Is the nuclear envelope a ‘generator’ of membrane?

Summary Early diplotene oocytes from Necturus maculosus ranging from 0.2 to 0.5 mm in diameter were examined by electron microscopy. In the smallest oocytes of this range, the cytoplasm is largely devoid of membranes, but contains primarily ribosomes and mitochondria. In slightly larger oocytes, smooth-surfaced cytomembranes first appear in the perinuclear cytoplasm. At this time, the outer layer of the germinal vesicle nuclear envelope (GVNE) shows frequent connections with long membranous lamellae that extend for considerable, but variable distances into the juxtanuclear ooplasm. The number of smooth membranous lamellae increases tremendously as the oocytes increase in diameter. In such oocytes as well, frequent continuities are observed between the outer membrane of the GVNE and many of the cytoplasmic membranes. Eventually, as the ooplasm becomes populated with extensive numbers of membranous lamellae, instances of continuity between the membranous lamellae and nuclear envelope now become sparse and eventually non-existent. The frequent connections observed between membranous lamellae and the outer membrane of the GVNE during a circumscribed interval of diplotene strongly implicate the GVNE in the generation of extensive amounts of cytoplasmic membrane. The ooplasm of larger oocytes in the size range indicated contain numerous Golgi complexes and large quantities of annulate lamellae most of which are positioned in the peripheral or subcortical ooplasm, as well as extensive quantities of smooth membranes of the endoplasmic reticulum and lipid droplets.     

The Origin and Fate of Annulate Lamellae in Maturing Sand Dollar Eggs

Electron micrograph evidence is presented that the nuclear envelope of the mature ovum of Dendraster excentricus is implicated in a proliferation of what appear as nuclear envelope replicas in the cytoplasm. The proliferation is associated with intranuclear vesicles which apparently coalesce to form comparatively simple replicas of the nuclear envelope closely applied to the inside of the nuclear envelope. The envelope itself may become disorganized at the time when fully formed annulate lamellae appear on the cytoplasmic side and parallel with it. The concept of interconvertibility of general cytoplasmic vesicles with most of the membrane systems of the cytoplasm is presented. The structure of the annuli in the annulate lamellae is shown to include small spheres or vesicles of variable size embedded in a dense matrix. Dense particles which are about 150 A in diameter are often found closely associated with annulate lamellae in the cytoplasm. Similar structures in other echinoderm eggs are basophilic. In this species, unlike other published examples, the association apparently takes place in the cytoplasm only after the lamellae have separated from the nucleus. If 150 A particles are synthesized by annulate lamellae, as their close physical relationship suggests, then in this species at least the necessary synthetic mechanisms and specificity must reside in the structure of annulate lamellae.     

The origin and fate of annulate lamellae in maturing sand dollar eggs

Electron micrograph evidence is presented that the nuclear envelope of the mature ovum of Dendraster excentricus is implicated in a proliferation of what appear as nuclear envelope replicas in the cytoplasm. The proliferation is associated with intranuclear vesicles which apparently coalesce to form comparatively simple replicas of the nuclear envelope closely applied to the inside of the nuclear envelope. The envelope itself may become disorganized at the time when fully formed annulate lamellae appear on the cytoplasmic side and parallel with it. The concept of interconvertibility of general cytoplasmic vesicles with most of the membrane systems of the cytoplasm is presented. The structure of the annuli in the annulate lamellae is shown to include small spheres or vesicles of variable size embedded in a dense matrix. Dense particles which are about 150 A in diameter are often found closely associated with annulate lamellae in the cytoplasm. Similar structures in other echinoderm eggs are basophilic. In this species, unlike other published examples, the association apparently takes place in the cytoplasm only after the lamellae have separated from the nucleus. If 150 A particles are synthesized by annulate lamellae, as their close physical relationship suggests, then in this species at least the necessary synthetic mechanisms and specificity must reside in the structure of annulate lamellae.     

