Ultrastructure of the ovary and oogenesis in the jellyfish Linuche ...

Marine Biology 114, 633-643 (1992)

Marine Biology

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1992

Ultrastructure of the ovary and oogenesis in the jellyfish Linuche unguiculata and Stomolophus meleagris, with a review of ovarian structure in the Scyphozoa K. J. Eckclbarger 1, 2 and R. Larson 3 1 Darling Marine Center, University of Maine, Walpole, Maine 04573, USA z Department of Animal, Veterinary and Aquatic Sciences, University of Maine, Hitchner Hall, Orono, Maine 04469, USA 3 U.S. Fish and Wildlife Service, 801 Gloucester Street, Federal Building, Room 334, Brunswick, Georgia 31520, USA Date of final manuscript acceptance: August 17, 1992. Communicated by J. Grassle, New Brunswick

Abstract. Ovarian structure and oogenesis has been ex-

amined in six scyphozoan species including the semaeostome Diplumularis antarctica Maas, 1908 (collected in 1987 in McMurdo Sound, Antarctic), the rhizostomes Cassiopea xamachana Bigelow, 1892 (collected in Belize in 1988), and Stomolophus meleagris L. Agassiz, 1862 (collected in Ft. Pierce Inlet in 1988), and the coronates Periphylla periphylla (Peron and Lesueur, 1810), Nausithoe atlantica Broch, 1914 (both collected in the Bahamas in 1988), and Linuche unguiculata (Schwartz, 1788) (collected in Nassau Harbor, Bahama Islands in 1989). Based on these findings and information on five other scyphozoan species from additional literature sources, at least two fundamentally different types of ovaries exist in the Scyphozoa. In semaeosotome and rhizostome species, oocytes develop in close association with specialized gastrodermal cells called trophocytes which may serve a nutritive function. However, coronate species lack trophocytes and oocytes develop freely in the mesoglea. The ovaries of S. meleagris and L. unguicuIata are used as models to represent the ultrastructural events occurring during oogenesis in species having trophocytes and those lacking them, respectively. In both L. unguiculata and S. meleagris, the ovaries arise as evaginations of the gastrodermis in the floor of interradial pouches. Germ cells appear to originate from endodermallyderived gastrodermal cells and migrate into the mesoglea prior to vitellogenesis. In L. unguiculata, the oocytes develop freely within the mesoglea throughout vitellogenesis, while in S. meleagris each oocyte maintains contact with specialized gastrodermal cells called trophocytes. In the vitellogenic oocytes of both species, numerous invaginations of the oolemma result in the formation of intraooplasmic channels throughout the ooplasm. These channels are intimately associated with cisternae of rough endoplasmic reticulum and may play some role in yolk precursor uptake by substantially increasing the surface area of the oocyte. Vitellogenesis is similar in both species and involves the autosynthetic activity of the Golgi complex and rough endoplasmic reticulum, and the heterosynthetic incorporation of yolk precursors through

receptor-mediated endocytosis. However, in the oocytes of S. meleagris, the trophocytes probably play a role in the transfer of nutrients from the gastrovascular cavity to the oocyte. The present study suggests that scyphozoans were among the first metazoans to develop ovarian accessory cells during their reproductive evolution. The trophocyte-oocyte association observed in some scyphozoans is similar to but structurally less complex than the trophonema-oocyte association described from anthozoans. Scyphozoan ovarian morphology helps support the view that the Scyphozoa share a closer phylogenetic relationship with the Anthozoa than with the Hydrozoa.

