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Notice: ©1980 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. (1980). An ultrastructural study of oogenesis in Streblospio benedicti (), with remarks on diversity of vitellogenic mechanisms in Polychaeta. Zoomorphologie, 94(3), 241‐263. doi:10.1007/BF00998204

euLO ~ \ Zoomorphologie 94,241 -263 (1980) Zoomorphologie © by Springer-Verlag 1980

An Ultrastructural Study of Oogenesis in Streblospio benedicti (Spionidae) , with Remarks on Diversity of Vitellogenic Mechanisms in Polychaeta

Kevin J. Eckelbarger* Harbor Branch Foundation, Inc.,RR I, Boxl96,Fort Pierce, Fla. 33450, USA

. Summary. The ultrastructural features of oogenesis were examined in the spionid Streblospio benedicti. Paired ovaries are attached to the genital blood vessels extending into the coe lomic space from the circumintes­ tinal sinus. The genital blood vessel wall is composed of flattened, peritoneal cells, large follicle cells and developing oocytes. Vitellogenesis occurs while the oocytes are attached to the blood vessel wall. Two morphologically distinguishable types of yolk are synthesized. Type I is synthesized first by an autosynthetic process apparently involving pinocytosis and the conjoined efforts of the Golgi complex and rough endoplasmic reticulum. Type II yolk appears later through a heterosynthetic process involving the infolding of the oolemma and the sequestering of materials from the blood vessel lumen by endocytosis. During this process, blood pigment molecules appear to be incorporated into endocytotic pits, vesicles and eventually the forming yolk body. The significance of heterosynthetic yolk formation to the general reproductive strategies of polychaetous is discussed.

A. Introduction

Streblospio benedicti Webster, 1879, is a larviparous, deposit-feeding, estuarine polychaete, widely distributed on the east and west coasts of North America and parts of Europe and South America (Foster, 1971). It has been described as an opportunistic species (Grassle and Grassle, 1974) and an indicator of organic pollution in the marine environment (Felice, 1959; Wass, 1967; Grassle and Grassle, 1974; Young and Young, 1978). Benthic populations of S. benedicti can reach enormous densities particularly in response to a variety of environmen­ tal perturbations. Experimental field studies have shown that densities exceeding 400,000 per m 2 can be attained within azoic sediment boxes in less than 10 days / (McCall, 1977).

• The author is grateful for the very capable technical assistance of Ms. P.A. Linley and the many stimulating discussions with Dr. Stan Rice. Contribution No. 156, Harbor Branch Foun­ dation, Inc.

0340-6725/80/0094/0241/$04.60 242 KJ. Eckelbarger

Streblospio benedicti is a semi-continuous spawner as defined by Olive and Clark (1978). In northern latitudes, S. benedicti reproduces primarily during the summer and fall months (Dean, 1965; Simon and Brander, 1967; Grassle and Grassle, 1974). In southern Florida waters, however, reproductive individ­ uals are present most of the year. Sexual maturity is reportedly reached in about a month following settlement (Grassle and Grassle, 1974). Developing eggs are incubated in dorsal brood pouches prior to the release of larvae into the plankton (Campbell, 1957; Dean, 1965; Collier and Jones, 1967). Oogenesis occurs rapidly with vitellogenesis taking place entirely within the ovary. Two types of yolk are found within the mature egg, one of which appears to be formed heterosynthetically, a feature recently reported for the first time in another polychaete (Eckel barger, 1979). Although a general description of larval development in Streblospio benedicti is available (Campbell, 1957; Dean, 1965), little is known of its reproductive biology and nothing concerning oogenesis. Due to the unusual mode of yolk synthesis and the scarcity of information regarding gametogenesis in this ecolog­ ically important polychaete, an ultrastructural investigation of oogenesis was undertaken.

B. Materials and Methods

Female specimens of Streblospio benedicti in various stages of sexual maturity were collected from the shallow water s of the Indi an River estuary in S1. Lucie County, Florid a. Genital segments were cut into small pieces and prepared for electron microscopy following the procedures outlined in Eckelbarger (1979). Thin sections of embedded tissue were cut on a Porter-Blum MT-2 ultramicro­ tome with a diamond knife, stained with aqueous, saturated uran yl acetate followed by lead citrate and examined with a Zeiss EM9-S2 electron microscope .

C. Observations

The ovaries of Streblospio benedicti are paired organs situated on the convoluted genital blood vessels which extend laterally into the coelomic space from the circumintestinal sinus in the middle and posterior body region . Oogenesis is unsynchronized within the ovary and therefore nearly all stages of development can be observed in histological sections. Mitotically-dividing gametogonia were never observed although they are assumed to exist. Spireme oocytes in the zygotene or pachytene phases of meiotic prophase are encountered commonly as are a variety of post-pachytene oocytes in various previtellogenic and vitellogenic stages.