An Electron Microscope Study of the Salamander Thyroid during Hormonal Stimulation

Cytological changes in thyroid glands following administration of thyroid-stimulating hormone (TSH), were studied in adult salamanders, Ambystoma tigrinum, Triturus torosus, and Triturus viridescens by electron and light microscopy. Thyroids from untreated salamanders contained large follicles, faintly basophilic colloid, low follicle cells with flattened nuclei, and scant, slightly basophilic cytoplasm. After TSH administration the cell height and nuclear volume increased. Cytoplasmic basophilia was markedly increased and follicle lumina were reduced. In electron micrographs, stacks of ergastoplasmic lamellae appeared near the nucleus occasionally in contact with the nuclear membrane. In more advanced stages of stimulation, lamellar arrays were largely replaced by small disoriented vesicles and larger vacuoles containing colloid-like material. Sections of obliquely oriented ergastoplasmic membranes contained rows of extremely fine particles. Microvilli increased in size and number and Golgi structures became more extensive. Homogeneous osmiophilic droplets increased in size and abundance. Some of the smaller droplets were seen associated with the Golgi zone. Droplets similar in size and density frequently contained closely packed, whorled lamellae. Mitochondria showed no structural changes but occurred in aggregates interposed between the nucleus and highly folded portions of the basal cell membrane.     

Differentiation of Acmaea digitalis oocytes with special reference to lipid-endoplasmic reticulum-annulate lamellae-polyribosome relationships

During initial stages of oogenesis, many nucleoli are adpressed to the inner membrane of the nuclear envelope. Small nucleolar fragments appear to traverse the pores of the nuclear envelope and accumulate in the perinuclear ooplasm as fibrogranular bodies. Mitochondria become closely associated with some of the fibrogranular bodies. In addition to ribosomes and polyribosomes that are present in small oocytes, lamellae of rough-surfaced endoplasmic reticulum (rER) increase greatly in number during early stages of differentiation. Some individual lamellae are attached at their ends to the outer membrane of the nuclear envelope. Many parallel lamellae of rER are then encountered as well as numerous circular profiles consisting of concentric loops of rER. Soon after the differentiation of the extensive system of rER, lipid droplets or spheres appear in the ooplasm and they are initially surrounded by many circular, concentric lamellae of rER. Initially, the number of concentric lamellae of rER surrounding a lipid droplet may vary from less than a dozen to more than two dozen. During middle and late phases of vitellogenesis, most of the lipid spheres that comprise the most numerous and significant component of the yolk are surrounded by only one or two concentric lamellae of rER (in some cases the lamellae are part rough-surfaced and part smooth-surfaced). In addition, annulate lamellae are then observed to be associated with a portion of the lipid droplet surface. The number of annulate lamellae that extend focally from the lipid sphere distally into the cytoplasm is variable; often two or three to more than a dozen lamellae. Small granules, many of which range from 6 to 12 nm and thin fibrils (approximately 2–3 nm in width) may be associated with the annulate lamellae. In addition, polyribosomes frequently appear to be continuous with the pore-associated material of the annulate lamellae. The ends of some annulate lamellae may extend as lamellae of the rER. The morphologic relationships and relationships and variations observed between the lipid droplets, rER, annulate lamellae, and polyribosomes during lipidogenesis in this oocyte are interpreted to support a recent hypothesis (Kessel, 1981a,b) that the pores of annulate lamellae may be involved in some manner with the processing of ribosomal subunits or precursors into functioning polyribosomes, and that their appearance in specific association with the surface of many lipid spheres and rER in the oocyte late in vitellogenesis may be related to the formation of additional functional polyribosomes necessary to complete the final synthesis of many lipid droplets that are present in the ooplasm of the full-grown oocyte.     

THE FINE STRUCTURE OF ANNULATE LAMELLAE

Certain lamellar structures have been described from snail (Otala) and clam (Spisula) oocytes, the acinar cells of amphibian (Ambystoma) pancreas, and from rat spermatids. These structures are alike in possessing numerous rings or annuli, resembling those in the nuclear membrane. Thus the name "annulate lamellae" has been proposed for them. It is suggested that they may function in the transfer of specificities from nucleus to cytoplasm.     