Introduction

Invertebrates offer excellent models for studies of the evolution of the metazoan ovary because they show great morphological diversity (see review of Adiyodi and Adiyodi 1983). Comparative studies of the ovary in "primitive" groups may provide insight into the evolution of this organ in higher organisms because their ovaries are usually structurally simple and often lack accessory cells (e.g. follicle and/or nurse cells) that could provide nutritive support for the developing oocytes. Lower metazoans (e.g. Porifera, Cnidaria) tend to have simple ovaries, and ultrastructural evidence suggests that the developing oocytes from a number of lower metazoan species play a significant role in yolk synthesis through an evolutionarily primitive autosynthetic process (Boyer 1972, Anderson 1974, Billinski 1976, Huebner and Anderson 1976, Gremigni 1979, 1983, Eckelbarger 1983, 1984, Gremigni and Nigro 1983, 1984, Weglarska 1987). This contrasts with the higher metazoa (e.g. Mollusca, Annelida, Arthropoda, Echinodermata) in which the ovaries are generally more complex and accessory cells frequently have a role in a heterosynthetic process of yolk formation (Adiyodi and Adiyodi 1983, Eckelbarger 1983). Unfortunately, our knowledge of invertebrate ovarian morphology is often based on only a single spe-

634 cies within any given taxonomic group. Interspecific studies are often lacking so single species models frequently prevail, resulting in broad generalizations being applied to an entire taxonomic group. In a recent paper (Eckelbarger and Larson 1988), the ultrastructural features of the ovary and vitellogenesis in the cosmopolitan semaeostome scyphozoan Aurelia aurita were described. We reported that the developing oocytes differentiate while in intimate contact with specialized gastrodermal cells we termed "trophocytes" and speculated that the trophocytes play a role in the transfer of nutrients to the oocytes during vitellogenesis in a manner similar to the trophonema cells in the anthozoan ovary (Larkman and Carter 1982). The oocytes appear to utilize a heterosynthetic process of yolk production, an unexpected observation in a primitive diploblastic metazoan. This was the first ultrastructural description of ovarian morphology and vitellogenesis in a scyphozoan and we assumed that the ovarian trophocyte-oocyte relationship in A. aurita was representative of all scyphozoans. However, recent studies of other scyphozoans indicate that our assumption was incorrect and that some species lack ovarian trophocytes. Using light and electron microscopy, we have examined the ovaries of six additional species from three scyphozoan orders, the Semaeostomeae, the Rhizostomeae, and the Coronatae. These include the semaeostome Diplumularis antarctica, the rhizostomes Cassiopeia xamachana and Stomolophus meleagris, and the coronates Periphylla periphylla, Nausithoe atlantica, and Linuche unguiculata. Trophocytes are associated with developing oocytes in the ovaries of each of the semaeostome and rhizostome species. Previously published investigations of oogenesis in five other semaeostomes and rhizostomes, including Cyanea capillata, C. palmstruchii, Chrysaora hysoscella, and Rhizostoma pulrno (Widersten 1965) and Pelagia noctiluca (Avian 1983, Rottini-Sandrini et al. 1983), reviewed recently by Lesh-Laurie and Suchy (1991), report similar observations. However, trophocytes are absent from the ovaries of the coronate species and oocytes develop freely within the mesoglea, an observation previously unreported in the Scyphozoa. In the present paper, we use Linuche unguiculata as a representative model to illustrate the general features of ovarian morphology and oogenesis in coronate scyphozoans. L. unguiculata is a c o m m o n circumtropical-subtropical species whose life history was recently described (Ortiz-Corps et al. 1987). We also present additional ultrastructural observations on selected features of oogenesis in Stomolophus meIeagris, a species having ovarian trophocytes similar to Aurelia aurita. S. meIeagris, popularly known as the "cabbage head jellyfish", occurs in the western Atlantic from Brazil to Cape Hatteras and from Panama to San Diego in the eastern Pacific (Larson 1976). Our results show that ovarian morphology has systematic importance within the Scyphozoa and offers new insight into the phylogenetic relationships between the cnidarian classes, Hydrozoa, Scyphozoa, and Anthozoa. The results also suggest that scyphozoans were probably among the first metazoans to develop true ovarian

