in vivo investigation of oocyte transit and maturation in a broadcast-spawning holothurian

In vivo investigation of oocyte transit and maturationin 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 2B72 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 undergoneby oocytes during transit in the gonoduct of the sea cucumber Holothuria leucospilota, fromovulation 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 thefollicle cells (ovulation). No germinal vesicle breakdown (GVBD) was visible and the oocyteswere 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 gonadbasis. The oocytes, which were still non-fertilizable, were then pressed forward through thefirst (proximal) section of the gonoduct. GVBD was completed during this rapid transit, butoocytes could not be fertilized unless they had soaked �20min 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 theentry of seawater. Hydration of the jelly coat was apparent (i.e., a 60% increase in oocytesurface area). Gamete release occurred in one powerful spurt B85min after the onset ofovulation. This oocyte maturation sequence is expected to occur in holothurian species withsimilar 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 releaseare determinant for the final maturation of reproduct-ive cells into fully competent gametes. With the ex-ception of echinoids, gametes released from the gonadvia the gonoduct in echinoderms are the culminationof a dependent series of cellular events triggered se-quentially by endogenous chemical messengers (Shi-rai & Walker 1988; Smiley 1990). Numerousinvestigations at the cellular level on surgically col-lected and/or laboratory-manipulated oocytes havedescribed the endocrine control of ovulation and oo-cyte meiosis by chemical or mechanical treatments inholothurians (e.g., Strathmann & Sato 1969; Ikegamiet al. 1976; Kishimoto & Kanatani 1980; Maruyama1980; Smiley 1984;Maruyama 1985; Smiley & Cloney1985; Maruyama 1986) and other echinoderms (e.g.,

Kanatani 1969; Davidson et al. 1982; Meijer & Guer-rier 1984; Davidson 1986; Giese & Kanatani 1987;Shirai &Walker 1988). In these studies, the process ofoocyte maturation is examined outside of its naturalsite 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 inmarine invertebrates. Widowati et al. (1995) conduct-ed a histological investigation of the oocytic pathwayin a mollusc (Pecten maximus LINNAEUS 1758), Hol-land (1988) studied the fine structure of oocyte mat-uration in one female crinoid (Oxycomanthusjaponicus MULLER 1841) by serial biopsy and histolo-gy, and Smiley & Cloney (1985) looked at ovulationunder a dissecting microscope in the sea cucumberStichopus californicus STIMPSON 1857. The processes ofovulation, maturation, and acquisition of fertilizabil-ity of the oocyte, relative to its in situ translocationfrom the gonad to the water column, remain largelyunexplored.

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

aAuthor for correspondence.

E-mail: [email protected]

Like several other species of holothurians,Holothu-ria leucospilota BRANDT 1835 displays distinct andclearly marked spawning postures (Hamel & Mercier1996a; Hamel et al. 2001; Mercier & Hamel 2002;Mercier et al. 2004; Hamel & Mercier 2004). Further-more, they possess a long gonoduct that facilitates theinvestigation of oocyte transit. The present study tookadvantage of these features to shed new light on theoocyte 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 cmcontracted length) were collected in B1m of wateron a fringing reef off Aruligo, Guadalcanal Island(912105900S, 15915005800E) on a monthly basis. Individ-uals were transferred within 30min to the laboratoryand maintained in 100L tanks containing coral sandand maintained under flow-through conditions(B150Lh�1) providing a natural food supply. Short-ly after the collection and several days before the ex-pected spawning date, all individuals were sexed bysampling a few gametes directly through the bodywall, using a 20 cm3 syringe and a 16gauge needle;males and females were kept in separate yet commu-nicating tanks. All studies were conducted on freshlycollected individuals.