J. Follicle Cells

The genital blood vessel wall (Figs. 1 and 2) is composed partially of large , interdigitating cuboidal or squamous follicle cells, derived from the peritoneum, which lie on a discontinuous layer of thin, sinuous peritoneal cells. The latter Oogenesis in Str eblospio benedict; 243

1

Fig. 1. Diagrammatic representation of transverse section through genital blood vessel of Streblospio benedicti illustrating morphological relationship between oolemma and perivasal ooplasm and ad­ jacent peritoneal cells and basal lamina in previtellogenic (I), early vitellogenic (2) and late vitelloge­ nic (3) oocytes

cells contain scattered thick and thin filaments and a few mitochondria. Nuclei or other organelles were never observed. The peritoneal cells rest on a thin basal lamina, as do the follicle cells where the latter come in contact with the vessel lumen. A part of the blood vessel wall is also occupied by developing germ cells that partially rest on the peritoneal cells and the underlying basal lamina but never on the follicle cells (Fig. 3). However, the follicle cells surround 244 K.J. Eckelbarger the majority of oocytes and partially encapsulate the entire ovary. The follicle , cell sheath is incomplete in places, permitting parts of some oocytes to contact each other. The surfaces of a few oocytes, particularly the larger ones, are in direct contact with the coelomic fluid. Within the blood vessel, a red, extracel­ lular blood pigment is present with a sufficiently high molecular weight to give it a distinctive, particulate appearance in the electron microscope (Figs. 2 and 24). The polymeric molecules measure 22-25 nm in diameter. Follicle cells contain large (4.5-6.0 11m), spherical nuclei with scattered patches ofperipheral, condensed chromatin and a single characteristic, nucleolus about 1.5 11m in diameter. The cytoplasmic surface of the nuclear envelope is covered with ribosomes. The most prominent feature of the cells is the presence of abundant rough endoplasmic reticulum (RER), frequently in the form of large, tightly packed whorls or parallel arrays (Fig. 4). Some free ribosomes are present as well as scattered oblong mitochondria with long, tubular cristae (Fig. 6). The follicle cells are also characterized by the presence of numerous organelles which morphologically resemble Iysosomes . They contain cytomem­ branes closely resembling mitochondria (Fig. 5). and RER (Fig. 6), enclosed by an isolation membrane. The enclosed mitochondria have a denser matrix than the mitochondria within the cytoplasm. In addition, one also observes heterogeneous, membrane bounded dense bodies containing an amorphous elec­ tron dense material, granules and a membranous component that frequently appears as a myelin configuration (membrane whorls) (see arrows, Fig. 6). From one to several Goigi complexes are frequently observed in close association with these inclusions (Fig . 7). The Golgi have a small number of saccules and are usually in close association with cisternae of the RER. Two products appear to be released from the mature face of the organelle in the form of numerous, small (50-70 nm), coated vesicles and larger (up to 200 nm) membrane-bounded electron dense droplets. Occasionally, multivesicular bodies are observed. Follicle cells lack cytoplasmic continuity with germ cells or other follicle cells but adjacent fol1icle cells are periodically attached by small desmosomes. Follicle cells remain in intimate association with most developing oocytes throughout oogenesis but never possess microvilli or other specializations of the plasmalemma. Specialized junctional complexes have not been observed between germ cells nor have intercellular bridges been encountered.

2. Previtellogenic Oocytes

These oocytes have a germinal vesicle diameter ranging from 9 to 20 11m. The smallest of these have little cytoplasm and a circular nucleus containing very sparse, scattered chromatin and occasionally a small , eccentric nucleolus (Fig. 4). The cytoplasm of large previtellogenic oocytes (Fig. 8) contains free ribosomes,

Fig. 2. Transmission electron micrograph (TEM) of genital blood vessel (B V) showing follicle cells with large nuclei (N) and underlying basal lamina (BL) and peritone al cells (P). x 10,800

Fig. 3. TEM through ovary showing previtellogenic oocytes (OC) att ached to the genital blood vessel (B V). Note surrounding follicle cells (FC). x 3,990 Oogenesi s in Str eblospio benedicti 245 246 K.J. Ecke1barger from one to several small Golgi complexes and scattered, small mitochondria with circular or oval profiles and tubular cristae. Associated with the mitochon­ dria in the perinuclear region are prominent, fibrogranular, basophilic aggregates (" nuage ") presumably of nuclear origin (Fig. 8, inset). As cell growth continues, the nucleolus enlarges and becomes bipartite with an inner fibrillar core and an outer granular cortex (Fig. 8, inset). There is a significant increase in the size and number of mitochondria in the ooplasm and a continuation of their association with basophilic aggregates. The oolemma is smooth in profile and adjacent follicle cells are apposed closely to the oocyte surface.