K.J. Eckelbarger and R. Larson: Comparative jellyfish oogenesis accessory cells during their reproductive evolution. We will describe the ovaries of the remaining scyphozoan order, the Stauromedusae, in a separate study because ovarian morphology in this group is highly specialized and quite unlike that observed in the other scyphozoan orders. Materials and methods Medusae of Linuche unguiculata (Schwartz, 1788) were collected by dip-net in Nassau Harbor, Bahama Islands, in June 1989 by P. Blades-Eckelbarger. Stomolophus rneleagris L, Agassiz, 1862 was collected by dip-net in Ft. Pierce Inlet on 31 October 1988. Cassiopea xamachana Bigelow, 1892 was hand-collected while skindiving in a shallow, mangrove-lined lagoon in Belize in May 1988. Periphylla periphylla (Peron and Lesueur, 1810) and Nausithoe atlantica Broch, 1914 were collected in the Bahamas in October 1988 using the manned submersible Johnson-Sea-Link (see Youngbluth 1984 for collection details). Diplumularis antarctica Maas, 1908 was collected by R. Harbison using scuba in McMurdo Sound, Antarctica in October 1987. For electron microscopy, pieces of the ovary were excised and fixed by immersion for 1 h at room temperature in 2.5% glutaraldehyde containing 0.3 M Millonig's phosphate buffer and 0.14 M sodium chloride. Tissue was then rinsed for 15 rain in three changes each of buffer wash (0.4 M Millonig's phosphate mixed 1:1 with 0.6 M sodium chloride) and postfixed for 1 h at room temperature in 1% osmium tetroxide buffered in 0.1 M Millonig's phosphate buffer and 0.38 M sodium chloride. Following fixation, tissue was rinsed in distilled water and dehydrated for 2 h in ascending concentrations of ethanol, transferred through two changes of propylene oxide over 10 min, and embedded in Epon. Thin sections were cut on a Porter-Blum MT2-B ultramicrotome with a diamond knife and stained for 10 min each with aqueous saturated uranyl acetate and lead citrate and examined with a Zeiss EM-9-S2 transmission electron microscope. Results The sexes are separate in both Linuche unguiculata and Stomolophus meleagris. In L. unguiculata (Fig. 1), eight bean-shaped, bluish-colored ovaries lie in the gastrovascular cavity peripheral to the stomach. Only a few developing oocytes are observed in any ovary at any given time. In S. meleagris (Fig. 2), the ovaries occur on four pleated membranes located on the floor of the stomach. Numerous oocytes in all stages of development arise in each ovary from a 1-cm wide band just distal to the gastric cirri.

Figs. 1 to 6. Five scyphozoan species. Fig. 1. Linuche unguiculata. Lateral view, live female (4 x life size). Fig. 2. Stomolophus meleagris. Lateral view, live female (1/4 life size). Fig. 3~Linuche unguiculata. Light microscopic cross-section through ovary showing solitary oocytes (OC) in the rnesoglea. GD: gastrodermis (1 500 x). Fig. 4. Stomolophus meleagris. Light microscopic cross-section through edge of ovary showing vitellogenic oocyte (OC) in close association with trophocyte cells (TR) of the gastrodermis (GD). MG: mesoglea; N: nucleus (2 200 x ). Fig. 5, Diplumularis antarctica. Light microscopic cross-section through vitellogenic oocyte (OC) in close association with trophocyte cells (TR) of the gastrodermis (GD). N: nucleus (2 500 x ). Fig. 6. Aurelia aurita. Light microscopic cross-section through vitellogenic oocyte (OC) associated with trophocyte cells (TR) of gastrodermis (GD). MG: mesoglea; N: nucleus (2 200 x )

K.J. Eckelbarger and R. Larson: Comparative jellyfish oogenesis

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Developing oocytes maintain an intimate relationship with gastrodermal trophocytes during vitellogenesis in Stomolophus meleagris (Fig. 4) and Diplumularis antarctica (Fig. 5) in a manner similar to that observed in Aurelia aurita (Fig. 6). The ultrastructural features of the oocytetrophocyte relationship in A. aurita were previously described (Eckelbarger and Larson 1988) and will not be repeated due to similarities among the three species. However, some salient ultrastructural features of oogenesis in S. meleagris will be presented in the present paper where relevant.