Morphology of the reproductive system

The female reproductive system is generallysubdivided 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 elongatedand sparsely branched, reddish ovarian tubules. Thegonad basis is the segment that joins the tubules tothe gonoduct. In non-spawning females (n5 28), thegonoduct measured 2.1–2.9 cm in length andB2mmin diameter. It can be further divided as shown inFig. 1. The first or proximal portion of the gonoduct

L

FC

GV

OT

GP

A

(1) (2) (3)

L

GV

B

L

C

L

JC

D

Fig. 1. Schematic representation of oocyte maturation and

transit through the gonoduct during spawning in

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

oocytes. T0 marks the onset of side-to-side sweeping

movements in spawning females. A. T0–15min. Mature

oocytes in the ovarian tubules (ot), just before ovulation.

fc, follicle cells; gv, germinal vesicles; gp, gonopore; l,

lumen. B. T0120min. Following ovulation, oocytes are

transferred from the ovarian tubules to the gonad basis,

where they are still not fertilizable. C. T0130min. 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,

although most are still not fertilizable unless preliminarily

soaked in seawater.D. T0155min. Oocytes are in the distal

section of the gonoduct, which begins to form a bulge

under the gonopore. Seawater begins to enter, hydration of

the jelly coat (jc) occurs, and the majority of oocytes

become fertilizable. Broadcast will occur within B15min.

82 Hamel & Mercier

Invertebrate Biologyvol. 126, no. 1, winter 2007

is defined as the slightly stiff, rather inflexible con-striction encompassed in the mesentery that becomesclearly visible when the gonad basis is filling with oo-cytes. This section does not perceptibly stretch duringspawning and generally measured B0.3–0.5 cm. Thesecond or distal section of the gonoduct extends fromthere to the gonopore. Its length was B1.8–2.4 cm.The gonopore is located on the dorsal anterior partof 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 between18:00 and 21:00 hours, a few days before or on thefull moon (Mercier & Hamel 2002). Before the ex-pected spawning evenings, mature females wereplaced in smaller 40L concrete tanks provided witha flow-through of B50Lh�1. No artificial spawninginduction methods were used. When possible, one ortwo 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 wereexamined over several monthly spawning periods.

The first indication of imminent spawning was theincreased locomotor activity. The speed of prospec-tive spawners was measured by assessing the distancetravelled in 1min intervals, using three replicatemeasures for each individual available during a givenspawning night. This was compared with the valuesrecorded similarly in foraging animals on non-spawn-ing nights. Displacements eventually ceased; they werefollowed by the typical lifting of the anterior part ofthe body and the onset of side-to-side sweeping move-ments, deemed Time 0 (T0). Each selected female wasmonitored separately from that moment on. A few(n55) were selected before the occurrence of theraised spawning posture in order to examine the stateof the reproductive system, especially the ovariantubules, before and soon after the first movements ofgametes (BT0–15min). Another group (n5 5) wasexamined after spawning (BT0170min).

Oocytes were collected surgically at intervals of10–15min from T0 until the end of the oocyte release,combining different individuals to obtain the com-plete series. The biopsy was performed through adorsal openingB2.5 cm behind the gonopore, whichexposed the length of the gonoduct and part of theovary. For each individual, the contracted length ofthe animal, the length and diameter of the gonadbasis, and the length and diameter of the proximaland distal gonoduct sections were noted. Evidence of

muscle band contractions and oocyte movementswere also recorded. Oocytes were sampled in theovarian tubules, gonad basis, and each section ofthe gonoduct, using a truncated disposable pipetteintroduced through a small incision. The diameter ofthe oocytes (n5 20–30) and the state of the germinalvesicle were noted on freshly collected gametes. Allstudied females were kept half immersed in seawaterduring the different procedures at a temperature ofB281C to minimize stress. Great care was taken toavoid contact of gametes with liquid sources (e.g.,coelomic fluid, seawater) during manipulations of thereproductive system.

The in vivo surgical methods described above aresimilar to previously used techniques that wereshown to have no significant impact on the behaviorand general health of the animals (Hamel & Mercier1996b, 1999). Indeed, holothurians are generally veryresilient; many of them use evisceration as a defensemechanism, and a high rate of organ regeneration isrecorded in most species (reviewed by Garcia-Arraras& Greenberg 2001). After the surgery, individualswere returned to the tanks to allow them to regener-ate. While the release of Cuvierian tubules was occa-sionally observed, no evisceration and no mortalitywere recorded during the study, and most of the an-imals were eventually released back to their naturalhabitat.

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 inthe maturation process include germinal vesicle mi-gration, germinal vesicle breakdown (GVBD), andacquisition of fertilizability. Maturation can only beascertained by normal cleavage and embryonic de-velopment after a successful fertilization (Meijer &Guerrier 1984).

