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Exogonadal oogenesis in a temperate holothurian.

Introduction

Oocytes in holothurians, as in other echinoderms, are produced in the ovary of the female; they are formed from reproductive cells called germ cells, in a process referred to as oogenesis. The very close link that exists between the germinal epithelium, the germ cells, and the growing oocytes is obvious from histological preparations in many echinoderms (see review in Giese et al., 1991). The production of new gametes is increasingly well understood in echinoderms, despite noticeable variations between different groups and species (Frick et al., 1996; Walker et al., 2005, 2006, and references therein). Briefly, the oocytes in holothurians are initially present as epithelial cells in the germinal epithelium and then bulge into the hemal sinus as they enlarge. A basal lamina lies between the oocyte base and the hemal sinus. As oogenesis progresses, the oocytes enlarge and the basal lamina is extended. The oocytes are growing from the basal lamina and hemal sinus toward the lumen of the gonadal tubules during this process (Frick et al., 1996).

The sea cucumber Cucumaria frondosa is a well known broadcast-spawner, and its oogenesis has been described in populations from various regions (Jordan, 1972; Coady, 1973; Hamel and Mercier, 1995, 1996b, 1999; Singh et al., 2001). The individuals examined during the present study followed the previously described pattern. However, whitish growths were found on the external epithelium of ovarian tubules in 5%-10% of samples collected off Newfoundland, Canada. A similar condition has previously been observed in C. frondosa and other species from various locations but was never fully characterized, except by an early Russian investigator who described it as a parasitic infestation (Djakonov, 1923). We hereby present histological, genetic, and ultrastructural evidence that these growths are in fact abnormal "exogonadal" oocytes. To our knowledge, this is the first description of its kind in the literature and should warrant further investigation into the process of oogenesis in holothurians.

Materials and Methods

Sampling and observations

Sea cucumbers of the species Cucumaria frondosa (Gunnerus, 1770) ranging in size from 5 to 30 cm (contracted length) were collected at various times between August 2005 and August 2006: (1) five samples were collected by divers in Admiral's Cove on the eastern coast of the Avalon Peninsula (Newfoundland; 47[degrees]5'N: 52[degrees]8'W) at depths of 10-15 m; (2) other samples were collected from 32 locations over the St. Pierre Bank (Newfoundland; 46[degrees]9'N: 55[degrees]11'W) by the CCGS Templeman at depths of 20-60 m. The specimens were either preserved in 100% ethanol, 4% formalin, or 3% glutaraldehyde. Some freshly collected specimens were kept alive in flow-through systems at the Ocean Sciences Centre (Memorial University).

Samples from St. Pierre Bank (n = 326) were only visually inspected for the presence or absence of white masses on the gonads. A total of 239 sea cucumbers from Admiral's Cove were examined more closely. Each of them was dissected and sexed. The affected gonadal tubules were measured, and the anomalies were counted and reported as the number of masses per unit of tubule surface. The size of the mass was measured to the nearest micrometer, and the number of separate entities per mass was assessed using the clearly visible germinal vesicle as a reference. All measurements and observations were done using a Nikon SMZ1500 stereo-microscope equipped with a DXM1200F digital camera and Simple PCI Imaging System, ver. 6.0.

Complementary analyses were performed on fresh specimens from Admiral's Cove. The perivisceral coelomic fluid (PCF) of 34 individuals measuring between 5 and 30 cm was filtered to look for the masses or their likely precursors. PCF from individuals with and without anomalies (n = 4 and 29, respectively) was compared. The PCF was collected directly through the anterior body wall by using a 50-cc syringe and a 16-gauge needle. The PCF aliquot (3-4 ml) was then filtered on 1-[micro]m Nitex, and all retained structures were observed under a light microscope. Subsamples from affected and non-affected individuals (n = 3 and 4) were treated with the dye Hoescht 33342, a vital nuclear stain, and observed under a Nikon Eclipse 80i fluorescence microscope.

