Evidence for Endocytotic Incorporation of Nutrients from the Haemal Sinus by the Oocytes of the Brittlestar Ophiolepis Paucispina

Echinoderm Biology, Burke e/ al. (eds), © 1988 Balkema, Rotterdam, ISBN 90 6191 7557 Evidence for endocytotic incorporation of nutrients from the haemal sinus by the oocytes of the brittlestar Ophiolepis paucispina

Maria Byrne Zoology Department, University College, Galway, Ireland

ABSTRACT: Ophiolepis pauci spina is a small brittlestar that produces large yolky eggs and has ovoviviparous development. Ultrastructural examination suggests that oogenesis is supported by exogenously derived nutrients. During oogenesis, oocytes evaginate into the genital haemal sinus. As the oocytes grow, they are s urrounded by the haemal s inus, although they remain separated from the haemal fluid by a basal lamina. Endocytotic activity is prevalent throughout oogenesis, indicating uptake of exogenous mate rial. The ER and Golgi complex appear to be involved in elaboration of the yolk bodies . It is proposed that yolk precursors formed outside the oocyte are delivered to the oocytes by the genital haemal sinus and that this sinus functions as an intragonada l nutrient stor age organ.

1 INTRODUCTION for a crinoid (Holland 1971) . Recent bio chemica l investigations with echinoids pro There is a growing body of evidence suppor vides clear evidence for the somatic origin ting the contention that the echinoderm of yolk precursors (Harrington & Ozaki 1986; haemal system plays a role in gametogenesis Shyu et al. 1986), but it is not known if as a soUrce of nutrients (Walker 1982; Fer the haemal system is a source of nutrients quson 1982; Kanatani & Nagahama 1983). As for echinoid oogenesis. s hown in several ultrastructural studies, Ophiolepis paucispina broods its young in the haemal sinus is closely associated with the genital bursae and as for other broo the developing oocytes of asteroids (Walker ding oph1uro1ds (Hendler 1 975 ; Strathmann & 1979 ; Schoenmakers et al.1981; Beijnink et Strathmann 1982) , this species is small (5 al. 1984), holothuroids (Davis 19 71;Smiley 7 mm disc diameter), is a simultaneous her & Cloney 1985) , crinoids (Davis 1971; Ho l maphrodite and has a 1m·, fecundity (2-70 land 1971) and ophiuroids (Davis 1971l. oocytes). Development of O. paucispina is For these echinodenns, the haemal fluid or ovoviviparous and embryogenesis appears to cells contained within the haemal sinus a re be completely supported by the nutrient re a potential source of nutrients. Addition serves present in the large yolky oocyte al evidence for a role of the haemal syst em (400 ~m diameter). There is a paucity of in providing nutrient material for gameto information on oogenesis in ophiuroids, genesis comes from autoradiographic, immu especially at the ultrastructural level nocytochemical and histochemical investiga (Walker 1982). The q uest ion of vitellogen tions of sea stars (Beijnink et al. 1 984; esis is particula rly interesting for dimin Ferguson 1984; Voogt et al. 1985; Beijnink utive speci es where size-associated e nerge & Voogt 1986). Although it appears that tic constraints may limit oocyte production vitellogenesis in many echinoderms is at (Chia 1974; Stea rns 1976). An ultrastruc l east partially dependent on yolk precur tural investigation of oogenesis of O. pau sors present in the genital haemal sinus cispina was undertaken to determine the (Walker 1982; Kanatani & Nagahama 19 83; vitellogenic mechanisms involved with the Voogt et al. 1985) , for the most part it is production of the large oocytes and the not known how t he oocytes internalize these role of the genital haemal sinus. precursors (Ferrand 1984 ; Beijnink et a l. 1984). Endocytosis of nutritive material 2. MATERIALS AND METHO DS is r eported for the oocytes of several echinoids (Bal 1970; Tsukahara 1970) and Specimens of Oph i o lep is paucispina were

