FAU Institutional Repository

http://purl.fcla.edu/fau/fauir

This paper was submitted by the faculty of FAU’s Harbor Branch Oceanographic Institute.

Notice: ©1988 Springer. This manuscript is an author version with the final publication available at http://www.springerlink.com and may be cited as: Eckelbarger, K. J., & Larson, R. L. (1988). Ovarian morphology and oogenesis in Aurelia aurita (Scyphozoa: Semaeostomae): ultrastructural evidence of heterosynthetic yolk formation in a primitive metazoan. Marine Biology, 100, 103‐115. doi:10.1007/BF00392960

Marine Biology 100, 103-115 (1988) Marine ::~~:~:':: Biology © Springer-Verlag 1988

Ovarian morphology and oogenesis in Aurelia aurita (Scyphozoa: Semaeostomae): ultrastructural evidence of heterosynthetic yolk formation in a primitive metazoan

K. J. Eckelbarger 1 and R. L. Larson 2

I Reproductive Biology Program, Harbor Branch Oceanographic Institution, 5600 Old Dixie Highway, Fort Pierce, Florida 34946, USA 2 Gelatinous Zooplankton Program, Harbor Branch Oceanographic Institution, 5600 Old Dixie Highway, Fort Pierce, Florida 34946, USA

Abstract The present study was undertaken because information on ovarian morphology and oogenesis in the lower In Aurelia aurita, the ovaries arise as horseshoe-shaped Metazoa could lead to a better understanding of the evo­ evaginations of the gastrodermis in the floor of four in­ lution of the invertebrate ovary. Also, Boyer (1972), in a terradial gastric pouches. Germ-cell islands arise within en­ study of oogenesis in a polyclad turbellarian, suggested dodermally-derived gastrodermal cells. Oocytes grow and that the oocytes of primitive organisms could be expected gradually bulge into the mesoglea while maintaining to synthesize their own yolk (autosynthesis) since they of­ physical contact throughout vitellogenesis with specialized ten have simple ovaries lacking accessory cells or other po­ cells called trophocytes. Ultrastructural changes suggest tential sources of yolk proteins. Autosynthetic yolk forma­ that these cells transport yolk precursors from the coelen­ tion appears to predominate in the lower Metazoa, teron to the oocytes in a manner similar to that reported whereas the oocytes of many higher animals appear to de­ for the trophonema cells of anthozoan ovaries. Vitellogen­ rive their yolk from extraovarian sources (heterosynthesis) esis involves both the autosynthetic activity of the oocyte (Anderson 1974, Eckelbarger 1983). The oocytes ofAurelia organelles (Golgi complex and rough endoplasmic re­ aurita might be expected to utilize autosynthetic yolk for­ ticulum) and the heterosynthetic incorporation of pre­ mation because the animal is a primitive diploblastic cursors through endocytotic processes involving both coat­ metazoan with a low grade of tissue organization (Hyman ed pits and vesicles and smooth-surfaced tubules. Ul­ 1940). Kessel (1968) and Anderson (1974) provided sup­ trastructural data suggest that different types or classes of port for this assumption by describing autosynthetic yolk yolk precursors enter the oocyte through the trophocytes formation in the oocytes of an unidentified trachyline hy­ and via the surrounding mesoglea. To our knowledge, this drozoan medusa and A. aurita, respectively, although the is the most primitive animal in which this type ofyolk syn­ latter author did not provide ultrastructural details. In this thesis has been described. The trophocyte-oocyte relation­ ship in oocytes of A. aurita is reminiscent of the tro­ phonema-oocyte relationship in anthozoans and supports the belief that the Anthozoa and Scyphozoa share a close phylogenetic relationship.

Introduction

Aurelia aurita is a cosmopolitan scyphozoan common in all seas except the Arctic (Larson 1976). Studies on the life cycles of cnidarians began with A. aurita (Sars 1829) and the gross morphology of the ovary has been described (Sie­ ORAL ARM bold 1839, Claus 1883, Widersten 1965, Russell 1970). Most information on the morphology of A. aurita dates back to the nineteenth century, and ultrastructural studies have been confined to non-reproductive tissues (see Heeger and Fig. 1. Aurelia aurita. Diagrammatic radial section, showing loca­ Moller 1987). tion of ovary 104 K.J. Eckelbarger and R. L. Larson: Ovarian morphology and oogenesis in A. aurita K. J. Eckelbarger and R. L. Larson: Ovarian morphology and oogenesis in A. aurita 105

