Comparative study of the ultrastructure of immune and normal phytohemagglutinin-treated lymphocytes]

Immune lymphocytes sorbed on the surface of the target cells were characterized during the period of the first three hours of combined incubation by the presence of the electron-dense matrix, abundance of mitochondria and lipids; small lymphocytes had disseminated ribosome organized into polysomes in the medium lymphocytes forming individual cysterns of the granular endoplasmic reticulum in the large lymphocytes, this indicating active protein synthesis by these cells. There were also revealed cells of plasmatic type. Cells incubated with the PHA for one hour represented a homogenous population of small lymphocytes of the same size as the clear cytoplasm containing free ribosomes and individual mitochondria. The proportion of the medium lymphocytes and the blasts increased with increase of the incubation period. These are cells with the clear cytoplasm freely disseminated polyribosomes in which no developed granular endoplasmic reticulum was sometimes revealed. The presence of two types of cells whose ultrastructure reflected their functional characteristics is discussed.     

Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary

We have studied the shape and size distribution of membrane-bound polysomes in somatotropes and mammotropes, which are the sources, respectively, of growth hormone and of prolactin in the rat pituitary. The observations were made in conventional electron micrographs of these cells in situ, where occasional surface or en face views of the rough endoplasmic reticulum allow the polysomes to be seen as rows of ribosomes arranged in distinctive patterns on the membranes. It is possible by this means to characterize the shape and number of ribosomes for the total population of bound polysomes in the respective cell types. The great majority of membrane-bound polysomes in these two cell types (81% in somatotropes, 78% in mammotropes) have an approximately circular shape and contain an average of 6.8 (somatotropes) or 6.5 (mammotropes) ribosomes, which is an appropriate size for translation of the polypeptide hormones produced by these cells. About 17% of the membrane-bound polysomes in somatotropes and 20% in mammotropes have a spiral shape, resembling somewhat the letter "G," and contain about eight to nine ribosomes in both cell types. The preponderance of circular polysomes on the rough endoplasmic reticulum of somatotropes and mammotropes suggests the possibility that ribosomes (or the 40S ribosomal subunit) may recycle on the polysome after the translation of growth hormone or of prolactin.     

COTTON EMBRYOGENESIS: THE EARLY DEVELOPMENT OF THE FREE NUCLEAR ENDOSPERM

An electron microscope study was made of the central cell and the development of the free nuclear endosperm surrounding the zygote and synergids during the first three days after pollination. The cytoplasm of the central cell, concentrated around the partially-fused polar nuclei, contains many ribosomes, mitochondria and large, dense, starch-containing plastids, some dictyosomes and lipid bodies, and long, single cisternae of rough endoplasmic reticulum (RER) that frequently terminate in whorls. Dense, core-containing microbodies are closely associated with the RER. After fertilization the cytoplasm of the 2-and 4-nucleate endosperm shows an increase in number of dictyosomes, and in amount of RER which becomes stacked in arrays of parallel cisternae. Cup-shaped plastids are associated with many long, helical polysomes. Perinuclear aggregates of dense, granular material also appear after fertilization. Granular aggregates and helical polysomes disappear after the first few divisions of the primary endosperm nucleus. During the second and third days of development there is an increase in dictyosome number and RER proliferation, and endosperm nuclei become deeply lobed. Concurrently, there is a sharp decline in the starch and lipid reserves of the central cell and elaborate transfer walls are formed at the micropylar end of the embryo sac and on the outer surface of the degenerating synergid. The transfer walls contain groups of small, membrane-bound vesicles, and are associated with large numbers of mitochondria and with the smooth endoplasmic reticulum.     

