J. Cell Sci. 4, 241-264 (1969) 241 Printed in Great Britain

SYNTHESIS AND DEPOSITION OF ENVELOPES (VITELLINE MEMBRANE, ) AND THE UPTAKE OF IN THE DRAGONFLY (ODONATA: AESCHNIDAE)

H. W. BEAMS AND R. G. KESSEL Department of Zoology, University of Iowa, Iowa City, Iowa, U.S.A.

SUMMARY Light and electron-microscope studies on dragonfly ovarioles reveal evidence that the precursor vitelline membrane and chorion secretions are synthesized within the follicle cells. It is suggested that the sequence of synthesis and deposition of the vitelline membrane occurs as follows. The vitelline membrane presecretion appears to be synthesized by the rough- surfaced endoplasmic reticulum, giving rise to intracisternal granules. These appear to migrate in the cisternae to the region of the Golgi complex where the endoplasmic reticulum loses most of its ribosomes and the intracisternal granules move into the Golgi region where they appear within small vesicles. These seem to find their way into the Golgi cisternae where they may be incorporated with the secretions from the Golgi cisternae to produce the definitive previtelline secretion. The previtelline secretion bodies are eventually discharged into the space between the oocyte and follicle cells, forming rows of secretion bodies between the microvilli. These fuse into progressively larger bodies until a complete membrane is established. Follicle cells actively secreting precursor vitelline membrane substance show many disk-shaped, relatively clear vesicles in the cytoplasm. After the vitelline membrane is laid down, the follicle cells take on an entirely different function; namely, the synthesis and deposition of the chorion. The first visible chorion secretion appears in profile as elongate dense bodies within the Golgi cisternae which tend to coil, and in so doing, expand the cisternae. As this occurs, the enlarged cisterna, loaded with concentric coiled secretion material, separates from the remainder of the Golgi cisternae and becomes free in the cytoplasm as a prechorion secretion body. These migrate to, and collect below, the surface of the cell where they are eventually ejected between the surface folds and become incorporated into the developing chorion. Uptake of yolk in the dragonfly seems to be predominantly by micropinocytosis. The oocyte surface during active vitellogenesis bears many pits which contain an extracellular material closely applied to the outer surface of the plasma membrane. Thin, radially oriented bristles are continuous with the inner surface of the plasma membrane in this region. The pits continue to invaginate until they are cut off from the plasma membrane and come to lie in the oocyte cortex as coated vesicles. These appear to lose their coats gradually and fuse with one another to produce definitive yolk spheres.

INTRODUCTION Relatively few studies have been concerned with the synthesis, transport and assembly of materials comprising the envelopes of insects (c.f. Okada & Wadding- ton, 1959; Bier, 1963; King & Koch, 1963; Engles & Drescher, 1964; Favard-Sereno, 1966; Hopkins & King, 1966). The developing of the dragonfly have proved to be exceptionally favourable material for a study of the morphogenesis of their 16 Cell Sci. 4 242 H. W. Beams and R. G. Kessel investing oocyte envelopes. Consequently, we are recording here the results of studies with both the light and electron microscopes on these cells with special reference to the synthesis, transport and assembly of the materials constituting both the vitelline membrane and chorion. Observations on yolk uptake in the oocyte are also included.

MATERIALS AND METHODS Female dragonflies (Aesckna sp.) were collected in Iowa City during the months of August and September. For electron-microscope study, ovaries were fixed for 1-2 h in ice-cold 3% phosphate-buffered glutaraldehyde (pH 7-3) and post-fixed in osmium tetroxide (Sabatini, Bensch & Barrnett, 1963). The ovaries were dehydrated rapidly in ascending grades of cold ethanol, treated with propylene oxide and embedded in Epon 812 (Luft, 1961). Thin sections were obtained with a Porter-Blum ultramicrotome and stained in uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). Sections were studied in RCA EMU 3D and 3G electron microscopes. Thick Epon-embedded sections of the ovarioles were stained with azure II and methylene blue (Richardson, Jarrett & Finke, i960).

