Invertebrate Biology 126(1): 81–89.

r 2007, The Authors

Journal compilation r 2007, The American Microscopical Society, Inc.
DOI: 10.1111/j.1744-7410.2007.00079.x

In vivo investigation of oocyte transit and maturation

in a broadcast-spawning holothurian

Jean-Fran@ois Hamel1,a and Annie Mercier2

1
Society for the Exploration and Valuing of the Environment (SEVE), Portugal Cove-St. Philips,
Newfoundland, Canada A1M 2B7
2
Ocean Sciences Centre (OSC), Memorial University of Newfoundland, St. John’s,
Newfoundland, Canada A1C 5S7

Abstract. A sequential in vivo approach was used to examine the transformations undergone
by oocytes during transit in the gonoduct of the sea cucumber Holothuria leucospilota, from
ovulation until fertilization competency. Spasms of the ovarian muscle bands, during the pre-
spawning locomotor activity of the females, coincided with the extrusion of oocytes from the
follicle cells (ovulation). No germinal vesicle breakdown (GVBD) was visible and the oocytes
were not fertilizable. As the animal began to display the anterior sweeping movements char-
acteristic of spawning, the oocytes streamed out of the gonad and were stored in the gonad
basis. The oocytes, which were still non-fertilizable, were then pressed forward through the
first (proximal) section of the gonoduct. GVBD was completed during this rapid transit, but
oocytes could not be fertilized unless they had soaked
20 min in seawater. In the second
(distal) section of the gonoduct, most oocytes were readily fertilizable; fertilization rates in-
creased noticeably after the formation of a bulge beneath the gonopore, which favored the
entry of seawater. Hydration of the jelly coat was apparent (i.e., a 60% increase in oocyte
surface area). Gamete release occurred in one powerful spurt B85 min after the onset of
ovulation. This oocyte maturation sequence is expected to occur in holothurian species with
similar anatomy and spawning behavior.
Additional key words: spawning, sea cucumber, Holothuria leucospilota, gonoduct

In many broadcast-spawning marine invertebrates,
the few hours or minutes that precede gamete release
are determinant for the final maturation of reproduct-
ive cells into fully competent gametes. With the ex-
ception of echinoids, gametes released from the gonad
via the gonoduct in echinoderms are the culmination
of a dependent series of cellular events triggered se-
quentially by endogenous chemical messengers (Shi-
rai & Walker 1988; Smiley 1990). Numerous
investigations at the cellular level on surgically col-
lected and/or laboratory-manipulated oocytes have
described the endocrine control of ovulation and oo-
cyte meiosis by chemical or mechanical treatments in
holothurians (e.g., Strathmann & Sato 1969; Ikegami
et al. 1976; Kishimoto & Kanatani 1980; Maruyama
1980; Smiley 1984; Maruyama 1985; Smiley & Cloney
1985; Maruyama 1986) and other echinoderms (e.g.,
a
Author for correspondence.
E-mail: hamel@seve.cjb.net
Kanatani 1969; Davidson et al. 1982; Meijer & Guer-
rier 1984; Davidson 1986; Giese & Kanatani 1987;
Shirai & Walker 1988). In these studies, the process of
oocyte maturation is examined outside of its natural
site of occurrence (i.e., the ovary).
In contrast, the normal sequence of events occur-
ring inside the gonad and along the gonoduct, just be-
fore gamete release, has rarely been examined in
marine invertebrates. Widowati et al. (1995) conduct-
ed a histological investigation of the oocytic pathway
in a mollusc (Pecten maximus LINNAEUS 1758), Hol-
land (1988) studied the fine structure of oocyte mat-
uration in one female crinoid (Oxycomanthus
japonicus MÜLLER 1841) by serial biopsy and histolo-
gy, and Smiley & Cloney (1985) looked at ovulation
under a dissecting microscope in the sea cucumber
Stichopus californicus STIMPSON 1857. The processes of
ovulation, maturation, and acquisition of fertilizabil-
ity of the oocyte, relative to its in situ translocation
from the gonad to the water column, remain largely
unexplored.
82 Hamel & Mercier

