Comparative organization of follicle, accessory cells and spawning anlagen in dynamic semelparous clutch manipulators, the urochordate Oikopleuridae

Authors

  • Philippe Ganot,

    1. Sars International Centre for Marine Molecular Biology, Bergen High Technology Centre, Thormøhlensgate 55, N-5008 Bergen, Norway
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  • Jean-Marie Bouquet,

    1. Sars International Centre for Marine Molecular Biology, Bergen High Technology Centre, Thormøhlensgate 55, N-5008 Bergen, Norway
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  • Eric M. Thompson

    Corresponding author
    1. Sars International Centre for Marine Molecular Biology, Bergen High Technology Centre, Thormøhlensgate 55, N-5008 Bergen, Norway
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(email Eric.Thompson@sars.uib.no).

Abstract

Background information. The urochordate appendicularians play a key trophic role in marine ecosystems and are the second largest component of zooplankton after copepods. Part of their success is due to their ability to undergo rapid population blooms in response to changes in primary productivity. Nonetheless, the reproductive biology of this important group remains poorly understood.

Results. In the present study, we investigated the organization of male and female germ and accessory somatic cells in the Oikopleuridae. We found that the structure of the ovary had been previously misconstrued as consisting of germ and accessory ‘cells’ interspersed together, whereas, in fact, the germline exists as a giant transparent syncytium. Somatic follicle cells, integral to regulation of the temporal progression of gametogenesis, could be classified into three types in females and two in males, and we characterized functional gap junctions between follicle cells and the germline syncytium in both sexes. The number of follicle cells per oocyte produced was much reduced in comparison with many commonly studied model organisms. We further identified a novel anlagen that permits spawning of the animal via rupture of the gonad wall, which is obligatory for the release of oocytes, but optional for the release of sperm that usually occurs via the spermiduct.

Conclusions. The organization of the female germline in the Oikopleuridae shares some features of meroistic oogenesis with the arthropod Drosophila, but the process of synchronous oogenesis in these semelparous organisms remains quite distinctive with respect to that previously characterized in the animal kingdom and certainly within the chordate phylum.

Abbreviations used:
BrdU

bromodeoxyuridine

Br-RNA

RNA transcripts that incorporated BrUTP

BrUTP

bromouridine triphosphate

DTC

distal tip cell

ITC

inner trinucleate cell

PGC

primordial germ cell

RT

room temperature

Introduction

The appendicularians have a pan-global distribution and form an important part of marine zooplankton communities. There are approx. 70 species, and of these all, with the exception of the coastal species Oikopleura dioica, are hermaphrodites. Appendicularians occupy a key phylogenetic position near the invertebrate—vertebrate transition, and the fact that O. dioica remains transparent throughout a very short life cycle (6 days at 15°C) makes it a useful model organism for chordate genetics. O. dioica can be maintained in laboratory culture throughout the year, and the sequencing of its very compact genome (70 Mb, 1 gene per 3–4 kb; Seo et al., 2001) is nearing completion.

To date, information on the germline and gametogenesis in appendicularians is limited, as it has been based essentially on conventional microscopic examinations of periodically collected field specimens. In nearly all species, the reproductive organs are positioned posterior to the digestive tract in the gonad cavity (see Supplementary Figure 1 for O. dioica, http:www.biolcell.orgboc098boc0980389add.htm), but in hermaphrodites the form of the testis and ovary, as well as their respective dispositions in the ovotestis, is variable (Fenaux, 1963). On the other hand, the cellular anatomy of the gonads is described as being much less variable. The testis contains proliferating nuclei arranged as a syncytium that will mature synchronously in Oikopleuridae and Fritillaridae into individual sperm (Martinucci et al., 2005). In addition to the germline, there are a few cells containing large nuclei that degenerate upon maturity in fritillarids (Martinucci et al., 2005). Two basic organizations have been noted within ovaries. The first consists of a central syncytium with large nuclei that is surrounded by a monolayer of developing oocytes. This is found among the Fritillaridae and the Kowalewskiidae. The second type, more characteristic of the Oikopleuridae, consists of an organization that was misinterpreted as germ and accessory ‘cells’ interspersed together. Within this latter organization, two subtypes have been observed: those that produce naked oocytes and those where oocytes are surrounded by a layer of follicle cells upon spawning (Fenaux, 1963). Sperm are released via a spermiduct, whereas oocytes are released by rupture of the gonad wall. Fertilization is external in appendicularians and emission of the gametes in the sea water immediately precedes the death of these semelparous organisms.

