NERVOUS CONTROL OF OVULATION AND EJACULATION

IN HELIX ASPERSA
EMILE GEOFFROY, ROBERT HUTCHESON AND RONALD CHASE
Department of Biology, McGill University, 1205 Ave. Docteur Penfield, Montre´al, Que´bec, H3A 1B1, Canada
(Received 16 December 2004; accepted 31 March 2005)

ABSTRACT
The ovotestis duct transports male and female gametes from the ovotestis, through the seminal vesicle,
and into the fertilization pouch-spermathecal complex. All these structures are innervated by small
branches of the intestinal nerve. Electrical stimulation of the nerve increased the rate at which
gametes were transported in the duct and caused autosperm to flow into the fertilization pouch-
spermathecal complex. These events were accompanied by stimulation-induced peristaltic contractions
along the duct and activation of the cilia lining the interior of the duct. Acetylcholine and serotonin
were identified as excitatory transmitters in this system, while FMRFamide was identified as a
muscle relaxant. The nervous control of ejaculation may contribute to optimizing the size of the ejacu-
late in a context of sperm competition, while the involvement of the nervous system in ovulation may
stem from a requirement for sensory integration that is peculiar to the Stylommatophora.

INTRODUCTION Chase et al., 2004). In the present paper, we describe motor func-
tions of the intestinal nerve and their effects on gamete move-
In hermaphroditic molluscs, the ovotestis is the source of both ments in the ovotestis duct of Helix aspersa.
oocytes and sperm. In Helix aspersa, the sperm cells differentiate
and mature through a sequence of stages that requires about 6
weeks to complete (Bloch & Hew, 1960), after which they MATERIAL AND METHODS
migrate to the seminal vesicle where they are stored for
mating. The oocytes, on the other hand, mature over a period Mature snails, Helix aspersa, were obtained from natural popu-
of undetermined length, and they remain within the ovotestis lations in California. They were maintained at 14 –178C under
until they are released for fertilization immediately prior to ovi- a light:dark cycle of 16 h:8 h. All experiments and all obser-
position (Tompa, 1984). As in most animals, sperm production vations were conducted during the animals’ scotophase. Three
is vastly greater than oocyte production. times weekly the animals were rinsed in water and fed an
A neuroendocrine mechanism for ovulation (the release of aqueous slurry comprising powdered grains, calcium, animal
oocytes) is well documented in certain opisthobranchs, notably protein, fat and vitamins. By these means, the animals were
Aplysia, and in a few basommatophoran pulmonates, notably kept in an active state. For most purposes, the snails were
Lymnaea (Chase, 2002). However, no chemical factor responsible housed in Lucite boxes (36  36  8 cm), 25 – 50 snails per
for either ovulation or oviposition has yet been identified in the box, and they were allowed to mate freely. Exceptionally, in
stylommatophoran pulmonates (Flari & Edwards, 2003). More- sperm release experiments, the snails were isolated to ensure
over, the mechanisms responsible for ejaculation (the release of maximum levels of sperm. The absence of a soft substratum pre-
sperm) have not been elucidated in any gastropod species. vented the snails from laying eggs. However, when experiments
The focus of this paper is the ovotestis duct, also known as the required snails that were ready to lay eggs, groups of 25 – 50
hermaphroditic duct or gonoduct (Tompa, 1984). It transports snails were placed in boxes containing 3 – 4 cm of clean, friable
the gametes from the ovotestis to the fertilization pouch- soil so that nest excavation could be observed. In our experience,
spermathecal complex (FPSC). The histological investigation approximately 50% of nest-digging snails lay eggs.
of Breucker (1964) showed that the lumen of the duct is For observations in vitro, the snails were anaesthetized by
surrounded by circular muscle, while the interior epithelium injection of 2 – 3 ml of isotonic MgCl2 into the posterior mantle
contains two dominant cell types, one of which is ciliated. column. After dissection, the pertinent organs and tissues
Following Tompa (1984) we refer to that part of the duct (Fig. 1) were pinned out in a dish and covered with saline
closest to the ovotestis as the ‘proximal’ part, and that part (Kerkut & Meech, 1966). The fertilization pouch-spermathecal
closest to the FPSC as the ‘distal’ part. complex (FPSC) was left embedded in the albumen gland.
The ovotestis and its duct is innervated by fine branches of the Nerve branches were labelled with Neurobiotin (Vector Labs)
intestinal nerve in Helix (Schmalz, 1914). One function of the using published protocols (Antkowiak & Chase, 2003). Briefly,
innervation is sensory. The number of mature oocytes is coded the nerve was cut close to the region of interest and the stump
in the firing frequencies of afferent nerve fibres, and this activity was sucked into a glass pipette filled with a buffered solution of
appears to operate as a permissive signal for oviposition (Antko- 8% Neurobiotin. Twenty-four hours later, the pipette was
wiak & Chase, 2003). Afferent signals from the ovotestis were removed and the tissue was fixed, then processed using the
also found to initiate central nervous system (CNS) motor Vectastain ABC kit (Vector Labs). The histology of the ovotestis
responses that resulted in an elevated level of cardiac output duct was examined using transverse paraffin sections cut to a
and more efferent traffic in the intestinal nerve, some of which thickness of 10 mm and sampled at intervals of 50 mm. The
was directed back to the ovotestis (Antkowiak & Chase, 2003; sections were stained with toluidine blue.
To stimulate the intestinal nerve, the cut end was sucked into
a glass pipette filled with saline. Pulse duration was 2 ms, with
Correspondence: R. Chase; e-mail: ronald.chase@mcgill.ca frequency and voltage adjusted as required (5 –50 V).