OBSERVATIONS Organization of ovariole The dragonfly ovary is of the panoistic type consisting of numerous ovarioles (Fig. 1). Each ovariole is surrounded by an outer flattened epithelial sheath and may be divided into different regions from the proximal to the distal end as follows: (1) a terminal filament which has a suspensory function; (2) a germarium which contains groups of cells from which the oogonia differentiate as well as newly differentiated oogonia; (3) vitellarium, consisting of the major portion of the ovariole and containing a series of oocytes in different stages of growth and development, each enclosed in a follicle which becomes progressively larger and more elongate toward the ; and (4) oviduct which constitutes the tube in which the mature oocytes are discharged from the vitellarium. The phase-contrast microscope clearly reveals the gross structure of the ovariole, relative size of the oocytes and their large nuclei and nucleoli (Fig. 1).

Follicle cell maturation Accompanying the growth of the young oocyte are two important cytological changes which occur more or less concomitantly. One is rapid growth of the follicle and the other the deposition of yolk. In the youngest oocytes, the follicle is composed of flattened squamous cells, but as the oocyte grows the follicle enlarges by rapid mitotic division and at the same time there occurs a marked increase in the size of the cells. With the advent of vitelline membrane deposition, the follicle cells have, or nearly have, attained their definitive size. They rest on a fibrous basement lamina (Fig. 4). Follicle cells active in the synthesis of previtelline membrane substance possess one or two nuclei, each usually containing a dense nucleolus (Figs. 2, 3). As Synthesis, deposition of insect egg envelopes 243 noted in the young oocytes, division of the follicle cells is generally by mitosis; however, in actively secreting cells, the binucleate condition sometimes observed has arisen either by mitotic division of the nucleus without an accompanying cytoplasmic division or else by amitosis. Amitosis has been described as occurring in insect follicle cells (cf. Raven, 1961). Other characteristics of the actively secreting follicle cells illustrated in light-microscope preparations are their deeply staining basophilic cytoplasm, presence of dense azure II-methylene blue-staining secretion granules and numerous, elongate or disk-shaped vesicles; the latter are located for the most part between the nucleus and free border of the cell (i.e. surface opposite the basement lamina, (Figs. 2, 3). The disk-shaped vesicles are roughly polarized with their long axes at right angle to the free border of the follicle cells, and electron micrographs demonstrate that they sometimes contain a coagulated material of low density and are generally free of previtelline secretion droplets (Fig. 13). Although not consistently demonstrated, the vesicles appear in some regions to possess a limiting membrane (Fig- !3)- During the stage of active secretion of the previtelline membrane substance, the follicle cells appear somewhat loosely spaced and rounded on their free surface, conditions which may represent a functional specialization associated with the trans- port of material between them. The relation of the actively secreting follicle cells to the oocyte is seen in Fig. 2. Here, the newly formed previtelline secretion droplets are arranged in rows which, as will be noted later, is probably due to the action of the microvilli from the surface of the follicle cells.

Vitelline, membrane synthesis and deposition As noted in the light-microscope preparations, with the onset of vitelline membrane synthesis, numerous azure II-methylene blue-staining bodies (secretion) are seen situated in faintly revealed, clear areas of the cytoplasm of the follicle cells (Figs. 2, 3). The electron microscope shows these to be Golgi bodies (dictyosomes) which appear to function in the segregation of previtelline membrane substance (Fig. 5). In actively secreting cells the Golgi bodies are numerous and scattered in the cytoplasm among the extensively developed, rough-surfaced endoplasmic reticulum (Fig. 5). They are more numerous in the region about the nucleus and free border of the cell than else- where. Electron micrographs depict the Golgi complex (Fig. 6) to be composed of smooth-surfaced cisternae, relatively large vesicles, small vesicles, large dense secretion bodies and adjoining cisternae of the endoplasmic reticulum; the last, upon approach- ing the Golgi region, become smooth-surfaced (Figs. 7-9). The endoplasmic reticulum often contains intracisternal granules, presumably synthesized by the ribosomes and transported through the cisternae to the region of the Golgi complex (Figs. 7-9). Here they apparently enter the region of the Golgi complex to be fabricated into secretion bodies destined to form the vitelline membrane (Figs. 6—9). How the secretory proteins are packaged by the Golgi complex into vitelline membrane secretion is not clear and experimental studies, such as labelling, seem necessary to establish this point. In any case, the secretion bodies apparently arise by budding from the cisternae of the Golgi bodies (Figs. 5, 6) and they eventually migrate to, and collect under, the plasma