To verify the competency of oocytes during theirtransit in the gonad basis and in the gonoduct, oo-cytes were collected as described above, in as manylocations as possible from every spawning femaleavailable, and fertilization assays were conducted.The trials were performed in 15 beakers filled with100mL of seawater (filtered and UV-treated) inwhich the sampled oocytes were carefully introduced(n5 20–30 per beaker). Three groups were fertilizedimmediately, the others were maintained at B281Cunder slight bubbling, and fertilization assays wereconducted 0.5, 1, 2, and 5 h after collection, usingtriplicates for each treatment. This protocol wasdevised because certain hormones known to play a

Oocyte maturation in holothurian 83


role in oocyte maturation in echinoderms were foundto require 2–4 h to be effective (Meijer et al. 1984;Maruyama 1985; Smiley et al. 1991). Spermatozoawere obtained surgically from three males and mixedtogether to provide a dry sperm preparation that wasmaintained at 10–121C, no more than 120min beforeusage. A drop of dry sperm was mixed in 1mL offiltered seawater and the concentration of spermato-zoa was measured with a hematocytometer. For theassay, the proper amount of sperm solution was add-ed to the beaker to obtain a final concentration ofB20,000 spermatozoamL�1. The mix was stirredgently for 15 s, and evidence of fertilization, such aselevation of fertilization envelope, the presence ofmale pronucleus, and embryonic development (two-cell or more) were monitored every 2–5min under alight microscope. Preliminary tests demonstratedthat nearly 100% fertilization and o2% polysper-mia (i.e., the presence of multiple male pronuclei inthe cytoplasm and/or abnormal cleavage) were ob-tained with this spermatozoa–oocyte ratio. Naturallyspawned oocytes typically show the male pronucleuswithin 10min after the addition of spermatozoa. Itwas thus assumed that delays � 20min 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 gonadbefore 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� 0:0136Lþ 0:0115BÞ � LB

Controls

Comparisons with gametogenetically mature non-spawning individuals were routinely made to deter-mine any effect the surgery and other manipulationsmight have on oocytes and on general behavior. Fur-ther comparisons were made at corresponding timeswith oocytes collected through the body wall, with a20 cm3 syringe and a 16 gauge needle, in the ovariantubules and in the bulging duct under the gonoporebefore spawning. The morphology and competencyof these oocytes were assessed according to the usualsamples. Overall, only three cases were noted wherehandling was found to interfere with the normalevents (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.

Results

Oocyte maturation

Forty-four of the 47 females studied showed aconsistent 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 theovary before pre-spawning activity were not fertiliz-able (Fig. 1A). Ovulation was characterized by theextrusion of the oocyte from follicle cells, a processthrough which oocytes became free in the lumenand assumed a more rounded shape. It took14.273.9min (mean7SD, n5 8) from the firstpre-spawning manifestation to complete ovulation,which began in a small group of tubules and spreadrapidly as a wave. The ovarian tubules displayedspasms (i.e., sudden contractions) and increasedin diameter from 1.5 to 2.1mm, as the oocytes werebeing freed in the lumen.Most females were not feed-ing but moved significantly more rapidly(345759 cmh�1; mean7SD) than during their nor-mal foraging activity (112760 cmh�1) (Student’s t-test, po0.05, n5 28).

The animals slowed and eventually ceased movingat 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 spreadfrom the tip to the base of each tubule, propellingthe oocytes toward the gonad basis. The gonad wasemptied two or three tubules at a time, as clearlyobserved in situ in four different females. It took15–25min to complete the transfer of all oocytesinto the gonad basis. The latter was distended tothree to four times its initial size, reaching a maxi-mum of 7–11mm in diameter (n5 14). At this stage,no sign of GVBD was noted and oocytes did notrespond 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 asingle file. They apparently became slightly squeezedwhen entering the gonoduct; there was a decelerationfollowed by a sudden acceleration. Oocytes collectedonward from this section had completed GVBD (Fig.1C), but could never be readily fertilized within theusual 10min after the addition of spermatozoa, al-though a small proportion (1179%) were fertilizedafter 20–25min (Table 1). The fertilization success ofthese oocytes increased significantly above 50% afterincubation in seawater for 0.5–5h (w2, po0.05,n5 485) (Table 1).