Cellular characteristics of immunolabeled luteinizing hormone releasing hormone (LHRH) neurons

This study utilized the preembedding immunocytochemical technique in order to identify LHRH-containing neurons in rat brain and define their ultrastructural characteristics. LHRH-containing neurons in the vertical limb of the diagonal band of Broca, medial septum, triangular nucleus of the septum and other regions were studied by taking ultrathin serial sections. These neurons had scant cytoplasm surrounding a centrally-located, spheroid, euchromatic nucleus. Neurosecretory granules were evenly distributed throughout the cell, but many tended to lie directly under the plasmalemma. The cytoplasm was organized in such a way that the most extensive portion of the rough endoplasmic reticulum was polar opposite to areas having high concentrations of Golgi complex, lysosome-like bodies, smooth endoplasmic reticulum and ribosomes. The perikarya had no axosomatic synapses but functional interaction via unspecialized appositions to the plasmalemma cannot be discounted. Many of the perikarya bore at least one cilium. Processes from immunonegative cells were occasionally observed to penetrate the cytoplasm of the LHRH perikaryon or its processes. At their points of origin, dendrites were found to be broadened processes containing many elements common to the cytoplasm: ribosomes, smooth endoplasmic reticulum, cristal and lamellar mitochondria, neurotubules, and an occasional alveolate caveola. Infrequently, some of the LHRH axons were partially myelinated. This method of studying serial-sectioned immunocytochemically-identified cells is suggested as a means of describing the cellular and subcellular characteristics of other specific peptide-containing cells.     

Biosynthesis of microsomal nicotinamide–adenine dinucleotide phosphate–cytochrome c reductase by membrane-bound and free polysomes from rat liver

1. NADPH-ferricytochrome c oxidoreductase (EC 1.6.2.3) was purified from the endoplasmic reticulum of rat liver cells. The methods, which involved digestion of membrane with Steapsin, a crude pancreatic extract containing diastase and trypsin, gel filtration and preparative electrophoresis on polyacrylamide, provided an enzyme with a high specific activity in good yield. 2. The incorporation of (14)C-labelled amino acids into the purified reductase by the incubation of various subcellular fractions was studied. The microsome fraction, bound polysomes, free polysomes and detergent-treated polysomes effected the synthesis of the enzyme. 3. The reductase that had been synthesized by the polysomes was tightly bound to preparations of smooth-surfaced endoplasmic reticulum that were added to the incubation medium. 4. Reductase activity could be detected on both free and detergent-treated polysomes. Evidence is presented to show that this activity was due, at least in part, to the presence on the ribosomes of nascent enzyme. The association of enzyme with detergent-treated polysomes did not appear to be due to contamination of the ribosomes with either membrane or cell sap but it is possible for such ribosomes to adsorb some enzyme. 5. The amount of reductase activity associated with the detergent-treated polysomes was increased when the rats from which the polysomes were derived had been previously injected with phenobarbitone. 6. The results are discussed with respect to their relevance for the question of the existence of two functionally different groups of polysomes in the liver and for current ideas on the biogenesis of membranes.     

Interactions of liver encoplasmic reticulum membranes and polysomes in vitro.

The interactions of various preparations of endoplasmic reticulum membranes and polysomes have been studied by means of a sandwich sucrose gradient that clearly isolates free ribosomes, smooth endoplasmic reticulum (S.E.R.) and rough endoplasmic reticulum (R.E.R.) from the microsomal fraction of rat liver homogenates. Reconstructed rough membranes separate well from the native R.E.R. but occupy the same position along the gradient as the S.E.R. and the rough membranes, stripped of their ribosomes by means of LiCl. Native R.E.R. and S.E.R. do not bind any added labeled polysomes at 0 degree C; previous treatment with LiCl does not modify the behavior of S.E.R. The presence of cell sap during the binding reaction does not increase polysome fixation by stripped-rough membranes but protects in some way the polysomes and preserves all their original functional capacity of amino acid incorporation into protein.     

Ultrastructural features of supramedullary neurons in Crenilabrus quinquemaculatus

The ultrastructural study of the supramedullary neurons in Crenilabrus quinquemaculatus shows that these cells are engaged in intense synthetic activity as is testified by the nuclear morphology, the plentiful rough endoplasmic reticulum and polysomes, and finally the remarkably developed Golgi complexes, many of them active. Moreover the cytoplasm of the supramedullary neurons shows numerous membrane-bounded bodies (1,800-4,000 A) containing electron dense material, more or less finely granular. Research is presently being carried out to establish the meaning of these electron dense bodies.     