Fine structural observations on nuclear maturation during spermiogenesis in Hydra littoralis

Cytodifferentiation during spermiogenesis in Hydra littoralis was studied at the fine structural level. Concentration of nuclear material as well as specific orientation of granular and filamentous nuclear elements are apparent in two regions of the early spermatid: where the nuclear envelope is in contact with mitochondrial membranes at one pole of the cell and at an opposite region where the nucleus is closely apposed to the plasma membrane. Ultimately the mass of condensed nuclear material becomes concentrated at the mitochondrial pole of the cell. Additional electron-dense material is extruded from the nucleus into a large vacuole which is in continuity with the nuclear membrane as well as associated with Golgi lamellae and vesicles. Eventually all residual cytoplasm is sloughed, leaving the nucleus, mitochondria, and flagellum. These observations are suggestive of nucleocytoplasmic interactions during development, especially influences of mitochondria and plasma membranes on chromatin condensation.     

OBSERVATIONS ON THE STRUCTURE OF RHODOSPIRILLUM MOLISCHIANUM

The lamellae of the bacterium Rhodospirillum molischianum originate as extensions of the cytoplasmic membrane into the cytoplasm of the cell. Initially, these extensions are narrow folds and occur independently of one another. The first lamellae to appear average about 80 A in width, representing one side of the infolded cytoplasmic membrane, or 160 A when the two sides of the fold are closely appressed. The 160-A lamellae increase in number and may associate to form larger lamellae, which represent varying degrees of association between adjacent folds. Later, the space within each fold increases; the two appressed regions of the cytoplasmic membrane in each fold separate to form distinct invaginations, and the lamellae observed at this stage are formed by an association of the sides of adjacent invaginations.     

An unusual membrane system in the oocyte of the ascidian Botryllus schlosseri

During vitellogenesis, oocytes of Botryllus schlosseri always exhibit an unusual system scattered in the cytoplasm. It consists of an association between a single fenestrated endoplasmic reticulum cisterna and one or a few smooth vesicles (cisterna vesicle association: CVA) containing a dense core facing the cisterna itself. The latter is smooth and perforated by numerous small pores (about 25 nm in diameter) in the area of association; towards the periphery, it extends into several branches with ribosomes bound to their membranes. In the vesicles, fibrillar material radiates from the dense core and is sometimes organized into a long, dense lamina. The membranes of both cisterna and vesicles appear to be coupled, but are in fact separated by a constant narrow space occupied by short densities. The presence in B. schlosseri of this unusual fenestrated membrane system contrasts with the absence of a typical porous cytoplasmic organelle, the annulate lamellae (ALs), which is widely distributed in female gametes. However, as in other animals, B. schlosseri oocytes possess intranuclear annulate lamellae (IALs) and vesicles. Comparative observations extended to the oocytes of the ascidian Ciona intestinalis have shown that the latter species exhibits typical ALs and IALs, but not the CVA. The morphology of the CVA is analysed here in detail, and similarities and differences with ALs are pointed out. Hypotheses regarding CVA function are discussed in terms of possible relations with ALs.     

Intranuclear membranous inclusions in oocytes of a viviparous teleost (Xiphophorus helleri).

Intranuclear inclusions were observed in oocytes of Xiphophorus helleri during prophase I. In osmium-fixed leptotene nuclei, the inclusions were made up of groups of membrane-limited vesicles or tubules with pale contents, situated near the inner nuclear membrane with which some of them exhibited apparent continuities. In zygotene nuclei, larger vesicles also appeared bounded by two or three membranes and containing tubules apparently invaginated from their walls. In pachytene-dictyate nuclei most vesicular bodies had a wall formed by stratified membranes, or were entirely made up of membranous whorls. In glutaraldehyde-osmium fixed material some of these myeline-like bodies showed a peculiar arrangement, consisting of concentric bands each containing thick inner dense lamellae 2-0-3-0 nm thick and a 5-0 nm outer lamella. It is suggested that these inclusion bodies arise from the inner nuclear membrane of oocytes when cells start to grow intensely during prophase I. The bodies seem to become more complex at late prophase, probably by association of individual vesicles and the occurrence of multiple membrane invaginations, which may be related to active metabolic phenomena taking place at this stage in oocytes.     

Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

The ultrastructural differentiation of the endoplasmic reticulum in choroidal epithelial cells of the chick embryo

During the development of the choroidal epithelium in the chick embryo, a substantial concentration of granular endoplasmic reticulum differentiates in the subnuclear cytoplasm of the epithelial cells. The formation of the membranous components of this organelle is preceded by the appearance of a dense, localized population of small, free polyribosomes. Subsequently, numerous membrane-bound vesicles appear in the perinuclear cytoplasm. These primordial ER vesicles measure from 0.1 μ to 0.5 μ or more and they originate from evaginations of the outer nuclear membrane. These vesicles commonly occur in successive rows situated around the margin of the nucleus, and they expand and/or interconnect to form incipient ER tubules. Most vesicles and early tubules are smooth to nearly smooth in appearance. With continued development nuclear evaginations cease, and ER tubules expand in Situ to form an elaborate, laminated system of 7–12 ‘bag-like’ cisternae. Throughout this period of expansive growth, small polyribosomes attach to the developing ER cisternae. As the ER cisternae progressively attain their granular appearance, the number of small, free polyribosomes diminishes. During later stages of development larger polyribosomes appear in association with the subnuclear concentration of ER, and the first accumulations of electron-dense material develop within cisternal spaces.     

ZYGOTE FORMATION IN ASCARIS LUMBRICOIDES (NEMATODA)

Ultrastructural observations of the in utero sperm of Ascaris lumbricoides reveal that it consists of a relatively clear, ameboid anterior region and a conical posterior region containing numerous surface membrane specializations, dense mitochondria, a lipid-like refringent body of variable size, and a dense nucleus which lacks an apparent nuclear envelope. No acrosomal complex was observed. Pseudopods emanating from the anterior cytoplasm make first contact with the primary oocytes and appear to be responsible for the localized removal of the extraneous coat covering the oolemma. Subsequently the gamete membranes interdigitate and finally fuse. Because this pseudopodial action appears similar to that reported for the acrosomal filaments in flagellated sperm, the anterior region of the Ascaris sperm is thought to serve an acrosomal function. Following gamete-membrane fusion, the sperm nucleus acquires a particulate appearance and becomes disorganized. Once inside the oocyte, the sperm cytoplasm consists of dense mitochondria, ribosomes, and vesicles derived from the surface membrane specializations. The refringent body, whose contents possibly contribute to the synthesis of ribosomes, is usually absent by the time the sperm cytoplasm attains a central position in the egg.     

Intranuclear annulate lamellae in oocytes of the tunicate,Styela partita

Summary Intranuclear annulate lamellae have been observed with the electron microscope in oocytes of the tunicate, Styela partita. Morphological evidence suggests that the annulate lamellae may arise by a specialized fusion process of individual vesicles. Intranuclear vesicles appear to be formed, in time, before differentiated annulate lamellae. It is also suggested that the position and structure of an annulus is in large part determined by the fusion of the vesicles. An annulus may be present as soon as two vesicles have completed their fusion process. Finally, it is again suggested on the basis of morphological evidence that the intranuclear vesicles are derived by the blebbing activity of the inner layer of the nuclear envelope.This investigation was supported by grants (RG-9229, 9230) from the National Institutes of Health, Public Health Service. The electron microscope facilities used were also supported by a grant (GM-05479) from the National Institutes of Health to Professor H. W. Beams.     

A POSSIBLE MECHANISM FOR THE MORPHOGENESIS OF LAMELLAR SYSTEMS IN PLANT CELLS

A mechanism for the formation of lamellar systems in the plant cell has been proposed as a result of electron microscope observations of young and mature cells of Nitella cristata and the plastids of Zea mays in normal plants, developing plants, and certain mutant types. The results are compatible with the concept that lamellar structures arise by the fusion or coalescence of small vesicular elements, giving rise initially to closed double membrane Structures (cisternae). In the chloroplasts of Zea, the cisternae subsequently undergo structural transformations to give rise to a compound layer structure already described for the individual chloroplast lamellae. During normal development, the minute vesicles in the young chloroplast are aggregated into one or more dense granular bodies (prolamellar bodies) which often appear crystalline. Lamellae grow out from these bodies. In fully etiolated leaves lamellae are absent and the prolamellar bodies become quite large, presumably because of inhibition of the fusion step which appears to require chlorophyll. Lamellae develop rapidly on exposure of the plant to light, and subsequent development closely parallels that seen under normal conditions. The plastids of white and very pale green mutants of Zea similarly lack lamellae and contain only vesicular elements. A specialized peripheral zone immediately below the double limiting membrane in Zea chloroplasts appears to be responsible for the production of vesicles. These may be immediately converted to lamellae under normal conditions, but accumulate to form a prolamellar body if lamellar formation is prevented, as in the case of etiolation and chlorophyll-deficient mutation, or when the rate of lamellar formation is slower than that of the production of precursor material (as appears to be the case in the early stages of normal development).     