84 Hamel & Mercier


Less than B9min was required for all the oocytesto move through the proximal into the distal sectionof the gonoduct. Upon collection, only 15711% ofthese oocytes were normally fertilized, whereas6778% of them were fertilized after 20–25min ofcontact with spermatozoa (Table 1). The fertilizationsuccess was even greater after incubation in seawaterfor 0.5–2h, but decreased significantly after 5 h (w2,po0.05, n5 165) (Table 1). The gonoduct progres-sively stretched to reachB15mm in diameter (i.e., ap-proximately seven times its initial size), while oocytesaccumulated and the bulging gonopore became vis-ible. Between 10 and 15min before gamete broadcast,the bulging allowed the entry of a small amount ofseawater through the gonopore, which mixed withoocytes inside the duct (Fig. 1D). The fertilizationrate immediately after collection was then muchhigher (72712%), and it reached nearly 100%when the oocytes were incubated for 0.5–2h in sea-water (Table 1). In most cases, fertilization successdecreased significantly after 5 h of incubation in sea-water (w2, po0.05, n5 165). These results are fairlyconsistent 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 fromthe reproductive tract and those collected throughthe body wall with a syringe at the correspondingtime and location (w2, p40.05; Table 1).

Oocytes in the gonad were an average of 9579mm(n5 246) in diameter. The thickening of the jelly coatincreased the total diameter to 123713mm (n5 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 thegonoduct after the entry of seawater and before thebroadcast. The gonoduct stretched, showing a21711% (n5 9) increase from its original lengthduring the accumulation and movement of oocytes.After gamete release, the sweeping movement and thegonadal muscle activity ceased, leaving the gonadamorphous and almost completely empty with onlya few pockets of unspawned mature oocytes.

Discussion

The huge body of information that exists today onthe processes involved in oocyte maturation haslargely been gathered from in vitro studies, which,by definition, must be uncoupled from the animalsystem. While great advances have been made, fun-damental discrepancies were reported even inside theEchinodermata, so that the pathway leading to ac-quisition of fertilization competence by an oocyte hasyet 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 therespective roles played by mechanical and hormonalprocesses during oocyte transit in the reproductivetract of a broadcast-spawning holothurian. Using se-quential observations and bioassays, the transloca-tion and maturation of oocytes were followed in realtime in naturally spawning individuals, therefore re-fining our understanding of the oocytic pathway andof the role played by the relatively long gonoduct inmany holothurians.

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 (n5 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

(after GVBD)

0 1179 55713 73712 66711 3477

In the distal section of the gonoduct (before

visible entry of seawater)

15711 6778 7776 85711 7276 4479

In the distal section of the gonoduct (after entry

of seawater)

72712 9475 9673 9772 9971 3779

(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 20–25min after contact with spermatozoa,instead of the usual B10min.



Data gathered during this study provide evidencethat the onset of gamete maturation is not only sup-ported by hormonal activity, as traditionally viewed,but also sustained by mechanical action. Ovulationcoincided with an increase in locomotor activity andspasms of the muscle bands along the ovarian tu-bules. A higher level of activity in preparation forbroadcast spawning was also noted in Isostichopusfuscus LUANIF 1875 (unpubl. data) and other hol-othurians (Battaglene et al. 2002). Contractions ofthe body associated with spawning have previouslybeen observed in sea stars (Kanatani 1970) and inseveral species of tropical holothurians (Bakus 1973;Hamel et al. 2001; Battaglene et al. 2002). Interest-ingly, a coupling between mechanical and hormonalaction has been proposed to occur in brachiopods:Stricker & Folsom (1997) suggested that detachmentof follicles from the ovarian germinal epitheliumstimulates 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 detachmentand trigger the onset of hormonal processes requiredfor maturation.