Like several other species of holothurians, Holothu-
ria leucospilota BRANDT 1835 displays distinct and
clearly marked spawning postures (Hamel & Mercier
1996a; Hamel et al. 2001; Mercier & Hamel 2002;
Mercier et al. 2004; Hamel & Mercier 2004). Further-
more, they possess a long gonoduct that facilitates the
investigation of oocyte transit. The present study took
advantage of these features to shed new light on the
oocyte maturation and release sequence during natu-
ral spawning events of holothurians, using a combi-
nation of in vivo observations and fertilization trials.
Methods
Collection and maintenance
Specimens of Holothuria leucospilota (20–25 cm
contracted length) were collected in B1 m of water
on a fringing reef off Aruligo, Guadalcanal Island
(91210 5900 S, 1591500 5800 E) on a monthly basis. Individ-
uals were transferred within 30 min to the laboratory
and maintained in 100 L tanks containing coral sand
and maintained under flow-through conditions
(B150 L h1) providing a natural food supply. Short-
ly after the collection and several days before the ex-
pected spawning date, all individuals were sexed by
sampling a few gametes directly through the body
wall, using a 20 cm3 syringe and a 16 gauge needle;
males and females were kept in separate yet commu-
nicating tanks. All studies were conducted on freshly
collected individuals.
Morphology of the reproductive system
The female reproductive system is generally
subdivided into three main sections: the gonad (i.e.,
ovary), the gonoduct (i.e., oviduct), and the gonop-
ore. The ripe female gonad is composed of elongated
and sparsely branched, reddish ovarian tubules. The
gonad basis is the segment that joins the tubules to
the gonoduct. In non-spawning females (n 5 28), the
gonoduct measured 2.1–2.9 cm in length and B2 mm
in diameter. It can be further divided as shown in
Fig. 1. The first or proximal portion of the gonoduct
(1) (2) (3)
A
GP
OT
FC
GV L
B

L
Fig. 1. Schematic representation of oocyte maturation and
transit through the gonoduct during spawning in GV
Holothuria leucospilota (not to scale). Sections of the
reproductive tract are divided into: (1) gonad basis (and
attached ovary), (2) proximal section, and (3) distal section
of the gonoduct. Transient deformations are illustrated by
dashed lines, and insets show an enlarged view of the C
oocytes. T0 marks the onset of side-to-side sweeping
movements in spawning females. A. T0–15 min. Mature
oocytes in the ovarian tubules (ot), just before ovulation.
fc, follicle cells; gv, germinal vesicles; gp, gonopore; l,
lumen. B. T0120 min. Following ovulation, oocytes are L
transferred from the ovarian tubules to the gonad basis,
where they are still not fertilizable. C. T0130 min. Oocytes
are transiting from the gonad basis through the proximal
section and into the distal section of the gonoduct, where
they will have completed germinal vesicle breakdown, D
although most are still not fertilizable unless preliminarily
soaked in seawater. D. T0155 min. Oocytes are in the distal
section of the gonoduct, which begins to form a bulge
JC
under the gonopore. Seawater begins to enter, hydration of
the jelly coat (jc) occurs, and the majority of oocytes
L
become fertilizable. Broadcast will occur within B15 min.