paper, we describe the ultrastructure of the ovary in canals radiate away from the distal perimeter of the A. aurita and present evidence that its oocytes utilize pouches. A thin membrane covers the subumbrellar sur­ heterosynthetic mechanisms of yolk formation during oo­ face of the pouch. A ring of thick subumbrellar mesoglea genesis. To our knowledge, this is the most primitive ani­ surrounds this membrane. The opening of the ring is mal in which this type ofyolk synthesis has been reported. referred to as the subgenital pit. The gastrodermis lining the floor of each gastric pouch evaginates to form a large, ribbon-like genital fold. Along Materials and methods the inner portion of the fold are numerous digitate pro­ cesses called gastric cirri. The gametes are distributed along Medusae of Aurelia aurita (10 to 15 em diam) were col­ the outer portion (Fig. I). The genital fold, within which lected in Nassau Harbor, Bahama Islands, in November the germ cells arise, is mostly flat with few secondary folds 1987. They arrived in the laboratory a day later and were in small medusae, but becomes more complex and convol­ maintained in filtered sea water at 20° to 23°C. The uted with sexual maturity (Fig. 3). The "ovary", is a meso­ gonads of the medusae were small and pale and there were glea-filled sac of gastrodermal cells into which developing few eggs in the oral arm brood pouches 48 h after col­ oocytes bulge as they develop (Fig. 39). The ovary is mostly lection. We began feeding the medusae newly hatched Ar­ free along its outer, abaxial margin, creating a sub genital temia sp. nauplii several times each day. After 4 d, the sinus between the ovary and the gastrodermis below. The ovaries were large and orange in color and large numbers subgenital sinus communicates with the coelenteron, and of eggs and developing embryos were present in the oral thus both surfaces of the gonad communicate with the gas­ arm brood-pouches. At this time, the gonads were removed trovascular cavity. and fixed for cytological study. For electron microscopy, pieces of the ovary were ex­ cised and fixed for I h at room temperature by emersion in Oogenesis 2.5% glutaraldehyde containing 0.2 M Millonig's phos­ phate buffer and 0.14 M sodium chloride. Tissue was then Oocytes arise from a germinal epithelium of endodermal rinsed for 15 min in three changes each of buffer wash origin consisting of islands of germ cells within surround­ (0.4 M Millonig's PO. mixed I: I with 0.6 M sodium ing gastrodermal cells. Three simultaneous stages of chloride) and postfixed for I h at room temperature in 1% oogenesis were observed in the ovaries of all specimens osmium tetroxide butTered in 0.1 M Millonig's phosphate examined: premiotic or spireme, previtellogenic and buffer and 0.38 M sodium chloride. Following fixation, tis­ vitellogenic. The majority of germ cells in these ovaries sue was rinsed in distilled water and dehydrated over a were early oocytes, but a variety of later stages were ran­ period of 2 h in ascending concentrations of ethanol, trans­ domly scattered throughout the gonad epithelium. No dis­ ferred through two changes of propylene oxide over a peri­ tinct population of mitotically-dividing oogonia was ob­ od of 10 min, and embedded in Epon. Thin-sections were served. The earliest oocytes are spherical cells in the zygo­ cut on a Porter-Blum MTl-B ultramicrotome with a dia­ tene/pachytene stage of meiosis with nuclei containing mond knife and stained for 10 min each with alcoholic synaptonemal complexes (Fig. 6). Most oocytes have nuclei saturated uranyl acetate and lead citrate and examined from 6 to 8,um in diameter containing a single nucleolus with a Zeiss EM 9-S2 transmission electron microscope. (Figs. 4, 7). Their cytoplasm is sparse, containing only free ribosomes, a few spherical mitochondria with tubular cris­ tae, and patches of fibrogranular material (nuage) of pre­ Results sumed nuclear origin (Fig. 8). A pair of centrioles, posi­ tioned at right angles to each other and associated with Ovarian morphology striated rootlets, are also common (Fig. 5). Previtellogenic oocytes, ranging in diameter from 15 to The sexes are separate in Aurelia aurita. The gonads lie in 20,um, begin cytoplasmic and nuclear growth resulting in the floor of interradial gastric pouches and appear clearly the formation of a prominent germinal vesicle. Oocytes at through the body wall as four horseshoe-shaped structures this stage protrude into the mesoglea, but still maintain (Figs. 1~3). The pouches communicate adaxially with the substantial contact with adjacent oogonia (Fig. 9). A thin mouth by a narrow tube, and a number of gastrovascular basal lamina covers the entire surface of the oocytes within

Figs. 2-8. Aurelia aurita. 2: Lateral view, live female; arrowhead indicates ovary (one-half life size). 3: Subumbrellar view of single ovary showing folded nature of germinal epithelium and large numbers of oocytes (arrowed) (7 x). 4: Light-microscope 1 ,urn section through germinal epithelium showing numerous young oocytes (*); YO: vitellogenic oocyte (2 200 x). 5: Two centrioles (C) and associated striated rootlet (R) in cortical ooplasm of young oocyte (27 000 x). 6: Early ooycte with synaptonemal complexes (arrowed); N: nucleus, SGS: subgenital sinus (8700 x ), 7: Small oocyte with prominent nucleolus (Nu) within its germinal vesicle (N), adjacent to larger early vitellogenic oocyte (OC) on right; arrowheads indicate small yolk bodies forming in cortical ooplasm; M: mitochondrion; SG: subumbrellar gastro­ dermis, SGS: subgenital sinus, TR: trophocyte (5550 x); 8: Early oocyte with perinuclear aggregations offibrogranular material (nuage, *); M: mitochondrion; N: nucleus, Nu: nucleolus (10200 x) 106 K.J. Eckclbarger and R. L. Larson: Ovarian morphology and oogenesis in A. aurita K. J. Eckelbarger and R. L. Larson: Ovarian morphology and oogenesis in A. aurita 107