to lack rigidity, often folding back against the oocyte surface rather than extending outward as in most invertebrate oocytes (Fig. 9). During early vitellogenesis in the oocytes of both L. unguiculata and Stornolophus rneleagris, the oolemma invaginates to form narrow ooplasmic channels that initially extend from the surface of the cell into the cortical ooplasm (Figs. 10, 25). In cross-section, these channels appear to be intra-ooplasmic membranes resembling smooth endoplasmic reticulum. As cell growth continues, the channels deepen until they penetrate into the perinuclear region (Fig. 24). Initially, the channels are occluded (Fig. 14), but as the oocyte grows they dilate to form complex intracellular spaces (Figs. 11, 24 and 25). Cisternae of RER (rough endoplasmic reticulum) become closely associated with the channels and closely parallel their contours (Figs. 11, 14, 27). Near the end of vitellogenesis, the ooplasmic channels disappear and the ooplasm is dominated by yolk bodies of varying sizes. In Linuche unguiculata oocytes, as the microvilli lengthen, a small perivitelline space appears between the surface of the oocyte and the overlying basal lamina (Figs. 15 to 17). As oocytes grow, this peritelline space progressively widens. Within this space and between the branching microvilli, one observes a network of loose, fibrous material (Fig. 17), similar to the microvillar glycocalyx. In oocytes of Stomolophus meleagris, microvilli

Ovarian morphology and oogenesis - Linuche unguiculata and Stomolophus meleagris The ultrastructural features of the ovaries and vitellogenesis in the three coronate species, Periphylla periphyIla, Nausithoe atlantica, and Linuche unguiculata are virtually indistinguishable so we will use L. unguiculata as representative. Oocytes appear to arise from within the gastrodermis and therefore presumably have an endodermal origin. Three stages of oogenesis were simultaneously observed within the ovary of any given individual including premeiotic or spireme, previtellogenic, and vitellogenic. Mitotically-dividing oogonia were never observed. Small primary oocytes migrate from the gastrodermis into the mesoglea when they reach about 30 to 40 gm in diameter. Previtellogenic oocytes at this stage have a prominent nucleus containing a single spherical nucleolus (Fig. 3). Oocytes remain solitary within the mesoglea throughout development and do not form specialized associations with gastrodermal cells. The ooplasm is generally devoid of organelles except for densely staining aggregations of fibrogranular material (nuage) that form distinctive patches in the perinuclear region of the ooplasm in intimate association with groups of mitochondria (Figs. 7 and 8). This material presumably enters the ooplasm through the adajcent nuclear pores. As the oocyte grows, these aggregations disperse throughout the ooplasm but continue a close spatial relationship with mitochondria. The mitochondria have dense matrices, mitochondrial granules, and tubular cristae (Fig. 8). Each oocyte is surrounded by a closely applied, thin basal lamina that separates it from the surrounding mesoglea (Fig. 9). In Linuche unguiculata, when oocytes initiate yolk synthesis, the oolemma begins to form highly irregular, branching microvilli covered by a thin, filamentous glycocalyx. The microvilli are of variable length and appear

Figs. 7 to 11. Linuche unguiculata. Fig. 7. Perinuclear aggregations of fibrogranular material (nuage, *) in previtellogenic oocyte. N: nucleus (26 600 x ). Fig. 8. Higher magnification of perinuclear nuage (*) in association with mitochondria (M) (30 000 x). Fig. 9. Cortical ooplasm of early vitellogenic oocyte showing flattened microvilli (MV) and the overlying basal lamina (arrowheads). MG: mesoglea (8 800 x ). _Fig.10_.Cortical ooplasm of early vitellogenic oocyte showing infoldings of the oolemma (arrowheads). MG: mesoglea (9 000 x ). Fig. 11. Two Golgi complexes(G) in close association with rough endoplasmic reticuhma(RER). IM: intraooplasmic membranes resulting from infoldings of the oolemma (30 500 x )