Interactions of liver endoplasmic reticulum membranes and polysomesin vitro

Summary The interactions of various preparations of endoplasmic reticulum membranes and polysomes have been studied by means of a sandwich sucrose gradient that clearly isolates free ribosomes, smooth endoplasmic reticulum (S.E.R.) and rough endoplasmic reticulum (R.E.R.) from the microsomal fraction of rat liver homogenates. Reconstructed rough membranes separate well from the native R.E.R. but occupy the same position along the gradients as the S.E.R. and the rough membranes, stripped of their ribosomes by means of LiCl. Native R.E.R. and S.E.R. do not bind any added labeled polysomes at 0°C; previous treatment with LiCl does not modify the behavior of S.E>R. The presence of cell sap during the binding reaction does not increase polysome fixation by stripped-rough membranes but protects in some way the polysomes and preserves all their original functional capacity of amino acid incorporation into protein.     

Ultrastructure of the indifferent gonad in male and female pig embryos.

Pig embryos aged 24 days were obtained from artifically inseminated sows for ultrastructural study of the indifferent gonads. Sex was identified by chromosome analysis. The gonads are composed in both sexes of three different tissues: the surface epithelium, the gonadal blastema and the mesenchyme. The surface epithelial cells contained elongate mitochondria, granular endoplasmic reticulum, free polysomes, the Golgi complex, fine filaments and coated vesicles. The primitive cords were continuous with the surface epithelium and the interior of the gonad was occupied by blastema cells. They had prominent nucleoli, elongate mitochondria, granular endoplasmic reticulum, the Golgi complex, free polysomes, some lipid droplets and occasionally circular smooth membrane profiles resembling the agranular endoplasmic reticulum. Individual primordial germ cells were seen in all parts of the gonad. They were roundish with prominent nucleoli, globular mitochondria, granular endoplasmic reticulum, free polysomes, the Golgi complex, coated vesicles, lipid droplets and dense bodies. Degenerating cells and cells having pseudopods were also encountered. In comparison to the gonad at the age of 22 days, the primordium had grown into a longitudinal roundish protrusion and the number of primoridal germ cells had increased. Histological and ultrastructural observations showed that the pig gonads at the age of 24 days were similar in both sexes.     

LIVER PARENCHYMAL CELL INJURY : I. Initial Alterations of the Cell Following Poisoning with Carbon Tetrachloride

The structure of the endoplasmic reticulum, plasma membrane, mitochondria, and Golgi apparatus of the liver parenchymal cell is strikingly altered within 1 hour following the administration of a single oral dose of carbon tetrachloride to rats. Progressive loss of glucose-6-phosphatase activity accompanies dispersal of the ergastoplasm. Electron microscopy reveals that these changes are associated with vacuolization of the cisternae of the granular endoplasmic reticulum, degranulation of its membranes, and the appearance of increased number of free ribosomes in the adjacent cytoplasmic matrix. Concomitantly, calcium enters the liver parenchymal cell and is sequestered by mitochondria. First increased at 30 minutes, calcium content is maximal at 1 hour and returns to normal at 2 hours. Although succinic and glutamic dehydrogenase activity patterns within the liver lobule are unaffected, liver cell mitochondria enlarge and some appear to fuse or assume cup-like configurations. Microvilli lining the space of Disse become irregularly indistinct and increasingly pleomorphic by 30 minutes when the plasma membrane becomes increasingly permeable to calcium. Golgi vesicles swell and discharge their granules during the period of poisoning studied. Although all the changes observed may be the result of direct interaction of carbon tetrachloride with the membranes of the cytoplasmic constituents of the liver parenchymal cell, the possibility that the irreversible changes observed in the granular endoplasmic reticulum may be due to the chemical interaction between the poison and this system is discussed.     

Colchicine effects on neurosecretory neurons and other hypothalamic and hypophysial cells,with special reference to changes in the cytoplasmic membranes

