (Fig. 2)
. Oxidized glutathione usually
did not produce spawning at less than 10-5 M.
Reduced glutathione, while producing small responses at
10-5 M, did not usually yield significant
response intensities until 10-4 M. APG, a
phenacyl derivative of reduced glutathione, was as effective as
oxidized glutathione in eliciting male spawning.
Two dipeptides, cys-gly and -glu-cys, corresponding to fragments of
glutathione, did not elicit spawning; however, -glu-cys appeared to
have an inhibitory effect. To test this, worms were transferred
according to usual assay procedures into a solution containing both
10-4 M -glu-cys and
10-5 M oxidized glutathione. Three of four
animals failed to spawn and the fourth produced only a small (visual
scale = +1) response. After being rinsed, the worms were tested in
10-5 M oxidized glutathione alone. All produced
spawning responses, with the animal that had produced the small
response previously now giving the most intense (+4) response.
Quantitative measurement of the sperm released by the four animals
(Fig. 3
) showed the difference to be significant (paired t test,
P<0.05).
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Video observations
Video observations demonstrated that multiple components of the
behavioral response of males to females (circling behavior, spawning,
and accelerated swimming) could be elicited by the pure nereithione.
Recordings were made of two swimming worms and a responsive quiescent
animal. All components of the response are illustrated in the example
shown in Fig. 4
. The animal had been swimming at 50 mm/s near the edges of the 65 mm
diameter container; upon application of 5 µl of
10-5 M nereithione near the swimming animal, it
immediately swam in several tight circles of ~20 mm diameter
(circling), resumed swimming around the dish, and circled once more
tightly (25 mm diameter), releasing large amounts of sperm (sperm
release) in small whitish clumps that began to disperse within seconds;
it then resumed swimming around the dish and at a faster speed (160
mm/s) than before (accelerated swimming). Animals showed none of these
behaviors in response to comparable applications of DSW, although a
slight hesitation (but not circling) sometimes occurred at the site of
application.
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Circling behavior could be elicited in the absence of an evident
spawning response. Thus, another animal curled its body dorsally in an
arc (Fig. 5
) and circled three times (20 mm diameter) but did not spawn in response
to 7.5 µl of 10-5 M nereithione. However, this
animal exhibited both the circling behavior (25 mm diameter) and
spawning when later tested with 100 µl 10-5 M
nereithione.
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When applied to a responsive nonswimming animal, 100 µl 10-5 M nereithione caused the animal to curve its body in a circle (~10 mm diameter), after which it began to swim around the dish and spawned profusely (+4) when it reentered the original site of chemical application. It was possible to see that the clumps of sperm were ejected with great force from the dorsal side of the animal at a point about two-thirds of the way from the head to the tail. Swimming behavior of this previously quiescent animal lasted approximately 1 min, comparable in duration to the accelerated swimming of the first animal described above. Circling, spawning, and swimming were elicited twice more from this animal by applications of 20 µl 10-5 M nereithione.
Electrophysiological response to nereithione
Multiunit spike activity was recorded from suction electrodes
applied to parapodia at various points along the worm. Spikes ranged
upward in magnitude to 100 µV. The multiunit activity consisted of
both slow (widths at half-peak of up to 10 ms) and fast spikes (widths
of 1–2.5 ms), as illustrated in Fig. 6
, which may correspond, respectively, to muscle potentials and to nerve
spikes from parapodial nerves or ventral nerve cord.
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Activity could be elicited by mechanical stimulation (e.g., applying
brief extra suction to the electrode, which was useful in determining
that the electrode was well-located to record activity). However, a
particularly effective stimulation could be achieved by application of
nereithione near the head of the animal while recording from parapodia
about two-thirds of the way from head to tail. In Fig. 7
, a couple of small fast spikes were elicited by application of 10 µl
DSW to the preparation; 10 µl 10-6 M
nereithione elicited ~20 s of large fast and slow spikes in several
short bursts, as well as numerous small spikes; and 10 µl
10-5 M nereithione activated a long-lasting
(~40 s) series of large spike bursts and almost a minute of small
spike activity. Similar responses could be obtained repeatedly by
rinsing off the preparation and applying nereithione again (data not
shown). Furthermore, similar responses to nereithione were obtained in
four of five preparations. The fifth preparation exhibited only
responses to tactile input (i.e., similar, but very brief fast spike
activity in response to DSW and nereithione applications).