The participation of somatic follicular cells and their reciprocal interactions with the germline is a conserved feature in gametogenesis (Estrada et al., 2003; Smith and Sinclair, 2004). Intimate connections between follicle cells and the oocyte are important in producing a mature oocyte of the correct size and content. During mammalian oocyte growth, thin membranous projections from the follicle cells penetrate the zona pellucida and make contact with the oocyte via gap junctions (Kidder and Mhawi, 2002). Metabolites, amino acids and nucleotides are passed from the follicle cells to the oocyte via these junctions (Eppig, 1991), and signals regulating meiotic maturation also transit via these connections (Fagbohun and Downs, 1991; Downs, 1995). Follicle cells can also have more specialized functions beyond their involvement in nutrition and maturation of the oocyte. The monolayer of Drosophila follicle cells that surround the egg chamber is composed of specialized subpopulations that synthesize the egg shell, produce yolk proteins for the oocyte and set positional cues that are interpreted in embryonic pattern formation (Deng and Bownes, 1998). Reciprocally, a localized Gurken signal originating from the oocyte is required at two junctures to induce first posterior and later dorsal and ventral fates in the population of follicular cells (Roth et al., 1995; Gonzalez-Reyes and St Johnston, 1998). In contrast with the numerous follicle cells surrounding the Drosophila egg chamber, only 5 pairs of somatic sheath cells surround each gonad arm in the hermaphrodite nematode Caenorhabditis elegans. These cells are closely associated with what begins as a syncytial germline, and they promote germline proliferation, exit from pachytene and male germ cell fate (McCarter et al., 1997). They also absorb excess female germ nuclei that are eliminated by programmed cell death (Gumienny et al., 1999), and are required for oocyte maturation and ovulation (McCarter et al., 1999). In appendicularians, the disposition and nature of the somatic cells and germlines remain poorly understood, particularly in those species producing naked oocytes. Semelparity introduces particular constraints on gametogenesis, such as synchronous meiosis, gamete maturation and spawning. These events are commonly decoupled both temporally and spatially in other model organisms, with multiple stages of gametogenesis co-existing in a single gonad. Semelparity also implies different regulatory strategies with regard to resource allocation in gamete formation.

In the present study, using confocal and electron microscopy with a number of cytoskeletal and germline molecular markers, we have delineated the organization of the male and female germlines of the dioecious O. dioica, as well as the associated somatic cell partners, and compare this with the hermaphroditic O. longicauda, O. fusiformis and O. labradoriensis. These studies reveal a shared syncytial-like organization of the gonad that we refer to as a coenocyst (P. Ganot, J.-M. Bouquet and E.M. Thompson, unpublished data). They further define the relationship of different populations of somatic follicle cells and additional accessory cells to the developing gametes in the different species. We have also identified a new spawning anlagen involved in the rupture of the gonad at maturity. The novel coenocystic organization of the oikopleurids may explain in part their ability as strong clutch manipulators to rapidly modulate numerical oocyte production as a function of fluctuating nutritional resources (Troedsson et al., 2002), and account in part for their capability to undergo rapid population increases in response to algal blooms (Uye and Ichino, 1995).

Results and discussion

Spawning in dioecious and hermaphroditic Oikopleuridae

In mature O. dioica, sperm is released via the spermiduct (Figure 1a and Supplementary Video 1, http:www.biolcell.orgboc098boc0980389add.htm), or alternatively via rupture of the gonad if the animal is stressed. Release of oocytes from females is obligatorily by rupture of the gonad wall (Figure 1b and Supplementary Video 2, http:www.biolcell.orgboc098boc0980389add.htm). Both the ovary and the testis are organized such that the germline nuclei share a common cytoplasm (Figures 1c and 1d). In the ovary, the germline differentiated into two populations of nuclei: polyploid nurse nuclei and meiotic nuclei arrested in prophase I. These have continuous cytoplasmic connections throughout the gonad cavity, forming a single giant multinucleate coenocyst (P. Ganot, J.-M. Bouquet and E.M. Thompson, unpublished data). The germline in the testis consists entirely of a uniform proliferating population of nuclei, which is in agreement with the study by Martinucci et al. (2005). Similar respective organizations were observed within the ovary and testis of the hermaphrodite, O. longicauda (Figures 1c and 1f).

Figure 1.

Hermaphroditic and dioecious Oikopleuridae

(a) Male ejecting sperm via the spermiduct (arrow). (b) O. dioica female releasing eggs through a rupture in the gonad epithelium. (c) Hermaphroditic O. longicauda, with testis (black asterisk) and ovary (white asterisk). (d, e, f) Confocal images of the organization of the gonad from O. dioica male (d) and female (e), and O. longicauda (f); F-actin, green; DNA, blue. The testis germline consists of a uniform population of proliferating nuclei (d, f), whereas the ovarian germline consists of giant polyploid nurse nuclei and meiotic nuclei (e, f). Scale bars are shown with units in μm.