Summary The morphological effects of colchicine on the entire neurosecretory (NS) tract and on various hypothalamic nuclei have been studied. The perturbation in axonal flow, indicated by the accumulation of NS material, coincide with fragmentation of the cytoplasmic membranes, i. e. the Golgi apparatus and the endoplasmic reticulum, whereas the neurotubules remain relatively well preserved. Autophagic destruction of NS material is observed along the entire length of the NS fibres. The rapid and systematic changes in the axoplasmic reticulum, known to store calcium, lead us to envisage a role for this system — similar to that of the sarcoplasmic reticulum — in controlling the transport of NS vesicles. The junctional zone between the stalk and the neural lobe seems to play a particular rôle in the transport of NS material to the posthypophysial terminals of the NS axons. Colchicine provokes an increase in dense-cored vesicles in most of the neurons of the other hypothalamic nuclei studied: arcuate, suprachiasmatic, periventricular and ventromedial. Membranous alterations are also observed in these sites. Colchicine administered to animals which were hypothyroid, castrated or adrenalectomized, reveals stimulated neurons, identified by their excessive content of dense-cored vesicles. These neurons display no specific localization, for they occur in all hypothalamic nuclei, irrespective of the stimulation. The frequency of stimulation of neurons of the periventricular nucleus is striking.     

Regional differences in the ultrastructure of Purkinje cells of the rat

Summary In the albino rat, perikaryal diameter, volume density of the granular endoplasmic reticulum and Golgi apparatus, and lumenal diameter of cisternae of the granular endoplasmic reticulum are larger in Purkinje cells of lobule Via (neocerebellum) than in those of lobule X (archicerebellum). In contrast, only the surface density of cisternae of the granular endoplasmic reticulum is larger in Purkinje cells of lobule X. The cisternae of granular endoplasmic reticulum are arranged into conspicuous Nissl bodies parallel to the nuclear membrane, but the content of ribosomes and polysemes is markedly less in lobule-X cells than in cells from lobule VI a. These results indicate qualitative and quantitative differences between the metabolically important organelles in Purkinje cells of the neo- and archicerebellum (cf. Larsell 1952).Supported by a grant from the Deutsche Forschungsgemeinschaft (La 184/7)     

The fine structure of continuous human neuroblastoma lines SK-N-SH,SK-N-BE(2), and SK-N-MC

Summary Cell cultures of the continuous human neuroblastoma lines SK-N-SH, SK-N-BE(2), and SK-N-MC at exponential and stationary growth phase have been examined by electron microscopy. At the level of fine structure these cells did not show typical neuronal differentiation such as extensive granular endoplasmic reticulum or neurites with microtubules and neurofilaments. Instead they were characterized by abundant free ribosomes, moderate Golgi complexes, and usually scant granular endoplasmic reticulum, features similar to the fine structure of early normal embryonic autonomic neurons. However, in several respects appearance of differentiated features of the neuroblastoma cells did not follow the pattern observed for normal neurons, suggesting noncoordinate, expression of neuronal phenotypic properties. First, an occasional neuroblastoma cell had as extensive granular endoplasmic reticulum as would be found at later stages in normal developing neurons. Second, the cellular processes of these neuroblastoma cells did not have the fine structure of developing or mature axons in vivo. Third, few dense core vesicles were found in SK-N-SH and SK-N-BE(2), though these organelles are numerous in early normal adrenergic neurons and the adrenergic character of these two lines is apparent from other studies that have demonstrated expression of neurotransmitter synthesizing enzymes (SK-N-MC is cholinergic). The fine structural characterization of these continuous human neuroblastoma cell lines will allow this parameter to be utilized with other approaches in future experimental studies. This work was supported by PSC-BHE Research Award 11612 from the City University of New York and in part by the National Cancer Institute Core Grant CA-08748 to the Sloan-Kettering Institute. E. N. B. was the recipient of a predoctoral fellowship under USPHS Training Grant GM02050.     

Changes in the brain neurons of the recipient around an embryonic nerve tissue transplant

Ultrastructure of adult neurons (sensomotor cortex) of recipient brain tissue localized in the vicinity of developing embryonic nervous tissue transplant has been studied. Partial dedifferentiation of pyramidal neurons has been revealed four days after the transplantation. The following changes were observed: dispersion and nearly complete absence of granular endoplasmic reticulum: appearance of a multitude of small mitochondria and disappearance of large ones; the presence of a large number of free polysomes; marked clearing of nuclei, and the presence of large nucleoli. No destructive processes in neurons were observed.     