Invertebrate Biology
vol. 126, no. 1, winter 2007
Oocyte maturation in holothurian
is defined as the slightly stiff, rather inflexible con-
striction encompassed in the mesentery that becomes
clearly visible when the gonad basis is filling with oo-
cytes. This section does not perceptibly stretch during
spawning and generally measured B0.3–0.5 cm. The
second or distal section of the gonoduct extends from
there to the gonopore. Its length was B1.8–2.4 cm.
The gonopore is located on the dorsal anterior part
of the body wall and remains difficult to localize be-
fore it bulges from the accumulation of oocytes.
Spawning observations and oocyte sampling
Spawning in H. leucospilota in the Solomon Is-
lands generally occurred around sunset between
18:00 and 21:00 hours, a few days before or on the
full moon (Mercier & Hamel 2002). Before the ex-
pected spawning evenings, mature females were
placed in smaller 40 L concrete tanks provided with
a flow-through of B50 L h1. No artificial spawning
induction methods were used. When possible, one or
two females were monitored every spawning night.
They were studied before and during the gamete re-
lease and until 1 h after the completion of the event,
on an opportunistic basis. A total of 47 females were
examined over several monthly spawning periods.
The first indication of imminent spawning was the
increased locomotor activity. The speed of prospec-
tive spawners was measured by assessing the distance
travelled in 1 min intervals, using three replicate
measures for each individual available during a given
spawning night. This was compared with the values
recorded similarly in foraging animals on non-spawn-
ing nights. Displacements eventually ceased; they were
followed by the typical lifting of the anterior part of
the body and the onset of side-to-side sweeping move-
ments, deemed Time 0 (T0). Each selected female was
monitored separately from that moment on. A few
(n 5 5) were selected before the occurrence of the
raised spawning posture in order to examine the state
of the reproductive system, especially the ovarian
tubules, before and soon after the first movements of
gametes (BT0–15 min). Another group (n 5 5) was
examined after spawning (BT0170 min).
Oocytes were collected surgically at intervals of
10–15 min from T0 until the end of the oocyte release,
combining different individuals to obtain the com-
plete series. The biopsy was performed through a
dorsal opening B2.5 cm behind the gonopore, which
exposed the length of the gonoduct and part of the
ovary. For each individual, the contracted length of
the animal, the length and diameter of the gonad
basis, and the length and diameter of the proximal
and distal gonoduct sections were noted. Evidence of
83
muscle band contractions and oocyte movements
were also recorded. Oocytes were sampled in the
ovarian tubules, gonad basis, and each section of
the gonoduct, using a truncated disposable pipette
introduced through a small incision. The diameter of
the oocytes (n 5 20–30) and the state of the germinal
vesicle were noted on freshly collected gametes. All
studied females were kept half immersed in seawater
during the different procedures at a temperature of
B281C to minimize stress. Great care was taken to
avoid contact of gametes with liquid sources (e.g.,
coelomic fluid, seawater) during manipulations of the
reproductive system.
The in vivo surgical methods described above are
similar to previously used techniques that were
shown to have no significant impact on the behavior
and general health of the animals (Hamel & Mercier
1996b, 1999). Indeed, holothurians are generally very
resilient; many of them use evisceration as a defense
mechanism, and a high rate of organ regeneration is
recorded in most species (reviewed by Garcia-Arraras
& Greenberg 2001). After the surgery, individuals
were returned to the tanks to allow them to regener-
ate. While the release of Cuvierian tubules was occa-
sionally observed, no evisceration and no mortality
were recorded during the study, and most of the an-
imals were eventually released back to their natural
habitat.
Oocyte diameter and competency
As described previously by Smiley et al. (1991),
ovulation is the phenomenon associated with the ex-
trusion of oocytes from follicle cells. Further steps in
the maturation process include germinal vesicle mi-
gration, germinal vesicle breakdown (GVBD), and
acquisition of fertilizability. Maturation can only be
ascertained by normal cleavage and embryonic de-
velopment after a successful fertilization (Meijer &
Guerrier 1984).
To verify the competency of oocytes during their
transit in the gonad basis and in the gonoduct, oo-
cytes were collected as described above, in as many
locations as possible from every spawning female
available, and fertilization assays were conducted.
The trials were performed in 15 beakers filled with
100 mL of seawater (filtered and UV-treated) in
which the sampled oocytes were carefully introduced
(n 5 20–30 per beaker). Three groups were fertilized
immediately, the others were maintained at B281C
under slight bubbling, and fertilization assays were
conducted 0.5, 1, 2, and 5 h after collection, using