Journal of Molluscan Studies (2005) 71: 393– 399. Advance Access Publication: 5 August 2005 doi:10.1093/mollus/eyi041
# The Author 2005. Published by Oxford University Studies on behalf of The Malacological Society of London, all rights reserved.
 E. GEOFFROY ET AL.

Figure 1. The hermaphroditic components of the reproductive system in

Helix aspersa. The ovotestis duct, together with the organs it connects, is
innervated by branches of the intestinal nerve. The approximate
location and relative density of each innervation field is indicated.

Duct contractions, sperm flow and gamete transport were

documented using a video camera mounted on either a com-
pound microscope or a dissecting microscope. Measurements
and rate calculations were made from taped records after
digitization (Scion Corp). To better visualize the proximal
part of the ovotestis duct, the ovotestis was desheathed and a
sliver of opaque plastic was inserted directly under the duct.
The tension generated by muscles lining the seminal vesicle was Figure 2. Variation in lumen area along the length of the distal ovotestis
measured isotonically using a force transducer (Grass Instruments, duct. Measurements in the graph are plotted from serial histological sec-
FT.03C). The amplified signal was low-pass filtered (corner tions sampled at intervals of 50 mm. Points marked in the graph as A and
frequency, 4 Hz) prior to digitization (Axon Instruments). B are illustrated in the micrographs above. The duct joins the FPSC at
Pharmacological treatments were done by injecting solutions 2.1 mm, which is represented here as the end point of the graph.
into the experimental dish using a hand-held pipette. The tip
of the pipette was positioned directly above the targeted tissue
at a distance of approximately 5 mm. All substances (Sigma)
were dissolved in saline and delivered in aliquots of either distinct organ, is actually part of the ovotestis duct; it demarcates
200 ml (acetylcholine, serotonin and FMRFamide) or 500 ml the proximal and distal segments of the duct. The duct is widest
(hexamethonium bromide and methysergide maleate). in the region of the seminal vesicle, more narrow near the ovotes-
To measure ciliary beat frequency, an incision was made tis and the FPSC. To quantify this form, we measured the duct
along the ovotestis duct and a glass coverslip was secured over lumen along the distal segment in one specimen. We found a
it to keep the duct’s inner walls exposed. The preparation was gradual reduction in lumen area, and then an expansion just
trans-illuminated on a compound microscope that was equipped before the duct connects to the FPSC (Fig. 2). At the point of
with a screen upon which images of the cilia were optically pro- maximum constriction, 450 mm from the FPSC, the area of
jected. An integrated record of ciliary beating within an area of the duct was reduced by 94% relative to its size near the
75 mm2 was obtained by positioning a cadmium sulphide photo- seminal vesicle. Under normal circumstances, this narrowing
cell (5– 500 KV) in front of the screen (Dalhamn & Rylander, of the distal duct would likely inhibit the movement of sperm
1962). The output of the photocell was low-pass filtered and as such prevent, or help to prevent, the emptying of the
(corner frequency, 20 Hz) prior to digitization. The records seminal vesicle. Consistent with this, the photomicrographs in
were then analysed for signal oscillation frequency using Figure 2 show the presence of a sperm mass in the centre of
Axoscope software (Axon Instruments). The person measuring the duct where it is wide (Fig. 2A), but the absence of sperm
frequency was unaware of the treatment condition. in the same duct where it is narrow (Fig. 2B).
Statistical analyses were performed using SPSS software, The innervation of the duct is summarized in Figure 1.
v. 11.0. For the effect of nerve stimulation on ciliary beat Although schematic, the drawing indicates the approximate dis-
frequency, an analysis of variance (ANOVA) was performed. tribution of the innervation along the duct and in the ovotestis
For post hoc comparisons between the baseline frequency and and FPSC. Illustrations of the most important areas of inner-
the frequencies recorded after stimulation, the Dunnett T3 test vation are shown in Figure 3. The proximal part of the duct is
was used; this test is not dependent upon equal variances across heavily innervated by fine nerve fibres that wrap around the cir-
groups. For the effect of serotonin on ciliary beat frequency, the cumference of the duct (Fig. 3A). These fibres are studded with
ANOVA was again employed; Levene’s test of equality of vari- varicosities that appear similar to those that have been identified
ances was insignificant (P . 0.10). Post hoc comparisons used as serotonergic release sites in Helix pomatia (Elekes, 1991; Kiss
Dunnett’s test (one-tailed). Gamete transport rates were ana- et al., 2003). By contrast, the seminal vesicle is sparsely inner-
lysed with a repeated-measures design of the ANOVA using the vated (Fig. 3B). The Neurobiotin label revealed a small
Huynh– Feldt within-subjects test. The within-subjects degrees number of nerve cell somata scattered on the nerve, and the
of freedom was adjusted using Mauchly’s test of sphericity. occasional nerve-cell process (Fig. 3C). These cells are evidently
identical to those described earlier as immunoreactive to FMRF-
amide antiserum and ‘grouped in true ganglia’ on this same
RESULTS nerve branch (Cardot & Fellman, 1983). They could be either
sensory or motor in function. Further along the ovotestis duct,
Anatomy and innervation of the ovotestis duct in the distal region linking the seminal vesicle with the FPSC,
As shown in Figure 1, the ovotestis duct connects the ovotestis a profusion of fine nerve branches envelopes the duct
with the FPSC. The seminal vesicle, while often considered a (Fig. 3D). The FPSC itself is only sparsely innervated, and