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DISCUSSION |
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Responses to nereithione have not been studied in other organisms;
however, responses to glutathione and its derivatives observed in other
animals indicate that possibly related receptors may be present in
animals as widely diverse as coelenterates and mammals. The
coelenterate Hydra exhibits feeding behavior in response to
reduced glutathione (12,
13)
. Oxidized glutathione is also a ligand at
the putative Hydra glutathione receptor (14,
15)
, but in
contrast to the spawning response of N. succinea, oxidized
glutathione antagonizes, rather than mimics, the response to reduced
glutathione (12,
16)
. In the present study, cys-gly had no effect, in
agreement with observations on Hydra that the -glutamyl
residue of glutathione is necessary for receptor interaction, without
which both antagonistic and agonistic properties are lost (16)
. The
importance of the -glutamyl residue in interactions with the
receptor is further supported in N. succinea by the
antagonistic effects of -glu-cys. S-methylglutathione has also been
shown to activate the Hydra behavioral responses (12,
16,
17)
and to compete for photoaffinity labeling of its putative receptor
by APG (18)
. The activity of APG in N. succinea is
consistent with this behavior and suggests that APG may also be useful
in photoaffinity labeling the nereithione receptor in N.
succinea. In rats and rabbits, oxidized glutathione promotes sleep
(19,
20)
. Brain glutathione undergoes circadian changes in mice (21)
and increases in the brain stems of sleep-deprived rats (22)
.
Intracerebroventricular infusion of either oxidized or reduced
glutathione increases both rapid eye movement (REM) sleep and non-REM
sleep in rats (19,
23)
. It will be interesting to determine whether
nereithione may have effects on either the Hydra or
mammalian glutathione-reactive systems and determine whether the
receptors for the responses may be related.
In N. succinea males, nereithione acts as both an arousal stimulus as well as a sperm-releasing agent. In the video recorded observations and in numerous visually observed responses, animals initially circled at the site of nereithione application, sometimes releasing a small amount of sperm, but only releasing massive amounts of sperm on swimming around more widely and reentering the nereithione solution (no doubt more diluted than when initially injected). The initial circling may serve to release chemical stimuli to activate the fictive (in this case) female to release eggs, with massive spawning by the male occurring only after her presence is confirmed by reentering the vicinity and again detecting nereithione. According to this concept, nereithione serves not only as a mate recognition signal and sperm-releasing agent, but also as an arousal stimulus, preparing the male to consummate the spawning encounter. Nuptial dances in the field should be studied more closely to determine whether this suggested approach, withdrawal, and final rendezvous are part of the dance pattern.
The electrophysiological preparation provides a test system in which
the receptors and functional effects of nereithione can be studied.
Electrophysiological responses to nereithione were rapid, reversible,
and repeatable. The electrophysiological activity corresponded well in
both sensitivity and duration to the observed behavioral responses:
Both spawning and electrophysiological activity were effectively
activated by 10-6 M nereithione, and the
electrophysiological response to 10-5 M
nereithione lasted almost a minute, similar to the duration of the
accelerated swimming phase of the behavioral response. The large spikes
during the initial 5 s may correspond to excitation of muscles
contracting to arch the body during initial circling. The responses
appear to be reflexly mediated, since application of the stimulus near
the head produced a rapid activation of activity two-thirds of the way
to the tail. Boilly-Marer and Lassalle (24,
25)
previously recorded
electrophysiological activity of the brains of N. succinea
in preparations consisting of only the head, prostomium, and anterior
six segments (approximately the anterior one-sixth of the animal) in
response to applications of crude coelomic fluid. Coelomic fluid from
mature females (but not from mature males or immature females)
activated long-lasting (several minutes) spiking activity in the brain
of males, with a latency of about 1 min. Activation of this activity
was dependent on the intactness of the swelled parapodial cirri in the
anterior six parapodia, suggested to be the sensory organs for this
chemical input. On the other hand, Townsend (10)
stated that the
"greatest sensitivity to egg-water [another source of nereithione]
was observed to be on the highly metamorphosed epitoke, just anterior
to the attenuated small posterior segments," a region not even
present in the preparations studied by Boilly-Marer and Lassalle (24,
25)
. Use of pure nereithione and the electrophysiological test system
described here should enable determination of the location(s) and
chemical sensitivity of the receptor(s) mediating the pheromone
responses.