3. Vitellogenic Oocytes

Vitellogenesis occurs while the oocytes are within the ovary and in contact with the genital blood vessel. Two morphologically distinguishable types of yolk are formed during vitellogenesis. Type I yolk is synthesized first and appears to be formed autosynthetically. Type II yolk appears later and apparently is formed heterosynthetically. In mature eggs, the size and number of Type II yolk granules are greater than that of the Type I variety. The smallest vitellogenic oocytes observed have a nuclear diameter of about 20 urn. Formation of the egg envelope begins at this time with the elaboration of short (150 nm), irregularly spaced, digitate or branching microvilli whose tips are covered by a dense glycocal yx that forms a thin , electron dense, outer layer (Fig. 9). The egg envelope initially forms in patches rather than uniformly over the surface of the oocyte. Small (150 to 200 nm), uncoated pinocytotic vesicles which contain a light to moderate electron dense flocculum, are com­ monly observed along the oolemma at this stage (Figs . 9-11). Golgi complexes are now restricted almost exclusively to the cortical ooplasm adjacent to the oolemma (Fig . II). Short, single strands of RER are observed throughout the ooplasm and gradually appear in parallel arrays (Fig. 12). The majority of the cisternae are flattened and lack an y obvious product at this stage. Rough ER cisternae are usually in close association with the immature face of each Golgi complex. Type I yolk synthesis is initiated by the appearance of swollen cisternae of RER (Fig. 13) containing an electron dense product that at times resembles a single coated vesicle about 100 nm in diameter (Fig . 14). Progressively larger,

Fig. 4. Edge of ovary showing follicle cells cont aining very prominent arrays of rough endo plasmic reticulum (ER) and sma ll previtellogenic oocyte with spherical nucleus (N) to left. x 4,130

Fig. 5. Follicle cell cyto plasm containing mitoch ondrion (arrow) surrounded by isolati on membrane and lysosome (eY) containin g remn ants of mitochondrion-like bod y (*). x 17,950

Fig. 6. Lysosomes with in follicle cell cytoplasm (arrows). Note whorl of RER (*) that is surro unded by an isolation membrane. M , mitochondrion. x 21,230

Fig. 7. G olgi complexes (G) and associated multivesicular body (arrow) in follicle cell cyto­ plasm. x 23,470 Oogenesis in Streblospio benedicti 247 248 K.J. Eckelbarger circular cisternae contammg larger more electron dense coated vesicles are observed throughout the ooplasm (Figs. 15 and 16). The smaller of these vesicles (Fig. 15) possesses a thin , electron dense cortex and an inner, fine fibrillar core. Further differentiation of these vesicles produces a droplet consisting of alternating electron dense and lucent concentric rings (Fig. 17) and finally a homogeneous electron opaque yolk droplet with a maximum diameter of about 3.5 11m (Figs. 16 and 18). Mature Type I yolk droplets lack any crystalline con­ figuration and are often somewhat irregular in outline. They are rarely en- countered in the perivasal ooplasm. . During Type I yolk formation cortically positioned Golgi complexes usually consisting offive or six parallel saccules are evenly distributed along the oolemma with the maturing face directed towards the center of the cells. Rough ER cisternae are often arranged along the immature face of the Golgi and transition vesicles between the two organelles are occasionally observed in favorable planes of section. In many instances, the contents of electron dense vesicles emerging from the mature face of the Golgi compl exes closely resemble the contents of the Type I yolk droplets described above (Figs. 11 and 18). . During Type I yolk synthesis, the oolemma in the perivasal region of the oocyte remains relatively smooth in profile (Fig. 19). Flattened, irregularly shaped, peritoneal cells are closely apposed to the surface of the oocyte except in a few areas where the oolemma is in direct contact with the basal lamina of the blood vessel. The thin basal lamina lines the blood vessel lumen and conforms closely to the irregular contours of the peritoneal cells and the oo­ lemma of each adjacent oocyte. Type II yolk synthesis occurs exclusively in the perivasal region of the oocyte and begins with an infolding of the oolemma (Fig. 20) resulting in a significant localized increase in the surface of the cell. Formation of these invaginations continues until most of the oolemma is in contact with the basal lamina of the blood vessel and the majority of the peritoneal cells no longer act as a barrier between the oocyte and the vessel lumen (Fig. 21). Figure 1 diagrammati­ cally illustrates the changes in the configuration of the oolemma during Type II yolk synthesis. Endocytosis, which accompanies the process of infolding, is heralded by the appearance of numerous coated pits and vesicles along the perivasal oolemma (Fig. 22). Pit formation is preceded by the appearance of molecules of blood pigment and often , but not always, by an electron dense flocculum between

Fig. 8. Two previtellogenic oocytes with large germinal vesicles (N) with bipartite nucleoli (NU) and fibrogranul ar mater ial (*) in ooplasm. Fe , nucleu s of follicle cells. x 4.800. Insert, bottom right : closeup of edge of nucleolus showing its fibrou s (F) and granular (G) components. x 30,600. Insert, upper left : closeup of fibro granul ar material (FG) and associated mitochondria. x 19,000