Comparative scanning and transmission electron microscopy studies of the ependyma of the central canal in the spinal cord of primates. I. Electron optical image of the ependyma in the central canal of the spinal cord of the callithrix monkey (Callithrix jacchus, Linné 1758)

The ependyma lining the central canal of the spinal cord of adult males and females monkey, Callithrix jacchus, was examined by scanning and transmission electron microscopy. The cross section of the lumen of the central canal are round, oval, or triangular. Light and dark ependymal cells, depending on the density of the cytoplasm, were found. The light ependymal cells are fewer than the dark cells. The ependyma cytoplasm contained numerous mitochondria, filamentous structures, one or more well-developed Golgi-complexes, vesicles of the smooth endoplasmic reticulum, ribosomes, lysosomes, multivesicular bodies, profiles of the rough endoplasmic reticulum, large osmophilic bodies, and microtubules. The nuclei of the ependyma cells usually have a simple, regular round or oval shape. They occupy a relatively large portion of the cell volume and lie in the central or mediobasal position. Some of the nuclei show deep invaginations into the karyoplasm. Most of the mitochondria occupy mainly the supranuclear portion of the apical cytoplasm. There are of the crista-typ. Ribosomes occur free in the cytoplasm, but some attached to the profiles of the rough endoplasmic reticulum or being arranged as polysomes. The filamentous structures are generally prominent cytoplasmic components and are distributed at the apical, lateral, or basal region of the ependymocytes. They are grouped into bundles and arranged in parallel arrays. Some of these bundles reach the plasmamembrane at the free lumina of the central canal, others take contact to the filamentous structures of the zonulae adherentes of the junctional complex below the free surface. The granular endoplasmic reticulum shows specializations. There profiles surrounding granular substances and widely distributed granulations in connection with the nuclear envelope. The functional significance of the deposition of these granulations is still unknown. The luminal surface of the ependymocytes bears many microvilli and cilia. The cilia are regularly arranged in cranio-caudal direction. Each cilium has the typical (9 + 2)-subfibres. The intercellular space at the surface of the ependymal layer shows a single zonula adherens or zonulae adherentes in the row. Tight junctions and gap junctions were not found in the material examined. Cell processes of liquor contacting neurons between adjacent ependyma cells, protruding into the lumen of the central canal, could be observed. The termination of these neurons contains accumulations of mitochondria in the central part, large amounts of vesicles, and small dense bodies. They have short microvilli and some stereocilia at the free surface.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructure of the haemocytes of Tetrodontophora bielanensis Waga (Collembola)

Five types of haemocytes: prohaemocytes, plasmatocytes, granular haemocytes, spherule cells and phagocytes, have been distinguished on the basis of ultrastructural studies. Prohaemocytes are ovoid cells with a simple structural organization. Plasmatocytes are larger; their cytoplasm contains well-developed rough endoplasmic reticulum, numerous mitochondria and free ribosomes. Granular haemocytes are the most numerous of the blood cells, characterized by the presence of electron-dense granules. The cytoplasm of spherule cells contains many spherules made up of filamentous material of medium electron density. Rough endoplasmic reticulum, free ribosomes and mitochondria are also found in the cytoplasm. Phagocytes are the largest haemocytes. Their cytoplasm contains an abundance of lysosomes and myelin structures. In addition to haemocytes, cells intermediate between plasmatocytes and granular haemocytes have been observed, which indicates that the granular haemocytes are derived from plasmatocytes.     

Segregation of specific classes of messenger RNA into free and membrane-bound polysomes

Analysis of mRNA populations from rat liver rough microsomes and free polysomes by homologous and heterologous cDNA . mRNA hybridization shows that the two mRNA populations are distinct, demonstrating that specific mRNA classes are efficiently segregated for translation in association with endoplasmic reticulum membranes. We estimate that approximately 90% of the mRNA in membrane-bound polysomes contains a diverse set of messengers with a minimum of 500--2000 different species although approximately 5--8 messengers may constitute 25--30% of the mRNA mass. The complexity of the mRNA population of free polysomes appears to be comparable to that estimated for total liver poly(A) + mRNA by other investigators, and is likely to be substantially greater than that of the bulk of bound mRNA. In addition, mRNA in free polysomes lacks the high abundance class characteristic of mRNA-bound polysomes. The substantial complexity of the bound mRNA population suggests that the segregation of polysomes in rough microsomes is not limited to a small class specialized in manufacturing secretory proteins, but extends to polysomes engaged in the synthesis of proteins for intracellular distribution. The segregation of specific messengers into the free and membrane-bound classes was abolished when polysome disassembly was induced by administration of ethionine. Thus, messenger RNA molecules themselves lacked the capacity for segregation, although they contain information for segregation which is expressed during translation. These findings are consistent with the presence of signal sequences in nascent polypeptides which determine the attachment of ribosomes to endoplasmic reticulum membranes.     