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 CONTROL OF OVULATION AND EJACULATION IN HELIX

Stimulus-induced contractions of the ovotestis duct

Electrical stimulation of the intestinal nerve consistently induced
contractions of the ovotestis duct provided sufficient voltage was
applied (.20 preparations). Quantitative measures of contrac-
tile responses were obtained by video for the proximal and
distal parts of the duct and by force transducer for the seminal
vesicle. We confirmed that the contractions are peristaltic by
measuring the duct width at specific locations over time
(Fig. 4). Peristalsis occurred in both the proximal and distal
parts of the duct, and it was always directed towards the
FPSC. To quantify the reliability of the response, we delivered
10 trains of electrical pulses (inter-train interval, 3 min) in a
single responsive preparation while monitoring the proximal
duct. Every stimulus train elicited a contraction, and the mean
reduction in duct diameter was 13.4% (SD + 1.7). In addition,
smaller contractions (,10% reductions) occurred irregularly
between stimulus trains, whereas no contractions occurred in
the 15 min prior to stimulation. It is noteworthy that contraction
latencies were consistently longer in the proximal duct than in
the distal duct. For the proximal duct, the mean latency was
4.4 s (N ¼ 10 trials), whereas in the distal duct the latency
was 1 s (Fig. 4).
A slight radial contraction of the seminal vesicle was seen under
the microscope following electrical stimulation of the intestinal
nerve. To confirm this observation, we measured the force
induced across the length of the seminal vesicle. As shown in
Figure 5, stimulation elicited a slow rise in tension that reached
a plateau after about 30 s. When the stimulus was terminated,
tension immediately began to return to baseline values.