triplicates for each treatment. This protocol was
devised because certain hormones known to play a
Invertebrate Biology
vol. 126, no. 1, winter 2007
84
role in oocyte maturation in echinoderms were found
to require 2–4 h to be effective (Meijer et al. 1984;
Maruyama 1985; Smiley et al. 1991). Spermatozoa
were obtained surgically from three males and mixed
together to provide a dry sperm preparation that was
maintained at 10–121C, no more than 120 min before
usage. A drop of dry sperm was mixed in 1 mL of
filtered seawater and the concentration of spermato-
zoa was measured with a hematocytometer. For the
assay, the proper amount of sperm solution was add-
ed to the beaker to obtain a final concentration of
B20,000 spermatozoa mL1. The mix was stirred
gently for 15 s, and evidence of fertilization, such as
elevation of fertilization envelope, the presence of
male pronucleus, and embryonic development (two-
cell or more) were monitored every 2–5 min under a
light microscope. Preliminary tests demonstrated
that nearly 100% fertilization and o2% polysper-
mia (i.e., the presence of multiple male pronuclei in
the cytoplasm and/or abnormal cleavage) were ob-
tained with this spermatozoa–oocyte ratio. Naturally
spawned oocytes typically show the male pronucleus
within 10 min after the addition of spermatozoa. It
was thus assumed that delays
20 min were associ-
ated with a need for the oocyte to finalize its matur-
ation before fertilization became possible.
Oocyte diameter was also examined in the gonad
before ovulation and throughout transit in the gono-
duct. The oocyte surface area (S) was calculated us-
ing the total diameter as both length (L) and breadth
(B) in micrometers (Narushin 2005):
S ¼ ð3:155  0:0136L þ 0:0115BÞ  LB
Controls
Comparisons with gametogenetically mature non-
spawning individuals were routinely made to deter-
mine any effect the surgery and other manipulations
might have on oocytes and on general behavior. Fur-
ther comparisons were made at corresponding times
with oocytes collected through the body wall, with a
20 cm3 syringe and a 16 gauge needle, in the ovarian
tubules and in the bulging duct under the gonopore
before spawning. The morphology and competency
of these oocytes were assessed according to the usual
samples. Overall, only three cases were noted where
handling was found to interfere with the normal
events (e.g., gamete movement, muscle contraction).
In these instances, the gametes were collected for fer-
tilization trials but in vivo observations were discard-
ed. No spontaneous ovulation was observed.
Invertebrate Biology
vol. 126, no. 1, winter 2007
Hamel & Mercier
Results
Oocyte maturation
Forty-four of the 47 females studied showed a
consistent oocytic pathway (i.e., ovulation, GVBD,
hydration of the jelly coat, and acquisition of ferti-
lizability) from the first sign of pre-spawning loco-
motor activity until the broadcast of gametes (Fig. 1).
Gametogenetically mature oocytes sampled in the
ovary before pre-spawning activity were not fertiliz-
able (Fig. 1A). Ovulation was characterized by the
extrusion of the oocyte from follicle cells, a process
through which oocytes became free in the lumen
and assumed a more rounded shape. It took
14.273.9 min (mean7SD, n 5 8) from the first
pre-spawning manifestation to complete ovulation,
which began in a small group of tubules and spread
rapidly as a wave. The ovarian tubules displayed
spasms (i.e., sudden contractions) and increased
in diameter from 1.5 to 2.1 mm, as the oocytes were
being freed in the lumen. Most females were not feed-
ing but moved significantly more rapidly
(345759 cm h1; mean7SD) than during their nor-
mal foraging activity (112760 cm h1) (Student’s t-
test, po0.05, n 5 28).
The animals slowed and eventually ceased moving
at the end of ovulation. They began to raise their an-
terior end in the typical sweeping motion (T0).
Spasms were replaced by peristaltic (i.e., wavelike)
contractions of the ovarian tubules that spread
from the tip to the base of each tubule, propelling
the oocytes toward the gonad basis. The gonad was
emptied two or three tubules at a time, as clearly
observed in situ in four different females. It took
15–25 min to complete the transfer of all oocytes
into the gonad basis. The latter was distended to
three to four times its initial size, reaching a maxi-
mum of 7–11 mm in diameter (n 5 14). At this stage,
no sign of GVBD was noted and oocytes did not
respond to any of the fertilization assays (Table 1;
Fig. 1B).
Before the gonad was completely empty, the oo-
cytes stored in the gonad basis initiated transit in a
single file. They apparently became slightly squeezed
when entering the gonoduct; there was a deceleration
followed by a sudden acceleration. Oocytes collected
onward from this section had completed GVBD (Fig.
1C), but could never be readily fertilized within the
usual 10 min after the addition of spermatozoa, al-
though a small proportion (1179%) were fertilized
after 20–25 min (Table 1). The fertilization success of
these oocytes increased significantly above 50% after
incubation in seawater for 0.5–5 h (w2, po0.05,
n 5 485) (Table 1).
Oocyte maturation in holothurian 85