557 collected from Conch Rey, Florida, U.S.A. FIGURE LEGENDS and from Southwater and Carrie Bow Cays, on the Belize Barrier Reef. The yolky Fig. 1. Cross section of the ovary of eggs of o. paucispina were difficult to Ophiolepis paucispina stained with AB/PAS. preserve for ultrastructural examination. The previtellogenic oocytes (PVO) stain and several fixation methods were employed, glue and early vitellogenic oocytes (EVO) including the prodedures detailed in stain pink. PAS+ yolk bodies are apparent Walker (1979) and Eckelbarger (1979). Fix in the mi d - vitellogenic oocyte (MVO) and ation however, was poor. Satisfactory late vitellogenic oocytes (LVO) are inten fixation was obtained with the method of sely PAS+. The oocytes have a PAS+ halo Buckland- Nicks et al. (1984). Freshly (arrow) and the haemal sinus (H) is filled collected o. paucispina were placed direct with PAS+ fluid. Paraffin section. Scale ly in 2.5~ glutaraldehyde in 0.45~m Milli : 50 ~m. pore filtered natura1 sea water without prior anaesthesia. The ovaries were dis Fig. 2. Low power cross section of the sected and placed in fresh fixative for 1 ovary showing the oute r and inner sacs hr at room temperature. This was followed separated by the genital coelom (C). The by a rinse in 2.5% sodium bicarbonate (pH outer sac consists of the coelomic per 7.2) and secondary fixation in 2% osmium itoneum (P), connective tissue (CT) and tetroxide in 1.25% sodium bicarbonate for the genital coelomic epithelium (E). The 1 hr at room temperature. Following fix inner sac sonsists of epithelium (E) and ation, the tissue was rinsed in distilled connective tissue containing the genieal water and dehydrated in increasing concen haemal sinus (GHS). The sinus surrounds trations of ethanol, transferred through a vitellogenic oocyte. BL, basal lamina: two changes of propylene oxide and embed G, Golgi complex; L, lipid; M, muscle; N, ded in Epon 812. Thin sections were nerves; Y, yolk. Scale=3 ~m. stained with 2% uranyl acetate followed by 2% lead citrate and viewed with a Zeiss Fig. 3. Golgi complex (G) and ER in the EM9-S2 electron microscope. cortical ooplasm of an early vitellogenic For light microscopy, specimens of O. oocyte. ER-projections (arrows) give rise paucispina were relaxed in a 1:1 mixture of to vesicles (V) which fuse with the forming 7% magnesium chloride and sea water for 10 face of the Golgi complex. Dila tions of min. The discs were fixed and decalcified the <501gi complex (D) give rise to vesicles in BGuin's fixative. the tissues were then (Gv) that fuse to form yolk bodies (y). dehydrated with ethanol, transferred to M, mitochondrion. Scale=0.5 ~m. xylene, embedded in paraffin and sectioned at 7~m thickness. The sections were Fig. 4. The oolemma of late vitellogenic stained with the alcian blue/periodic acid oocytes have numerous microvilli (MV) and Schiff's method (AB/PAS) for carbohydrates. endocytotic invaginations (arrows). BL, basal lamina; E, epithelium; C, genital 3 RESULTS coelom; GHS, genital haemat sinus; L, lipid; M, muscle; N, nerve; Y, yolk body. 3.1 structure of the ovary Scale=2 ~m.