Stimulus-induced activation of cilia

Stimulation of the intestinal nerve also elicited an increase in the
beat frequency of the cilia that line the interior of the ovotestis
duct. In the single preparation represented by Figure 6A,
inter-beat intervals were measured in seven periods of 50 s
duration (N ¼ 83 intervals in each period). The initial measure-
ment after stimulus onset was delayed 10 s to avoid contraction
artefacts, and all subsequent measurement periods were simi-
larly separated by 10 s intervals. Electrical stimulation of the
nerve caused a significant increase in beat frequency
(ANOVA: F6, 589 ¼ 193.4, P , 0.0001). Beat rates were still

Figure 3. Innervation patterns as revealed by Neurobiotin. A. Radial

fibres surrounding the proximal ovotestis duct. Note the numerous var-
icosities. B. Seminal vesicle. Arrowheads point to fine fibres. C. Cell
somata (arrowheads) associated with the main nerve branch as it tra-
verses the seminal vesicle. Arrows point to a process emanating from
one of the somata. D. Profuse innervation of the distal ovotestis duct.

Figure 4. Peristalsis in the distal ovotestis duct. The duct width is plotted
at two points in a single preparation. The point represented by filled
this is mostly in the area of the fertilization pouch. Note that a circles is located 500 mm from the seminal vesicle; the point represented
bifurcation of the intestinal nerve provides separate nerve by open circles is located 1200 mm from the seminal vesicle. Stimulation
branches to the distal and proximal parts of the duct, the of the intestinal nerve begins at time 0 (pulses at 10 Hz, 2-s trains at
latter including the seminal vesicle (Fig. 1). 0.2 Hz).

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 E. GEOFFROY ET AL.

Acetylcholine (2  10 – 7 M) caused contractions of the ovotes-

tis duct that were equivalent in magnitude to those induced by
nerve stimulation (Fig. 7A). However, in contrast to the peristal-
tic pattern of contraction induced by nerve stimulation, the
contractions induced by acetylcholine occurred simultaneously
over the entire length of the exposed duct. This difference
implies that peristalsis is dependent on a precise spatial-temporal
Figure 5. Example of contraction of the seminal vesicle elicited by pattern of acetylcholine delivery that results from the branched
stimulation of the intestinal nerve. Tension was measured using a force
transducer. Electrical stimulation at 0.7 Hz.
pattern of innervation. Serotonin (5-hydroxytryptamine,
2  10 – 7 M) also caused contractions, and these were more
persistent than those caused by acetylcholine (Fig. 7A).
The peptide FMRFamide (2  10 – 7 M) had a pure relax-
significantly elevated above baseline 4 min after cessation of the ation effect. The latency of action for FMRFamide was longer
stimulus (P , 0.0001), but they fell to pre-stimulus levels 5 min than for the contractions produced by acetylcholine and sero-
after stimulation. tonin (Fig. 7A). To determine whether activity in the intestinal
nerve was capable of relaxing the duct, we blocked the actions of
both acetylcholine (with hexamethonium, 5  10 – 7 M) and
Pharmacology serotonin (with methysergide, 5  10 – 7 M), and we measured
the duct’s response to nerve stimulation over a period sufficiently
To test the effect of serotonin on cilia in the ovotestis duct, prep-
long to reveal the effects of FMRFamide. Under these condi-
arations were bathed in saline for 5 min and the beat frequencies
tions a pure relaxation phenomenon was observed (Fig. 7B).
were calculated for the final 30 s. Then, either saline as a control,
or serotonin at one of three concentrations was applied to the
preparation, and beat frequencies were again determined at
the end of 5 min. This protocol was repeated in five preparations
for each treatment group. The results (Fig. 6B) clearly demon-
strate an excitatory effect of serotonin (ANOVA: F3,16 ¼ 82.5,
P , 0.0001). All three concentrations of serotonin (0.1, 1.0
and 10  10 – 6 M) induced a beat frequency increase that was
larger than that induced by saline alone (P , 0.0003).

Figure 6. Nerve stimulation and serotonin both increase the cilia beat Figure 7. Pharmacology of duct contraction and relaxation. A. Changes
frequency. A. Electrical stimulation of the intestinal nerve at 5 Hz for in width of the proximal duct following application of FMRFamide,
60 s. Asterisks indicate a significant difference in relation to baseline fre- acetylcholine (ACh) and serotonin (5-HT). All transmitters, 2  10 – 7 M.
quency (P , 0.0001). Error bars are 1 SD. B. Serotonin treatment. Bars B. The effect of nerve stimulation during blockade of acetylcholine
show the mean change in frequency after application of either saline or (by hexamethonium) and serotonin (by methysergide), both at
serotonin (each treatment, N ¼ 5). Error bars are 1 SD. Asterisks indi- 5  10 – 7 M. Stimulation in all conditions, 5 Hz for 20 s. Data points
cate a significant difference in relation to the saline control (P , 0.0001). are means from four trials in the indicated condition.