Histology

Fresh specimens collected from Admiral's Cove were analyzed by light microscopy (LM) and transmission electron microscopy (TEM). Pieces of gonadal tubules displaying the white masses were fixed in a 3% glutaraldehyde solution in cacodylate buffer (0.1 mol [l.sup.-1], pH 7.8) at 4 [degrees]C and rinsed in cacodylate buffer before post-fixation in a 1% osmium tetroxide solution in the same buffer at 4 [degrees]C for 1 h. After a final buffer wash, they were dehydrated through a graded series of ethanol and transferred into Spur resin. For LM analysis, thin sections (1 [micro]m) were cut with a Reichert OmU2 microtome equipped with a glass knife, stained with a 1:1 mixture of 1% azur II and 1% methylene blue solutions, and observed and photographed with a Leitz Laborlux S light microscope equipped with a Nikon Coolpix 990 camera. For TEM analysis, ultrathin sections (90 nm) were cut with a Leica UCT ultramicrotome equipped with a diamond knife. Sections were contrasted with uranyl acetate and lead citrate and observed with a Zeiss LEO 906 E transmission electron microscope.

Genetic analysis

A few masses from the Admiral's Cove samples were removed with a sterile scalpel, and their genomic DNA was extracted with a DNeasy Tissue kit (Qiagen) following the manufacturer's instructions. DNA from the nuclear small ribosomal subunit (18S rDNA) was amplified in three overlapping fragments of about 600 nucleotides each with primers from Eeckhaut et al. (2000). PCR was performed using Ready-To-Go PCR beads (Amersham Biosciences) in a Thermal iCycler (Bio-Rad). After an initial denaturation of 130 s at 94 [degrees]C, 35 cycles of 30-s denaturation step at 94 [degrees]C, a 45-s annealing step at 56 [degrees]C, and a130-s elongation step at 72 [degrees]C were executed. Amplification products were purified with the QIAquick PCR Purification kit (Qiagen) and sequenced with the Big Dye Terminator ver. 3.1 Cycle Sequencing kit (ABI) in a Prism 3100 genetic analyser (ABI). To find related species, the sequence was checked, using the BLAST tool, against the GenBank database (Altschul et al., 1990). The nucleotide sequence obtained in this study was deposited in GenBank under the accession number AM422388.

Results

Observations

The masses are white or slightly pinkish with a size varying between 50 and 6000 [micro]m (Fig. 1). In specimens from all locations, their presence was recorded with a prevalence of 6.3% to 9.4%, regardless of the depth of collection. Specifically, 23 of the 326 individuals from St. Pierre Bank were affected (i.e., 7.1%). The five batches from Admiral's Cove presented a prevalence of 5/53 (9.4%), 3/44 (6.8%), 2/31 (6.5%), 4/64 (6.3%), and 3/47 (6.4%). Similar masses were also reported from females of Cucumaria frondosa from the Barents Sea (Russia) and C. japonica from eastern Russia (E. Gudimova, Polar Research Institute of Marine Research and Oceanography, pers. comm.). They were also observed in C. frondosa collected in 1993-1994 by scallop trawls in the Gulf of St. Lawrence, as well as in Psolus fabricii along the eastern coast of Canada (unpubl. data). The masses occurred singly, in groups or in rows, and exclusively in female individuals.

[FIGURE 1 OMITTED]

Detailed examination of the specimens from Admiral's Cove revealed that each mass was composed of one to six units (Fig. 2), later identified as oocytes (see below). Oocytes in the mass were usually in a comparable state of gametic development (Fig. 1B), although a single individual could possess masses with vitellogenic exogonadal oocytes (EOs hereafter) and others with smaller previtellogenic ones. The smallest masses were composed of EOs measuring 7-100 [micro]m and the largest of EOs between 900 and 1000 [micro]m. Larger masses generally comprised more oocytes (Fig. 2). Within a single individual, EOs showed a normal size-frequency distribution with dominant classes between 300 and 700 [micro]m in diameter (Fig. 3). The range of sizes observed in EOs is consistent with previously published data on normal oocytes/eggs in C. frondosa (Hamel and Mercier, 1996a, b).