Gonads contain specialized somatic tissues which are specific to male or female gametes. The sex determination pathway is unknown in Oikopleuridae, but since O. dioica is the only dioecious species, it was of interest to compare the anatomy of their gonads with the hermaphroditic ovotestis. In toto staining of the F-actin network with phalloidin—FITC in O. dioica readily identified the spermiduct, but also revealed a novel anlagen we refer to as the ‘moustache’ which is implicated in rupture of the gonad wall for gamete release (Figure 2, part I). The moustache was present in both males and females. Precursors of the spermiduct and moustache could not be identified during proliferation of the germline in the gonad before day 3 (Figure 2, part I, g), and first became evident after sexual differentiation around the transition from day 3 to 4. At this time they were present as an undifferentiated field of cells, enriched in F-actin, located near the dorsal junction of the gonad and the trunk (Figure 2, part I, h). In day 5 males, 1 day prior to spawning, the spermiduct could clearly be distinguished from the moustache (Figure 2, part I, i). A similar disposition of the spermiduct and moustache was observed in the hermaphroditic species O. longicauda, O. fusiformis and O. labradoriensis (Figure 2, part II). In the first two species the moustache was located over the testis, whereas in the third it spanned both the ovary and testis. Under normal conditions, where no stress is applied to the animals, the testis is first emptied of its contents via the spermiduct prior to release of the oocytes via rupture of the gonad wall (Fenaux and Gorsky, 1983). Independent of its location, a single moustache anlagen is sufficient for release of both sets of gametes from the ruptured hermaphroditic gonad. This field of somatic cells which differentiate inside the gonad epithelium into a spermiduct and a moustache is thus specific to hermaphrodite and male dioecious Oikopleuridae. The absence of the spermiduct in female O. dioica might then imply that the male germline is required for spermiduct differentiation.

Figure 2.

Specialized spawning anlagen

Part I: confocal images of O. dioica male (a, b, e, f, h, i), female (c, d) and juvenile (g); F-actin, green; DNA, blue. (af) Day 6 specimens; (gi) as indicated. F-actin labelling shows the spermiduct (arrows in b, e, i) and a novel anlagen, the ‘moustache’ (all images, except g). The moustache is present in both sexes, whereas the spermiduct is found only in the male (compare a and b with c and d). The moustache is a gonad-specific anlagen that opens the epithelium when gametes are mature, releasing them into the environment (e, f). During development, neither the moustache nor the spermiduct can be detected by F-actin staining before day 3 (g). After sexual differentiation at days 3–4, a precursor field of undifferentiated cells can be identified. Subsequently, the moustache and the spermiduct are distinguishable at day 5 in males (h, i). Part II: left-hand panel (from top to bottom): in toto pictures of O. fusiformis, O. longicauda and O. labradoriensis stained for DNA; each ovotestis (to the left of the trunk) has two compartments, the ovary (identified by giant nurse nuclei) posteroventral to the testis. Right-hand panel (labelling as in part I): same species as above with enlargement of the ovotestis, emphasizing the conserved spermiduct (arrow) and moustache structures (see sketch). Scale bars are shown with units in μm.