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 CONTROL OF OVULATION AND EJACULATION IN HELIX

Control trials, conducted before and after treatment with the sperm was initially present in the seminal vesicle (Fig. 9). In
receptor antagonists hexamethonium and methysergide, such cases, the sperm accumulated in the FPSC and, with pro-
resulted in a typically fast contraction followed by a late, weak longed stimulation (1 h) it passed through the FPSC and
relaxation. entered the saline bath through an incision in the spermoviduct
(uterus).
To investigate the contributions of different nerve branches to
The release of gametes sperm release, lesions were made selectively to either the branch
innervating the seminal vesicle or the branch innervating the
The movement of gametes was examined at two locations: one distal ovotestis duct (Fig. 1). Five preparations were evaluated
just proximal to the ovotestis and the other on the distal ovotestis in each lesion group, together with a control group in which
duct. These sites allowed us to investigate the release of gametes there was no lesion. The duration of sperm flow to the FPSC
from the ovotestis and the seminal vesicle, respectively. To maxi- was measured throughout a 30-min period of intestinal nerve
mize the probability of ovulation, snails were selected for in vitro stimulation. For the control group, the mean duration of flow
observations after being found motionless at the bottom of a nest was 26.0 + 4.0 (SE) min; for the seminal vesicle branch lesion,
that they had excavated in the soil, since nest-digging behaviour 2.6 + 1.4 min; and for the distal duct lesion, zero. It is apparent
normally precedes oviposition. After dissection, the movement of from these results that the seminal vesicle and the distal ovotestis
gametes out of the ovotestis was videotaped before, during and duct must be activated together to produce an ejaculation.
after electrical stimulation of the intestinal nerve (5 Hz for In all instances, the release of gametes was accompanied
20 min). Even before stimulation, large numbers of sperm and by pronounced peristaltic contractions of the duct. Whereas
immature oocytes were seen exiting the ovotestis. This obser- the rate of flow from the ovotestis was constant during nerve
vation is consistent with the results from a separate experiment stimulation, the flow of sperm in the distal duct was sometimes
in which we examined the contents of the ovotestis, in different intermittent, even during continuous stimulation. In the latter
snails, before and after oviposition. In that experiment we cases, sperm flow appeared impeded at a point approximately
noted a similar flushing of the ovotestis in association with ovi- mid-length along the distal duct segment, probably due to the
position (Reynolds & Chase, unpublished). However, fewer narrowing of the duct lumen in this region (Fig. 2).
than four mature oocytes (diameter .100 mm) were released
by nerve stimulation in any of the in vitro preparations
(N ¼ 5), despite the fact that the ovotestes contained numerous
mature oocytes.
To calculate transport rates, four gametes (either sperm or
immature oocytes) from each preparation were tracked as they
passed through the field of view (travel lengths, 850–1130 mm).
Different gametes were tracked in each 20-min treatment
period. As shown in Figure 8, transport rates increased, on
average, 2.7 times during nerve stimulation (Fig. 8), represent-
ing a significant effect of nerve stimulation (ANOVA:
F1.068 ¼ 34.4, P , 0.004).
In 10 additional preparations, also using snails that had exca-
vated a nest, observations similar to those described above were
made throughout 6 h or more of continuous nerve stimulation
(5 Hz). Again, stimulation was accompanied by a major flushing
of sperm, immature oocytes and other unidentified material
from the ovotestis, but no substantial number of ripe oocytes.
Stimulation of the intestinal nerve reliably caused sperm to
flow out of the seminal vesicle and into the FPSC whenever

Figure 9. Nerve stimulation causes sperm to flow from the seminal

vesicle. Video frames recorded during stimulation of the intestinal
Figure 8. Nerve stimulation increases gamete transport rates in the nerve at 10 Hz during 2-s trains at 0.2 Hz. The preparation is trans-
proximal ovotestis duct. Four gametes (sperm or oocytes) were tracked illuminated through the albumen gland, making the sperm appear
in each of five preparations. Points represent mean transport rates dark. The distal ovotestis duct is outlined in the top frame. Abbreviations:
before, during and after electrical stimulation of the intestinal nerve. FPSC, fertilization pouch – spermathecal complex; SV, seminal vesicle.

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 E. GEOFFROY ET AL.