Figs. 12 to 20. Linuche unguiculata. Fig. 12. Golgi complex (G) in association with rough endoplasmic reticulum (RER) and nascent yolk body (Y) (25 300 • ). Fig. 13. Two Golgi complexes (G) with Golgi-derived product fusing (arrowheads) to nascent yolk body (Y) (30 500 • ). Fig. 14. Intraooplasmic membranes (IM) bounding occluded intercellular channel in an early vitellogenicoocyte. Note the parallel cisternae of rough endoplasmic reticulum (RER) (33 000• Fig. 15. Cortical region of early vitellogenic oocyte showing irregular microvilli (MV) and overlying basal lamina (arrowheads). MG: mesoglea (14 000 x ). ~ 16. Expanded perivitelline space between oocyte surface and overlying basal lamina (arrowheads). MG: mesoglea; MV: microvilli (9 000x). Fig. 17. Perivitelline space filled with irregular microvilli (MV) and flocculent material (*). MG: mesoglea (9 500 x ). Fig. 18_.Endocytotic pits (arrowheads) forming along the oolemma of vitellogenic oocyte. MV: microvilli;Y: yolk body (24 000 x ). Fig. 19. Flocculent material on surface of oolemma where endocytotic pits are forming (arrowheads). MV: microvilli; Y: yolk body (24 000 x ). Fig. 20. Endocytotic pits and vesicles forming along surface of vitellogenic oocytes (arrowheads). MV: microvilli; Y: yolk body (27 000 x ) Figs. 21 to 29. Figs. 21-23. Linuche unguiculata. Yolk bodies from a mature oocyte. (16 500 x ). Figs. 24-29. Stomolophus meleagris. Fig. 24. Intraooplasmic channels (arrowheads) in central and perinuclear ooplasm of vitellogenic oocyte. N: nucleus (5 000 x ). Fig. 25. Intraooplasmic membranes (IM) bounding intracellular channels in cortical ooplasm. Y: yolk body. (7 600 x ). Insert: Annulate lamellae in ooplasm of early vitellogenic oocyte (14 500 x). Fig. 26. Perivitellinespace filled with fibrillar material (*). Note thin overlying basal lamina (arrowheads). MG: mesoglea (12 000 x ). Fig. 27. Intracellular channel (*) with parallel cisternum of rough endoplasmic reticulum (RER) and adjacent yolk body (Y) (28 500 x ). Fig. 28. Perivitellinespace (*) of viteUogenicoocyte and intracellular channel (arrowheads). MG: mesoglea (12 000x). F~g. 29. Coated pits and vesicles forming (arrowheads) along surface of vitellogenicoocyte. Y: yolk body. Note intracellular channels (*). MG: mesoglea (15 000 x )


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Fig. 30. Diagrammatic synopsis of the ultrastructural features of oogenesis in the oocytes of Linuche unguiculata and Stomolophus meleagris. (1) Previtellogenicstage showing migration of fibrogranular material (nuage) through nuclear pores into perinuclear ooplasm and association with mitochondria. (2) Previtellogenic stage in which nuage has dispersed into central ooplasm, Golgi complexes and rough endoplasmic reticulum (RER) have appeared, and the oolemma is invaginating to form intraooplasmic channels.

(3) Early vitellogenic stage in which yolk is being synthesized by Golgi-RER, and ooplasmic channels and closely associated RER cisternae are observed throughout the cell. The surface of the oocyte is covered with endocytotic pits which migrate into the cortical ooplasm. (4) Vitellogenicoocyte filled with yolk bodies, Golgi complexes, and RER. Although endocytotie activity is still apparent along the surface of the cell, the ooplasmic channels have disappeared. Nu: nuage; N: nucleus

tend to be shorter (Fig. 28), and deep, intermittent perivitelline spaces or extracellular crypts appear which are filled with fibrillar material similar to that composing the surrounding basal lamina (Figs. 26, 28). As differentiation continues, Golgi complexes appear in the ooplasm in close association with a single cisternum of R E R (Figs. 11 and 12). Yolk synthesis begins when oocytes reach approximately 70 gm in diameter. Transition vesicles appear between cisternae of the R E R and the forming face of the Golgi complexes, and nascent yolk bodies are released as secretory vesicles along the maturing face of the Golgi (Figs. 11 to 13). The secretory products from several Golgi complexes appear to fuse to form progressively larger yolk bodies (Fig. 13). Concomitant with the initiation of Golgi activity, coated pits and vesicles appear along the oolemma in the oocytes of both Linuche unguiculata (Figs. 18 to 20) and Stomolophus meIeagris (Fig. 29). Pit formation in L. unguiculata is preceded by the appearance of a dense, fibro-