Follicle and accessory cells associated with the coenocystic gonad

In most common model organisms used to study oogenesis, including Drosophila, Xenopus and mammals, a large number of somatic follicle cells surround each maturing oocyte. This contrasts with the limited number of sheath cells surrounding the germline in the nematode C. elegans. Gap junctions are, in part, responsible for the exchange of information between somatic follicle cells and maturing gametes, and they are also present between proximal sheath cells and oocytes in C. elegans (Hall et al., 1999). In O. dioica, the arrangement of follicle cells showed some similarities with that of C. elegans in that the follicle cells were peripheral to the coenocyst and relatively few in number per maturing oocyte (Figures 3–5). Females had a monolayer of follicle cells lining both the inner and outer surface of the coenocyst (Figure 3a and see Supplementary Figure 1 for definition of inner and outer surface, http:www.biolcell.orgboc098boc0980389add.htm). The coenocyst proper exhibited a ramified network of numerous membrane invaginations (Figures 3b–3d, and see also Figure 4e) that was contiguous with the extracellular space of the follicle cells. Outer follicle cells were located directly under the monolayer epithelium covering the gonad compartment. The outer and inner follicle cells exhibited distinct morphological characteristics (Figures 3b and 3c), and so are referred to as type I and II follicle cells respectively. Male type I follicle cells differed from female type I cells by the presence of a large cytoplasmic vacuole (Figure 3e). They also showed structures that resembled gap junction connections with the inner germline cyst, a hallmark shared with female type I follicles (Figures 3d and 3f). A functional definition of gap junctions is that they allow adjacent cells to share small molecules (<1 kDa). To test this function, we performed local injections of BrUTP (bromouridine triphosphate; 0.62 kDa), a molecule known to diffuse very poorly through membranes, into the female coenocyst or male syncytium (Figure 4). Br-RNA (RNA transcripts that had incorporated BrUTP) were rapidly detected in nuclei throughout the coenocyst/syncytium within 5 min of injection, and were also detected with similar kinetics in type I follicular nuclei in both males and females. Br-RNA was never detected in the overlying epithelial nuclei or in tissues adjacent to the gonad, even after chase times of 90 min (data not shown), ruling out the possibility of passive diffusion of BrUTP through the plasma membrane of the coenocyst/syncytium. This demonstrated the passage of BrUTP from the coenocyst/syncytium to the type I follicle cells in both sexes, in support of the presence of functional gap junctions between these compartments. Thus the follicle cells identified in both males and females present morphological characteristics of follicle cells described in other model systems. The gonad is enclosed by a monolayer epithelium with follicle cells surrounding the central syncytial germline. Communication between the single germline cell and the follicles can be achieved through direct passage of small molecules via the gap junctions or by paracrine release in the extracellular space (Figure 4e). Such a strategy is well adapted to the synchronous control of germline differentiation throughout the entire gonad.

Figure 3.

Peripheral follicle cells (type I and II) of O. dioica

Plastic section of a day 5 female O. dioica ovary stained with Toluidine Blue (a) and transmission electron micrographs of day 5 female (bd) and male (e, f) gonads. (a) On the exterior and interior surfaces of the coenocystic germline, two distinct cell types were identified. At the outer surface, beneath the epithelium (black arrowheads), is a discrete layer of cells (white single arrows) and, on the internal side, adjacent to the gonad cavity, a few inner cells (double white arrows) can be seen. We refer to these as type I and II follicle cells, respectively. (b) Detail of a type II follicle cell showing distinct nuclear morphology compared with type I follicle cells (c). (d) Magnification of the interface between a follicle cell (right) and the germline coenocyst (left) showing distinct plasma membranes between the two compartments. The extracellular region between the coenocyst and the follicle cells consists, in part, of invaginations that are ramified throughout the coenocyst. (e) In male O. dioica, cells in a similar location to type I follicle cells in the female are observed and these contain a characteristic large vacuole. (f) Magnification of the intercellular space between the follicle cells and the germline shows structures resembling gap junctions. Inset: a junction at higher magnification. The same junctions were also observed between female type I follicles and the coenocyst (black arrows in d and f).

Figure 4.

Connections between the germline and type I follicles

(ad) BrUTP (0.62 kDa) was injected into the germline cytoplasm of day 5 male (a) and female (bd) gonads and BrdU incorporation into de novo-transcribed RNAs was monitored by immunofluorescence (Br-RNA, red; DNA, blue). Br-RNAs were detected in germline nuclei, as expected, both in males [small nuclei in (a)] and females [polyploid nurse nuclei in (b, d)]. Br-RNAs were also detected in the type I follicle nuclei (arrows, ad), but not in the epithelial cell nuclei (arrowheads, ad), indicating passage of BrUTP from the germline compartment to type I follicles, but not to epithelial cells. The image in (a) is a projection of confocal image stacks from the superficial epithelium of the testis, through the follicle layer and into the germline syncytium. (bd) Sections through the gonad with the epithelium at the top. (c) Transmission image corresponding to the immunostaining in (d). (e) Scheme of the disposition of epithelial cells, type I follicle cells and the differentiated coenocystic germline nuclei in the ovary of O. dioica. Interactions between the follicle cells and the coenocyst are mediated by gap junctions and a ramified extracellular network that penetrates throughout the coenocyst. Scale bars, 10 μm.