In affected females, densities of exogonadal masses varied from a maximum of 5/[mm.sup.2] down to 1 in the entire gonad. Their attachment to the external epithelium of the gonadal tubules was relatively weak, allowing for easy removal with a scalpel or forceps, especially from preserved tissues. Compared to an average normal intragonadal fecundity of 9000-12000 oocytes (i.e., maximum quantity of full-grown oocytes susceptible to be released during the spawning period), the EOs reached a maximum of about 2752 in a single individual. Although masses could be observed all along the tubules, the highest densities were found around the apex. Degenerating masses were observed on occasion, along with previtellogenic and vitellogenic EOs.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Masses of EOs were observed in individuals as small as about 12 cm long (contracted length). However, sub-adults with undifferentiated gonads never bore such masses; the smallest individuals examined were 5 cm long. The total number of EOs increased significantly from 0 to 2752 with increasing size of the sea cucumbers ([chi square], P < 0.01, n = 17). No EOs were observed in sea cucumbers under 10 cm in length, whereas 5 [+ or -] 5 EOs were found in individuals measuring 11-15 cm, 56 [+ or -] 44 EOs in individuals of 16-20 cm, 245 [+ or -] 110 EOs in individuals measuring 21-25 cm, and an average of 1257 [+ or -] 945 EOs in the largest (>25 cm) individuals (values are mean [+ or -] SE, n = 7).

No variation in the size structure or the dominant size mode was observed between the EOs and normal oocytes found in the lumen of the gonadal tubules ([chi square], P < 0.001, n = 2752 and 475, respectively). These data were gathered from heavily infested individuals in which enough oocytes were analyzed to allow comparison just before the spawning in March 2006. Individuals collected after the breeding season still showed the presence of masses of vitellogenic EOs, whereas most vitellogenic oocytes were no longer present in the lumen.

In some cases, high densities of EOs were correlated with a decreased proportion of oocytes in the lumen of the gonadal tubules to a point where castration was observed. In five individuals collected in March 2006, prior to spawning, the lumen of some of the heavily affected tubules contained very few previtellogenic oocytes, or none at all, whereas the rest of the gonad was filled with vitellogenic oocytes. These sections of heavily affected tubules looked dry, with a thinner peritoneum and a very circumvoluted germinal epithelium.

Examination of the perivisceral coelomic fluid (PCF) and the epithelium of most internal organs in contact with PCF (including body wall and muscle bands) in individuals with and without EOs did not reveal the presence of oocytes or any of their known precursors except on the ovarian tubule themselves.

Histology

The EOs grow outside the gonadal tubule on the peritoneum toward the perivisceral coelomic cavity. There is no evidence of any linkage with the germinal epithelium at any of the observed stages of oogenesis; direct contact is solely with the peritoneum of the ovarian tubule. Parts A and C of Figure 4 illustrate the ovarian tubule with normal oocytes in the lumen; parts B and D show the EOs growing among cells of the visceral peritoneum toward the coelomic cavity. Both oocyte types are composed of germinal vesicles, nucleoli, and follicular cells. The follicular cells in the EOs are located exclusively between two oocytes (Fig. 4B, D, E), whereas they occur between the peritoneum and the oocytes and on individual oocytes under normal conditions. Lateral and basal junctions between EOs and the peritoneum show circumvolutions and a close association (Fig. 5). The EO does not touch the gonadal haemal sinus; a peritoneum always separates it from the sinus (Fig. 5C, D).

TEM observations of the EOs and comparison with normal oocytes show that the cortical area of a normal oocyte is surrounded by a vitelline coat (Fig. 6A), whereas the vitelline coat is absent and the cortical granules are few and therefore rarely apparent in EOs (Fig. 6B). The EOs seem to possess a more clearly defined, thicker, and less circumvoluted external membrane (Fig. 6A, B). Furthermore, the cytoplasm is characterized by abundant oil droplets in normal oocytes, and by numerous yolk granules in the exogonadal counterparts (Fig. 6C, D). TEM sections of the normal oocytes (Fig. 6C) also show the presence of numerous electron-clear circular vacuoles of various sizes, generally between 0.5 and 5 [micro]m in diameter. A few denser vacuoles are present, which are also rounded with a diameter varying between 1 and 2 [micro]m. Comparison with EOs (Fig. 6D) shows a dominance of yolk granules that appear more abundant than in normal oocytes (ca. 10 per 100 [[micro]m.sup.2]). They are ovoid, electron-clear, and measure about 1-2 [micro]m in diameter. Their particularity is the presence of up to four electron-dense bands. The yolk granules are often shattered, appearing as a light hole (Fig. 6D, arrows).