Fig. 9. Newly form ing egg envelope (EE) and pinocytotic vesicles (arrows). An adjacent follicle cell appears above. x 33,870

Fig. 10.Electron dense pinocytotic vesicles (arrows) beneath egg envelope. x 18,000

Fig. 11. Go lgi complex in cortical ooplasm of early vitellogenic oocyte. A rrow indicates small Type I yolk body. x 21,900 Oogenesis in Str eblospio benedicti 249 250 K.J. Eckelbarger the basal lamina and the oolemma adjacent to where a coated pit is destined to form (Fig. 22). During coated pit formation, the dense flocculum adheres closely to the free surface of the pit. Pigment molecules are visible within the invagination but are obscured near the oolemma by the adhered material. In some instances, pits contain pigment molecules but little or no flocculent material (Fig. 25). The pits range from 300 to 350 nm in diameter and usua lly have a thin (25 nm) filamentous border on the cytoplasmic side of the membrane. Newly formed pit-derived coated vesicles (endosomes) are approximately the same diameter as the coated pits. The flocculum-filled endosomes usually possess three distinct layers including an outer, 25 nm filamentous coat, a middle 7.5 nm plasmalemma and a flocculent inner layer lining the vesicle, ranging from 70 to 85 nm in thickness (Fig. 22, insert). The texture and electron densit y of this inner layer is indistinguishable from that of the material adhering to the surface of the forming coated pits. Endosomes deeper into the cytoplasm have lost their outer coating. Often, the central regions of these vesicles are occupied by a number of recognizable blood pigment molecules (Figs. 23 and 24). Eventually, the flocculent contents condense to form a body with a homogeneous electron dense core (Figs. 25-27). These bodies then fuse with larger Type II yolk bodies (Fig. 27). These larger and presumably more mature yolk droplets consist of a solid or ring-shaped central electron dense core and a flocculent cortex (Figs. 27 and 28). The cortex usually contains elongated crystalline bodies composed of parallel dense bands spaced at intervals of 28 nm, intercepted at right angles by thinner bands spread at intervals of 15 nm (Figs. 28 and 29). In addition, granular particles similar in size (22 to 25 nm) and shape to the blood pigment molecules seen in the blood vessel lumen , coated pits and coated vesicles, are also observed occasion­ ally in this region of the developing yolk droplets (Fig. 27). Mature Type II yolk spheres are often irregularly shaped, uniformally electron dense (Fig: 31) and generally have a crystalline-like appearance consisting of faintl y visible parallel striations (Fig. 30). During the active period of endocytosis, the ooplasm of the entire oocyte, but particularly that of the perivasal region, rapidly and rather abruptly develops

Fig. 12. Early vitellogenic oocyte showing large nucleolus (NU) and parallel cistern ae of RER (ER). x 5,770

Fig. 13. Profiles of dilated RER . A rrolV indicates electron dense flocculum within ER. x 28,500

Fig. 14. Coated-vesicle-like body within ER cisterna. x 33,250

Fig. 15. Swollen ER cisternae containing forming Type I yolk material adjacent to mature Type I yolk body (Y) . x 23,750

Fig. 16. Type I yolk in early (I), intermediate (2) and late (3) stages of formation. x 21,700

Fig. 17. Type I yolk bod y formin g within ER cisterna . x 44,500

Fig. 18. Golg i complex secreting vesicles (arrolVs) similar in app earance to Type I yolk bodies (Y). x 29,850 Oogene sis in Streblospio benedicti 252 K.J. Eckelbarger

an extensive system of smooth ER. There is also a simultaneous appearance of CJ. and f3 glycogen particles and a noticeable reduction of free ribosomes and RER. Spherical or oval-shaped lipid droplets appear during the later stages of vitellogenesis and reach a maximum diameter of 0.5 urn (Fig. 31). Aside from smooth ER, cellular organelles are seldom observed in the periva­ sal ooplasm during Type II yolk synthesis. Mitochondria were occasiona lly en­ countered as were a few microtubules but Golgi complexes and RER were rare in this region. Mature oocytes are irregular in shape and have a nuclear diameter of from 31 to 35 urn. The egg envelope is comparatively thin (less than 150 nm) and composed of three layers penetrated by short (ca. 150 nm) digitiform microvilli that bifurcate at their base and lie nearly parallel to the oolemma (Fig. 32). The inner layer is composed of a filamentous, slightly electron dense material from 35 to 55 nm in thickness. The midd le layer is similar to the inner layer but is thinner (25 to 30 nm) and more electron dense. The outer region is a loosely organized 55 nm layer of glycocalyx strands that arise from the tips of the microvilli . Cortical granules have never been observed in the oocytes of Streblospio benedicti.