The ovary wall consists of two parts, the Fig. 5. Endocytosis: The process starts a inner and outer sac, and these are separa coated pit (CP) which is invaginated to ted by the genital coelom (Figs. 1,2,4). form an omega figure (arrow) and finally Each sac is composed of three layers of internalized forming a coated ve sicle (CV). tissue. The outer sac consists of the The genital haemal sinus (GHS) contains visceral peritoneum, a central connective granulated haemal fluid (HF) and fibrils tissue laye r and the genital coelomic (F). A, amoebocyte; BL, basal lamina; MV, epitheLium. The inner sac is composed of microvillus. Scale=0.5 ~m. the internal coelomic epithelium, a conne ctive tissue layer that contains the haemal Fig. 6. Detail of the endocytotic invagin sinus and the germinal epithelium. ati on shOWing the fuzzy coat along the The genital haemal sinus is an extra cytoplasmic surface (FC) and the flocculent cellular matrix consisting of granules, material attached to the extracellular small diameter striated collagen fibrils surface (FM) of the coated pit. Scale: and unstriated fibrils (Fig. 5). Amoeb6 0.1 ~m. cytes and phagocytes are occaSionally

558 559 encountered in the haemal sinus. The hae posess a large germinal vesicle and the mal sinus is delimited on either side by a ooplasm contains free ribosomes, mitochon basal lamina that is often tortuous in out dria, lipid droplets and microvilli are ela line, although the inner basal lamina is borated along the oolemma (Figs. 2-5). straight when adjacent to vitellogenic The yolk bodies of early vitellogenic 00 oocytes (Figs. 5,6). The germinal epithe cytes are positioned near a Golgi complex. lium lies adjacent to the inner basal lam It appears that this organelle and the ER ina and usually contains oocytes at differ are involved in the elaboration of the yolk ent stages of oogenesis and oosorption. bodies (Fig. 3). The process starts with small expansions of the ER cisternae that 3.2 Histochemistry of vitellogenesis project towards the Golgi complex. These projections often have an external coat and The tissue layers comprising the inner and their detachment gives rise to coated ves outer sacs of the ovary cannot be discerned icles. A flocculent material is contained with the light microscope. However, part within the vesicles and they appear to fuse of the ovarian wall and a layer adjacent with the forming face of the Golgi complex. to the oocytes corresponding to the loca An electron-dense material is evident with tion of the haemal sinus, exhibits a PAS+ in dilations of the mature Golgi cisternae tinctoral response (Fig. 1). Oogenesis is and these dilations give rise to vesicles continuous with oocytes at different sta containing proteid yolk. The Golgi vesi ges of development present in the ovary cles fuse to form yolk bodies and through (Fig. 1). Because the yolk bodies are out vitellogenesis add to exist.ing yolk PAS+, the process of yolk deposition can bodies by accretion. Newly formed and small be followed with the AB/PAS method. Pre yolk bodies have a granular appearance and vitellogenic oocytes (10-50 ~m diameter) are variable in their electron-density. stain blue with AB/PAS and the onset of the The yolk material condenses and the cyto vitellogenic period is marked by the pre plasm of the mature oocyte is dominated by sence of pink yolk bodies in the ooplasm yolk bodies that are uniformly electron (Fig. 1). Early vitellogenic eggs (50 dense and 2-5 ~m in diameter (Figs. 2,4). 