Genetic analysis

From the sequencing, an 18S rRNA gene sequence of 1664 nucleotides was obtained and checked against the GenBank database, using the BLAST tool to find related species. Although no data were available in the databank for Cucumaria frondosa, the sequence matched the three first species on the list with 99% of similarity; those species are Aslia lefevrei [AY133480], C. elongata [AY133479], and C. sykion [Z80950], indicating the holothurian origin of the DNA extracted.

[FIGURE 4 OMITTED]

Discussion

Whitish masses found on the external epithelium of ovarian tubules in 5%-10% of sea cucumbers of the species Cucumaria frondosa, collected off Newfoundland (Canada), were examined in this study. Histological, genetic, and ultrastructural analyses provide strong evidence that these masses are abnormal exogonadal oocytes (EOs), warranting further investigation into the process of oogenesis in holothurians. The fact that similar observations were made in C. frondosa and other holothurians from many temperate locations evokes the possibility of an anthropogenic disturbance.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Identification

Very similar white spots were described covering gonads of Cucumaria frondosa from Kola Bay in Murmansk, Russia (Djakonov, 1923, see table VI and figures 1, 6). The investigator assigned this phenomenon to a parasitic infestation and went on to describe a new gregarine species, Diplodina gonadipertha (Djakonov, 1923). Numerous gregarines indeed parasitize holothuroids and spatangoid echinoids, with 22 known species, including D. gonadipertha, associated with 16 echinoderm hosts (Jangoux, 1990; Eeckhaut et al., 2004). Most of these hosts are deposit-feeding species, but some suspension-feeding sea cucumbers are also infected, presumably through the respiratory current (Jangoux, 1990). Gregarines can be subdivided into two groups: cephalines and acephalines. Trophozoites in the cephaline gregarines assume a substructure in which a small part of the anterior end, the protomerite, is delimited from the nucleated posterior portion, the deuteromerite, by a septum (Perkins, 1991). Acephaline gregarines lack this septum and are fixed against the host cell surface by means of a mucron. The latter consists in a flattening of the anterior part of the parasite with associated fibrillar material and microtubules. Another feature of gregarine trophozoites is the presence of two subpellicular membranes lying under the plasma membrane. Finally, the surface of the individuals is longitudinally folded, and each fold has either one or two layers of microtubules.

The white masses investigated in the present study do not display any of the gregarine structures (i.e., septum, mucron, subpellicular membranes). Both microscopic and genetic analyses strongly support the conclusion that the masses found on the ovarian tubules of some females of C. frondosa are oocytes. Sequencing of the 18S rRNA gene revealed a 99% affinity of the masses with three holothurian species, whereas microscopic observations clarified the nature of the structures: (1) their size is equivalent to that of normal oocytes present in the lumen of the tubules, (2) they display a germinal vesicle containing one or several nucleoli, and (3) this germinal vesicle lies within a developed cytoplasm filled with yolk granules containing a crystalloid structure, as reported in several other echinoderm species (Smiley, 1990). Cortical vesicles bearing a biphasic matrix are also present in the cytoplasm. Finally, the structures were discovered only in female individuals having reached sexual maturity.

Abundance and size structure

Our results suggests that the bigger the sea cucumbers the more abundant the EOs. The EOs are apparently not released during spawning events, so masses accumulate over successive years. Nonetheless, the size structure of the EOs is normally distributed, suggesting the degeneration and disappearance of the larger, presumably older, EOs. This hypothesis is supported by the observation of partially degraded EOs attached to the surface of some ovarian tubules.

The partial castration observed in cases of high abundances of EOs suggests an adverse effect on normal oogenesis. One or a few of the following situations may be occurring: (1) a predetermined pool limits the number of oocytes synthesized per surface area, be it inside or outside of the tubule; (2) there is local competition for nutrients during maturation; (3) the presence of abundant exogonadal development alters the tubule to a point where it can no longer support normal gamete maturation.