the mesoglea and is continuous with the basal lamina which occur in greater abundance than on adjacent en­ covering the adjacent endoderm cells. Their ooplasm ap­ dodermal cells (Fig. IS). Smooth endocytotic pits and ves­ pears darker in histological sections due to the presence of icles are also commonly observed along this surface high numbers of free ribosomes. Numerous mitochondria (Fig. 19). The cytoplasm contains mitochondria, glycogen are also present along with clusters of perinuclear nuage, particles in the (3-conformation (Figs. 20-21) and large an occasional small Golgi complex, and parallel arrays of numbers of membrane-bounded inclusions up to 1.0,um in smooth membranes resembling smooth endoplasmic re­ diameter (Figs. 16, 20). These inclusions contain a fibrous, ticulum (SER) (Fig. 10). The oolemma is irregular in out­ occasionally striated core, that appears to result from the line and few microvilli are observed (Fig. 9). fusion ofGolgi-derived vesicles (Fig. 20). When oocytes reach 40 to 50 ,urn in diameter, they have The earliest indication of yolk deposition is observed moved almost entirely into the mesoglea, but still retain in oocytes approximately 75 ,urn in diameter. At least three contact with a group of adjacent endodermal cells which types of storage products have been observed in vitello­ we have termed "trophocytes" (Fig. 11). The trophocytes genic oocytes, including membrane-bounded yolk bodies, are distinguished from surrounding germ cells by their flat­ glycogen deposits and lipid droplets. Ultrastructural ob­ tened appearance and dark-staining qualities. As differen­ servations indicate that yolk bodies originate from three tiation continues, the trophocytes gradually become the sources: (I) from the uptake of precursors in the region of only cells in the germinal epithelium in contact with the oocyte-trophocyte contact; (2) from the uptake of pre­ developing oocyte (Fig. 12). The trophocytes are closely cursors along the surface of the oocyte in contact with the apposed to the contours of the oocyte oolemma (Fig. 13), mesoglea, (3) from the combined synthetic efforts of the but no specialized junctions were observed between the Golgi complex and rough endoplasmic reticulum (RER). cells. An irregular intercellular gap of 30 to 70 nm in width Yolk-like bodies consisting of 0.3 to 0.5,um membrane­ persists between them throughout oocyte development bound inclusions containing an electron-dense, granular (Fig. 21). Gradually, the trophocytes become the only cells product, appear first in a narrow organelle-free zone of positioned between the developing oocyte and the sub­ ooplasm adjacent to the trophocytes (Figs. 7, 13, IS, 16,21, genital sinus (Fig. 13). The oolemma becomes elaborated 22, 30). Similar bodies are occasionally observed in other into a series of elongate, interdigitating microvilli that fold cortical regions of the oocytes in vitellogenic oocytes, but over on the surface of the oocyte (Figs. 22, 24, 25, 27). Cis­ are less common (Figs. 35-36). These inclusions appear to ternae of the smooth endoplasmic reticulum increase in originate from the fusion of numerous uniformly-sized number and some form expanded, vesicular-like profiles (O.I,um diam), coated, endocytotic pits and vesicles that (Fig. 14). Simultaneously, the germinal vesicle migrates appear in great numbers along the oolemma (Fig. 21). Very close to the oolemma where the oocyte contacts the dif­ few oocyte microvilli are observed along the oolemma ferentiating trophocytes (Fig. IS). where it contacts the majority of the trophocytes (Fig. 21). At the beginning of vitellogenesis, the trophocytes However, microvilli appear at the periphery of the oocyte­ reach their final stage of differentiation, forming a thin trophocyte zone ofcontact where thin extensions ofthe tro­ plate of cells that covers about 15% of the oocyte surface phocytes cover the surface of the oocyte (Fig. 22). (Fig. IS). The trophocytes are slightly depressed into the Larger. more electron-dense yolk bodies appear simul­ germinal epithelium (Figs. 23, 30) and remain in intimate taneously in other regions of the oocyte (Figs. 26, 27). In contact with the developing oocyte throughout yolk syn­ these early vitellogenic oocytes, endocytotic activity of the thesis. Trophocyte nuclei are positioned laterally at the oocyte is evident where the trophocytes contact the egg edge of the plate (Fig. 16), while the remaining cell bodies surface (Fig. 24) and along the oocyte-mesoglea boundary meet in the center where they form tortuous, interdigitat­ (Fig. 25). Along the mesogleal border, the coated vesicles ing lateral cell boundaries with adjacent cells (Fig. 17). appear to fuse and form nascent yolk bodies (Fig. 26). In These junctions consist of small apicolateral septate junc­ addition, profiles of generally smooth-surfaced tubules of tions followed by gap junctions extending about one-third various sizes arise near the oolemma and appear to fuse the length of the plasmalemma (Figs. 17, 18). The re­ with small growing yolk bodies (Fig. 27). In deeper regions maining two-thirds of adjacent cell membranes lack any of the ooplasm, Golgi complexes and closely associated cis­ specialized junctions (Fig. 17). The apical surface of each ternae of rough endoplasmic reticulum appear to be active cell is covered by branching microvilli (Figs. 16, 17, 19) in the synthesis ofadditional yolk bodies (Figs. 28, 29).

Figs. 9-14. Aurelia aurita. 9: Small oocyte (OC) protruding into mesoglea (MG); oocyte still remains closely associated with smaller oocytes (*) within the germinal epithelium (3000 x). 10: Parallel smooth membranes (arrowed) in perinuclear region of young oocyte; N: nucleus; *: nuage (26600 x). 11: Small oocyte (OC) in mesoglea (MG) in close association with flattened, darkly staining, newly differentiating trophocytes (arrowed); note small oocytes (*) still within germinal epithelium; N: nucleus (4800 x). 12: Light microscopic thick-section of small oocyte (OC) in mesoglea (MG) in close association with darkly-staining group oftrophoeytes (arrowed); N: nucleus; SGS: subgenital sinus (3 700 x). 13: Close-up of newly differentiating trophoeytes closely associated with oocyte (OC); single arrowheads indicate close apposition of thin, darkly-staining trophocyte to adjacent oocyte; double arrowheads indicate small yolk inclusions along oocyte-trophocyte border; *: trophocyte nuclei; N: oocyte nucleus; SGS: subgenital sinus (5700 x). 14: Perinuclear region of ooplasm in small oocyte showing smooth membrane cisternae (arrowed) and expanded, vesicular elements (V); N: nucleus (6 500 x)

K.J. Eckelbargcr and R. L. Larson: Ovarian morphology and oogenesis in A. aurita 109