DISCUSSION injecting various substances into snails, as has been done success-
fully in other gastropod molluscs. Regrettably, despite multiple
The results of this investigation establish two motor roles for the attempts, neither the injection of CNS homogenates from Helix
innervation of the ovotestis duct, namely, control of the radial nor the injection of hormone-containing bag cells from Aplysia
musculature and control of cilia beating. These functions comp- was successful (Geoffroy, Sharma & Chase, unpublished).
lement a sensory role that was described earlier (Antkowiak & We similarly failed to elicit oviposition by injecting serotonin
Chase, 2003). The nerve branches at issue here are tiny, the (Reynolds & Chase, unpublished). Additional trials were con-
largest of them measuring only 20 mm in diameter (Antkowiak ducted in vitro combining nerve stimulation with the application
& Chase, 2003). Nevertheless, gastropod nerves commonly of CNS homogenates or FMRFamide or freshly drawn blood
contain mixes of sensory and motor fibres (Chase, 2002), from ovipositing snails, again without eliciting ovulation.
and even the aforementioned branch contains 3,025 axons These results might reflect the absence of an ovulation
(Antkowiak & Chase, 2003). hormone in the Stylommatophora. Alternatively, they may
The cilia that are abundantly present on the interior walls of indicate a complex interplay between endocrine and nervous
the snail’s ovotestis duct are generally assumed to assist in trans- controls that cannot be adequately investigated in simple exper-
porting gametes (Breucker, 1964). Consistent with this, we iments. It must also be acknowledged that the in vitro procedures
found that nerve stimulation increased both ciliary beat rates used by ourselves and others necessarily disrupt the normal
and gamete transport rates. While peristaltic contractions were blood circulation and the internal hydrostatic skeleton, either
the most visible manifestation of nervous activity, we also discov- of which could be important for ovulation.
ered a more subtle relaxation effect. It is difficult to know how
contractions and relaxations are actually patterned in vivo, but
it is reasonable to expect that the passage of large oocytes and Control of ejaculation
large sperm masses would be facilitated by contractions
pushing objects into regions of the duct that were already Sperm are much smaller than oocytes and are not attached to
dilated. We imagine that this scenario could be realized by the any immobile structure, hence they probably move freely into
appropriately timed excitation of separate nerve fibres media- the seminal vesicle once they have fully matured. Their passage
ting contraction and relaxation. out of the seminal vesicle is impeded by two factors. First, they
All the transmitters implicated in the aforementioned motor are stored not as isolated individuals, but as a high-density
effects are well known in gastropod molluscs (Chase, 2002). mass formed of entangled and sticky tails. Second, the movement
Acetylcholine, for example, is the typical fast transmitter for of this mass is blocked in the distal ovotestis duct by the narrow-
muscles. Serotonin’s mediation of ciliary activation is well estab- ing of the channel, as described in this paper.
lished (Audesirk, McCaman & Willows, 1978), as is the ability Our results indicate that efferent discharges in the intestinal
of FMRFamide to relax muscles (Lehman & Greenberg, nerve are necessary and sufficient to release sperm from the
1987). The contractile effect of serotonin is less common, but it seminal vesicle. Although we were able to evoke release by
has been reported in the salivary duct of Helix pomatia (Kiss stimulation of the entire nerve, it is probable that under normal
et al., 2003). More intriguing is how the employment of these circumstances neurons innervating the seminal vesicle and
several transmitters is orchestrated to produce a coherent effect neurons innervating the distal ovotestis duct are activated sepa-
on duct function. Again, our interpretations are limited by our rately, something that was beyond our technical ability. The
inability to stimulate selected fibres comprising the nerve most efficient scenario would have the seminal vesicle contract
branches of interest. Based on studies in Aplysia, however, we first to release a quantity of sperm and thus apply pressure on
expect that even a single muscle might be innervated by motor the constriction in the distal duct. Shortly thereafter, peristaltic
fibres releasing two fast transmitters and multiple peptide modu- contractions in the distal duct would move the sperm mass past
lators (Keating & Lloyd, 1999). the point of obstruction. Finally, the duct would dilate to allow
even greater flow. Dilation is likely to result from a combination
of the moving sperm mass itself, the elasticity of the duct muscle
Control of ovulation and the relaxing action of FMRFamide. It is noteworthy that
the constraint imposed on sperm movements in the distal ovotes-
Although we were able to influence the rate of gamete transport,
tis duct is similar to that operating on bile in the mammalian bile
we were unsuccessful in triggering ovulation. In fact, only rarely
duct. In the latter case, a structure known as the sphincter of
did we observe ripe oocytes passing through the ovotestis duct.
Oddi limits passage of bile into the duodenum. Bile passes
The vast majority of ripe oocytes evidently remained attached
through only when there are rhythmic contractions of the
to the walls of the acini regardless of the strength, patterning
sphincter (Toouli et al., 1983).
or duration of nerve stimulation. In Aplysia, where the process
of ovulation has been well studied, the ovotestis is not innervated
and the oocytes are released solely through the activity of an
Significance of nervous controls
ovulation hormone (Coggeshall, 1970; Dudek et al., 1980). It is
unclear whether the Aplysia hormone acts by contracting The nervous control of ejaculation may be important for permit-
muscles on the acini or by acting on the bonds that attach the ting a variable quantity of sperm to be transferred. Theory pre-
oocytes to the acini walls (Dudek et al., 1980). dicts, and empirical investigations in some taxa confirm, that
There is strong evidence for peptide ovulation hormones in males regulate their ejaculate size according to the character-
several species of opisthobranchs and basommatophoran pulmo- istics of their partner or the risk of competition (Birkhead &
nates (Chase, 2002), and immunocytochemical work in Helix Møller, 1998). Although such a regulation has not yet been veri-
aspersa suggests that a homologous peptide may be present in fied in molluscs (Baur, Locher & Baur, 1998), if it were to occur
the CNS of this species (Van Minnen, Schallig & Ramkema, it would probably require mediation by the nervous system. For
1992). Other studies, however, suggest that egg-laying in the example, a simple mechanism to control the size of the ejacula-
Stylommatophora is controlled by steroid hormones (Takeda, tion would be to vary the degree of neuromuscular excitation at
1979). Authors also disagree about whether the putative egg- the seminal vesicle. In a similar vein, selection for robust inner-
laying hormone is released from the ovotestis, the CNS or the vation of the seminal vesicle is predicted if it enhances a snail’s
tentacles (see Flari & Edwards, 2003). To clarify the situation ability to transfer large numbers of sperm (Greeff & Michiels,
with respect to Helix, we attempted to induce egg-laying by 1999).