Using extracted oocytes, Smiley (1984, 1988) andMaruyama (1985, 1986) determined that a radial nervefactor (RNF) acted through the follicle cells to pro-duce a maturation-inducing substance (MIS) leadingto GVBD in holothurians. Specifically, Maruyama(1985) noted that follicle cell-free oocytes in H. leuco-spilota never underwent GVBD when exposed toRNF extracts from holothurian tissues. The presentin vivo study hints at a different scenario as (1) GVBDwas completed well after extrusion from the folliclecells and (2) GVBD was never found to occur in oo-cytes collected in the lumen of the ovarian tubules orin the gonad basis after ovulation but before passagethrough the proximal constricted section of the ovi-duct, no matter how long they were left to soak inseawater. Two hypotheses can be offered: (1) the ac-tion of RNF through follicle cells must be combinedwith mechanical activation (i.e., passage through theconstricted duct, or pipetting in the case of in vitrostudies) and (2) RNF acts through another pathwayinside the gonoduct. Incidentally, oocytes of the seastar Pisaster ochraceus BRANDT 1835 (Schuetz 2000)and several sea cucumbers, including H. leucospilota(Maruyama 1986), did respond to sea star RNF byundergoingGVBD even when denuded of their folliclecells, suggesting that other ovarian components mayindeed produce the MIS. Such inconsistencies empha-size that in vitro studies of oocytes, which can involvevarious manipulations (i.e., squeezing, pipetting,rinsing, soaking in seawater and other media, centrif-

ugation, injection, etc.), provide data that are valuablefor 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 studiesin 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 seastars (Kanatani 1969), crinoids (Holland 1988), andsea cucumbers (Smiley et al. 1991). Additionally, ithas been widely assumed that, following GVBD,mei-osis of sea star and sea cucumber oocytes proceededwithout further arrest until extrusion of the two polarbodies, 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 withthe inducing substance, thus exposing oocytes tomany concurrent stimuli. Here, we clearly showedthat oocytes sampled from the ovary after ovulationhad not yet completed GVBD and could never befertilized, 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 recentstudy showed that an arrest in the metaphase I oc-curred after GVBD within the sea star ovary, beforerelease, when hormonal induction was triggered in-side the body cavity of the female to simulate naturalspawning (Harada et al. 2003). Release of this arrestwas induced by an intracellular pH increase when theoocyte was spawned into seawater. This sequencebears more similarities to the one reported herethan all previous in vitro studies. An arrest betweenGVBD 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 mostsuccessful development (Gould & Stephano 2003).

Walker (1975) mentioned that the gonoduct in As-terias vulgaris VERRILL 1866 was capable of extremeexpansion. The same was found in H. leucospilotawhen oocytes pushed into and accumulated withinthe gonad basis. The swelling of the gonad basis, andpossibly the action of cilia of the gonoduct as describedin H. scabra by Mary Bai (1980), seemed to build mo-mentum to allow forced passage through the muchstiffer proximal section of the gonoduct. A clear vel-ocity change was observed during this transit, afterwhichmost oocytes had completedGVBD, apparentlyvia some kind of mechanical stimulus. Mechanical

86 Hamel & Mercier


induction of GVBD and ensuing maturation havebeen reported in oocytes of sea stars (Guerrier et al.1988) and holothurians (Maruyama 1981) after shak-ing and pipetting, respectively. The latter phenomenonis similar to the ‘‘squeezing’’ observed in the proximalsection of the gonoduct.

Although they had completed GVBD after squeez-ing through the first segment of the duct, only15711% of oocytes were competent immediatelythereafter. 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 ofseawater in the final maturation of oocytes. Specific-ally, acquisition of full competency occurred duringthe 10–20min interval between the first noticeableentry of seawater in the duct and the broadcast. In-terestingly, B20min is also the minimum time need-ed for some of the oocytes, having completed GVBD,to show evidence of fertilizability (i.e., when mixedwith spermatozoa in seawater). This delay possiblyallows extracellular ions to act directly or indirectlyon the oocytes. The role of calcium in various stagesof 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 arecent review that in vitro demonstration may notnecessarily hold in vivo (Whitaker 2006). A generalassumption is that ions (calcium, potassium, and so-dium) are sufficiently abundant within the ovarianfluid to allow completion of the maturation process.However, Harada et al. (2003) showed that the in-crease in intracellular pH necessary to completemetaphase I was blocked inside the sea star ovary,preventing the completion of maturation until releaseinto seawater. The present work showed that expos-ure to seawater naturally occurred inside the hol-othurian oviduct, allowing the oocytes to becomereadily fertilizable just before being broadcasted inthe water column, thus minimizing dispersion beforefertilization. This is important as males generallyspawn first in H. leucospilota and many other speciesof 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 seawatersuggest that optimum fertilization will occur soonafter release.