Middle and late-stage vitellogenic oocytes continue to associated oocytes during vitellogenesis. Our ultrastructur­ form yolk-like inclusions at the oocyte-trophocyte contact al observations demonstrate that the "nurse cells" do not zone (Fig. 30), but the most active yolk-body accumulation maintain cytoplasmic continuity with the developing is observed elsewhere in the cell. Numerous coated pits oocyte by way ofintercellular bridges and therefore do not and vesicles form along the oocyte oolemma (Fig. 31), constitute the classical definition of nurse cells (Anderson which are about twice the diameter of those forming along 1974, Huebner and Anderson 1976). Nurse cells are gener­ the oocyte-trophocyte border (0.2,um vs 0.1 ,urn). Large ally abortive germ cells, but we are uncertain as to the numbers of smooth-surfaced tubules of variable length are origins of these cells in A. aurita. Follicle cells are a second observed along the oolemma which appear to pinch off be­ cell type often associated with developing eggs, but they tween the bases of the microvilli (Fig. 32) and fuse to small generally surround the oocyte and are of somatic origin cortical yolk bodies (Figs. 32-34, 36). Some of these tu­ (Anderson 1974, Huebner and Anderson 1976). Because of bules appear to be dense-cored (Fig. 36). Yolk granules of their uncertain lineage, we have chosen the term "tropho­ increasing diameter appear to arise from the fusion of cyte" for these specialized cells. smaller yolk bodies (Fig. 37). In oocytes over 100,um, The function of the trophocytes in Aurelia aurita cannot abundant stores of glycogen appear throughout the be determined conclusively through ultrastructural studies ooplasm (Figs. 33, 37), while in oocytes nearing maximum alone. They do not contain the cellular organelles usually diameter (175 ,urn), small quantities of lipid droplets ab­ associated with protein synthesis, such as rough en­ ruptly appear (Fig. 35). Large numbers of annulate la­ doplasmic reticulum, and have only a few Golgi com­ mellae are also common in middle- and late-stage vitello­ plexes. They do contain membrane-bounded inclusions genic oocytes from the perinuclear to the cortical regions of which appear to originate from the Golgi complexes, but the oocyte (Fig. 38). their function is unknown. The presence of gap junctions The microvilli of vitellogenic oocytes continue to in­ between these cells suggests that they have an integrated crease in number and reach 3 to 4,um in length (Fig. 31). function, because such junctions are intercellular pathways The microvilli tend to become highly branched, with their of low-molecular weight molecules (Lowenstein 1979) and distal portions either projecting outwards into the meso­ provide metabolic exchange essential for cell coordination glea or lying parallel to the surface of the oocyte. No well­ and communication (Staehelin 1974, Revel 1978). defined egg envelope or extracellular coat is observed. The Several types of evidence suggest that the trophocytes oocyte surface is covered with unevenly spaced, irregular are involved either in the production of or the transport of microvilli covered by a thin filamentous glycocalyx. yolk precursors to the oocytes: (I) The trophocytes differ­ Although the overlying basal lamina (Fig. 31) resembles an entiate just prior to vitellogenesis and remain in close as­ extracellular egg coat, we do not believe it is a product of sociation with developing oocytes throughout vitellogen­ the egg. Fig. 39 diagrammatically summarizes the ul­ esis; (2) they have distal surfaces adorned with microvilli trastructural events associated with vitellogenesis in the and are endocytotically active, indicating the capacity for oocytes ofAurelia aurita. uptake of materials from the coelenteron; (3) their basal Our observations of live individuals in the laboratory surfaces are intimately associated with the oocyte oolemma indicate that the period of vitellogenesis can be less than where extensive endocytotic activity by the oocyte is ob­ 4 d in length when ample food is available. served and yolk bodies first arise; (4) the intercellular junc­ tions between adjacent trophocytes suggest that they may Discussion be involved in the maintenance of selective permeability between the oocyte and the coelenteron; this suggests that General features of oogenesis in four scyphozoans, includ­ the trophocytes function as a conduit for the transfer of ing Aurelia aurita, were described at the light microscope yolk precursors from the coelenteron to the oocytes in a level by Widersten (1965), but no studies of vitellogenesis manner similar to that reported in anthozoans by special­ in this group have been reported. Widersten described the ized endodermal cells collectively termed the trophonema migration of oocytes into the mesoglea and the association (Hertwig and Hertwig 1879). of vitellogenic oocytes with specialized endodermal cells in In many anthozoan ovaries, trophonemal cells establish Cyanea capillata and A. aurita. He termed these cells relatively complex associations with developing oocytes "nurse cells", and suggested that they nourish the closely following oocyte migration into the mesoglea (Schmidt and

Figs. 15-22. Aurelia aurita. 15: Group of trophocyte cells (TR) adjacent to early vitellogcnic oocyte; note large germinal vesicle of oocyte (N) and yolk bodies (arrowed) deposited in ooplasm; Nu: nucleolus; SGS: subgenital sinus (5800 x). 16: Higher magnification of trophocyte, showing laterally-displaced nucleus with small nucleolus (Nu); arrowheads indicate yolk bodies in ooplasm of adjacent oocyte (OC); insert: enlargement of membrane-bound trophocyte inclusion; SGS: subgenital sinus (9100 x ). 17: Apicolateral junctions between adjacent trophocytes; arrowheads indicate gap junction (12 600 x). 18: Enlargement of septate junction (SJ) between adjacent trophocytes (55500 x). 19: Distal border of trophocyte showing microvilli (MY) and smooth-walled endocytotic pits and vesicles (arrowed) (25 300 x). 20: Cytoplasm of trophocyte showing Golgi complexes (G), associated membrane-bound inclusions (*) and clusters of {i-glycogen granules (G L) (23 000 x ). 21: Close apposition of cell membranes between trophocyte (TR) on left and oocyte on right; note yolk granules (Y) and coated vesicles (arrowed) in ooplasm; G L: glycogen (34 300 x). 22: Trophocyte cell extension (TR) over surface of oocyte microvilli (MY); note basal lamina covering surface of trophocyte (arrowed); MG: mesoglea; Y: yolk body (24000 x) 110 K.J. Eckelbarger and R.L. Larson: Ovarian morphology and oogenesis in A. aurita K. J. Eckelbarger and R. L. Larson: Ovarian morphology and oogenesis in A. aurita III