16-2 244 H. W. Beams and R. G. Kessel membrane at the free border of the cell (Figs. 10-13). Here they are eventually discharged between the microvilli by a process of reverse micropinocytosis (Fig. 10) and come to lie in rows within the space between the oocyte and follicle cells (Figs. 11-13). Their arrangement in rows is apparently effected by the microvilli which are well developed on the surface of the follicle cells at this time. As maturation of the oocyte continues, more and more secretion bodies collect in the space between the oocyte and follicle cells. Those first secreted begin to fuse into progressively larger ones until all of them have joined giving rise to a dense continuous membrane (Fig. 4). Successive stages in the fusion of the secretion droplets to form the continuous vitelline membrane are illustrated in Figs. 2-4 and 11 and 12.

Vitellogenesis Before proceeding to a description of the origin and deposition of the chorion, a few observations on vitellogenesis seem appropriate. A detailed study of yolk deposition in the dragonfly has not been made, but preliminary observations indicate that in this organism, as in a number of other insects, the yolk is synthesized elsewhere than in the oocyte and incorporated into it by the process of micropinocytosis (Telfer, 1954, 1961, 1965; Roth & Porter, 1962, 1964; Kessel & Beams, 1963; Anderson, 1964; Favard- Ser6no, 1964; Stay, 1965; King & Aggarwal, 1965; Hopkins & King, 1966). Micro- pinocytosis is extremely active in this organism from the onset of vitellogenesis until its completion (Figs. 14, 15). Present in the region between the follicle cells and the oocyte, in addition to the dense precursor vitelline membrane droplets, is a layer of loosely arranged, small and irregularly shaped bodies of coagulated material (Figs. 14, 15). This same material appears to be located in spots on the oocyte plasma membrane and may constitute the precursor protein yolk substance. In fact, the pits between the microvilli of the oolemma are coated with a material of similar structure and density, and it is probable that the pinocytotic vesicles (coated vesicles) represent the method of transport of this presumed precursor protein yolk into the oocyte. As reported by Roth & Porter (1962, 1964), the pits are composed of three layers, listed from the outside inward as follows: coated layer, plasma membrane, and bristle layer (Figs. 14, 15). As the pinocytotic vesicles become detached from the surface they migrate inward and fuse with one another to form relatively large bodies which eventually become the dense definitive yolk spheres.