D. Discussion

I. Follicle Cells and Lysosomes

King et al. (1969), in their ultrastructural study of vitellogenesis in Spirorbis suggested that yolk precursors are pinocytotically absorbed by the oocytes from the coelomic fluid and assembled into yolk by an autosynthetic process. Potswald (1972) postulated that the perivasal cells of Spirorbis , in addition to chlorocruorin production, might synthesize yolk precursors destined for the developing oocytes in much the same manner as liver cells in man y vertebrates. The circulatory system would then mediate in the movement of these materials from the digestive system to the coelomic fluid. Since Streblospio benedicti oocytes apparently obtain macromolecular yolk precursors directly from the circulatory system, cells capable of synthesizing these materials are presumably associated with the circulatory system. Based on the results of the present stud y, the follicle cells would be likely candidates for such a cell type. Their fine structure indicates that they are undergoing intense protein synthesis and they are intimately asso­ ciated with both the circulatory system and the ovary. In addition, no analogous

Fig. 19. Perivasal region of two previtellogenic oocytes (OC) illustratin g barrier formed by peritoneal cells (arroll's) between oocytes and genital blood vessel lumen (BV) . Fe , follicle cell. x 11 ,650

Fig. 20. Perivasal region of early vitellogenic oocytes showing infolding of oolemma and initiation of endocytosis. The adjacent peritoneal cells (arroll's) no longer provide a significant barrier between the vessel lumen and oolemma. B V, blood vessel. x 9,600

Fig. 21. Perivasal region of oocyte in advanced stages of vitellogenesis showing extensive surface area of oolemma that is in contact with lumen of blood vessel (BV). Arro ll's indicate perit oneal cells. x 5,400 Oogenesis in Strcb lospio benedicti 253 254 K.J. Eckelbarger cell type has been observed in the wall of the genital blood vessel in male S. benedicti (pers. obs.) suggesting an exclusive function in the female. They are ultrastructurally similar in some ways to the perivasal cells of Spirorbis (Potswald, 1969) except that there is no indication of an hematopoietic function. The presence of lysosomes and dense bodies in the follicle cells of Streblospio benedicti and the periva sal cells of Spirorbis (Potswald, 1969) are a prominent feature of both cell types but their significance is not entirely clear. Lysosomes, myelin figures and dense bodies have been noted in a wide variety of cells and correlated with an assortment of activities and conditions (see Buckland­ Nicks and Chia , 1976, for review). The predominant presence of mitochondria and RER within the numerous Iysosomes observed in S. benedicti follicle cells suggests a selective process of autophagy. Although autophagy of endogenous components is common in cells undergoing physiological or pathological regres­ sion, it is also a normal occurrence in differentiating cells (Arstila and Trump, 1968) and in metabolically active cells (Napolitano, 1963) and other non-regress­ ing tissues (Dean, 1977). It has also been hypothesized that Iysosomes may function in normal cells to isolate and degrade cellular organelles no longer needed (Locke and Collins, 1965). Potswald (1969) suggested this might be true of a cell that has more than one synthetic function and cited the perivasal cells of Spirorbis as a possible example. However, if the follicle cells of S. benedicti possess more than one function, that additional function(s) has not been ascertained.

2. Yolk Uptake M echanis;ns The presence of intense endocytotic activit y in the oocytes of Streblospio benedicti is interpreted as selective uptake ofprotein (yolk precursors) from the circulatory system and is remini scent of many insect oocytes (Telfer, 1961, 1965; Roth and Porter, 1964; And erson, 1969). Roth and Porter (1964) hypothesized that

Fig. 22. Perivasal region of oocyte in early stages of Type II yolk formation. Between the blood vessel (BV) basal lamina (BL) and the oolemma of oocyte one can see an electro n dense flocculurn and blood pigment molecules (*). Note the coated pits (arrows) forming along the oolemma. x 24,600. Insert : high magnification of coated vesicle showing three layered constructi on. x 74,600

Fig. 23. Uncoated vesicle from perivasal ooplasm containing flocculum in its cortical region and blood pigment molecules centrally. x 70,600

Fig. 24. Blood pigment molecules shown at same magnification as tho se shown in Fig. 23. x 70,600

Fig. 25. Coated pit (arrow) containing blood pigment molecules. A small Type II yolk droplet ( Y) is also illustrated . x 23,750

Fig. 26. Vesicle containing blood pigment molecules (arrow) adjacent to dense-cored Type II yolk body (Y) . x 28,500

Fig.27. Small Type II yolk droplets (yl) in apparent process of fusing with large Type II yolk body (y2). Ar rows indicate granules resembling blood pigment molecules in cortical regions of yolk dropl ets. x 40,700 Oogenesis in Streblospio benedicti 255 256 K.J. Eckelbarger

