Sensory innervation of the ovotestis in the snail Helix aspersa
Department of Biology, McGill University, 1205 Ave Docteur Penfield, Montreal, Quebec, H3A 1B1, Canada
* Author for correspondence (e-mail: ronald.chase{at}mcgill.ca)
Accepted 22 July 2003
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Summary |
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Key words: oviposition, clutch size, oocyte, ovotestis, sensory innervation, land snail, Helix
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Introduction |
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Obviously, as clutch size increases, so too does the energetic cost to the
female function. It is important to note, however, that while clutch sizes
vary, each oviposition event is coupled to a fixed minimum cost. Factors
contributing to the fixed cost of egg laying include the search for adequate
soil conditions (travel distances of up to 15 m in Helix pomatia;
Pollard, 1975), nest
excavation and the generation of hydrostatic pressure to expel the egg mass
(Perrot, 1938
). Since the
fixed costs are high, it is reasonable to attribute the infrequency of
oviposition to the fixed costs. The cost per egg can be reduced, however, by
producing large clutches. Furthermore, as clutch size increases, so too does
the offspring survival rate because large clutches are less prone to
desiccation (Bayne, 1968
). On
the other hand, predation risks may favour more frequent depositions of
smaller clutches at multiple sites. Ultimately, the number of eggs laid by an
individual is determined by balancing energetic costs against reproductive
benefits. We propose that oviposition events occur when the clutch size
surpasses a minimum value for which the reproductive benefit exceeds the total
of fixed and variable costs. Therefore, a prerequisite for egg laying should
be the availability of the threshold number of mature oocytes. In the present
study, we address the question of how H. aspersa is able to monitor
its oocyte production to ensure the presence of this minimum number of gametes
before it initiates the costly cascade of physiological events that results in
oviposition.
In 1914, Schmalz described the innervation of the ovotestis (OT) in H.
pomatia by a branch of the intestinal nerve. The functional significance
of this innervation, however, remains unknown. Similarly, innervation of the
mammalian ovary has been described in several species, including pigs
(Majewski et al., 2002), rats
(Burden and Lawrence, 1977
) and
humans (Hill, 1962
), but
information regarding the function of such innervation is sparse. Anatomical
clues and lesion studies suggest a sensory role possibly related to follicular
recruitment (Aguado, 2002
),
steroidogenesis (Kawakami et al.,
1981
) or blood flow regulation
(Ojeda and Lara, 1989
). Here,
we describe further details of the innervation of the ovotestis in H.
aspersa, and we demonstrate a sensory function. We conclude that sensory
endings in the gonad respond to the volumetric increase that occurs with
oocyte maturation and that the neural signal is relayed to the central nervous
system (CNS) via a low-frequency, tonic discharge of afferent
spikes.
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Materials and methods |
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Morphology and innervation
The innervation of the gonad was examined after labelling the ovotestis
branch of the intestinal nerve with neurobiotin (Vector Labs, Burlington, ON,
Canada). The nerve branch was cut at its point of entry into the ovotestis.
The distal nerve stump was sucked into a glass capillary that was then filled
with 8% neurobiotin in 0.1 mol l-1 Tris buffer (pH 7.6).
Infiltration was allowed to continue for 24 h. After fixation in 4%
paraformaldehyde, the preparation was incubated for 16 h in 4% Triton X-100 in
0.1 mol l-1 phosphate-buffered saline (pH 7.4). For visualization,
we used the Vectastain ABC kit (Vector Labs). Preparations were initially
examined as wholemounts, then cut as 20 µm frozen sections.
For electron microscopy, a nerve segment was fixed in 5% glutaraldehyde in
0.1 mol l-1 sodium cacodylate (pH 7.2) and dehydrated in acetone.
Epon sections were stained with 4% uranyl acetate and Reynolds lead citrate
(Reynolds, 1963).
Photomicrographs were digitized, then analysed using Sigma Scan software (SPSS
Inc., Chicago, IL, USA). The perimeter of every fibre profile was individually
traced using a mouse-driven cursor. For each profile, we calculated the
diameter (feret diameter) of a hypothetical circular object with an area
equivalent to the measured profile.