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Nervous control may also be important for the correct timing of BAUR, B., LOCHER, R. & BAUR, A. 1998. Sperm allocation in the
ejaculation, which is critical for effective sperm transfer. The sper- simultaneously hermaphroditic land snail Arianta arbustorum. Animal
matophore is formed, and begins to fill with sperm, only after Behaviour, 56: 839–845.
intromission. Interestingly, the delay between intromission and BIRKHEAD, T.R. & MØLLER, A.P. 1998. Sperm competition and sexual
spermatophore filling differs substantially between species. In selection. Academic Press, London.
Helix pomatia the spermatophore is filled and transferred within BLOCH, D.P. & HEW, H.Y.C. 1960. Schedule of spermatogenesis in
4.5 min of intromission (Lind, 1973), whereas for H. aspersa the the pulmonate snail Helix aspersa, with special reference to histone
comparable interval is 300 min (Adamo & Chase, 1988). Since transition. Journal of Biophysical and Biochemical Cytology, 7: 515–531.
the spermatophore of H. aspersa does not leave the epiphallus BREUCKER, H. 1964. Cytologische Untersuchungen des
before 2 h (Adamo & Chase, 1988), it is likely that it is still Zwitterganges und des Spermoviduktes von Helix pomatia
L. Protoplasma, 58: 1–41.
being filled with sperm at this time. On the other hand, the
spermatophore of H. pomatia is full after about 1 min. Whatever CARDOT, J. & FELLMAN, D. 1983. Immunofluorescent evidence of
an FMRFamide-like peptide in the peripheral nervous system of
the adaptive significance of these differences in timing, we the gastropod mollusc Helix aspersa. Neuroscience Letters, 43: 167–172.
imagine that they are mediated by differences in either the
CHASE, R. 2002. Behavior and its neural control in gastropod molluscs. Oxford
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While it remains to be seen whether the Stylommatophora
CHASE, R., ANTKOWIAK, T., GEOFFROY, E. & WEATHERILL,
have an ovulation hormone (Flari & Edwards, 2003), the fact D. 2004. Why the ovotestis of Helix aspersa is innervated. Acta Biologica
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(Coggeshall, 1970), invites a comparison between these two gas- COGGESHALL, R.E. 1970. A cytologic analysis of the bag cell control
tropods. Our results indicate that the innervation in Helix is at of egg laying in Aplysia. Journal of Morphology, 132: 461– 486.
least contributory to ovulation, whereas in Aplysia ovulation is DAGUZAN, J. 1981. Contribution à l’élevage de petit-gris: Helix aspersa
controlled entirely by a peptide hormone (Chase, 2002). The Müller. Annales des Zootechnie, 30: 249– 272.
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different environmental requirements for egg-laying. Aplysia measured with a photo-sensitive cell. Nature, 196: 592– 593.
lays eggs repeatedly throughout the reproductive season (as DUDEK, F.E., INJEYAN, H.S., SOUTAR, B., WEIR, G. & TOBE,
many as 27 separate episodes in 4 months), and as many as S.S. 1980. The ovotestis of Aplysia californica: anatomy and egg
140,000 eggs can be deposited in a single mass (MacGinitie, release. Canadian Journal of Zoology, 58: 2220–2229.
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presence of a solid object on which to attach the egg mass. In body region and neural sheath of the snail, Helix pomatia, ganglia:
contrast, Helix lays eggs only once or twice in an equivalent an electron microscopic immunocytochemical study. Neuroscience,
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only about 100 (Daguzan, 1981; Antkowiak & Chase, 2003). FLARI, V.A. & EDWARDS, J.P. 2003. The role of the endocrine
Whereas the sea provides a relatively constant environment for system in the regulation of reproduction in terrestrial pulmonate
Aplysia, on land Helix must deposit its eggs in soil that is gastropods. Invertebrate Reproduction and Development, 44: 139 –161.
friable, rich in calcium, moist, but not flooded. Also, since the GREEFF, J.M. & MICHIELS, N.K. 1999. Sperm digestion and
overall egg production is much less for Helix than for Aplysia, reciprocal sperm transfer can drive hermaphrodite sex allocation to
Helix takes the greater risk with each oviposition event; more- equality. American Naturalist, 153: 421–430.
over, it probably expends more energy (Antkowiak & Chase, KEATING, C. & LLOYD, P.E. 1999. Differential modulation of motor
2003). An observation that highlights the snail’s cautious neurons that innervate the same muscle but use different excitatory
approach to oviposition is that a snail will often excavate a see- transmitters in Aplysia. Journal of Neurophysiology, 82: 1759–1767.
mingly perfect nest but then leave it without depositing eggs. It KERKUT, G.A. & MEECH, R.W. 1966. The internal chloride
were as if a final ‘go’ signal is not detected. Together, the afore- concentration of H and D cells in the snail brain. Comparative
mentioned considerations imply that egg-laying in Helix is Biochemistry and Physiology, 19: 819–832.
dependent on an exacting set of internal and external conditions, KISS, T., HIRIPI, L., PAPP, N. & ELEKES, K. 2003. Dopamine and
and that the role of the nervous system is to sense and integrate serotonin receptors mediating contractions of the snail, Helix pomatia,
salivary duct. Neuroscience, 116: 775 –790.
the signals that collectively trigger egg-laying. Although Aplysia
also processes sensory signals, we suggest that the innervation of LEHMAN, H.K. & GREENBERG, M.J. 1987. The actions of
FMRFamide-like peptides on visceral and somatic muscles of the
the ovotestis in Helix provides an additional degree of control. snail Helix aspersa. Journal of Experimental Biology, 131: 55 –68.
LIND, H. 1973. The functional significance of the spermatophore and
ACKNOWLEDGEMENTS the fate of spermatozoa in the genital tract of Helix pomatia
(Gastropoda: Stylommatophora). Journal of Zoology, 169: 39– 64.
Emile Geoffroy and Robert Hutcheson contributed equally to MAC GINITIE, G.E. 1934. The egg-laying activities of the sea hare,
this work. We thank David Rogers for helping to obtain the Tethys californicus (Cooper). Biological Bulletin, 67: 300–303.
photomicrograph shown in Figure 3D. We also thank Tomasz SCHMALZ, E. 1914. Zur Morphologie des Nervensystems von Helix
Antkowiak and Annie Reynolds for valuable discussions. pomatia L. Zeitschrift für wissenschaftliche Zoologie, 111: 506 –568.
Supported by a grant to R. C. from NSERC Canada. TAKEDA, N. 1979. Induction of egg-laying by steroid hormones in
slugs. Comparative Biochemistry and Physiology, 62A: 273–278.
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