Fig. 28. Type II yolk body (Y) showing dense core and crystalline configuration in cor tical region . x 25,900 Fig. 29. High magnification of cortex of Type II yolk body showing crystalline configuration. x 65,300 Fig. 30. Type II yolk body showing crystalline appe arance of cortical region. x 34,200 Fig. 31. Cortical region of mature oocyte showing egg envelope (E£), Type II yolk bodies ( Y), lipid droplets (L) and scattered glycogen part icles (G). x 24,900 Fig. 32. Higher magnification of mat ure egg envelope showing bifurcated microvilli (MV) and three layered construction. x 126,000

"coated pits" observed in the post-blood meal oocytes of the mosquito Aedes aegypti were morphological manifestations of gross protein absorption by the cells. Since then the phenomenon has been reported widely in the oocytes of other groups (for discussion and review see Norrevang, 1968; And erson, 1969, 1974). Although significant pinocytotic activit y related to yolk synthesis has Oogenesis in Streblospio benedicti 257 been reported in the Ophryotrocha labronica (Emanuelsson, 1969), Spirorbis borealis (King etal., 1969) and Mercierella enigmatica (Sichel, 1966), in each instance proteosynthetic organelles within the oocytes participated in the assembly of yolk. There was no indication of Golgi or ER contribution to Type II yolk synthesis in the oocytes of S. benedicti. Three basic processes have been described (using terminology of Schechtman, 1955) by which oocytes acquire deutoplasmic components during vitellogenesis: (I) autosynthesis, by which yolk is produced by the oocyte itself; (2) heterosyn-, thesis, in which yolk is produced in extraovarian sites and transported to the oocyte; or (3) both auto- and heterosynthesis. Autosynthetic yolk production is widespread among the invertebrates and is common among the Spiralia (see Anderson, 1974, and Huebner and Anderson, 1976, for review). Heterosynthetic yolk formation is characteristic of many insects, amphibians, fish, reptiles and birds (Anderson, 1974). "Mixed" yolk synthesis in which both autosynthetic and heterosynthetic processes are involved in the formation of a single yolk body is recognized in some annelids, crustaceans, fish, amphibians and numerous insects (see Eckelbarger, 1979, for review). A recent study of vitellogenesis in the oocytes of the reef-building polychaete Phragmatopoma lapidosa (Eckelbarger, 1979) described the formation of two morphologically distinct kinds of yolk, one by an autosynthetic process and the other by a heterosynthetic process. From the present study, it appears a similar phenomenon occurs in Streblospio benedicti oocytes. Neither of these examples fits the" mixed" auto- and heterosynthetic category of yolk formation because published accounts describe the formation of a single type of yolk utilizing this process. To the author's knowledge, no other published works have reported the formation of two (or more) morphologically distinct kinds of yolk by separate autosynthetic and heterosynthetic processes. There has been much discussion of the morphological and functional signifi­ cance of coated vesicles in oocytes (see discussions and review by Anderson, 1969 and 1974). It has been speculated that the presence of a spiked outer coating around endocytotic vesicles may function to keep the population of vesicles from re-fusing with the oolemma «Huebner and Anderson, 1972) and that it possesses specific binding sites for the attachment of vitellogenin (Yusko and Roth, 1976). It has also been suggested (Chapman-Andresen, 1965; Ander­ son, 1969) that the variation in the size of the endocytotic pits may reflect the kind of substances that are being internalized from the microenvironment. In the present study at least two types of pits were recognized during Type II yolk formation. Although both contained blood pigment and were the same diameter, only one contained an electron dense flocculum. It is interesting to note for comparative purposes that the perivasal coated pits contributing to Type II yolk synthesis in Phragmatopoma lapidosa oocytes (Eckelbarger, 1979) possessed a granular, outer (cytoplasmic) coating and were relatively small (120-160 nm) whereas those of Streblospio benedicti had a spiked outer surface and were 300-350 nm in diameter. Type II yolk in both species is morpholog­ ically similar except for the crystalline nature of S. benedicti yolk granules. The uncoated pinocytotic vesicles observed along the egg envelope in other regions of S. benedicti oocytes presumably represent sequestered materials des­ tined for Type I yolk synthesis or for maintenance or growth of the cell. 258 K.J. Eckelbarger