Electrophysiology
Nerve recordings were obtained from reduced preparations consisting of the
CNS, the albumen gland, the seminal vesicle and the OT embedded in the
digestive gland (Fig. 1). The
entire preparation was pinned down in a Sylgard-coated recording dish
comprising a CNS chamber, an OT chamber and a central gap
(Fig. 1). The intestinal nerve
was desheathed and passed through small slits in the Sylgard walls of the
central gap that were subsequently sealed with Vaseline. During recordings,
either saline or an isotonic sucrose solution was perfused through the central
gap at a flow rate of approximately 10 ml min-1, while the
recording chambers were always filled with snail saline
(Kerkut and Meech, 1966).
|
To avoid possible artefacts associated with cut nerves, we recorded from the intact intestinal nerve using en passant glass suction electrodes. To assess whether recorded spikes were afferent or efferent, it was necessary to record simultaneously from at least two locations on the nerve (Fig. 1). In the OT recording chamber, one distal electrode and one proximal electrode (hereafter referred to as SV-D and SV-P, respectively) were placed on the nerve adjacent to the seminal vesicle portion of the hermaphroditic duct. There was no branching of the nerve between SV-D and SV-P, and nearly all recorded action potentials were detected at both electrode sites. A third central electrode was placed on the nerve in the CNS recording chamber (CNS electrode). In some experiments, a fourth electrode was placed in the OT chamber to monitor activity from the proximal stump of the pericardial branch of the intestinal nerve (PC electrode). The amplified signals were digitized using the Digidata 1322A converter and viewed using Axoscope 8.0 software (Axon Instruments, Union City, CA, USA).
A naive participant identified afferent spikes in the records. SV-D and SV-P traces were viewed simultaneously on a computer screen with an expanded time scale. A moveable vertical line provided by Axoscope was used to assess the intervals between pairs of spikes when both members of the pair were judged to be from the same unit based on waveform similarities and inter-spike intervals. A spike that appeared first at SV-D and second at SV-P was identified as afferent, whereas a spike appearing first at SV-P followed by SV-D was identified as efferent (see Fig. 4B). Since the number of afferent spikes was much less than the number of efferent spikes, the latter was determined by subtraction. First, the total number of spikes with amplitudes exceeding 100% of the noise level was counted using Mini Analysis software (Synaptosoft, Decatur, IL, USA). Then, the number of identified afferent spikes was subtracted from the total to give the number of efferent spikes. To facilitate the identification of afferent spikes, most of the efferent activity was suppressed during recording periods devoted exclusively to assessing afferent discharges; this was accomplished by perfusing sucrose through the gap. The directional nature of spikes in the pericardial nerve branch was not considered because these recordings were obtained from the proximal nerve stump; Mini Analysis was used to obtain the total spike count.
|
Tactile stimulation
To test for sensory responses, a wooden applicator stick was held by a
micro manipulator, and its cut end (2 mm diameter) was lowered onto the centre
surface of the OT. The probe covered approximately 50% of the OT surface area.
Responses were evoked when the probe was moved laterally across the surface of
the OT using a knob on the manipulator. A back-and-forth movement was
employed, the total duration of which was 3 s. Punctate stimuli of shorter
duration were ineffective.
Oocyte and egg counts
After completing the electrophysiological recordings, fine forceps were
used to break apart the connective tissue covering the OT. Suction from a 1 ml
micropipette was used to collect the OT homogenate and transfer it to a test
tube. By using coded tubes and examining homogenates in groups of two or more,
the oocytes were counted without bias. Small volumes (1.4 ml) of the OT
extract were successively transferred to a shallow glass dish and viewed under
a dissecting microscope at 20x magnification. A grid system was used to
ensure that each oocyte was counted only once. Each oocyte was measured on two
perpendicular axes and the resulting mean was taken as the cell's diameter.
Only mature oocytes at the vitellogenic stage (diameter >100 µm) were
counted (Griffond and Bolzoni-Sungur,
1986). Sperm were not counted because they are stored in the
seminal vesicle not the OT.
To ensure an adequate representation of snails with high oocyte counts, some individuals were selected after they exhibited nest-excavating behavior (a prelude to egg laying), since oviposition is performed only by snails with a large store of mature oocytes. To obtain nest-excavating snails (hereafter referred to as `diggers'), a group of approximately 50 snails was transferred to a Lucite box that contained 5-7 cm of moist potting soil and sand in a 1:1 mixture. Diggers were removed before they could oviposit, and they were used for electrophysiological experiments within 3-5 days.
Counts of oviposited eggs were obtained after placing snails on soil as described above. Once a snail had departed from its nest, the deposited eggs were removed, washed and counted.