Table 1. Fertilization rate of oocytes collected from different sections of the reproductive tract. Assays were conducted
immediately upon collection and after various periods of incubation in seawater at 281C. Data are presented as mean
%7standard deviation (n 5 60–90). Data in parentheses show the results obtained with oocytes collected through the
body wall with a syringe at the corresponding time/location. GVBD, germinal vesicle breakdown.

Location of oocytes Incubation in seawater (h)

0 0 0.5 1 2 5
In the ovary (before ovulation) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
In the ovary (after ovulation) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
In the gonad basis (before GVBD) 0 0 0 0 0 0
In the proximal section of the gonoduct 0 1179 55713 73712 66711 3477
(after GVBD)
In the distal section of the gonoduct (before 15711 6778 7776 85711 7276 4479
visible entry of seawater)
In the distal section of the gonoduct (after entry 72712 9475 9673 9772 9971 3779
of seawater) (6879) (8978) (9871) (9277) (10070) (2576)
In the water column just after broadcast 9672 9772 9871 9572 9671 3274
0 h of incubation in seawater but the presence of male pronucleus was noted 20–25 min after contact with spermatozoa,
instead of the usual B10 min.

Less than B9 min was required for all the oocytes
to move through the proximal into the distal section
of the gonoduct. Upon collection, only 15711% of
these oocytes were normally fertilized, whereas
6778% of them were fertilized after 20–25 min of
contact with spermatozoa (Table 1). The fertilization
success was even greater after incubation in seawater
for 0.5–2 h, but decreased significantly after 5 h (w2,
po0.05, n 5 165) (Table 1). The gonoduct progres-
sively stretched to reachB15 mm in diameter (i.e., ap-
proximately seven times its initial size), while oocytes
accumulated and the bulging gonopore became vis-
ible. Between 10 and 15 min before gamete broadcast,
the bulging allowed the entry of a small amount of
seawater through the gonopore, which mixed with
oocytes inside the duct (Fig. 1D). The fertilization
rate immediately after collection was then much
higher (72712%), and it reached nearly 100%
when the oocytes were incubated for 0.5–2 h in sea-
water (Table 1). In most cases, fertilization success
decreased significantly after 5 h of incubation in sea-
water (w2, po0.05, n 5 165). These results are fairly
consistent with the fertilization rates observed in nat-
urally broadcasted oocytes (Table 1).
Similar fertilization rates were recorded when com-
paring gametes that were collected surgically from
the reproductive tract and those collected through
the body wall with a syringe at the corresponding
time and location (w2, p40.05; Table 1).
Oocytes in the gonad were an average of 9579 mm
(n 5 246) in diameter. The thickening of the jelly coat
increased the total diameter to 123713 mm (n 5 51)
just before broadcast. This corresponds to an in-
crease of the surface area by B60%. The most sig-
nificant increase occurred in the distal section of the
gonoduct after the entry of seawater and before the
broadcast. The gonoduct stretched, showing a
21711% (n 5 9) increase from its original length
during the accumulation and movement of oocytes.
After gamete release, the sweeping movement and the
gonadal muscle activity ceased, leaving the gonad
amorphous and almost completely empty with only
a few pockets of unspawned mature oocytes.
Discussion
The huge body of information that exists today on
the processes involved in oocyte maturation has
largely been gathered from in vitro studies, which,
by definition, must be uncoupled from the animal
system. While great advances have been made, fun-
damental discrepancies were reported even inside the
Echinodermata, so that the pathway leading to ac-
quisition of fertilization competence by an oocyte has
yet to be fully understood (Kishimoto 2003; Voro-
nina & Wessel 2003). This study has adopted a dif-
ferent, in vivo, approach to shed new light on the
respective roles played by mechanical and hormonal
processes during oocyte transit in the reproductive
tract of a broadcast-spawning holothurian. Using se-
quential observations and bioassays, the transloca-
tion and maturation of oocytes were followed in real
time in naturally spawning individuals, therefore re-
fining our understanding of the oocytic pathway and
of the role played by the relatively long gonoduct in
many holothurians.