Schafer 1980). The trophonema is believed to have a nutri­ species, large interconnected oocyte-nurse cell complexes tive function (Larkman and Carter 1982, Larkman 1983) form, which eventually result in the maturation ofonly one and perhaps represents the early stages of follicle or nurse or a few definitive oocytes at the expense of the nurse cells cell evolution. Trophonemata maintain intimate contact through absorption or engulfment. In other species, non­ with developing oocytes through cell junctions and the in­ germinal accessory cells are engulfed during oogenesis terdigitation of long oocyte "cytospines" with endodermal (Campbell 1974, Beams and Kessel 1983, Spracklin 1984). cell processes (Larkman 1983). Several features of the tro­ The similarities in oocyte development in the Anthozoa phocytes in the ovaries ofAurelia aurita are reminiscent of and Scyphozoa and the gastrodermal origin of the germ the trophonema-oocyte relationship in anthozoans. In both cells in both classes, support the belief that they share a instances, oocytes retain a close association with special­ close phylogenetic relationship (Thiel 1966, Barnes 1980). ized endoderm cells during vitellogenesis after entering the Kessel (1968) concluded that yolk formation occurred mesoglea. In both groups, the oocyte germinal vesicle mi­ through the autosynthetic activity of the Golgi complex grates closer to this zone of endodermal cell contact. Tro­ and RER in the oocytes ofa trachyline medusa. He did not phocytes and trophonema cells each contain glycogen and observe any accessory cell association with developing lipid droplets and the ooplasm immediately adjacent to oocytes and saw little significant endocytotic activity dur­ them contains characteristic vesicles. However, in the an­ ing vitellogenesis. The present study provides evidence that thozoan Actinia fragacea, Larkman (1983) described the the oocytes of Aurelia aurita derive yolk through both an vesicles as small and empty and did not attribute their autosynthetic process involving the activity of its own or­ origin to endocytosis. The inclusions we report in the ganelles (Golgi complex and RER), and the heterosynthet­ oocytes ofAurelia aurita contain an electron-dense product ic incorporation of high molecular weight proteins (vitel­ resembling yolk and appear to originate from the fusion of logenin?) through receptor-mediated endocytosis. endocytotic vesicles. We have concluded that they are not Coated vesicles are considered to be a distinct mecha­ cortical granules, for they never achieve wide distribution nism for the incorporation of specific large molecular beneath the oolemma. In an autoradiographic study of weight proteins into the oocyte (Anderson 1974, Wild oogenesis in Actinia fragacea, Larkman and Carter (1982) 1980). These specialized structures form by the infolding of reported that the trophonemata more actively incorporated the oolemma and are thought to retain receptor sites and a tritiated leucine than surrounding endodermal cells, sug­ cytoplasmic coat consisting primarily of clathrin for the gesting that they convey nutrients from the coelenteron to sequestration of yolk precursors (Woodward and Roth the oocytes. 1978). Different types of endocytotic activity appear simul­ The trophonema-oocyte relationship in anthozoans is taneously in different regions of the oocyte surface in A u­ more highly evolved than the trophocyte-oocyte relation­ relia aurita during vitellogenesis. This includes differences ship in Aurelia aurita. Larkman and Carters' (1982) de­ in the respective size of endocytotic vesicles, their mor­ piction of the intimate association of the two cell types is phology, and ultimate fate. The yolk bodies that arise in clearly more complex than the simple cell-cell apposition the trophocyte-oocyte zone are morphologically different in A. aurita. In A. aurita, no cell junctions between tro­ from those formed from the uptake of materials from the phocytes and oocytes and, aside from some convolutions of mesoglea, and result from the fusion of small (0.1 ,urn) en­ apposing cell surfaces, no complex interdigitation of oocyte docytotic vesicles. Cortical yolk bodies in other regions of oolemma and trophocyte cell processes occur. This suggests the oocyte are morphologically different and result from that the germ cell-accessory cell relationship in scy­ the combined fusion of larger (0.2 ,urn) endocytotic vesicles phozoans is evolutionarily more primitive than that of an­ and numerous smooth-surfaced tubules. While most thozoans. oocytes possess only one type and size of endocytotic ves­ Oogenesis and ovarian morphology in the Hydrozoa is icle, the oocytes of the spider Lebistes reticulatus (Droller very different from that observed in either the Anthozoa or and Roth 1966), the cockroach Periplaneta americana (An­ Scyphozoa. Germ cells arise ectodermally in hydrozoans derson 1964), four species of sea urchins (Tsukahara 1970), (Campbell 1974) and the oocytes often maintain complex and a variety of annelids (Eckelbarger 1984) possess two associations with somatic cells or other germ-line cells types which differ in size, fate of the ingested material, and quite unlike that seen in other cnidarian classes. In many time of appearance. .. Figs. 23-30. Aurelia aurita. 23: Early vitello genic oocyte containing small yolk bodies (Y) and in close association with trophocytes (arrowed); MG: mesoglea; N: germinal vesicle; SGS: subgenital sinus (2 200 x ). 24: Coated pits (arrowed) forming along oolemma of oocyte (OC); note overlying trophocyte (TR) containing large mitochondrion (M); MV; oocyte microvilli (32 000 x). 25: Coated vesicles (arrowed) adjacent to microvilli (MV) in early vitello genic oocyte (38 000 x ). 26: Small yolk bodies (Y) and associated vesicles of possible endocytotic origin (arrowed); note similar vesicle within bottom yolk body (arrowed) (26800 x). 27: Yolk bodies (Y) in cortical ooplasm of early vitellogenic oocyte; note elongated tubules (arrowed); MV: microvilli (30 000 x). 28: Several Golgi complexes (G) surrounded by variety of membrane-bound inclusions formed by fusion of Golgi-derived vesicles (*) (25 300 x). 29: Golgi complex (G) and associated nascent yolk body (Y) formed by fusion of Golgi-derived vesicles (31700 x). 30: Light microscopic thick-section of late vitellogenic oocyte with large germinal vesicle (N) and single nucleolus (Nu); note dark-staining group of trophocytes (TR) and adjacent zone of small yolk bodies in adjacent ooplasm (between arrowheads); SGS: subgenital sinus (2800 x) 112 K.J. Eckelbarger and R.L. Larson: Ovarian morphology and oogenesis in A. aurita K. J. Eckelbarger and R. L. Larson: Ovarian morphology and oogenesis in A. aurita 113