granular flocculum at localized regions along the oolemma (Fig. 18). As the pits form, this material is internalized as endocytotic vesicles enter the cortical ooplasm. Coated pits and vesicles observed in the oocytes of S. rneleagris are devoid of this flocculent material (Fig. 29). We saw no evidence that coated vesicles fuse directly with Golgi-derived yolk bodies. Mature oocytes contain numerous spherical yolk granules that vary in morphology but generally possess a thin, relatively homogeneouslystaining outer cortex and a highly vesiculated core resembling numerous lipid droplets (Figs. 21 to 23). In middle to late-stage vitellogenic oocytes of S. meleagris, parallel cisternae of annulate lamellae are observed (Fig. 25, insert). Fig. 30 provides a diagrammatic synopsis of the ultrastructural features of oogenesis in the oocytes of Linuche unguiculata and Stomolophus meleagris. Table 1 summarizes our present knowledge regarding the presence or absence of trophocytes in scyphozoan ovaries.

K.J. Eckelbarger and R. Larson: Comparative jellyfish oogenesis Table 1. Scyphozoan ovaries and accessory cells Order Species

Trophocytes present

Source

Order Semaeostomeae Aurelia aurita

Yes

Pelagia noctiluca

Yes

Diplumularis antarctica Cyanea capillata Cyanea palmstruchii Chrysaora hysoscella

Yes Yes Yes Yes

Eckelbarger and Larson 1988, Widersten 1965 Rottini-Sandrini 1983, Avian 1983 Present study Widersten 1965 Widersten 1965 Widersten 1965

Order Rhizostomeae Cassiopea xamachana Stomolophus rneleagris Rhizostoma pulmo

Yes Yes Yes

Present study Present study Widersten1965

Order Coronatae Linuche unguiculata Periphylla periphylla Nausithoe atlantica

No No No

Present study Present study Present study

Discussion

This study establishes the existence of at least two fundamentally different types of ovaries in the Scyphozoa. In the semaeostome and rhizostome species we and other workers have examined, developing oocytes form a close spatial relationship with specialized endodermal cells we have termed "trophocytes" (Eckelbarger and Larson 1988). One exception is the report by Smith (1936) who believed oocytes developed freely in the mesoglea in the rhizostome, Cassiopea.~'ondosa. However, this study was conducted using standard paraffin histology which physically disrupts the delicate oocyte-trophocyte junctions (personal observation) and probably caused developing oocytes to break free from the inner gastrodermaI wall. Also, one of Smith's figures, Fig. 6, appears to show many developing oocytes still attached to the gastrodermis, indicating the likely existence of oocyte-trophocyte associations. Trophocyte-like cells also have been reported for the semaeostome and rhizostome scyphozoans, Cyanea capillata, C. palmstruehii, Aurelia aurita, Chrysaora hysoscella, and Rhizostoma pulmo but were called "nurse cells" by Widersten (1965). However, trophocytes do not communicate with the oocytes by way of intercellular bridges and therefore do not constitute the modern definition of nurse cells (Anderson 1974). Similar cells were collectively termed the "para ovular body" in the ovary of the semaeostome, Pelagia noctiluca (Avian 1983, Rottini-Sandrini et al. 1983). However, in the ovaries of the three coronate species we examined, no trophocytes were observed and oocytes developed freely within the mesoglea. The differences observed in scyphozoan ovarian structure reflect phylogenetic relationships rather than differences in habitat or rate of egg production. The semaeostome and rhizostome species we examined have similar ovaries yet live in diverse habitats ranging from