100 ~m diameter) stain pale pink and as Endocytotic activity is conspicuous at yolk accumulates, the colour deepens. Late the surface of vitellogenic oocytes through vitellogenic oocytes are characterised by out vitellogenesis (Figs. 4,5). Coated pits their inte nse and homogeneous PAS+ response, (150-200 nm in diameter) are abundant at staining a deep magenta. At the end of the bases of microvilli with up to 3 coated oogenesis the oocytes are 400 ~m in dia pits per ~m of oocyte surface. The cyto meter. plasmic surface of the pits has a fuzzy ap pearance and flocculent material adheres to 3.3 Ultrastructure of vitellogenesis the extracellular surface (Fig. 5). The pit and adherent material appear to invagi The oocytes of O. paucispina undergo vitel nate to form an omega figure and are then logenesis unaccompanied by somatic cells. internalized forming a coated vesicle (Fig. Each oocyte is surrounded by the haemal 6). Coated vesicles are present near the sinus throughout vitellogenesis, although oolemma. They appear to loose their fuzzy is s e parated from the haemal fluid by the coat soon after internalization and their inner basal lamina (Figs. 2,4,5). This in fate could not be followed. vestment of haernal fluid is formed by eva gination of the oocytes from the germinal 4 DISCUSSION layer into the haemal sinus. It appears that the oocytes bulge into the sinus as The organization of the ovary of o. pauci they enlarge and thereby develop an evagi spina is similar to that described for other. nation in the sinus. Their growth causes ophiuroids (Davis 1971; Patent 1976). the inner basal lamina to unfold and atten Somatic cells do not accompany the oocytes uate, thereby following the contours of of o. paucispina during vitellogenesis; un the oocyte. In contrast, the outer basal like the ophiuroid Gorgonocephalus caryi in lamina is thicker and plicated (Figs. 4,5). which the vitellogenic oocytes are surroun The vite llogenic oocytes contain numer ded by follicle cells (Patent 1976) . ous Golgi complexes in the cortical oopla As found for other echinoderms (Walker sm composed of 7-9 cisternae (Fig. 3). A 1982; Beijnink & Voogt 1986), the haemal single cisterna of ER is usually associated fluid of o. paucispina is PAS+ and vitello with the forming face of the Golgi complex. genesiS involves the accumUlation of PAS+ All the ER in the oocyte appears to be yolk granules in the oocytes. This accumu smooth, perhaps due to loss of attached lation is thought to be due to transfer of ribosomes during fixation. The oocytes nutritive PAS+ material stored in the sinus

560 to the developing oocytes (Walker 1982) . extracellular matrices (Wilkie 1984), it is During vitellogenesis, the oocytes of O. possible that the viscosity of the haemal paucispina develop in an evagination of the fluid may decrease to assist local trans haemal sinus and are surrounded by haemal location. Alternatively, the coelomic fluid fluid throughout vitellogenesis. For O. may be the transport medium, whereby nutri paucispina, it appears that yolk precursors tive material present in the coelomic fluid are largely extraovarian in origin and ap or in coelomocytes is delivered to the gonad pear to be a component of the haemal fluid. through the genital coelom (Ferguson 1982). During oogenesis potential yolk precursors The material is then taken from the coelom present in the haemal sinus need only to by the genital haemal sinus and stored for pass through the inner basal lamina and the use during vitellogenesis. Recent work on prevalence of endocytotic activity suggests echinoid vitellogenin synthesis provides that the oolemma of O. paucispina is well evidence for the somatic origin and coelomic equipped with receptor sites to internalize translocation of yolk precursors. In the precursor material. From ultrastruc Dendraster excentricus, vitellogenin is syn tural and histological evidence, it appears thesised by coelomocytes and secreted into that the oocytes of Ophioderma panamensis the coelomic fluid by these cells (Harring and Gorgonocephalus caryi are also surroun ton & Ozaki 1986) and in Strongylocentrotus ded by haemal fluid (Davis 1971; Patent purpuratus, vitellogenin synthesis occurs 1976), however this is not the case for in the intestine and gonads (Shyu et al. ophiopteris papillosa (Davis 1971). As for 1986) . O. pausispina, crinoid oocytes also develop For O. paucispina it appears that yolk in an evagination of the genital haemal precursors are probably delivered to the sinus (Davis 