The cytoskeletal organization of both epithelial and follicle somatic cells was distinct from that of the germline cyst. In female O. dioica, the F-actin network did not completely enclose either the meiotic or polyploid nurse nuclei, but left an open network throughout the coenocyst. In contrast, there was a clear continuous subcortical actin border enclosing both individual follicular and epithelial cells (Figure 5). Within these cells numerous actin stress fibres could also clearly be seen (e.g. Figure 5, part II, b). The microtubule network of epithelial and follicle cells displayed a typical interphase appearance very different from that observed within the interior of the coenocyst. In the latter compartment, only reduced localized aggregations of tubulin staining were observed (Figure 5, part II, c). This difference in tubulin staining was reproducible using three different pan anti-tubulin antibodies under a variety of fixation conditions; although, at present, we cannot rule out the possibility that the differential staining patterns were a result of the differential stability/composition of the germline microtubule network as opposed to it simply being drastically reduced. A role for the follicle cells in partially organizing the actin cytoskeleton of the coenocyst was suggested in experiments where vitellogenesis was induced prematurely in females. Under standard culture conditions, pro-oocytes will begin to increase rapidly in volume around day 5.5. However, after differentiation of the female germ nuclei at the day 3 to 4 transition, it is possible to prematurely stimulate very rapid oocyte growth as a response to stress, such as mechanical prodding. Under these conditions, a rapid increase in the volume of the coenocyst occured and thick cables of actin were observed to project deeply into the coenocyst from anchorage points associated with type I follicle cells (Figure 5, part II, d and e). Under standard culture conditions, the increase in the volume of the coenocyst is more gradual and these actin cables were not apparent.

Figure 5.

Organization of cytoskeletal elements in the O. dioica gonad

Part I: micro-dissected day 5 gonads with the surrounding epithelium. F-actin, green; microtubules, red; DNA, blue. (a) Confocal cross-section through the different layers of the gonad: the coenocystic germline, type I follicle cells (arrow) and the epithelium (arrowhead). (b) The tubulin network is detected only in the follicle (arrow) and the epithelial (arrowhead) cells. (c) Superficial top view of the epithelium monolayer, including the moustache (top left). (d) Detail of the microtubule network of a single epithelial cell with a branched filiform nucleus. Part II: micro-dissected gonad where the epithelium has been manually removed (staining as in part I). (a, b) The microtubule network of type I follicle cells (delineated by cortical F-actin), in females (a) and males (b). (c) When immunofluorescence was performed on sections, microtubule networks were still only detected in the somatic follicles, never in the surrounding germline, precluding a problem of antibody penetration. (d, e) A network of thick actin cables projecting from the type I follicle cell layer (arrows) into the coenocyst becomes apparent after induction of oocyte growth by stress, whether the animal is immature (d) or maturing (e). Scale bars are shown with units in μm.

In O. dioica, we also identified another distinctive cell that was surrounded by the coenocyst (Figure 6). Present in both males and females, this cell exhibited a continuous sub-cortical actin network surrounding three large nuclei, leading to it being called the ITC (inner trinucleate cell). The ITC was detectable from very early in gametogenesis at a time when germ cells were proliferating prior to their differentiation. As for follicle cells, the ITC was not stained for the germline marker vasa and appeared to be of somatic origin (Figure 6h). There was only one ITC per gonad. At present we are unable to propose any specific function for the ITC. In C. elegans, the DTC (distal tip cell), located at the end of the distal gonad arm, maintains nuclei within the gonad syncytium in a state of mitotic proliferation. Nuclei that migrate beyond the signalling range of the DTC enter meiosis (Kimble and White, 1981). The positioning of the ITC with respect to the germline nuclei in the O. dioica coenocyst indicates that it is improbable that the ITC has a homologous function with that of the DTC of C. elegans, as there is no evidence of any gradient effect with respect to differentiation in the coenocyst relative to the placement of the ITC. The ITC is comparable with, to a limited extent, the perioesophageal body (Okada and Yamamoto, 1999) identified in the ascidian Ciona intestinalis, a representative of a sister class of the Appendicularia within the Urochordata. The perioesophageal body is located just beneath the ventral margin of the oesophagus, adjacent to the developing gonad. This body was composed of wedge-shaped cells of a single type, surrounding a central cavity. Nuclei were large with a prominent nucleolus, and cytoplasmic features suggested an active secretory function. The ITC was initially located near the digestive tract of O. dioica (Figures 6e–6g), but in contrast with the perioesophageal body it was always a part of the gonad proper. The ITC did not increase in cell number and size as did the perioesophageal body, and came to occupy an inner position within the coenocyst as the gonad developed. However, the presence of polyploid nuclei in the ITC is consistent with a secretory function, since in many organisms, and in O. dioica in particular, polyploidization is a feature of secretory cells (Ganot and Thompson, 2002).

Figure 6.