COELENTERON

GASTRODERMIS

4

SUBUMBRELLAR GASTRODERMIS

SUBUMBRELLAR EPIDERMIS Fig. 39. Aurelia aurita. Diagrammatic reconstruction of transverse section through ovary, based on light- and electron-micrograph observa­ tions. Various stages of oogenesis are indicated by numbers: (1) Oocyte begins moving into mesoglea from germinal epithelium; (2) endodermal cells in germinal epithelium begin differentiating into trophocytes, and oocyte begins forming parallel arrays of membranes resembling smooth endoplasmic reticulum; (3) oocyte completes movement into mesoglea but retains association with trophocytes, the germinal vesicle migrates closer to the trophocytes and yolk synthesis begins; (4) oocyte involved in synthesis of yolk through activity of Golgi complex and RER and uptake of precursors through endocytosis; (5) late-stage vitellogenic oocyte (175 .um diam) containing large quantity of yolk. Note formation of yolk bodies in relatively organelle-free zone of ooplasm adjacent to trophocytes in Stages 3-5

Figs. 31-38. Aurelia aurita. 31: Coated pits and vesicles (arrowed) forming along oolemma of vitellogenic oocyte; note long microvilli (MV) and overlying basal lamina (BL) (24 000 x). 32: Profiles of cortical tubules possibly in the process of forming (between two arrowheads) and fusing (single arrowheads) with small cortical yolk bodies (Y); MV: microvilli (22 600 x ). 33: Yolk bodies (Y) and associated tubular profiles (single arrowheads) and small nascent yolk droplets (double arrowheads) in apparent process offusing with large yolk bodies (Y); note coated vesicle in upper left of figure (double arrowheads); GL: glycogen (30500 x ), 34: Tubules (arrowed) adjacent to and in apparent process of fusing with small yolk bodies (Y) in cortical ooplasm (30 000 x ). 35: Cortical ooplasm containing numerous tubules (arrowed), small nascent yolk bodies (Y) and lipid droplets (L); MV: microvilli; note similarity of yolk bodies to those in Figs. 13 and 22 (20000 x). 36: Dense-cored tubules and smaller yolk droplets (arrowed) apparently in process offusion with large yolk body (Y)(25700 x). 37: Small nascent yolk bodies (*) in various stages of maturity; note dense-cored tubules (arrowed) and glycogen deposits (GL) (28500 x). 38: Array of annulate lamellae in ooplasm of late vitellogenic oocyte (24 500 x ) 114 K. J. Eckelbarger and R. L. Larson: Ovarian morphology and oogenesis in A. aurita

Profiles of tu bular bodies similar to those in the oocytes and the Turbellaria (see Gremigni and Nigro 1984), but it of Aurelia aurita are less common in the oocytes of other also occurs in many higher Metazoa in combination with animals, but still are viewed as a mechanism for the intra­ heterosynthetic processes (see Anderson 1974, Eckelbarger cellular transport of extracellular products (Dumont 1978, 1983). Some workers suggest that the presence or absence Selman and Wallace 1982). The origin of these tubules is of auto- or heterosynthetic yolk in the oocytes of various often undetermined, but they have been attributed to the turbellarians could indicate taxonomic position and/or smooth endoplasmic reticulum (Cummings and King evolutionary history (Gremigni 1979, 1983, Gremigni and 1970), the fusion of endocytotic vesicles (Kessel and Nigro 1983, 1984). In contrast, Anderson (1974) concluded Ganion 1979), or to the Golgi complex (Selman and Wal­ that it was impossible to construct an evolutionary classifi­ lace 1982). In the oocytes of A. aurita, we believe the tu­ cation of vitellogenic mechanisms. This conclusion is sup­ bules originate as infoldings of the oolemma in a process ported by comparative studies ofoogenesis in polychaetous akin to endocytosis, because they occur only in the cortical annelids (Eckelbarger 1983, and in press). In the Poly­ ooplasm and not in association with Golgi complexes, and chaeta, vitellogenic mechanisms are better correlated with fuse with developing yolk bodies. They are not viewed as breeding pattern than taxonomic position, with species sectional artifacts created by surface infoldings, because having the shortest vitello genic periods utilizing hetero­ they often occur rather deep in the cortical ooplasm and synthetic mechanisms and those with the longest periods are invariably observed in the apparent process of fusing utilizing autosynthetic mechanisms (Eckelbarger 1983, with yolk bodies. Regardless of their origins, they appear to 1984). The high levels of endocytotic activity in the oocytes participate in the formation ofyolk. Differences in size and of Aurelia aurita may be an adaptation to accelerate the type of oocyte endocytotic vesicles has been attributed to uptake ofyolk precursors and therefore the rate ofegg pro­ differences in the composition of material being ingested duction. (Anderson 1974). We suggest that different types or classes The oocytes of Aurelia aurita never develop an ex­ ofyolk precursors are being imported into the oocytes from tracellular coat or complex egg envelope found on most in­ the trophocytes and mesoglea. Trophocytes presumably vertebrate eggs. The egg envelope consists of simple, import precursors directly from the coelenteron. The origin branching microvilli that fold over on the egg surface and of precursors in the mesoglea may be the gastrodermal resemble, in general, those of anthozoan oocytes. An­ cells. The population of amoebocytes within the mesoglea thozoans also lack an extracellular coat, but the oocytes are probably are not trophic cells since they are small, do not unique in developing long, often stiff microvilli found in a contain significant storage products (lipid and glycogen) large number of species (Schmidt and Schafer 1980). The and appear to be primarily phagocytic. Widersten (1965) microvilli often cluster together to form spiral aggregates, hypothesized that the mesoglea might be involved in the which Schroeder (1982) suggested provides protection for nutrition ofoocytes in scyphozoans. the egg. Oocyte microvilli ofA. aurita lack significant num­ The presence of an extensive system of parallel smooth bers of microfilaments and presumably are absorptive. membranes in the oocytes of Aurelia aurita is very similar Schmidt and Schafer (1980) referred to the presence of to that reported by Kessel (1968) in an unidentified trachy­ "vitelline membranes" surrounding anthozoan oocytes, but line medusa. Kessel commented that this system represents Larkman (1983) suggested that the structure described a unique ooplasmic component in animal oocytes. The probably represents a basal lamina that covers nearly all function of these membranes in the trachyline medusa and cell surfaces, particularly those exposed to mesoglea. In the the oocytes of A. aurita is unknown. Kessel postulated that oocytes of A. aurita, we observed a similar basal lamina the membranes were derived from the invagination of the closely associated with the oocyte surface which strongly oolemma, but we found no support for this theory. The resembles the extracellular coat common to many inverte­ membrane system in A. aurita resembles SER and appears brate oocytes. Kessel (1968) illustrated a thick filamentous early in small oocytes, often in the perinuclear or central membrane surrounding the oocytes of a hydrozoan rather than the cortical region of the cell, suggesting a dif­ medusa, but was uncertain as to its origin. ferent origin. The association of these membranes with Numerous stacks of annulate lamellae occur in the RER suggests some synthetic function in the oocytes of oocytes of Aurelia aurita in the middle to late stages of both animals. vitellogenesis. To our knowledge, this organelle has never Boyer (1972) suggested that the autosynthetic method been reported for anthozoan oocytes. Kessel (1968) report­ of vitellogenesis probably characterized primitive animals, ed small aggregations of annulate lamellae in late-stage because they generally do not have well developed ovaries oocytes in a hydrozoan medusa which were primarily pe­ and accessory cells. Many workers agree that autosynthesis ripheral in distribution. The organelle occurs in the oocytes probably represents the original method of yolk formation of many invertebrates and has been postulated to function in the Metazoa (Anderson 1974, Bilinski 1976, Huebner in the release, packaging, or assembly of stored de­ and Anderson 1976, Gremigni 1979, 1983, Eckelbarger velopmental information and the processing and assembly 1983, 1984, Gremigni and Nigro 1983, 1984, Weglarska of functioning polyribosomes (Kessel 1985), 1987). Autosynthetic yolk formation occurs in many lower Acknowledgements. The authors thank P. A. Linley for technical Metazoa including the Porifera (Diaz et al. 1975, Gallissian assistance. This paper is Contribution No. 669 of Harbor Branch and Vacelet 1976), Cnidaria (Kessel 1968, Anderson 1974), Oceanographic Institution. K.J. Eckelbargcr and R. L. Larson: Ovarian morphology and oogenesis in A. aurita 115