1971; Holland 1971). ovary by the coelomic system and the genital This study provides the first ultrastruc haemal system serves as an intragonadal nu tural evidence of endocytosis of yolk pre trient storage site. That the haemal sys cursors from the genital haemal sinus by tem functions as an insect fat body absor an eChinoderm oocyte. Similar selective bing nutritive material from the coleomic uptake of yolk precursors from a circula fluid is suggested by the results of sever tory system is reported for the oocytes of al investigations (Ferguson 1984; Jackson & insects and polychaetes (Anderson 1974; Fontaine 1984; Beijnink & voogt 1986). The Eckelbarger 1979,1984). Although the grow genital haemal sinus of O. paucispina ap ing body of evidence suggests that vitello pears to function primarily to support oogen genesis in many echinoderms depends on exo esis, and as emphasised by Walker (1982), genously derived yolk precursors (Walker this sinus should be regarded as a unique 1982; Kanatani & Nagahama 1983; voogt et al. componenet of the haemal system. For O. 1985; Harrington & Ozaki 1986; Shyu et al. paucispina, the relationship between the 1986), the apparent absence of endocytosis oocyte and the genital haemal sinus may be is noted for the oocytes of several aster enhanced to facilitate the produc tion of oids (Beijnink et al. 1984; Ferrand 1984; large yolky oocytes. Aisenshadt & Vassetzky 1986). Based on ev The fate of the coated vesicles formed by idence from autoradiographic and immunocy endocytosis could not be followed, but it is tochemical investigations, vitellogenesis presumed that they contain yolk precursors of the asteroid Asterias rubens is consi that are encorporated into the yolk bodies. dered to be supported by nutrients stored It is not known if the uptake material is in the genital haemal sinus (Voogt et al . delivered to the ER and Golgi complex for 1985). In a recent ultrastruc tural inves processing, or if they add their contents tigation of the oocytes of A. rubens, the directly to the yolk bodies. Although the apparent absence of endocytosis is sugges ER and Golgi complex appear to be involved ted to be due to the low frequency of in formation of the yolk bodies, their con coated pit formation (Beijnink et al. 1984). tribution to the yolk material is not known. Digestive processes are the ultimate A strikingly similar relationship between source of nutrients for oogenesis but, the rough ER and Golgi complex is described for origin of the yolk precursors and the path the oocytes of the ophiuroid Ophioderma pan way of their delivery to the genital haemal amensis (Kessell 1968) and also for holothu sinus of O. paucispina are not known. The roid and asteroid oogenesis (Kessell 1966; vitellogenic material may -be taken from the Aisenshtadt & Vassetzky 1986). This suggests gut and delivered to the genital haemal that the prevalence of smooth ER in the sinus through haemal pathways. The haemal oocytes of O. paucispina may be caused by system however, with its gelatinous haemal the loss of attached ribosomes during fix fluid appears poorly adapted for translo ation, as noted for some polychaete oocytes cation of nutrients. On the other hand, (Eckelbarger pers. comm.). given the unusual properties of echinoderm Endocytosis of proteins is a rapid series

561 of events and coated pits a r e usually Zellforsch. 111: 1-14. short-lived (Dautry-Varsat 1984). For Beijnink, F.B ., C.I·I. Walker & P.A . Voogt fibroblasts, coated pits have a life time 1984. An ultrastructural study of r e l a estimated to be one minute (Bretscher 1987). tionships between the ovarian haemal If the coated pits of O. paucispina oocytes system, follicle cells, and primary are also short-lived, then the prevalence oocytes in the sea star, Asterias rubens. of endocytotic profiles a long the oolen~a Cell Tissue Res. 238: 339-347. suggests that incorporation of material Beijnink, F.B., J.J.S. Broertjes , F. Bra nds from the genital haemal sinus is intense & P.A . Voogt 