Chorion formation After formation of the vitelline membrane, the follicle cells no longer secrete precursor vitelline membrane substance, but turn to an entirely different function; namely, the secretion and laying down of the chorion which differs both morpho- logically and chemically from the vitelline membrane (King, i960). Light-microscope observations of azure II-methylene blue-stained preparations show the chorion to be more deeply stained than the vitelline membrane (Fig. 4). Scattered within the follicle cell cytoplasm and aggregated just below the cell surface are many small bodies which under the electron microscope are revealed to be precursor chorion secretion droplets (Figs. 16—21). A condition consistently observed in follicle cells actively secreting Synthesis, deposition of insect egg envelopes 245 precursor chorion is the rather wide and vacuolated intercellular spaces (Fig. 4). These probably function in the transport of materials from the haemocoel to the space between the follicle cells and oocyte. Here too (Fig. 4) may be observed the relationship between the oocyte, vitelline membrane, developing chorion and follicle cells. Electron micrographs of the developing chorion show that it is composed of two components; an outer relatively narrow and homogeneous dense layer and an inner, thicker filamentous layer (Figs. 16, 17). With respect to the oocyte surface, the thin, homogeneous layer represents the endochorion while the thicker, filamentous layer represents the exochorion. We have not observed the formation of the endochorion layer and the results reported here refer to the secretion and fabrication of the outer layer only (exochorion). It is not clear how the exochorion differs from the endo- chorion, but both layers are secreted by the follicle cells. Perhaps in the transition from vitelline membrane to chorion secretion a thin layer composed of elements of both membranes is formed, the endochorion. Figure- 17 is a low magnifi- cation electron micrograph of a follicle cell illustrating the relative amount and distribution of the endoplasmic reticulum, Golgi bodies, mitochondria, secretion droplets and chorion. At this stage, the cell contains extensive amounts of rough- surfaced endoplasmic reticulum and the Golgi bodies are numerous. A detailed study of the Golgi complex reveals it to be composed of the usual components; namely, smooth-surfaced cisternae, large vesicles, and small vesicles (Figs. 16,18-20). However here as in vitelline membrane secretion, it should be pointed out that not all of the components of the Golgi complex are consistently well displayed. Whether or not this is due to inadequate fixation or to a characteristic of the Golgi body is not known. The Golgi complex is immediately surrounded by cisternae of the endoplasmic reticulum, but unlike the condition of previtelline membrane synthesis, intracisternal presecretion bodies are not sufficiently numerous to appear significant. Hence, intracisternal precursor proteinaceous chorion secretion, if present, is not in a state of formed bodies, but probably exists as a diffuse material. Where the cisternae of the endoplasmic reticulum adjoin the Golgi complex, they lose their ribosomes and appear closely related to small vesicles which enter, and become a part of the Golgi complex (Figs. 16, 18-21). The small vesicles probably contain ribosomal-synthesized proteinaceous substance which will somehow enter the Golgi cisternae where it will be mixed with material synthesized by the Golgi cisternae and fabricated into pre- chorion secretion granules. It is clear that the prechorion secretion filaments make their first appearance within the cisternae of the Golgi saccules and vesicles (Figs. 16, 18- 20). How the filaments are discharged from the cisternae, if they are discharged, is difficult to determine; do they pinch off from the periphery of the exposed Golgi cisternae and round up (Figs. 19, 20), or does an entire Golgi cisterna together with its intracisternal secretion become dissociated on the 'maturing face' from the remaining cisternae? In any case, as the filamentous precursor secretion body moves away from the Golgi cisternae, it undergoes coiling and gives rise to bodies of the type seen in Figs. 16-21. Several secretion bodies may be present in the region of a single Golgi complex and some may be more tightly coiled and dense than others (Figs. 16- 21). 246 H. W. Beams and R. G. Kessel Because the developing chorion at this stage is so tightly apposed to the follicle cell surface, it is difficult to determine the nature of the follicle cell-surface modification, but it appears that the microvilli have been replaced with infoldings of the plasma membrane. The coiled filamentous presecretion bodies migrate to the region below the free surface of the cell where they contact the plasma membrane between and at the base of the folds (Figs. 22-25). Eventually they are ejected from the cell and upon becoming free appear to uncoil. The elongate filaments, which probably vary in length, migrate between the folds of the plasma membrane to join and become incorporated into the chorion (Figs. 22-25). I* is difficult to be certain of the orienta- tion of the filaments making up the exochorion. In Fig. 25 most of them are cut transversely, but some are sectioned longitudinally. Electron-microscope studies have not been made on the completely formed chorion of the newly laid dragonfly egg; however, this has been done for the grasshopper egg (Slifer & Sekhon, 1963).