The significance of blood pigment uptake in Streblospio benedicti oocytes is unknown although it raises intriguing questions as to the developmental, physiological or ecological significance to the developing embryo or . The author is unaware of any published reports of blood pigment incorporation in the oocytes of other , although Fox (1949) reported the presence of protohaem in the eggs of the sabellid polychaete Sabella spallanzanii. Demonstration of autosynthetic yolk production in oocytes based on ultrastructural studies and morphological interpretations alone, can be diffi­ cult because of the complex and often subtle processes involved. However, in both Phragmatopoma lapidosa and Streblospio benedicti, Type I yolk appears to be synthesized through the conjoined efforts of the Golgi complex and RER. Significant pinocytotic activity related to Type I yolk synthesis occurs in S. benedicti oocytes, but little or none was observed in the oocytes of P. lapidosa. It has been demonstrated previously that maternal blood proteins (yolk precursors) can traverse the basal lamina in insect oocytes (Telfer, 1961; Roth and Porter, 1964). In Phragmatopoma lapidosa (Eckelbarger, 1979), endocytotic uptake of presumed yolk precursors occurred despite the intervening basal la­ mina between the vessel lumen and the oocyte. The large, extracellular blood pigment molecules, however, were restricted to the lumen and never were observed on the coelomic side of the basal lamina. In Streblospio benedicti, however, the basal lamina underlying each oocyte does not act as a barrier to pigment movement from the vessel lumen into the oocyte. On the other hand, blood pigment was never observed within the follicle cells or peritoneal cells lining a blood vessel, suggesting that these cells or their underlying basal lamina act as a selective barrier to the pigment. Potswald (1969), in his ultrastruc­ tural study of the synthesis and secretion of chlorocruorin from the perivasal cells of Spirorbis, also demonstrated that the blood vessel basal lamina underly­ ing the perivasal cells was selectively permeable to pigment during transport of the pigment from the perivasal cells to the blood vessel lumen by " reverse pinocytosis ". However, the basal lamina underlying the myoepithelial cells of the blood vessel did serve as a barrier to pigment movement. Nakao (1974) reported some extravasal hemoglobin particles up to 22 nm in diameter in the intercellular spaces between peritoneal cells in the blood vessel walls of Nereis japonica but suggested that most hemoglobin particles were retained within the vessel lumen by the basal lamina. In all polychaetes, yolk precursors must be derived ultimately from the products of digestion. In some species, these nutrients are stored in the body wall, gut , peritoneum and coelomic fluid, and are destined to be passed to the oocytes for assembly into yolk droplets during vitellogenesis (see Eckelbarger, 1976, for review). From these storage sites, nutrients must make their way to the site ofthe ovary and into the developing oocytes generally via the coelomic fluid. There are four processes by which nutrients can enter the developing oocytes. In the first, low molecular weight precursors present in the coelomic fluid apparently enter the oocyte through the well-developed microvilli by a process not rendered visible with ultrastructural techniques (i.e., no endocytotic activity in evidence). Biosynthesis of yolk bodies then occurs within the oocyte using autosynthetic processes that utilize these diffused materials. This process Oogene sis in Streblospio benedicti 259 is common particularly in the Nereidae as evidenced by the ultrastructural and autoradiological studies of Dhainaut on Nereis pelagica (1966, 1968, 1970a and b), N. diversicolor (1967) and Perinereis cultrifera (1976), but is suspected also in the terebellid Nicolea zostericola (Eckel barger, 1976). In the latter study, some minor pinocytotic activity was reported but it was not certain to what degree, if any, it contributed to yolk synthesis. All of the species listed above undergo "solitary oogenesis" (Korschelt and Heider, 1902) in that no accessory cells are associated with the oocytes during vitellogenesis. Signifi­ cantly, Fischer (1979) recently presented immunological evidence for a vitelloge­ nin-like protein in the coelomic fluid of Nereis virens, a finding that will undoubt­ edly be repeated in other " solitary" species. In the second process, yolk precursors in the form of macromolecules enter the oocyte by endocytosis that is expressed by the appearance of coated pits and vesicles along the oolemma. Spirorbis borealis (King et aI., 19'69; Potswald, 1972) and Polydora ligni (pers. obs.), both species that undergo "solitary oogen­ esis", exemplify this process. In each case, however, the heterosynthetic materials taken into the oocyte are assembled subsequently into mature yolk bodies by the proteosynthetic machinery of the oocyte. In the third category, accessory cells provide yolk precursors or other mate­ rials directly to the oocyte via intercellular bridges. In Ophryotrocha labronica (Emanuelsson, 1969),vitellogenesis is a complex process in which yolk precursors in the form of multivesicular bodies and dense bodies, are passed to the oocyte. In Diopatra cuprea, Anderson and Huebner (1968) speculated that the attached chain of nurse cells probably contributed free ribosomes to the oocyte for possible use during autosynthesis of yolk. Both species would be categorized by Korschelt and Heider (1902) as undergoing "riutrimentary oogenesis" since accessory cells (nurse cells) are associated with the oocytes. The fourth process involves" pure" heterosynthetic yolk production in which large molecular weight yolk precursors from extraovarian sources are taken up by endocytosis and directly assembled into yolk bodies without the participa­ tion of the prot eosynthetic organelles of the oocyte. Although this process is found in many insects and vertebrates, . no polychaetes have been found that are totally dependent upon the metabolic resources of other cells to produce their stored yolk material (Schroeder and Hermans, 1975). However, the oocytes of Phragmatopoma lapidosa and Streblospio benedicti apparently utilize this process in the assembly of one type of yolk body whereas a second type is produced by an autosynthetic process. They are also the only species reported whose oocytes derive yolk precursors directly from the circulatory system.