641 oligotrophic, under-ice (e.g. Diplumularis antarctica) to eutrophic, subtropical (e.g. Aurelia aurita and Stomolophus meleagris) environments. Similarly, the coronate species range from the deep-sea Periphylla periphylla to the shallow water Linuche unguiculata. Although scyphozoans are believed to be iteroparous, the number of eggs produced per day is highly variable depending on species. Neritic semaeostomes (e.g.A. aurita) produce large numbers of eggs per day ( > 100) (Larson unpublished observation). Although egg production in mesopelagic coronate and semaesostome scyphozoans has not been specifically investigated, Larson (1986) speculated that they produced eggs very slowly. This suggestion is substantiated by observations of submersible-collected deep-sea coronates (P. periphylla, AtolIa sp., Nausithoe atlantica) which produced only a few eggs per day (Larson unpublished observation). Some of these medusae were relatively large (> 5 cm diameter) in contrast to L. unguiculata (only 2 cm diameter), which can produce in excess of 100 eggs d - 1 (Kremer et al. 1990). In our earlier paper on oogenesis in Aurelia aurita (Eckelbarger and Larson 1988), we described the structural relationship between developing oocytes and the ovarian trophocytes and concluded that the trophocytes functioned as a conduit for the transfer of yolk precursors from the coelenteron to the oocytes in a manner similar to that of the trophonemal cells of anthozoans (Larkman and Carter 1982). Four types of evidence were presented to support this conclusion: (1) the trophocytes differentiate just prior to vitellogenesis and remain in close association with developing oocytes during vitellogenesis; (2) they have distal surfaces adorned with microvilli and are endocytotically active, indicating the capacity for uptake of materials from the coelenteron; (3) their basal surfaces are intimately associated with the oocyte oolemma where extensive endocytotic activity by the oocyte is observed and yolk bodies first arise; (4) the intercellular junctions between adjacent trophocytes suggest that they may be involved in the maintenance of selective permeability between the oocyte and the coelenteron. Ultrastructural evidence from our comparative studies suggests that the ovarian trophocytes of all four species in the present study are homologous and probably serve a similar function. In the differentiating oocytes of Aurelia aurita (Eckelbarger and Larson 1988), yolk bodies arose from three separate sources, judging from ultrastructural observations: (1) from the uptake of precursors in the region of oocyte-trophocyte contact through receptor-mediated endocytosis; (2) from the endocytotic uptake of precursors along other surfaces of the oocyte that have direct contact with the mesoglea, and; (3) from the combined synthetic efforts of the Golgi complex and RER. Based on morphological grounds, similar mechanisms of yolk synthesis appear to be at work in the oocytes of Stomolophus meleagris, Diplumularia antarctica and Cassiopea xamachana, species we have identified with trophocytes. However, in the three coronate species lacking trophocytes, yolk synthesis appears to originate solely fiom the activity of the Golgi-RER system and the uptake of precursors from the mesoglea through receptor-