DISCUSSION Considerable confusion exists in the early literature concerning the identification and origin of insect oocyte envelopes (see Slifer, 1937 for discussion). Much of the debate has centred around the question '... as to whether the follicle cells or oocytes play the more active role in the secretion of the vitelline membrane' (King & Koch, 1963). This is due, in part, to the difficulty of adequately fixing and sectioning maturing oocytes and to the limiting resolving power of the light microscope. Most of the evidence indicates that the vitelline membrane of insects is produced by the follicle cells (e.g. Raven, 1961; King & Koch, 1963; Favard-Sereno, 1966); however, in some organisms it is said to be a product of the oocyte (Okada & Waddington, 1959), or a combination of both the oocyte and follicular cells (Hopkins & King, 1966). King & Koch (1963) have presented good evidence that in Drosophila mutants, 'tiny' and 'female sterile', the vitelline membrane may be laid down adjacent to cells other than the oocyte, a condition strongly implicating the follicle cells as the source of the vitelline membrane. It is commonly observed that the vitelline membrane forms from a collection and fusion of dense globules in the space between the oocyte and follicle cells. However, except for a short note by Favard-Sereno (1966) clear ultrastructural evidence for the synthesis and deposition of the insect oocyte membranes (vitelline, chorion) has not been revealed. King & Koch (1963) have described in Drosophila follicle cells, during the period of active secretion of the vitelline membrane, small spherical sudanophil bodies sur- rounded by concentric layers (whorls) of rough-surfaced endoplasmic reticulum. These have been termed 'epithelial bodies' and the lipid component is thought to be derived from the surrounding endoplasmic reticulum. Since the epithelial bodies are present only during the period of the laying down of the vitelline membrane, it is suggested that they contribute in some unknown way to its formation. In Bombus, two kinds of follicular cells are present: a common, large one, thought to be active during the secretion of albuminous yolk and a less common, narrow one Synthesis, deposition of insect egg envelopes 247 which presumably secretes protein that is transferred to the oocyte before albuminous yolk secretion starts (Hopkins & King, 1966). Hopkins & King (1966) observed the vitelline membrane to be composed of more than one layer. Such a layering of the vitelline membrane has not been observed in this study. However, probably great variation in its gross morphology occurs in different species of insects. Of special interest in connexion with the results reported here are the observations recorded by Favard-Sereno (1966) dealing with the origin and structure of the egg membranes of Gryllus. She observed that the follicle cells show two stages of secretion: the first being the secretion of the vitelline membrane and the second, the secretion of the chorion. In stage one, the follicle cells show a large amount of rough-surfaced endoplasmic reticulum and a typical dictyosome or Golgi body. Within the cisternae of the Golgi complex are seen dense previtelline secretion droplets which are eventually elaborated from the cisternae of the Golgi complex and migrate to the surface of the cell, where they are extruded and come to lie in the space between the oocyte and follicular cells. These extracellular bodies are thought to fuse, forming the vitelline membrane. During the secretion of the chorion, the Golgi saccules become highly vacuolated and filled with a dense fibrous material. This material is detached from the Golgi saccules and migrates to the apical end of the cell where it is discharged into the space between the follicle cells and vitelline membrane. This brief account by Favard- Ser^no (1966) on the origin and deposition of the oocyte membranes in the cricket concurs in a general way with that described in this paper, and it is the first description to show clearly the origin of egg membrane presecretion substance within the Golgi saccules. It seems well established that the exocrine secretion of the guinea-pig pancreas is synthesized by the rough-surfaced endoplasmic reticulum and transported in its cisternae to the condensing vacuoles of the Golgi complex where it is converted to zymogen granules (Palade, Siekevitz & Caro, 1962; Caro & Palade, 1964; Jamieson & Palade, 1967a, b). In general, other secretions containing a high percentage of protein have been described as originating in a similar manner (e.g. Beams & Sekhon, 1966; Kessel, 1966). On the other hand, equally good evidence has been presented that the Golgi complex in some cells is the site of synthesis of complex carbohydrate and, also, that protein synthesized by the endoplasmic reticulum may be joined to complex carbo- hydrate in the Golgi region (cf. Neutra & Leblond, 1966a, b). Since both the vitelline membrane and chorion are composed of protein, carbohydrate and lipid (King & Koch, 1963), it is not unreasonable to su^,j:-; :hat perhaps the protein component is synthe- sized by the rough-surfaced endoplasmic reticulum and the carbohydrate component by the Golgi complex. It is apparent that the Golgi complexes (and probably the endoplasmic reticulum as well) in the dragonfly follicle cells are concerned with the elaboration of at least two distinct cell products during the course of their life history. Instances in which the Golgi complexes of a single cell take part in the formation of more than one kind of secretion are not numerous. However, in polymorphonuclear leucocytes, Bainton & Farquhar (1966) described two distinct types of granules both of which were 248 H. W. Beams and R. G. Kessel produced by the Golgi complex. However, they are elaborated at different times during maturation of the cell and originate from different 'faces' of the Golgi complex. Vitellogenesis in insects has been extensively studied and it has been demonstrated conclusively by Telfer (1954, 1961, 1965) that the precursor proteinaceous yolk substance in the saturniid moths is synthesized outside the ovary and transported to it in the haemolymph. Telfer suggests that the blood proteins reach the oocyte surface by diffusion through the spaces between the follicle cells and that they combine selectively with certain carrier molecules and enter the ooplasm within pinocytotic vesicles which transform into yolk spheres. The general conclusions of Telfer have been confirmed by Roth & Porter (1962) and Stay (1965) and preliminary radioauto- graphic experiments suggest that in the mosquito the precursor proteinaceous sub- stance may be formed in the mid-gut where it is transported in the haemolymph to the oocyte (Roth & Porter, 1964). Roth & Porter (1962, 1964) were apparently the first to illustrate the mechanism of protein uptake by insect oocytes. Between the microvilli of the oocyte they observed numerous pit-like invaginations of the oolemma. Four to 7 h after a blood meal, marked changes occur in the periphery of the oocyte; the microvilli increase in length and many new pits are formed by invagination of the plasma membrane. The pits are composed of a layer or coat of protein on their concave extracellular side, plasma membrane, and a layer of bristles on their convex or protoplasmic side. Mature pits are pinched off from the oocyte surface into the cortical ooplasm and become known as coated vesicles. Eventually, the coated vesicles lose their bristles and fuse into large dense yolk spheres. This is essentially the same basic process of yolk uptake as is herein described for the dragonfly. Of possible physiological significance is the fact that during active vitellogenesis in the dragonfly, a well-developed space occurs between the follicle cells (Telfer, 1961, 1965; Roth & Porter, 1964; Stay, 1965; King & Aggarwal, 1965). This condition, as pointed out by Telfer (1961, 1965) and Roth & Porter (1964), is probably an adaptation of the follicle for transport of protein from the haemolymph to the oocyte surface. Consistently demonstrated in the follicle cells, mainly during the vitelline membrane secretion period, are numerous relatively clear disk-shaped vesicles. A coagulated material of low density is present within them and they appear to be lined by a membrane. Probably the disk-shaped vesicles are comparable to those reported in the 'green gland' of the crayfish (Anderson & Beams, 1956), urinary bladder epithelium (Rhodin, 1963) and ureter epithelium (Hicks, 1965). Porter, Kenyon & Badenhausen (1967) have observed that upon injection of ferritin into the urinary bladder it even- tually appears in the disk-shaped vesicles, strongly suggesting that the latter are at some time connected with the surface. Porter et al. (1967) suggest that the disk-shaped vesicles are derived from a fusion at the margins of deeply invaginated plaques on the plasma membrane. Since so little is known of the function of these bodies further discussion of them here seems unwarranted. This study was supported by research grants (HD-00699; RG-5706; GM-09229; RG-5479) from the National Institutes of Health, U.S. Public Health Service and the National Science Foundation (G-9879). Synthesis, deposition of insect egg envelopes 249