Invertebrate Biology
vol. 126, no. 1, winter 2007
86
Data gathered during this study provide evidence
that the onset of gamete maturation is not only sup-
ported by hormonal activity, as traditionally viewed,
but also sustained by mechanical action. Ovulation
coincided with an increase in locomotor activity and
spasms of the muscle bands along the ovarian tu-
bules. A higher level of activity in preparation for
broadcast spawning was also noted in Isostichopus
fuscus LUANIF 1875 (unpubl. data) and other hol-
othurians (Battaglene et al. 2002). Contractions of
the body associated with spawning have previously
been observed in sea stars (Kanatani 1970) and in
several species of tropical holothurians (Bakus 1973;
Hamel et al. 2001; Battaglene et al. 2002). Interest-
ingly, a coupling between mechanical and hormonal
action has been proposed to occur in brachiopods:
Stricker & Folsom (1997) suggested that detachment
of follicles from the ovarian germinal epithelium
stimulates the secretion of an inducing substance,
leading to oocyte maturation. Hence, body move-
ments and ovarian contractions in Holothuria leuco-
spilota could provoke or assist follicular detachment
and trigger the onset of hormonal processes required
for maturation.
Using extracted oocytes, Smiley (1984, 1988) and
Maruyama (1985, 1986) determined that a radial nerve
factor (RNF) acted through the follicle cells to pro-
duce a maturation-inducing substance (MIS) leading
to GVBD in holothurians. Specifically, Maruyama
(1985) noted that follicle cell-free oocytes in H. leuco-
spilota never underwent GVBD when exposed to
RNF extracts from holothurian tissues. The present
in vivo study hints at a different scenario as (1) GVBD
was completed well after extrusion from the follicle
cells and (2) GVBD was never found to occur in oo-
cytes collected in the lumen of the ovarian tubules or
in the gonad basis after ovulation but before passage
through the proximal constricted section of the ovi-
duct, no matter how long they were left to soak in
seawater. Two hypotheses can be offered: (1) the ac-
tion of RNF through follicle cells must be combined
with mechanical activation (i.e., passage through the
constricted duct, or pipetting in the case of in vitro
studies) and (2) RNF acts through another pathway
inside the gonoduct. Incidentally, oocytes of the sea
star Pisaster ochraceus BRANDT 1835 (Schuetz 2000)
and several sea cucumbers, including H. leucospilota
(Maruyama 1986), did respond to sea star RNF by
undergoing GVBD even when denuded of their follicle
cells, suggesting that other ovarian components may
indeed produce the MIS. Such inconsistencies empha-
size that in vitro studies of oocytes, which can involve
various manipulations (i.e., squeezing, pipetting,
rinsing, soaking in seawater and other media, centrif-
Invertebrate Biology
vol. 126, no. 1, winter 2007
Hamel & Mercier
ugation, injection, etc.), provide data that are valuable
for elucidating cellular processes but that do not nec-
essarily reflect the actual in situ sequence.
In fact, the maturation sequence determined dur-
ing this in vivo study of H. leucospilota differs some-
what from most previous accounts of in vitro studies
in echinoderms. The breakdown of the follicular en-
velope (i.e., ovulation) is usually reported to be con-
comitant with or closely followed by GVBD in sea
stars (Kanatani 1969), crinoids (Holland 1988), and
sea cucumbers (Smiley et al. 1991). Additionally, it
has been widely assumed that, following GVBD, mei-
osis of sea star and sea cucumber oocytes proceeded
without further arrest until extrusion of the two polar
bodies, whether or not fertilization occurred (e.g.,
Maruyama 1985; Yamamoto 1997). However, hor-
monal stimulation naturally occurs inside the ovary,
whereas the standard procedures in these experi-
ments involve oocytes being isolated from the ovary,
placed in seawater, and then treated or injected with
the inducing substance, thus exposing oocytes to
many concurrent stimuli. Here, we clearly showed
that oocytes sampled from the ovary after ovulation
had not yet completed GVBD and could never be
fertilized, either immediately or after soaking in sea-
water for r5 h. Furthermore, oocytes did not ac-
quire full competency until some time after GVBD,