Structure of the ITC

(ag) Within the open F-actin (green) network of the female coenocyst resides a separate cell, outlined by complete cortical actin labelling (a, projection of confocal serial sections) enclosing large nuclei (b, single section). DNA is labelled blue. These appear to be three distinct nuclei giving rise to the name ‘inner trinucleate cell’ (ITC) (arrowheads, ch). The ploidy of these nuclei has not been determined, although based on size they are likely to be polyploid (when compared with neighbouring polyploid nurse nuclei). The ITC is present in females (c) as well as in males (d). Before sexual differentiation, the ITC is readily observed near the gut apparatus (e) and can be distinguished from the germline nuclei as early as day 1 of development (f, g). (h) Both the ITC (arrowhead) and follicle cells (arrows) were negative for germline-specific vasa staining (arrows); vasa mRNA (green) and DNA (blue). Scale bars are shown with units in μm.

The end result of oogenesis in O. dioica is the release of naked oocytes arrested in metaphase I of meiosis (Holland et al., 1988). The follicle cells, rather than having a specific association with a given oocyte, appear to play a more general supportive role to the entire coenocyst. Therefore, we were interested to explore the origin of follicle cells which surround individual oocytes upon their release in other oikopleurid species, such as O. longicauda (Figure 7). In this species the organization of the ovary as a coenocyst with polyploid nurse nuclei sharing the same cytoplasm as meiotic nuclei in prophase I resembled that of O. dioica. However, we also observed an additional population of nuclei interspersed in the ovary. These nuclei differed from the meiotic nuclei in their chromatin texture, and occasionally mitotic figures, stained positively for histone H3 (phospho-Ser10), were observed among them (Figures 7a–7c), in comparison with the meiotic nuclei that were not stained with this marker. They also differed from the nuclei in type I and II follicle cells. Initially, this interspersed population of nuclei did not reveal any specific association with particular meiotic nuclei, but as oogenesis proceeded they began to associate progressively with the periphery of growing oocytes (Figures 7d and 7e). We refer to these as type III follicle cells, restricted to those species that spawn oocytes surrounded by a monolayer of follicle cells.

Figure 7.

Type III follicle cells in the ovotestis of O. longicauda

(ac) In addition to the giant polyploid nuclei and the presumptive meiotic nuclei (white arrowheads), a third type of nucleus (pink arrowheads) was present in the immature coenocyst ovary of O. longicauda. These nuclei were small, appeared diploid based on size, and had a chromatin texture different from that of the presumptive meiotic nuclei (c). They also differed from the peripheral follicle cells (arrows). The histone H3 (phospho-Ser10) (H3S10P) marker for mitosis and prophase I recognized a few dividing nuclei in the coenocyst (one pink arrowhead shows labelled H3S10P nuclei pairs in a and c), as well as proliferating sperm precursors (red arrowhead at bottom in a), but not the presumptive meiotic nuclei. (d, e) When the ovarian vitellogenic phase commenced and individual oocytes with their meiotic nuclei (white arrowheads) were discernable in the coenocyst, this third population of nuclei (pink arrowheads) gradually gathered around the growing oocyte [in (e) the oocyte and its ring canal are depicted with a dashed line]. We interpret these nuclei to represent a third type of follicle cell, surrounding the oocytes of O. longicauda upon maturation and release. Colour coding for the different fluorescence labelling is indicated for each image. Scale bars, 10 μm.

Oogenesis and folliculogenesis in appendicularians compared with solitary ascidians

In the various Oikopleuridae we have examined, the overall organization of the gonads with respect to the disposition of the germline and follicle cells is summarized in Figure 8. In all of these species, the gonad is located in proximity to the digestive tract and formation is initiated in the region of the tail-trunk junction. The germline invariably forms a syncytium; in the testis, this consists of a uniform population of proliferating nuclei, whereas in the ovary, meiotic nuclei in pro-oocytes are linked via ring canals to a common cytoplasm shared by polyploid nurse nuclei also of germline origin (P. Ganot, J.-M. Bouquet and E.M. Thompson, unpublished data). type I and II follicle cells surround the coenocyst in both the ovary and the testis of all species and, in O. dioica, naked oocytes are released at maturity. In O. longicauda, O. fusiformis and O. labradoriensis, a third type of follicle cell is found interspersed within the ovarian coenocyst. This type III follicle cell comes to surround growing oocytes and, upon release, the mature oocytes are encircled by a monolayer of these cells.

Figure 8.