One question regarding the role of nereithione as a pheromone in
N. succinea is whether it is active in a low enough
concentration that it is likely to function as a pheromone (26)
.
Pheromones are often active at concentrations in the range of
1–10 x 10-12 M [e.g.,
Euplotes mating pheromone (27)
and Aplysia
attractin (3)
], although other pheromones are known to act in the µM
range (e.g., uric acid in Platynereis dumerilii) (28,
29)
.
In the quantitative spawning assays described here, the lowest
concentration that elicited spawning was 10-7 M.
Although useful for quantitative comparison between different
stimulants, the method of exposing the animal to the test chemicals
(gently dropping the animal into it) differs from the more natural
swimming encounter, possibly hindering the arousal sequence and
inhibiting the response. With swimming animals, application of as
little as 5 µl of 10-5 M nereithione into a
swimming volume of 20 ml elicited spawning. The amount of dilution of
the nereithione at the moment of response (due to diffusion and mixing
from the animal's swimming movements) is unknown, but if we assume
dilution into a quarter of the volume (i.e., 5 µl into 5 ml), then
the effective concentration is ~10-8 M. The
threshold level would be less than this. In pilot experiments with
lower concentrations, a 10-fold lower concentration was usually
ineffective at eliciting spawning, although circling behavior was
frequently observed.
Although these minimal effective concentrations are higher than
several other pheromones, they are nevertheless appropriate for a
polychaete spawning pheromone. The function of this pheromone is to
elicit circling and spawning behavior in the vicinity of the eggs being
released by the female. Female N. succinea have been shown
to release enough pheromone to activate many males (9
10
11)
. Unlike an
attractant that must be detectable at considerable distances (e.g.,
meters in the case of Aplysia attractin) and therefore
becomes greatly diluted with distance, it would be counterproductive if
the pheromone elicited spawning more than the 10 to 20 mm distance away
indicated by the circling diameter of N. succinea males.
Since the concentration of a diffusing solute decreases in proportion
to the cube of its distance from a point source (crude approximation of
a worm), a concentration of 10-12 M at a
distance of one meter would correspond to a concentration of
10-6 M at a distance of 10 mm from the worm,
more than enough to elicit spawning. To elicit spawning, the observed
sensitivity is therefore probably enough, and this calculation suggests
that the female needs to release less material than required to achieve
a concentration of 10-12 M nereithione at a
distance of 1 meter. Given the multiple behavioral components already
demonstrated to be elicited by nereithione, it is possible that
nereithione may also act as an attractant at the lower concentrations
present at distances greater than 10 mm, a possibility that should be
investigated in future studies.
Identification of the water-borne pheromones in several species [a
mollusc (Aplysia; 3), a protozoan (Euplotes spp.;
4,
5,
6,
30
), and a polychaete, N. succinea] should
facilitate investigations of pheromone-mediated mechanisms. All of
these pheromones are peptides and there is no homology between their
sequences; however, one curious similarity between these pheromones is
their high cysteine content. Nereithione ([cys][-glu-cys-gly])
is fully 50% cysteine, and the Aplysia and
Euplotes pheromones each have 6 cysteine residues out of
their 38 to 58 amino acid full-length sequences. The significance for
pheromonal function of cysteines and resultant disulfide bonds in
regulating their synthesis, stabilizing the pheromones in the
environment, or interacting with their receptors should become evident
as additional water-borne pheromone structures are determined and their
functions in N. succinea and other organisms are
investigated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication October 14, 1998.
Revision received December 5, 1998.
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REFERENCES |
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