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Fig. 1. Phase-contrast photomicrograph of living ovariole showing terminal filament (tf), germarium (gm), vitellarium (vi) and interfollicular cells (ifc). X35. Fig. 2. Follicle cells actively engaged in the synthesis of the vitelline membrane. Note rounded surface of cells facing the oocyte, disk-shaped clear vesicles (dv), small secretion droplets and the alignment in rows of previtelline secretion bodies (vm) within the space between the follicle cells and oocyte. Present also are small yolk bodies just underneath the oocyte surface (os) and larger yolk spheres more deeply situated in the ooplasm (ys). Epon section, azure II-methylene blue stain, x 2500. Fig. 3. Follicle cell near the end of vitelline membrane formation. Note small secretion granules (sg) which appear to be enclosed in less dense areas, probably the Golgi bodies, disk-shaped relatively clear vesicles, nearly complete fusion of vitelline secretion bodies (tim) to form the vitelline membrane and peripheral region of oocyte con- taining yolk spheres (ys). Epon section, azure II-methylene blue stain, x 5000. Fig. 4. Photographs of more mature oocyte displaying the relative positions of its components. They are listed from the outside inward as follows: basement lamina (Jj[), follicle cell with small secretion granules, intercellular space (is), developing dense chorion membrane (an), vitelline membrane (vm) and periphery of oocyte including yolk spheres (ys). Epon section, azure II-methylene blue stain, x 2000. Synthesis, deposition of insect egg envelopes 252 H. W. Beams and R. G. Kessel