confirming that further activation is involved in mat-
uration along the gonoduct. Interestingly, a recent
study showed that an arrest in the metaphase I oc-
curred after GVBD within the sea star ovary, before
release, when hormonal induction was triggered in-
side the body cavity of the female to simulate natural
spawning (Harada et al. 2003). Release of this arrest
was induced by an intracellular pH increase when the
oocyte was spawned into seawater. This sequence
bears more similarities to the one reported here
than all previous in vitro studies. An arrest between
GVBD and further maturation is useful as it is gen-
erally accepted that fertilization of oocytes before ex-
trusion of the first polar body yields the most
successful development (Gould & Stephano 2003).
Walker (1975) mentioned that the gonoduct in As-
terias vulgaris VERRILL 1866 was capable of extreme
expansion. The same was found in H. leucospilota
when oocytes pushed into and accumulated within
the gonad basis. The swelling of the gonad basis, and
possibly the action of cilia of the gonoduct as described
in H. scabra by Mary Bai (1980), seemed to build mo-
mentum to allow forced passage through the much
stiffer proximal section of the gonoduct. A clear vel-
ocity change was observed during this transit, after
which most oocytes had completed GVBD, apparently
via some kind of mechanical stimulus. Mechanical
Oocyte maturation in holothurian
induction of GVBD and ensuing maturation have
been reported in oocytes of sea stars (Guerrier et al.
1988) and holothurians (Maruyama 1981) after shak-
ing and pipetting, respectively. The latter phenomenon
is similar to the ‘‘squeezing’’ observed in the proximal
section of the gonoduct.
Although they had completed GVBD after squeez-
ing through the first segment of the duct, only
15711% of oocytes were competent immediately
thereafter. The fact that a larger proportion of oo-
cytes became competent as seawater entered the dis-
tal section of the duct further emphasizes the role of
seawater in the final maturation of oocytes. Specific-
ally, acquisition of full competency occurred during
the 10–20 min interval between the first noticeable
entry of seawater in the duct and the broadcast. In-
terestingly, B20 min is also the minimum time need-
ed for some of the oocytes, having completed GVBD,
to show evidence of fertilizability (i.e., when mixed
with spermatozoa in seawater). This delay possibly
allows extracellular ions to act directly or indirectly
on the oocytes. The role of calcium in various stages
of oocyte maturation has been examined in echino-
derms, mostly in terms of transients and channels,
with little reference to the role of external ions (Voro-
nina & Wessel 2003). It has also been pointed out in a
recent review that in vitro demonstration may not
necessarily hold in vivo (Whitaker 2006). A general
assumption is that ions (calcium, potassium, and so-
dium) are sufficiently abundant within the ovarian
fluid to allow completion of the maturation process.
However, Harada et al. (2003) showed that the in-
crease in intracellular pH necessary to complete
metaphase I was blocked inside the sea star ovary,
preventing the completion of maturation until release
into seawater. The present work showed that expos-
ure to seawater naturally occurred inside the hol-
othurian oviduct, allowing the oocytes to become
readily fertilizable just before being broadcasted in
the water column, thus minimizing dispersion before
fertilization. This is important as males generally
spawn first in H. leucospilota and many other species
of holothurians (Hamel et al. 2001; Battaglene et al.
2002; Mercier et al. 2004, 2007). The lower fertiliza-
tion rates obtained after 5 h of incubation in seawater
suggest that optimum fertilization will occur soon
after release.
The penetration of seawater in the oviduct before
spawning may also play another role by helping to
saturate the muscle bands with calcium, enabling
them to void gametes with a single or a few power-
ful contractions. Again, ions present in seawater have
been shown to play similar roles. Kanatani & Shirai
(1969, 1970) indicated that calcium ions seemed to
87
provoke contractions of the ovarian wall, which dir-
ectly forced out the freely movable oocytes in sea
stars. Furthermore, Shirai & Walker (1988) men-