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Results |
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We examined the OT branch of the intestinal nerve in cross sections at the level of the seminal vesicle or just before it enters the OT. Here, the nerve measures only 20 µm in diameter but it contains 3025 fibre profiles (Fig. 3A). Two types of fibres appear to be partitioned in distinct regions of the nerve. The smaller of these regions is approximately 5 µmx5 µm, contains approximately 2250 fibre profiles and is surrounded by darkly stained glial processes (Fig. 3B). Nearly all of these profiles measure <0.25 µm in diameter. Larger fibres comprise the remaining 80% of the cross-sectional area. Because many of the larger profiles are irregularly shaped, the dimensions of all fibres were measured as feret diameters. Overall, 57% of the fibres have diameters of <0.20 µm; the largest diameters are 2.1 µm. It is likely, based on numerous studies in other animals, that the smaller fibres serve sensory functions, while the larger fibres have motor functions.
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Afferent discharges
To address a possible sensory role of OT innervation, we first examined the
effect of OT stimulation on spike activity in the intestinal nerve. Using
SV-D, SV-P and CNS electrodes (Fig.
1), we recorded a significant increase in spike frequency
following tactile stimulation of the OT
(Fig. 4A). Quantification of
afferent spikes was performed on the basis of spike timing at SV-P and SV-D
electrodes (Fig. 4B). Our
reason for preferring this laborious procedure over the alternative of
recording from a distal nerve stump is that early recordings of the latter
type exhibited prominent bursting patterns and much greater spike rates than
did recordings from an intact nerve. We concluded that the nerve lesion caused
a persistent increase in excitability, yielding anomalous results. With the
intact nerve, OT stimulation resulted in a 20-fold increase in afferent spike
frequency (Fig. 4C).
Concomitantly, we observed a 40-fold increase in overall spike frequency at
the CNS electrode. These results indicate a sensory function for the OT
innervation.
To further elucidate the sensory role of innervation we examined spontaneous afferent spike rates in relation to the number of mature oocytes present in the OT. The frequency of afferent spikes varied from 1.0 spikes min-1 with 31 oocytes present to 79.2 spikes min-1 with 118 oocytes present. In a sample of 14 snails, the afferent spike rate was strongly correlated with the number of oocytes in the OT (Fig. 5A). A linear regression analysis on log-transformed data revealed a highly predictive relationship (r2=0.812, P<0.001; Fig. 5B). This result suggests that information regarding oocyte number in the OT reaches the CNS via the intestinal nerve. Furthermore, we found a significant relationship between log afferent spike frequency and log efferent spike frequency (r2=0.623, P<0.001; Fig. 5C), suggesting that afferent spikes originating in the OT reach the CNS and cause increased firing in neurons that project back to the OT. Fig. 5A,B shows that animals selected as nest diggers had significantly higher oocyte counts than did non-diggers. Diggers had a mean (±S.E.M.) oocyte count of 116.2±4.3 while non-diggers had a mean oocyte count of 53.8±14.2. These numbers are in line with the 86.9±2.7 fertilized eggs deposited during 104 oviposition events observed in our laboratory colony of snails. Notably, we observed a jump in the frequency of afferent activity when the number of oocytes approached 87 (Fig. 5A). The mean afferent spike frequency in all animals with fewer than 87 oocytes was 4.23 spikes min-1, whereas the mean frequency in animals with oocyte counts exceeding 87 was 44.5 spikes min-1. Thus, the data suggest the presence of a threshold oocyte value below which afferent spike activity is low and above which spike frequency increases substantially.
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Experimental inflation of the ovotestis
Because oocytes grow to large sizes (up to 250 µm), at which point their
diameters exceed that of the acini and ducts within which they develop, it can
be assumed that they will eventually exert pressure on the innervated walls of
the acini and ducts. Therefore, to examine the mechanism of sensory
excitation, we artificially inflated the OT to simulate the expected
volumetric expansion (N=3). First, we cut the hermaphroditic duct at
the junction of the OT and the seminal vesicle. We then inserted a cannula
into the distal portion of the duct and injected fast green dye (0.01 g per
100 ml saline) into the OT. For the experiment illustrated in
Fig. 6, we used an animal that
had very few (17) oocytes. Injections of 10 µl or 20 µl were made
incrementally at 5 min intervals. As can be seen by the traces shown in
Fig. 6A, the combined afferent
and efferent activity increased significantly following the injections. When
afferent spikes were analysed separately (raster display in
Fig. 6A;
Fig. 6B), the effect of OT
inflation was even more apparent. While the afferent spike frequency prior to
OT inflation was 4.5 spikes min-1, this value increased 10-fold to
47 spikes min-1 after a total of 40 µl saline had been injected.