Fig. 5. Low-magnification electron microgTaph of follicle cell in the initial stages of vitelline membrane secretion (vs). Observe an extensive development of rough- surfaced endoplasmic reticulum (er) and numerous Golgi bodies (gb). Mitochondria (ni) show no usual features, x 25 000. Synthesis, deposition of insect egg envelopes 254 H- w- Beams and R- G- Kessel

Fig. 6. Detailed fine structure of Golgi bodies: Golgi cisterna (gc), Golgi vesicles (gv), small vesicles (sv), vitelline secretion (vs), microtubule (mt). x 50000. Figs. 7-9. Surrounding the Golgi bodies are numerous cisternae of the rough-surfaced endoplasmic reticulum which contain intracistemal granules {ig). Where the endo- plasmic reticulum adjoins the Golgi body, it becomes smooth-surfaced and the intracistemal granules are probably budded into the region of the Golgi complex. Vitelline secretion bodies are formed within and budded from the Golgi cisternae. Fig. 7, x 20000; Fig. 8, x 72500; Fig. 9, x 54000. Synthesis, deposition of insect egg envelopes 255- 256 H. W. Beams and R. G. Kessel

Fig. 10. Vitelline presecretion bodies (vs) apparently in the process of becoming ejected from the cell. Many microtubules (mi) are present, particularly in the cortex of the cell, x 50000. Fig. 11. Stage of active secretion of vitelline membrane substance. Note vitelline secretion bodies at periphery of follicle cell (vs), rows of secretion bodies in space between follicle cell and oocyte (vm), microvilli (mv). On surface of oocyte at right in picture are surface pits (sp), flocculated material attached to and near the surface of the oocyte (/TO) and coated vesicles (cv). x 19000. Fig. 12. Later stage in vitelline membrane deposition showing the fusion of the secretion bodies (vm) (compare with Fig. 3, vm). x 13500. Synthesis, deposition of insect egg envelopes

Cell Sci. 4 H. W. Beams and R. G. Kessel

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Fig. 13. Displayed in the follicle cell actively secreting vitelline membrane substance are the following: copious amounts of rough-surfaced endoplasmic reticulum (er), numerous Golgi bodies (gb), vitelline secretion droplets, both in the region of the Golgi body and near the surface of the cell (vs), extracellular large vitelline bodies (vm) and microvilli (mv). Present are numerous disk-shaped vesicles {dv) which appear to contain low-density material and in some cases appear surrounded by a limiting membrane (Im). x 19000. Synthesis, deposition of insect egg envelopes 259

Figs. 14, 15. Surface of oocyte active in yolk uptake. Shown are surface pits (arrows), flocculated material (Jm), coated vesicles (cv) and enlarging yolk spheres (Fig. 14, ys). Microvilli are also present on the surface (mv). Fig. 14, x 34000; Fig. 15, x 72500.

17-2 260 H. W. Beams and R. G. Kessel

Figs. 16-21. Portions of follicle cells actively secreting prechorion substance (cs). Fig. 17 is an electron micrograph showing the relationship of oocyte to its surrounding membranes. Present within the follicle cells are numerous Golgi bodies (gb), chorion presecretion bodies (cs), rough-surfaced endoplasmic reticulum (er). Figs. 16, 18-21 show in more detail the origin of the prechorion secretion with the Golgi cisternae (gc). The secretion seems to be laid down within the Golgi cisternae as dense filaments

(continued on p. 262) Synthesis, deposition of insect egg envelopes 262 H. W. Beams and R. G. Kessel

(Figs. i6, i8-2i,£c). The secretion apparently leaves the cisternae, either by budding (Figs. 19, 20, gc), or by a rounding up of the whole cisterna which becomes segregated as a secretion body composed of concentric layers of dense material (Figs. 19, 20, cs). In the peripheral region of the Golgi complex are many small vesicles (sv) which appear to be derived from the rough-surfaced endoplasmic reticulum and utilized by the Golgi cisternae in fabricating the secretion body. Figs. 16, 17 display the fine structure of the endochorion (enc) and the exochorion (exc). Fig. 16, X5oooo;Fig. 17, X25000; Figs. 18, 19 and 21, x 50000; Fig. 20, x 72500. Synthesis, deposition of insect egg envelopes 263