Identification of helical polyribosomes in sections of mature skeletal muscle fibers

Summary The localization and configurations of ribosomes in mature white skeletal muscle fibers of the rat were investigated. Differential visualization of ribosomes and glycogen granules was obtained by fixation with glutaraldehyde only and staining of the sections with uranyl acetate. Ribosomes are then electron dense and glycogen granules electron transparent. Their identity was ascertained by selective extractions of ribonucleic acid and polysaccharide.The vast majority of the ribosomes is not membrane-bound. They are located intermyofibrillarly (predominantly at the level of the I-bands), beneath the sarcolemma, and in the paranuclear cones of sarcoplasm. Occasionally short stretches of granular reticulum occur, often as characteristic double walled vesicles with ribosomes on the inner membrane only.Three main types of polysomal configurations are observed: rosettes of 4 to 6 ribosomes, helical arrays, and whorls of up to about 25 probably membrane-bound ribosomes. The average number of ribosomes in the extended helical configurations is estimated to be about 60. It is argued that these helices represent the polysomes instrumental in the synthesis of the large subunits of myosin. It is emphasized that helical polyribosomes are by no means typical of striated muscle, but probably represent a common configuration of large free polysomes.With the technical assistance of Tineke J. Hoogenboezem.     

Polysomes and intracisternal accumulations in enucleate sieve elements of rice (Oryza sativa L.)

Polysomes in sieve elements of rice (Oryza sativa L.) were studied with the electron microscope. The polysomes were found on the rough endoplasmic reticulum (ER) present in immature sieve elements and also on the cisternae of aggregated ER in the parietal layer of mature, enucleate sieve elements. In the immature sieve elements the ER cisternae existed as narrow profiles while in the mature sieve elements the ER cisternae were considerably dilated and contained a fibrillar material and, occasionally, electron-opaque inclusions. In addition to the aggregated ER, single profiles of ER were found applied to the lateral walls and also the sieve plates. These cisternae also bore ribosomes and were separated from the plasmalemma by a narrow, dense space. In the mature sieve elements much of the surface of the ER membranes was covered with polysomes. The dimensions of the polysomes are described and the possibility that they contribute to the formation of the fibrillar material in the intracisternal space is discussed.Abbreviations ER endoplasmic reticulum     

Ribosome loading, but not protein synthesis, is required for estrogen stabilization of Xenopus laevis vitellogenin mRNA.

We have examined the effect of protein synthesis and of ribosome loading on the estrogen-mediated stabilization of hepatic Xenopus laevis vitellogenin mRNA. Removal of estradiol-17 beta from the culture medium, which destabilizes vitellogenin mRNA, does not alter the density of ribosomes on polysomal vitellogenin mRNA, or change the proportion of vitellogenin mRNA associated with the endoplasmic reticulum. Cycloheximide, which inhibits elongation, without changing the density of ribosomes on vitellogenin mRNA, does not block estrogen-mediated stabilization. In contrast, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, (MDMP), which inhibits initiation, greatly reduces the density of ribosomes on vitellogenin mRNA, and completely blocks estrogen-mediated stabilization. Vitellogenin mRNA in MDMP treated cells is degraded at a rate similar to that seen when untreated cells are transferred from medium containing estrogen to estrogen-free medium. This suggests that a ribosome-associated degradative system may not be responsible for vitellogenin mRNA degradation. The failure of estrogen to stabilize vitellogenin mRNA in MDMP-treated cells is not due to the release of vitellogenin mRNA from the endoplasmic reticulum. Vitellogenin mRNA in MDMP-treated cells remains associated with the endoplasmic reticulum in small polysomes containing 3-5 ribosomes. These data demonstrate that maintaining a high density of ribosomes on vitellogenin mRNA, but not continuing protein synthesis, is necessary for estrogen-mediated stabilization of vitellogenin mRNA.