THE FINE STRUCTURE OF THE CILIA FROM CTENOPHORE SWIMMING-PLATES

The ctenophore swimming-plate has been examined with the electron microscope. It has been recognized as an association of long cilia in tight hexagonal packing. One of the directions of the hexagonal packing is parallel to the long edge of the swimming-plate and is perpendicular to the direction of the ciliary beat. All the cilia in the swimming-plate are identically oriented. The effective beat in the movement of the swimming-plate is directed towards the aboral pole of the animal, and this is also the side of the unpaired peripheral filament in all the cilia. The direction of the ciliary beat is fixed in relation to the position of the filaments of the cilia. The swimming-plate cilium differs from other types of cilia and flagella in having a filament arrangement that can be described as 9 + 3 as opposed to the conventional 9 + 2 pattern. The central filaments appear in a group of two "tubular" filaments and an associated compact filament. The compact filament might have a supporting function. It has been called "midfilament." Two of the peripheral nine filaments (Fig. 1, Nos. 3 and 8) are joined to the ciliary membrane by means of slender lamellae, which divide the cilium into two unequal compartments. These lamellae have been called "compartmenting lamellae." Some observations of the arrangement of the compartmenting lamelae indicate that they function by cementing the cilia together in lateral rows. The cilia of the rows meet at a short distance from each other, leaving a gap of 30 A only. The meeting points are close to the termini of the compartmenting ridges. An electron-dense substance is sometimes seen bridging the gap. Some irregularities are noted with regard to the arrangement of the compartmenting lamellae particularly at the peripheral rows of cilia. In many cilia in these rows there are small vesicles beneath the ciliary membrane.     

Some observations on spermatogenesis in Gelastocoris oculatus (Hemiptera) with the aid of the electron microscope

The nuclear cap in the spermatogonial and early spermatocyte cells of Gelastocoris is an aggregate of closely packed mitochondria with their long axes perpendicular to the nuclear membrane. Eventually in the early growth period, the mitochondria move from the cap and appear to become more or less equally distributed in the cytoplasm where they remain until their fusion in the spermatid to form the nebenkern. The Golgi complex consists of clusters of lamellae and vesicles, the Golgi bodies. Granules form within the vesicles, increase in size, move from their place of origin and become distributed at random in the cytoplasm. They are the pro-acrosomal granules and at the end of the growth period fuse to form the proacrosome, about which Golgi bodies collect. The Golgi bodies, however, never fuse into an acroblast. At one end of the oval-shaped pro-acrosome is a small dark body and a less dense vesicle the future of which is uncertain. The dark body eventually occupies a position at the tip of the acrosome. The pro-acrosome, after moving to the side of the nucleus opposite the nebenkern, differentiates into the acrosome which elongates into a tail-like structure. The nuclear membrane of some spermatocytes may appear wave-like in cross section, with the crest and trough different in appearance. Near the membrane and in the troughs of the waves large clusters of granules are frequently present. Similar clusters may be found elsewhere in the cytoplasm. Presumably they had their origin near the membrane but this is not conclusive. Bodies of indeterminate origin and structure may be present in the cytoplasm. They could be lysosomes but evidence is lacking. In late spermatocytes and in spermatids, a group of ten or twelve granules is present. They are smaller than the pro-acrosomal granules, are always closely associated and pass as a group into the tail. Their significance is unknown. The endoplasmic reticulum is typical of cells in general. There are no granule accumulations within the vesicles as in some secretory cells. Vesicles of various shapes and sizes are present within the centrosphere of the first meiotic division. While their location is similar to that of the centriole, the identity of the vesicles is uncertain.     