Literature cited Kessel. R. G. (1968). Electron microscope studies on developing oocytes of a coelenterate medusa with special reference to Anderson, E. (1964). Oocyte differentiation and vitellogenesis in vitellogenesis. J. Morph. 126: 211-248 the roach Periplaneta americana. J. Cell BioI. 20: 131-152 Kessel, R. G. (1985). Annulate lamellae (porous cytomembranes): Anderson, E. (1974). Comparative aspects of the ultrastructure of with particular emphasis on their possible role in differen­ the female gamete. In: Bourne, G. H., Danielli, J. F., Jean K. tiation of the female gamete. Devl BioI. I: 179-233 W. (eds.) Aspects of nuclear structure and function. Academic Kessel, R. G., Ganion, L. R. (1979). Localization of horseradish Press, New York, p. 1-70. (Int. Rev. Cytol. Suppl. No.4) peroxidase in the panoistic dragonfly ovary. 1. submicrosc. Barnes, R. D. (1980). Invertebrate zoology. W. B. Saunders Co., Cytolll: 313-324 New York Lark man, A. U. (1983). An ultrastructural study of oocyte growth Bilinski, S. (1976). Ultrastructural studies on the vitellogenesis of within the endoderm and entry into the mesoglea in Actinia Tetrodontophora bielanensis (Waga) (Collembolla). Cell Tissue fragacea (Cnidaria, Anthozoa). J. Morph. 178: 155-177 .. Res. 168: 399-410 Larkrnan, A. U., Carter, M. A. (1982). Preliminary ultrastructural Boyer, C. B. (1972). Ultrastructural studies ofdifferentiation in the and autoradiographic evidence that the trophonema of the sea oocyte of the polyclad turbellarian, Prosteceraeus j7oridanus. J. anemone Actinia fragacea has a nutritive function. Int. 1. In­ Morph. 136: 273-296 vertebrate Reprod. Dev. (Amsterdam) 4: 375-379 Beams, H. w., Kessel, R. G. (1983). Cnidaria. In: Adiyodi, K. G., Larson, R. J. (1976). Marine flora and fauna of the Northeastern Adiyodi, R. G. (eds.) Reproductive biology of invertebrates, United States. Cnidaria: Scyphozoa. NOAA natn. mar. Fish. Vol. I. Oogenesis, oviposition, and oosorption. John Wiley & Servo tech. Rep. U. S. Dep. Commerce 397: 1-18 Sons, New York, p. 31-66 Loewenstein, W. R. (1979). Junctional intercellular communi­ Campbell, R. D. (1974). Cnidaria. In: Giese, A. C., Pearse, J. S. cation and the control of growth. Biochim. biophys. Acta 560: (eds.) Reproduction of marine invertebrates. Academic Press, 1-65 (1979). New York, p. 133-199 Revel. J. P. (1978). Morphological and chemical organization of Claus, C. (1883). Untersuchungen tiber die Organisation und Ent­ gap junctions. Proceedings of the Ninth International Congress wicklung der Medusen. Haase, Prag, Leipzig on Electron Microscopy, Toronto, Vol. 3, p. 651-658. (Cited af­ Cummings, M. R., King, R. C. (1970). The cytology of the vitello­ ter Huebner and Injeyan 1981) genic stages of oogenesis in Drosophila melanogaster. II. Ul­ Russell, S. F. (1970). The medusae of the British Isles. II: Pelagic trastructural investigations on the origin of protein yolk Scyphozoa. Cambridge University Press, Cambridge spheres. J. Morph. 130: 467-478 Sars, M. (1829). Bidrag til Sodyrenes Naturhistorie af M. Sars, Diaz, J.-P., Connes, R., Paris, J. (1975). Etude ultrastructurale de cando theol. Forste Halfte med sex illuminerede Steen­ l'ovogcnese d'une demosponge: Suberites massa Nardo. BioI. tryktafler. 8: 17-26, Bergen. (Cited after 1977) cell. 24: 105-116 Schmidt, H., Schafer, W. G. (1980). The anthozoan egg: trophic Droller, M. J., Roth, T. F. (1966). An electron microscope study of mechanisms and oocyte surfaces. In: Tardent, P., Tardent, R. yolk formation during oogenesis in Lebistes reticulatus Guppy. (eds.) Developmental and cellular biology of coelenterates. 