The penetration of seawater in the oviduct beforespawning may also play another role by helping tosaturate the muscle bands with calcium, enablingthem to void gametes with a single or a few power-ful contractions. Again, ions present in seawater havebeen shown to play similar roles. Kanatani & Shirai(1969, 1970) indicated that calcium ions seemed to

provoke contractions of the ovarian wall, which dir-ectly forced out the freely movable oocytes in seastars. Furthermore, Shirai & Walker (1988) men-tioned that oocyte release from the ovary was inhibit-ed in calcium-free seawater as muscles could notcontract. While earlier studies on sea stars emphasizethe role of gonadal wall muscles in the expulsion ofgametes (Shirai et al. 1981), that role was predomi-nantly played by the gonoduct in H. leucospilota.Indeed, the gonads were already empty at the timeof 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). Asthe gonoduct is not muscularized, this bulgingpossibly provides momentum for the broadcast,which is sometimes powerful enough to propel thegametes out of the tanks. Nonetheless, seawater maystill affect the gonads and provoke contractions thatindirectly contribute to gamete release. The concur-rent contraction of the body wall could also play arole, as proposed by Kanatani (1970) for sea stars.

A third function of seawater during the finalpreparation for broadcast is evidenced by theB60% increase in oocyte surface area in the distalsection of the gonoduct, with the hydration ofthe jelly coat. Larger oocytes and/or oocytes withthicker jelly coats were shown to be preferentiallyfertilized in sea urchins (Vogel et al. 1982; Epel 1991;Levitan & Irvine 2001; Podolsky 2001). This is a sig-nificant advantage for species that broadcast smalloocytes. Indeed, a female ofH. leucospilota can storemore of the smaller, gametogenetically mature,oocytes in the gonad and release larger oocytes thatare readily fertilizable to maximize its reproductivesuccess.

This study revealed that oocytes in H. leucospilotaconsistently required B85min 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 (Smiley1990). However, the stress of capture and the induc-tion methods used in aquaculture prompted the re-lease of fully fertilizable oocytes in B1 h byholothurians that did not show any prior signs ofspawning activity (Reichenbach 1999; Hamel et al.2001; Mercier & Hamel 2002; Hamel & Mercier2004). It is worth mentioning that the sequential mat-uration of oocytes reported in the present study isassociated with a gamete transit that culminates in aforceful final broadcast after the formation of a bulgeunder the gonopore. As this is a rather commontrend (Mosher 1982; Hamel et al. 2001; Battagleneet al. 2002; Mercier et al. 2007), it is expected that



similar results could be observed in other holothuri-ans. Species that display a different spawning behav-ior involving the release of a continuous stream ofoocytes (McEuen 1988; Hamel & Mercier 1996a) arelikely to depart, at least in some aspects, from theoocytic pathway described here.

The present study has shown that the ovulation,transit, and maturation of oocytes are complexinterlinked processes that are influenced by behaviorand anatomical particularities. Subtle elements such asshifting between two types of muscular contractions, asmall constriction in the gonoduct, and entry of sea-water through the gonopore play decisive roles inoocyte maturation in H. leucospilota. We thereforeconclude that, in the search for comprehensiveinformation on gamete biology in broadcast spawn-ers, the assessment of in vivo processes is a significantcomponent. A recent review on drug research and bi-ological systems stressed the crucial link between invitro findings and in vivo investigations (Lipinski &Hopkins 2004). The long gonoduct in H. leucopsilotaand other holothurians may well provide an oppor-tunity to further investigate cellular processes occur-ring at various stages of oocyte maturation within livesystems.

Acknowledgments. We would like to thank the staff ofICLARM (now WorldFish Center) in Solomon Islandsfor their help during the work that was performedthere, as well as Dr. S. Smiley (University of Alaska) forhelpful exchanges during the early stages of manuscriptpreparation and two anonymous reviewers for providingjudicious comments and suggestions.

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