3. Vitellogenesis and Reproductive Strategies

Anderson (1974) commented that despite the wealth of information available on vitellogenesis it has not been possible thus far to organize a classification of this process from an evolutionary point of view. However, Boyer (1972) in her ultrastructural study of vitellogenesis in the polyclad turbellarian Prosthe­ ceraeusfloridanus, suggested that the highly independent process of autosynthesis 260 K.J. Eckelbarger observed in the oocytes of this animal may represent the original method of yolk synthesis in Metazoan oocytes. Based on the limited number of comprehen­ sive ultrastructural studies on polychaete oogenesis published thus far (see Eckel­ barger, 1979, for review), it would appear that no clear correlation can be drawn between mechanisms of yolk formation and phylogenetic status within this group. A more reasonable approach might be to set aside issues of phylogenetic position and compare mechanisms of vitellogenesis in various species to their respective reproductive strategies. Reproductive strategies in polychaetes have been delineated into three types (Olive and Clark, 1978): 1. monotelic (=sernel­ parous) species in which individuals breed only once in their lifetime, releasing all their gametes in one or a few large batches and frequently die afterwards ; 2. polytelic ( = iteroparous) species that like monotelic species release all their gametes in one or a few large batches but survive to breed again the following year ; and 3. continuous or semi-continuous species in which individuals breed several times in their lifetime, releasing gametes in a number of small broods over an extended breeding season. If one examines the available literature it appears that monotelic spawners such as the onuphid Diopatra cuprea (Anderson and Huebner, 1968), the nereids Nereis diversicolor (Dhainaut, 1967) and N. pelagica (Dhainaut, 1970a), Per­ enereis cultrifera (Dhainaut, 1976) and the terebellid Nicolea zostericola (Eckel­ barger, 1976), all utilize some form of autosynthetic yolk production during oogenesis. Semi-continuous and some polytelic spawn ers tend to undergo vitello­ genesis much more rapidly than monotelic species and as a result appear to utilize various degrees of heterosynthetic yolk production. In Spirorbis, King et al. (1969) and Potswald (1972) reported significant pinocytosis by the free­ floating oocytes during vitellogenesis that the former authors considered as evidence of uptake of yolk precursor molecules from the coelomic fluid. Ema­ nuelsson's (1969) study of vitellogenesis in Ophryotrocha suggests that a nurse cell pro vides yolk precursors to each developing oocyte. Phragmatopoma lapidosa (Eckelbarger, 1979) and Streblospio benedicti (present study) both utilize a more direct form of heterosynthesis by sequestering yolk protein from the circulatory system for direct assembly into yolk bodies . Preliminary, unpublished research by the author on the capitellid Capitella capitata Type I (Grassle and Grassle, 1976)and the spionid Polydora ligni, both semi-continuous spawners and oppor­ tunistic (Grassle and Grassle, 1974) and pollution indicator species (Grassle and Grassle, 1974, 1976; Young and Young, 1978) indicate that both species utilize some form of heterosynthetic yolk production. The oocytes of C. capitata remain in the ovary during vitellogenesis and are surrounded by large proteosyn­ thetic follicle cells which produce yolk precursors that are absorbed by the oocyte by endocytosis. The oocytes of P. ligni leave the ovary prior to vitellogen­ esis and float freely within the coelomic fluid where endocytosis occurs. There is wide variation in the reproductive physiology of polychaetes (partic­ ularly , vitellogenic mechani sms) that reflects the heterogeneity of their respective life histories and reproductive strategies. On a morphological level, each species is confronted with specific life history requirements or demands that must be satisfied, such as the number of eggs produced in each brood, the number Oogenesis in Str eblospio benedicti 261 of broods per lifetime, quantity and qu ality of yolk needed per egg and the length of oogenesis, to nam e a few. The physiolo gical and metabolic needs of a monotelic spawner such as Nicolea zostericola in which vitellogenesis extends for an estima ted period of 5-6 months (Eckelbarger, 1974, 1975) must be signifi­ cantly different from Str eblospio benedicti, a species in which vitellogenesis occurs in less than two weeks. It seems reasonable th en that a mechanism of extraova­ rian synthesis ofyolk precursors and uptake ofthese materials in a macromolecu­ lar form by the oocytes would provide a faster and more efficient means of yolk assembly in species undergoing rapid vitellogenesis. In the polychaetes studied thus far , this appears to be the case. A broader study of the mechanisms of yolk synthesis in polychaetes having a wide variety of reproductive strategies should provide a better opportunity to understand the evolution and adaptive significance of these mechanisms.

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Received October 23, 1979