Oogenesis in the terrestrial hermit crab Coenobita clypeatus (decapoda,anomura). I. Previtellogenic oocytes

Ultrastructural study of previtellogenic oocytes found in cystlike clusters scattered throughout the length of the bilobed ovary of the hermit crab Coenobita clypeatus shows a high nuclear:cytoplasm ratio. Large, round nuclei containing synaptinemal complexes serve as good temporal markers for identification of previtellogenic oocytes. The cytoplasm contains many smooth-membraned vesicles filled with granules and probably of nuclear origin. In addition to its complement of Golgi complexes, endoplasmic reticulum, mitochondria, and free ribosomes, the cytoplasm also contains stacks of annulate lamellae, a feature not previously described for decapod oocytes. Typically, the previtellogenic oocyte with its accumulation of ribosomes has the appearance of a nonsynthetic cell preparing to go through a metabolic transition.     

Fine structure of the developing oocytes in adultArgas (Persicargas) arboreus (Ixodoidea: Argasidae)

The structure of the developing oocytes in the ovary of unfed and fed femaleArgas (Persicargas) arboreus is described as seen by scanning (SEM) and transmission (TEM) electron microscopy. The unfed female ovary contains small oocytes protruding onto the surface and its epithelium consists of interstitial cells, oogonia and young oocytes. Feeding initiates oocyte growth through the previtellogenic and vitellogenic phases of development. These phases can be observed by SEM in the same ovary.The surface of isolated, growing oocytes is covered by microvilli which closely contact the basal lamina investing the ovarian epithelium and contains a shallow, circular area with cytoplasmic projections and a deep pit, or micropyle, at the epithelium side. In more advanced oocytes the shell is deposited between microvilli and later completely covers the surface.Transmission EM of growing oocytes in the previtellogenic phase reveals nuclear and nucleolar activity in the emission of dense granules passing into the cytoplasm and the formation of surface microvilli. The cell cytoplasm is rich in free ribosomes and polysomes and contains several dictyosomes associated with dense vesicles and mitochondria which undergo morphogenic changes as growth proceeds. Membrane-limited multivesiculate bodies, probably originating from modified mitochondria, dictyosomes and ribosomal aggregates, are also observed. Rough endoplasmic reticulum is in the form of annulate lamellae. During vitellogenesis, proteinaceous yolk bodies are formed by both endogenous and exogenous sources. The former is involved in the formation of multivesicular bodies which become primary yolk bodies, whereas the latter process involves internalization from the haemolymph through micropinocytosis in pits, vesicles and reservoirs. These fuse with the primary yolk bodies forming large yolk spheres. Glycogen and lipid inclusions are found in the cytoplasm between the yolk spheres.     

ELECTRON MICROSCOPIC STUDY OF THE CYTOPATHOLOGY OF ECHO VIRUS INFECTION IN CULTIVATED CELLS

The cultivated monkey kidney cell is subject to changes when infected with ECHO viruses 6, 9, and 19. The electron microscope reveals three stages of infection: (a) initial stage. The nucleus appears granular with chromatin condensation on the nuclear envelope. The cytoplasm contains electron transparent vesicles and vacuoles forming nests. (b) Intermediate stage. The nucleus seems to diminish, appearing more pycnotic and displaced toward the periphery. The cytoplasm is filled with electron transparent vacuoles and vesicles, and dense masses as well as some spiral bodies are seen. The mitochondria retain their shape. Dense particles are seen, which are possibly of viral nature. (c) Final stage. The nucleus is contracted to a narrow strip close to the cellular membrane or is completely destroyed. The cytoplasm shows no apparent changes. Crystals are frequently observed in cells infected with ECHO viruses 6 and 19, consisting of dense particles with an average diameter of 14.4 mµ ranging from approximately 13.2 to 15.6 mµ for ECHO virus 6, and 14.5 mµ ranging from approximately 12.5 to 16.5 mµ for ECHO virus 19. These particles are clustered in hexagonal packages forming angles of 75° and 105°. The particles in most crystals are arranged in rows separated by a constant distance, the latter varying from one crystal to another and being approximately 1.5 and 2.5 times the distance between particles. Other particles were observed which, however, are not considered to be of viral nature.