642 mediated endocytosis, as exemplified by Linuche unguiculata. In this species, the Golgi complex and R E R are actively involved in the synthesis of yolk bodies in a manner similar to that observed in the oocytes ofA. aurita. In addition, endocytosis is involved in the production of other yolk bodies, perhaps independent of the GolgiRER-derived yolk. In S. meleagris, there was detectable endocytotic activity, but it was at a much lower level than that observed in L. unguiculata oocytes. It is difficult to account for the morphological differences in yolk bodies in the oocytes ofL. unguiculata and S. meleagris, but they might be related to differences in mechanisms of yolk synthesis. The presence of intra-ooplasmic channels in scyphozoan oocytes is a peculiar feature unique to cnidarians. Kessel (1968) was the first to describe the channels in an ultrastructural study of oogenesis in an unidentified trachyline hydromedusa. He termed them "intra-ooplasmic cytomembranes" and speculated that they represented infoldings of the oolemma but declined to speculate on their functional significance. In our study of oogenesis in Aurelia aurita (Eckelbarger and Larson 1988), we reported similar cytomembranes but suggested that they represented a system of smooth endoplasmic reticulum rather than infoldings of the oocyte surface. In view of our present observations of the oocytes of five additional jellyfish species, it is likely that our interpretation was incorrect and that the cytomembrane system in A. aurita arises from surface invaginations as Kessel originally hypothesized (Kesse11968). We believe the interpretational differences between our A. aurita study and the present one are due to variation in the quality of tissue fixation. We support Kessel's original hypothesis and assume that this feature is common to the oocytes of other scyphozoans. The function of these intra-ooplasmic channels is unknown. In his study of hydromedusan oogenesis, Kessel (1968) noted that yolk bodies were transiently connected to the intraooplasmic cytomembrane system during vitellogenesis but he did not suggest a direct involvement with vitellogenesis. Although we observed a close association of RER cisternae with these infoldings, we could see no correlation between them and the developing yolk bodies in the oocytes of Linuche unguiculata and Stomolophus meleagris. Also, we saw no evidence of endocytotic activity associated with the channels. However, the infoldings significantly increase the surface area of the oocyte and might provide a mechanism for the rapid and efficient diffusional incorporation of low molecular weight precursors from the mesoglea during vitellogenesis. In our paper on oogenesis in Aurelia aurita (Eckelbarger and Larson 1988), we suggested that comparisons of ovarian morphology and oogenesis within the Cnidaria could provide additional insight into the phylogenetic relationship between the Hydrozoa, Scyphozoa, and Anthozoa. In the Hydrozoa, oocytes arise ectodermally and oogenesis is significantly different from that observed in other cnidarians. Like sponges (Harrison and de Vos 1991) hydrozoans lack specialized accessory cells and developing oocytes are believed to derive nutrients from somatic or other germ-line cells through several methods, including phagocytosis (Campbell 1974, Beams and

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OC

Fig. 31. Diagrammatic representations of cross-sections through tile ovaries of selected scyphozoans and anthozoans. (A) Oocytes (OC) developing freely within the mesoglea (MG) in the ovary of coronate scyphozoans. GD: gastrodermis. (B) Developing oocyte (OC) attached to specialized gastrodermal cells, the "trophocytes" (stippled), in the ovaries of semaeostome and rhizostome scyphozoans. (C) Developingoocyte(OC) in closeassociationwith specialized gastrodermal cells, the "trophonema" (stippled), in the ovary of some anthozoans

Kessel 1983, Spracklin 1984, Thomas and Edwards 1991). In contrast, oocytes have a gastrodermal origin in both the Anthozoa and Scyphozoa, a feature that supports the belief that that the two groups share a close phylogenetic relationship (Thiel 1966, Barnes 1980). Most anthozoan oocytes develop in close assocation with gastrodermal cells that are collectively termed the "trophonema" (Larkman and Carter 1982, Larkman 1983, Fautin and Mariscal 1991). Experimental evidence has confirmed their nutritional function (Larkman and Carter 1982). The anthozoan trophonema-oocyte association is similar to but structurally more complex than the trophocyte-oocyte relationship we have described in some scyphozoan species. In those anthozoans having trophonemata, the specialized gastrodermal cells of the trophonema protrude into the cortical ooplasm and interdigitate with the oocyte microvilli (Larkman and Carter 1982). This suggests that the Anthozoa and Scyphozoa are closely related but that the germ-accessory cell relationship in scyphozoans is evolutionarily more primitive than that of the anthozoans. Scyphozoans were perhaps the first metazoans to form true oocyte-accessory cell complexes during their reproductive evolution. Fig. 31 depicts the structural differences observed in the ovaries of most anthozoans and semaeostome, rhizostome, and coronate scyphozoans.


Acknowledgements. The authors thank P. Blades-Eckelbarger for assistance with specimen preparation for electron microscopy, ultramicrotomy, and darkroom work, S. Tyler and K. Edwards for use of the Electron Microscopy Center in the Zoology Department of the University of Maine, and the officers and crew of the research vessel R. V. "Seward Johnson" (Harbor Branch Oceanographic Inst.). The manuscript was substantially improved by the suggestions of two anonymous reviewers. Partial support was provided by N. S. E grant OCE-877922 to KJE. This paper is contribution no. 254 of the Darling Marine Center, University of Maine.

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