Model for the organization of appendicularian gonads

With the exception of O. dioica, all appendicularians are hermaphrodites. In the ovotestis, an ovary and testis are juxtaposed with different species-specific topological arrangements (Fenaux, 1963). The dioecious spawning structures (moustache and spermiduct) are also present in hermaphrodites. Within the ovary of O. dioica, the germline adopts a novel structure, the ‘coenocyst’, where meiotic and germinal polyploid nuclei are enclosed in the same unique cell. The somatic follicle cells, divided into type I and II classes, are found at the periphery of the coenocyst. O. dioica produces naked oocytes, whereas O. longicauda (and others) spawn oocytes that are covered with a monolayer of follicle cells. This third follicle cell type Is recognizable within the coenocyst at early stages of oogenesis. At later stages of oocyte growth, these follicular cells surround the oocyte. The actin fibres upon which the coenocyst is organized are represented by wavy lines.

The origin of the PGCs (primordial germ cells) that colonize the gonad is unknown in the Appendicularia. In the adult ascidian, Ci. intestinalis, the gonad is also located in close association with the digestive system. In tracing the origin of the gonad, vasa-positive cells were detected in the endodermal strand of the tailbud in embryos and larvae, and the cells migrated from the post-metamorphic debris of the resorbed tail into the gonad rudiment (Takamura et al., 2002). Some evidence suggests that the most posterior B7.6 blastomeres are the precursors of the PGCs. These blastomeres house the CAB (centrosome-attracting body) (Hibino et al., 1998), a novel structure resembling the germ plasm that has been described in other organisms (Iseto and Nishida, 1999) and is also known to be the precursor of the posterior part of the larval endodermal strand (Nishida, 1987). In contrast with ascidians, appendicularians do not resorb the tail upon metamorphosis, but maintain this structure in the adult, suggesting plasticity among the urochordates in strategies for recruiting PGCs into the developing gonad rudiment. This, of course, does not preclude the migration of PGCs from the tail into the gonad rudiment in appendicularians, but it is intriguing that in Ci. intestinalis, removal of the tail prior to metamorphosis delays, but fails to eliminate, the development of normal gonads and germ cells (Takamura et al., 2002). This suggests some compensatory mechanism for germ cell formation in Ci. intestinalis, although it is unknown whether this alternative pathway may in any way resemble mechanisms used in appendicularians.

There seems to be considerable differences in the cellular mechanisms generating oocytes in solitary ascidians, exemplified by Ci. savignyi (Sugino et al., 1987), compared with that delineated above for the Oikopleuridae. Rather than forming a coenocyst, individually cellularized oocytes migrate out of the germinal epithelium and contain a readily identifiable germinal vesicle. They are not associated with polyploid nurse nuclei of germline origin. Folliculogenesis commences with the appearance of the chorion rudiment in the region where the oocyte remains attached to the germinal epithelium, and the maturing oocyte is surrounded by outer follicle cells and primary follicle cells. The primary follicle cells then subsequently differentiate into inner follicle cells that continue to line the outside of the oocyte, as well as test cells located underneath the vitelline coat in pockets on the oocyte surface. Upon ovulation the mature oocyte is released with test cells and inner follicle cells, whereas the outer follicle cells remain behind. Thus the coenocystic nuclear organization during oogenesis in the Oikopleuridae resembles more the meroistic oogenesis of the arthropod Drosophila, rather than that of the sister class ascidians. The external location of the transparent Oikopleura gonad, combined with the wide variability in the number of oocytes it is able to produce from this novel coenocystic organization, make it an accessible and intriguing model organ, with original cellular perspectives, for mechanistic studies of comparative oogenesis in a chordate.

Materials and methods

Animal collection and culture

O. dioica were maintained in laboratory culture at 15°C (Chioda et al., 2004). Under such conditions, animals reach maturity and spawn at an average age of 6.5 days. O. labradoriensis specimens were collected from fjords around Bergen, Norway. O. longicauda and O. fusiformis were obtained from the bay of Villefranche-sur-Mer, France, fixed and kindly supplied by Dr Gabriel Gorsky and Mr Fabien Lombard (Observatoire Oceanologique, Villefranche-sur-Mer, France).

Chemicals and antibodies

Rabbit polyclonal anti-[histone H3 (phospho-Ser10)] (H3S10P, Upstate, 06-570) and rat Ig2a anti-BrdU (bromodeoxyuridine) (Accurate Chemical, BU1/75) antibodies were used at a final dilution of 1:100. Sheep anti-pan-tubulin antibody (Cytoskeleton, anti-αβ-tubulin, ATN02) was used at final dilution of 1:25. Secondary antibodies, anti-rabbit (Chemicon), anti-rat (Jackson Immunoresearch) and anti-sheep (Abcam), all conjugated to Rhodamine Red-X, were at a final dilution of 1:1000. Phalloidin-Alexa Fluor 488 (Molecular Probes) was dissolved as a 200 units/ml stock solution in methanol and incubated with the primary and secondary antibodies at a final concentration of 0.6 units/ml.