1. Cell BioI. 28: 209-225 Elsevier/North Holland Biomedical Press, Amsterdam, Dumont, J. N. (1978). Oogenesis in Xenopus laevis (Daudin). VI. p.41-46 The route of injected tracer transport in the follicle and de­ Schroeder, T. E. (1982). Novel surface specialization on a sea veloping oocyte. J. expo Zool. 204: 193-217 anemone egg: "spires" of actin-filled microvilli. J. Morph. 174: Eckelbarger, K. J. (1983). Evolutionary radiation in polychaete 207-216 ovaries and vitello genic mechanisms: their possible role in life Selman, K., Wallace, R. A. (1982). Oocyte growth in the sheeps­ history patterns. Can. J. Zoo I. 61: 487-504 head minnow: uptake of exogenous proteins by vitellogenic Eckelbarger, K. J. (1984). Comparative aspects of oogenesis in oocytes. Tissue Cell 14: 555-571 polychaetes. Fortschr. Zoo I. 29: 123-148 Siebold, Th. V. (1839). Beitrage zur Naturgeschichte der wir­ Eckelbarger, K. J. (In press). Oogenesis and female gametes. bellosen Tiere. I. Uber Medusa aurita. Neue Schr. naturf. Ges. Mikrofauna mar. Danzig 94: 1-23 Gallissian, M. F., Vacelet, J. (1976). Ultrastructure de quelques Spracklin, B. W. (1984). Oogenesis in Tubularia larynx and Tubu­ stades de l'ovogenese de Spongiaires du genre Verongia (Die­ laria indivisa (Hydrozoa, Athecata). Ph. D. dissertation, Uni­ tyoceratida). AnnIs Sci. nat. (ser. Zool.) 18: 381-404 versity ofNew Hampshire, Durham, New Hampshire, USA Gremigni, V. (1979). An ultrastructural approach to planarian Staehelin, L. A. (1974). Structure and function of intercellular taxonomy. Syst. Zool. 28: 345-355 junctions. Int. Rev. Cytol. 39: 191-283 Gremigni, V. (1983). Platyhelminthes-Turbellaria. In: Adiyodi, K. Thiel, H. (1966). The evolution ofscyphozoa a review. In: Rees, W. G., Adiyodi, R. G. (eds.) Reproductive biology of inverte­ J. (ed.) The Cnidaria and their evolution. Academic Press, New brates. John Wiley & Sons Ltd., New York, p. 67-107 York, p. 77-117 Grernigni. V., Nigro, M. (1983). An ultrastructural study ofoogen­ Thiel, M. E. (1977). Bibliographie: Bd 2. Spongiara, Coelenterata, esis in a marine triclad. Tissue Cell 15: 405-415 Echinodermata. 11. Abt.: Coelenterata. 2. Buch, Scy­ Gremigni, V., Nigro, M. (1984). Ultrastructural study of oogenesis phomedusae. 8. Lieferung. Bronn's Kl. Ordn. Tierreichs p. 80 in Monocelis lineata (Turbellaria, Proseriata). Int. J. Inverte­ Tsukahara, J. (1970). Formation and behaviour of pinosomes in brate Reprod. Dev. (Amsterdam) 7: 105-118 the sea urchin oocyte during oogenesis. Dev. Growth Differen­ Heeger, Too Moller, H. (1987). Ultrastructural observations on prey tiation 12: 53-64 capture and digestion in the scyphomedusa Aurelia aurita. Weglarska, B. (1987). Yolk formation in Isohypsibius (Eu­ Mar. BioI. 96: 391-400 tardigrada). Zoomorphologie 107: 287-292 Hertwig, 0., Hertwig, R. (1879). Die Aktinien anatomisch und his­ Widersten, B. (1965). Genital organs and fertilization in some Scy­ tologisch mit besonderer Berticksichtigung des Ner­ phozoa. Zool. Bidrag. Upps. 37: 45-57 venmuskelsystems untersucht. Jena. Z. Naturw. 13: 457-640 Wild, A. E. (1980). Coated vesicles: a morphologically distinct sub­ Huebner, E., Anderson, E. (1976). Comparative spiralian oogen­ class of endocytotic vesicles. In: Ockelford, C. D., Whyte, A. (eds.) esis - structural aspects: an overview. Am Zool. 16: 315-343 Coated vesicles, Cambridge University Press, Cambridge, p. 1-24 Huebner, E., Injeyan, H. (1981). Follicular modulation during Woodward, M. P., Roth, T. F. (1978). Coated vesicles: characteri­ oocyte development in an insect: formation and modification zation, selective dissociation, and reassembly. Proc. natn. Acad. of septate and gap junctions. Devl BioI. 83: 101-113 Sci. U.SA 75: 4394-4398 Hyman, L. H. (1940). The invertebrates: Protozoa through Cteno­ Date offinal manuscript acceptance: July 7, 1988. phora. McGraw-Hill Book Co., New York Communicated by J. M. Lawrence, Tampa