1984. Immunocytochemi cal throughout vitellogene sis. This makes the demonstration of vitellogenic substances oocyte of O. paucispina a good subject .lith in the haemal system of the sea star, which to study the process of endocytosis. Asterial rubens . Mar. Biol. Lett. 5: In conclusion, the large oocyte of O. 30:i-333. paucispina is produced through endocytotic Beijnink, F.B. & P.A. Voogt, 1986 . The incorporation of yolk p recursors derived aboral haemal system of the sea s t ar, from a somatic source and the genital hae Asterial rubens (Echinodermata, Asteroi mal sinus functions as the proximate source dea): An ultrastructural and histochem and store of these precursors. The results ical study. Zoomorphology 106: 49-60. of this investigation together with the re Bretscher, M.S. 1987. How animal cells sults of other studies (Walker 1982; Bei move. Sci. Amer. 257: 72-90. jnink et al. 1984; Smiley & Cloney 1985; Buckland-Nicks, J., C.W. Walker & F .-S. Beijnink & Voogt 1986; Harrington & Ozaki Chia 1984 . Ultrastructure of the male 1986 ; Shyu et al. 1986} , suggests that ex reproductive system and of spermatoge n traovarian vitellogenesis may be common in esis in the viviparous brittle-star , echinoderms. Amphipholis squamata. J. Morphol. 179: 243-262, ACKNOWLEDGEMENTS Chia, F.-S. 1974. Classification and adap tive significance of developmental pat I thank Dr. K.J. Eckelbarger and Dr. S. terns in marine invertebrates. Thal. Smiley for critically r eading a previous Jugos. 19: 12 1-130. version of the manuscript and for construc Dautry-Varsat, A. & H.F. Lodish 1984 . How tive comments from the proceedings review receptors bring proteins and particles board. Dr. J. Costello and Ms. J. Brodie into cells. Sci. Amer. 250: 52-58. also read the manuscript. This report is Davis, H.S. 1971. The gonad wall of the contribution number 213 of the Smithsonian Echinodermata: a comparative study based Marine Station Link Port, no. 600 of the on electron microscopy. M.Sc. Thesi s Harbor Branch Oceanographic Institution, Univ. California, San. Diego. 90 pp . no. 213 of the "Investigations of Marine Eckelbarger, K.J. 1979. Ultrastructura l ShallOl,-Water Ecosyste ms Reef and Mangrove evidence for both autosynthetic and heter Studies-Belize" supported by an Exxon Cor osynthetic yolk formation in the oocytes poration grant to Dr. K. Ruetzler (Smith of an annelid (Phragmatopoma lapidosa: sonian Institution), and no. 297 of the polychaeta). Tissue & Cell 11: 425-443. Ga lway School of Marine Science. Eckelbarger, K.J. 1984 . Comparative aspects of oogenesis in polychaetes. In Polychaete REFERENCES Reproduction: Progress in Comparative Reproductive Biology (eds . Fischer A & Aisenshtadt, T.B. & S.G. Vassetsky 1986. H-D. Pfannenstiel), pp. 123-148. Gustav Fine structures of oocytes and accessory Fischer, Verlag. ce lls of the ovary in the starfish Ferguson, J.C. 1982. Nutrient transloca Patiria (Asterina) pectinifera at differ tion. In Echinoderm Nutrition. (eds. ent stages of oogenesis and after I-meth Jangoux, M. & J.M. Lawrence), pp. 373 yladenine-induced maturation . Develop. 393. Balkema , Rotterdam. Growth and Differ. 28: 449-460. Ferguson, J.C. 1984 . Translocative Anderson, E. 1974. Comparative aspects of functions of the enigmatic organs of star-' the ultrastructure of the female gamete. fish-the axial organ , hemal vessels, Tie In Aspects of Nuclear Structure and demann's bodies, and rectal caeca: an Function . (eds. G.H. Bourne, J.F. Daniel autoradiographic study. Biol. Bull. 166: l i & K.W. Jean), Int . Rev. Cytol. Suppl. 140-155. 4: 1-70. Academic Press, New York. Ferrand, J.G. 1984. Les mechanismes de La Bal, A.K. 1970. Ultrastructural changes in vitellogenese chez les ast.erides. Sym the accessory-cells and the oocyte sur bioses 16: 245-267. face of the sea urchin Strongylocentrotus Harrington, F.E. & H. Ozaki 1986. The ma droebachiensis during vitellogenesis. Z. jor glycoprotein precursor in echinoids

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