Immunofluorescence

For the detection of histone H3 (phospho-Ser10), BrdU or actin (phalloidin staining), animals were fixed at room temperature (RT) in 6% paraformaldehyde/0.1 M Mops (pH 7.4)/0.5 M NaCl/5 mM EGTA/0.2% Triton X-100 and then placed at 4°C overnight. For tubulin detection, coupled with actin staining, animals were fixed at RT in 4% paraformaldehyde/0.25% glutaraldehyde (Sigma, EM grade)/0.1 M Hepes (pH 7)/0.4 M dextrose/10 mM EGTA/10 mM MgSO4/0.2% acrolein in cacodylate buffer/0.2% Triton X-100 for 1 h. After both types of fixation, samples were washed 3 times (5 min per wash at RT) in PBST [PBS (pH 7.4)/0.1% Tween], blocked in PBST/1% BSA (Roche) for 1 h at RT and incubated at 4°C for 3–5 days with primary antibodies. Samples were then washed 3 times in PBST, post fixed in 3% paraformaldehyde/PBS (pH 7.4) for 30 min at RT, washed 3 times in PBST, blocked for 1 h at RT in PBST/1% BSA and incubated for 48 h at 4°C with secondary antibodes diluted in PBST/1% BSA. Samples were washed 3 times in PBST, incubated for 10 min in PBST/Topro-3 (dilution, 1:1000) at RT to counterstain DNA, washed in PBS and mounted in Vectashield medium. For actin staining alone, samples were fixed and washed as above, and then incubated in PBST/1% BSA supplemented with phalloidin—Alexa Fluor 488 (0.6 units/ml) for 3 h at 4°C. Samples were then washed, counterstained and mounted as above.

For whole-mount detection of vasa mRNA, a cDNA probe (a gift from Dr Lisbeth Olsen, Molecular Biology Institute, University of Bergen, Norway), corresponding to a 500 bp region encompassing the DEAD box, was digoxigenin-labelled and in situ hybridization was carried out as described by Spada et al. (2001).

Microinjection of BrUTP

BrUTP was dissolved in 140 mM KCl/2 mM Pipes (pH 7.4) at a final concentration of 20 mM and Phenol Red was added for visual monitoring of the injection. The BrUTP solution was microinjected directly into the gonad of day 4 and day 5 animals anaesthetized with MS222 (Sigma) under a Nikon SMZ46 stereomicroscope using Oxford micromanipulators (5:1 reduction) with an aluminosilicate glass injection needle (WPI) connected to an Eppendorf 5246 injector. Animals were fixed 10–90 min after BrUTP injection. Neo-transcribed RNAs that had incorporated BrUTP (Br-RNA) were detected using anti-BrdU antibody.

Image analysis and processing

Nikon SMZ1550 stereomicroscope and associated software was used to acquire light microscopic images and time-lapse videos. Confocal images were obtained with a Leica TCS-SP confocal microscope and Leica v2.5 software. Images were processed with Zeiss Image Browser, Adobe Photoshop v7.0 and Illustrator v10 software.

Electron microscopy

Animals were fixed in 1.5% glutaraldehyde/0.2 M sodium cacodylate (pH 7.4)/1.6% NaCl, and the fixative was replaced once. After 3 h at RT, samples were washed 3 times (10 min per wash) in 0.2 M sodium cacodylate buffer supplemented with stepwise decreasing NaCl concentrations (1.6%/1.2%/0.5%). Samples were then incubated in 1% OsO4/0.2 M sodium cacodylate for 1 h in the dark at 4°C before being rinsed in 0.2 M, 0.15 M and 0.1 M sodium cacodylate buffer. Samples were dehydrated stepwise in 10%, 30%, 50%, 70%, 90% and 100% ethanol, with the incubation at 100% repeated 3 times. Embedding was carried out in borate tubes: ethanol was replaced with propylene oxide (2 times, 5 min) followed by a 1:1 mixture of propylene oxide/PolyBed {PolySciences; 12 g of PolyBed 812/24.7 g of DDSA (dodecenyl succinic anhydride)/1.0 ml of DMP-30 [2,4,6-tri(dimethylaminomethyl) phenol]} for 3 h, then with a 1:3 mixture of propylene oxide/PolyBed for 10 h and finally in 100% PolyBed overnight. Samples were embedded in fresh PolyBed mix and left to polymerize at 65°C for 2 days. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined with a JEOL JEM-1230 transmission electron microscope.

Acknowledgements

We thank the staff of the Appendicularia culture facility for supplying animals. This work was supported by a grant from the Norwegian Research Council.

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