tioned that oocyte release from the ovary was inhibit-
ed in calcium-free seawater as muscles could not
contract. While earlier studies on sea stars emphasize
the role of gonadal wall muscles in the expulsion of
gametes (Shirai et al. 1981), that role was predomi-
nantly played by the gonoduct in H. leucospilota.
Indeed, the gonads were already empty at the time
of spawning (i.e., the oocytes had gathered in the dis-
tal section of the oviduct). A similar pattern was ob-
served in I. fuscus and H. scabra (unpubl. data). As
the gonoduct is not muscularized, this bulging
possibly provides momentum for the broadcast,
which is sometimes powerful enough to propel the
gametes out of the tanks. Nonetheless, seawater may
still affect the gonads and provoke contractions that
indirectly contribute to gamete release. The concur-
rent contraction of the body wall could also play a
role, as proposed by Kanatani (1970) for sea stars.
A third function of seawater during the final
preparation for broadcast is evidenced by the
B60% increase in oocyte surface area in the distal
section of the gonoduct, with the hydration of
the jelly coat. Larger oocytes and/or oocytes with
thicker jelly coats were shown to be preferentially
fertilized in sea urchins (Vogel et al. 1982; Epel 1991;
Levitan & Irvine 2001; Podolsky 2001). This is a sig-
nificant advantage for species that broadcast small
oocytes. Indeed, a female of H. leucospilota can store
more of the smaller, gametogenetically mature,
oocytes in the gonad and release larger oocytes that
are readily fertilizable to maximize its reproductive
success.
This study revealed that oocytes in H. leucospilota
consistently required B85 min to achieve competen-
cy, from the onset of ovulation to the final broadcast.
Shorter and longer delays have previously been re-
ported for hormonally activated ovulation, GVBD,
and maturation to occur in holothurians (Smiley
1990). However, the stress of capture and the induc-
tion methods used in aquaculture prompted the re-
lease of fully fertilizable oocytes in B1 h by
holothurians that did not show any prior signs of
spawning activity (Reichenbach 1999; Hamel et al.
2001; Mercier & Hamel 2002; Hamel & Mercier
2004). It is worth mentioning that the sequential mat-
uration of oocytes reported in the present study is
associated with a gamete transit that culminates in a
forceful final broadcast after the formation of a bulge
under the gonopore. As this is a rather common
trend (Mosher 1982; Hamel et al. 2001; Battaglene
et al. 2002; Mercier et al. 2007), it is expected that
Invertebrate Biology
vol. 126, no. 1, winter 2007
88
similar results could be observed in other holothuri-
ans. Species that display a different spawning behav-
ior involving the release of a continuous stream of
oocytes (McEuen 1988; Hamel & Mercier 1996a) are
likely to depart, at least in some aspects, from the
oocytic pathway described here.
The present study has shown that the ovulation,
transit, and maturation of oocytes are complex
interlinked processes that are influenced by behavior
and anatomical particularities. Subtle elements such as
shifting between two types of muscular contractions, a
small constriction in the gonoduct, and entry of sea-
water through the gonopore play decisive roles in
oocyte maturation in H. leucospilota. We therefore
conclude that, in the search for comprehensive
information on gamete biology in broadcast spawn-
ers, the assessment of in vivo processes is a significant
component. A recent review on drug research and bi-
ological systems stressed the crucial link between in
vitro findings and in vivo investigations (Lipinski &
Hopkins 2004). The long gonoduct in H. leucopsilota
and other holothurians may well provide an oppor-
tunity to further investigate cellular processes occur-
ring at various stages of oocyte maturation within live
systems.
Acknowledgments. We would like to thank the staff of
ICLARM (now WorldFish Center) in Solomon Islands
for their help during the work that was performed
there, as well as Dr. S. Smiley (University of Alaska) for
helpful exchanges during the early stages of manuscript
preparation and two anonymous reviewers for providing
judicious comments and suggestions.
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