These data are consistent with the idea that the high levels of afferent
spiking observed in animals with high oocyte counts are caused by a volumetric
expansion of the OT associated with oocyte growth.
|
CNS efferent reflex
Given that the OT branch of the intestinal nerve responds to sensory
stimulation at the periphery, we looked for an associated reflex output from
the CNS. The intestinal nerve branches twice prior to its innervation of the
OT, and the largest of these early branches innervates the pericardium
(Schmalz, 1914). We recorded
from the central stump of the severed pericardial branch while mechanically
stimulating the surface of the ovotestis (N=6).
Fig. 7A shows that stimulation
induced a surge in spike frequency detectable at the CNS, seminal vesicle and
pericardial electrodes. In the OT branch, the afferent activity increased
4-fold after stimulation, while the concurrent efference increased 6.7-fold
(Fig. 7B). In the central stump
of the pericardial nerve branch, the spike rate increased 3.8-fold
(Fig. 7B). These results
demonstrate that OT sensory stimulation effectively elicits a reflexive
response from the CNS. Moreover, it is noteworthy that the efferent response
was sustained for more than 10 min following the brief stimulus.
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Discussion |
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Structure of the nerve
It is remarkable that the OT branch of the intestinal nerve, which measures
only 20 µm in diameter, should contain 3025 axon profiles. This is made
possible by the inclusion of many fibres that approach the smallest size
reported for any animal (0.05-0.1 µm). Another intriguing aspect of the
morphology is that the total number of profiles far exceeds that expected from
the size of the nerve, in relation to the sizes and number of other nerves,
and the number of neurons in the CNS. The central neuronal population in
Helix aspersa is estimated to be 40 000 neurons
(Chase, 2001
; this value
excludes the small olfactory interneurons in the procerebrum). Thus, as a
first approximation, the number of axon profiles in the OT nerve is
approximately 8% of the number of neurons in the CNS. However, because the
circumference of the OT nerve is much less than 8% of the total circumference
of all nerves combined, there is an apparent paradox. Possibly, the majority
of the axons belong to peripheral sensory cells that, inexplicably, were not
labeled when the OT nerve was back-filled with neurobiotin. Another possible
explanation is provided by Pin and Gola
(1984
), who demonstrated, by
Lucifer Yellow injections and novel electrophysiological experiments, that
certain central neurons in H. pomatia send as many as 25 axons, in
parallel, into the intestinal nerve. If this were generally true, then the
3025 axons in the OT would represent only 121 central neurons. However, this
would raise the question, why do the axons branch so profusely before reaching
their target organ?
A readiness signal
For the reasons presented in the Introduction, it would appear that H.
aspersa delays laying eggs until it has a sufficient store of ripe
oocytes. Our results demonstrate a close association between the number of
ripe oocytes and the rate of spontaneous afferent discharge in the OT nerve
branch (Fig. 5). Furthermore,
there is a sharp increase in afferent activity when the oocyte count exceeds
87, which is the mean number of eggs actually laid by snails in our laboratory
colony. We conclude that one function of the OT innervation is to signal the
availability of a minimum quantity of ripe oocytes. Since oviposition requires
that certain other conditions are also satisfied (e.g. soil composition and
environmental moisture), we view the OT afferent activity as providing a
permissive signal for oviposition, not a trigger.
The results from our OT inflation experiment
(Fig. 6), together with the
anatomical data, suggest that the fine terminals of the OT nerve branch
contain stretch-sensors capable of monitoring oocyte growth. The oocytes
within the OT grow substantially as they mature from the early oogonia stage
to the final vitellogenic stage, increasing 10-fold in diameter and 1000-fold
in volume (Griffond and Bolzoni-Sungur,
1986). It is likely that this growth, which can occur concurrently
in over 100 oocytes, results in a stretching of the acinar walls and the
neuronal processes that lie within. From
Fig. 2, it can be seen that the
diameter of the largest oocytes (250 µm;
Fig. 2A) is greater than the
diameter of a typical acinus (
170 µm;
Fig. 2C). However, the oocytes
are nonetheless able to move in the acini and in the hermaphroditic duct
because their shape is plastic. Similar examples of volumetric expansion
causing sensory discharges in gastropod nerves have been reported for the
anterior gut of Aplysia (Susswein
and Kupfermann, 1975
), the pro-oesophagus of Lymnaea
(Elliott and Benjamin, 1989
)
and the prostate gland of Lymnaea
(De Boer et al., 1997
).