Possible origin

The origin of the EOs is unclear. Their unusual location within the cells of the visceral peritoneum of the ovarian tubules suggests that these oocytes are abnormally transported here following their origin in the germinal epithelium on the other side of the hemal sinus (Inoue and Shirai, 1991; Frick et al., 1996). Instead of moving toward the lumen as described by Frick et al. (1996), these abnormal oocytes may have been diverted toward the coelomic cavity across the hemal sinus. Studies of Synaptula hydriformis showed that oocytes naturally rupture the perivisceral peritoneum to be released directly into the coelom (Clark, 1898; Estabrooks, 1984). However, no evidence was found of EOs passing through the gonadal tubule peritoneum of C. frondosa, and histological preparations did not reveal any link between the germinal epithelium, the hemal sinus, and the EOs. Moreover, the bond between the EOs and the peritoneum is rather weak; lightly scraping them is often enough to remove the cells from the peritoneum's surface, leaving no visible scar. Conversely, no evidence was found to suggest that these EOs could transit through the coelomic fluid and settle on the gonadal tubules where they would mature. A coelomic origin would likely result in settlement on various other organs, but atypical oocytes were observed only on ovarian tubules. Furthermore, filtration of the PCF before and after the breeding season did not reveal the presence of oocytes or any likely precursors. The possibility nonetheless remains that our samplings and observations were insufficient to ascertain the role of the coelom and PCF in the transit of germ cells. Frick et al. (1996) presented a detailed description of the association between the emerging oogonia, the germinal epithelium, the hemal system, and the follicular cells. On the basis of this report, it is not impossible that EOs could develop from the peritoneum at the interface of the coelom.

Conclusion

The structures reported in this study have been seen only in holothuroid echinoderms. If EOs occur in other echinoderms, they would probably not appear as they do on the anatomically simple holothurian gonad, which is devoid of an outer sac, unlike the gonad of asteroids and echinoids, for instance (Giese et al., 1991). Thus, comparable EOs would be present in the genital coelom of other echinoderms, not in the perivisceral coelom as described here.

The fact that holothurians with exogonadal masses liable to be EOs were found in many locations suggests that the synthesis of EOs may be linked to anthropogenic activities. Although some of the affected specimens were collected from presumably pristine sites, sensitivity to low levels of pollution cannot be discounted. Alternately, the phenomenon could be induced by a normal but uncommon condition that may be uniquely related to the structure of the holothurian gonads. Further investigation of this atypical oogenesis may shed new light on the origin and pathway of germ cells and on the maturation process leading to fully mature vitellogenic oocytes.

Acknowledgments

We warmly thank C.W. Walker (University of New Hampshire) for helpful advice, S. Smiley (University of Fairbanks, Alaska) for information on gregarines, M. Sewell (University of Auckland) for very useful suggestions, as well as J.S. Pearse (University of California, Santa Cruz) and an anonymous reviewer for comments on the original manuscript. We also wish to acknowledge the assistance of the OSC Field Services for samplings in Admiral's Cove and the Department of Fisheries and Oceans (St. John's) for samplings on the St. Pierre Bank area. This research was partly funded by the National Science and Engineering Research of Canada (NSERC) and the Canada Foundation for Innovation (CFI), as well as by the Commission Universitaire pour le Developpement (CUD) and FRFC contracts (No 2.4.583.05F) in Belgium.

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JEAN-FRANCOIS HAMEL (1), PIERRE BECKER (2), IGOR EECKHAUT (2), AND ANNIE MERCIER (3,*)

(1) Society for the Exploration and Valuing of the Environment (SEVE), 21 Phils Hill Road, Portugal Cove-St. Philips, Newfoundland A1M 2B7, Canada; (2) University of Mons-Hainaut, Laboratoire de biologie marine, 6 avenue du Champ de Mars, B-7000 Mons, Belgium; and (3) Ocean Sciences Centre (OSC), Memorial University of Newfoundland, St. John's, Newfoundland A1C 5S7, Canada

Received 30 January 2007; accepted 9 May 2007.

* To whom correspondence should be addressed. E-mail: amercier@mun.ca

Abbreviations: EO, exogonadal oocyte; PCF, perivisceral coelomic fluid.
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Author:Hamel, Jean-Francois; Becker, Pierre; Eeckhaut, Igor; Mercier, Annie
Publication:The Biological Bulletin
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Date:Oct 1, 2007
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