The frequency of afferent spikes is strongly associated with the frequency of efferent spikes conducted towards the OT (Fig. 5C), suggesting that, as the oocytes mature, the heightened afferent signal mediates the increased efferent signal. Here, we are discussing only the spike activity that occurs continuously and spontaneously; in the section below, we discuss the phasic responses following mechanical stimulation. If the efferent activity is continuous in vivo, it is probably not related to an imminent oviposition event. Rather, the efferent fibres might release a trophic factor that mediates the final stages of oocyte maturation. Or, the efference could provide a motor command to maintain tonus in the radial muscles surrounding the hermaphroditic duct, thereby retaining the oocytes until ovulation.
An ovulation signal
While the presence of mature eggs causes a tonic afferent discharge in the
OT branch of the intestinal nerve, tactile stimulation causes a phasic
response (Figs 4,
7). Just as the tonic afferent
discharge causes a tonic efferent discharge, the phasic afferent response
causes a phasic efferent, or reflexive, discharge
(Fig. 7). In both cases, the
physical isolation of the CNS in our recording dish allows us to rule out
mediation by diffuse chemical signals. We speculate that the natural stimulus
for the phasic response is ovulation or, specifically, the passage of oocytes
from their sites of attachment on the walls of the acini into distal segments
of the hermaphroditic duct. We further propose that the sensory signal
generated by the movement of oocytes is responsible for orchestrating
subsequent events in the multi-stage process of oviposition, in a manner
similar to that proposed for the control of egg laying in Aplysia
(Cobbs and Pinsker, 1982;
Ter Maat and Ferguson,
1996
).
Oviposition is a process that requires nearly 48 h of heightened metabolic
activity (Perrot, 1938). A
snail spends the majority of this time with its head beneath the soil surface
in an extended, forward position. Thus, in addition to meeting the metabolic
load, an ovipositing snail must increase its cardiac output to elevate its
internal hydrostatic pressure. Cardiac output also plays an essential role in
the mobilization of calcium for eggshell calcification
(Tompa, 1984
). Just prior to
oviposition, bound and unbound fractions of blood calcium increase by
approximately 60% (Tompa and Wilbur,
1977
). As in vertebrate and other molluscan species, the heart of
H. aspersa is myogenic but subject to central nervous control
(Chase, 2002
). Therefore, to
achieve the elevated heart rate necessary to accommodate the hydrostatic and
metabolic demands of oviposition, CNS excitatory output to the heart is
expected to increase. Consistent with this view, our experiments detected a
dramatic acceleration of activity in the pericardial branch of the intestinal
nerve following brief mechanical stimulation of the OT
(Fig. 7). Also, electrical
stimulation of the OT branch of the intestinal nerve produces a robust
increase in heart beat amplitude (D. Weatherill, unpublished).
The OT of H. aspersa is another target of the efferent reflex
elicited by mechanical stimulation. Studies in marine bivalves indicate that
gonadal innervation is implicated in the maturation of oocytes and the
induction of ovulation (Matsutani and
Nomura, 1987; Ram et al.,
1996
). Studies in our laboratory are attempting to elucidate the
role of efferent activity in the OT nerve branch of Helix. Thus far,
neurobiotin labelling has revealed dense, varicose innervation of the distal
hermaphroditic duct. Also, results indicate that efferent spikes, elicited by
stimulation of the intestinal nerve, increase cilia beat frequencies and cause
radial contractions in the hermaphroditic duct (E. Geoffroy, unpublished).
These effects are consistent with a motor role for OT innervation in promoting
the movement of oocytes from the OT to the fertilization chamber during the
early stages of egg laying.
As we briefly summarized in the Introduction, there exists a substantial
body of literature describing innervation of the mammalian ovary (reviewed in
Aguado, 2002). A handful of
such reports has characterized the innervation as sensory on the basis of
anatomical clues, immunohistochemical results and lesion studies
(Kummer et al., 1990
;
Majewski, 2002; Price and Mudge,
1983
). However, to our knowledge, the results presented here
constitute the first physiological evidence for sensory innervation of the
gonad in any species. The sensory innervation of the OT in H. aspersa
may provide insights into the function of analogous innervations in humans and
other mammalian species.
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Acknowledgments |
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