Electron Microscope Autoradiographic Study Research Articles

Patterns of DNA and RNA synthesis during the development of ovarian nurse cells in Gerris najas (Heteroptera)

The spatial and temporal patterns of nucleic acid synthesis are described as observed in various regions of the germarium from the third instar up to the adult. The nurse cells proliferate in the third instar and undergo endopolyploidization in the fifth instar, as revealed by [ 3H]thymidine autoradiography and Feulgen-scanning cytophotometry. No indication of DNA under-replication or amplification has been found. RNA synthesis occurs throughout the development of the trophocytes but is highest in the fifth instar. Ultrahistochemical and electron microscopic autoradiographic studies revealed a mass transfer of ribonucleoprotein from the nucleus into the cytoplasm, where the material accumulates on the nuclear envelope in the form of electron-dense bodies. The results are briefly discussed with respect to the role of endopolyploidy in cells of high gene activity.

The biosynthesis of fibroin: III. Involvement of the Golgi apparatus in transport and secretion—An electronmicroscope autoradiographic study

The intracellular transport of fibroin and its secretion have been studied in the silk gland of Bombyx mori incubated in vitro. Sites of synthesis and transport were determined radioautographically by pulse-chase labelling with tritium-labelled glycine. The label, initially located in the rough endoplasmic reticulum (RER) is found in the complexes and the fibroin globules of the Golgi apparatus before it accumulates in the cell-lumen border and further on reaches the compact cylinder of silk in the lumen. Study of the relative distribution of the radioactivity in the gland has allowed us to use a compartment model, developed in the appendix, to calculate the mean residence times of a molecule in the RER and the Golgi apparatus. They were found to be respectively 35 and 13 min. The involvement of the Golgi apparatus in the transport of secreted protein is discussed by comparing our results with current knowledge on fibrous protein transport and secretion.

Synthesis and transport of newly formed proteins in dendrites of rat hippocampal pyramid cells. An electron microscope autoradiographic study

The synthesis and transport of newly synthesized proteins in dendrites of rat hippocampal pyramid cells were investigated. Labelled leucine was injected into the left lateral ventricle and the hippocampal region was processed for light and electron microscopic autoradiography. To differentiate between the silver grains originating from ‘sedentary’ or ‘migratory’ proteins, the radioactivity in dendritic areas free of ribosomes and rich in ribosomes was determined separately. Several conclusions were reached. (1) Protein synthesis in dendrites takes place mainly in the proximal parts although a slight synthetic activity can be observed along the whole dendritic tree as well. (2) Newly synthesized proteins are transported toward the distal dendritic region; the data obtained suggest that cisterns of the smooth endoplasmic reticulum and the microtubular system may be involved in this transport. (3) Two phases of dendritic transport may be distinguished; a fast phase with a rate of 100–200 mm/day and a slow phase with a rate of 2.8–10 mm/day. It also seems probable that the majority of the proteins newly synthesized in dendrites are transported by the slow phase.

Regeneration of Oligodendroglia During Recovery from Demyelinating Disease

Infection of mice with the JHM strain of mouse hepatitis virus causes demyelination as a result of a cytolytic infection of oligodendroglia. In recovery, animals show remyelination, which could result either from surviving oligodendrocytes extending their territory or by generation of new oligodendroglia. Electron microscopic autoradiographic studies with 3H-labeled thymidine demonstrate that the cells associated with remyelination are newly generated oligodendroglia.

Export of protein from the osteoclast as studied by electron microscopic autoradiography.

The present electron microscopic autoradiographic study includes a quantitative analysis of osteoclasts in vitro using tritiated leucin as a protein tracer. A significant increase in the grain density over the ruffled border and the underlying resportion zone was demonstrated two hours post pulse whereas the grain density of the remaining cytoplasm was relatively constant. This indicates a transport of newly synthesized protein from the osteoclast to the extracellular resorption zone. Earlier histochemical and biochemical experiments suggest that the exported protein may represent lysosomal enzymes to be used in the extracellular bone degradation.

Fasciola hepatica: A light and electron microscope autoradiographic study of incorporation of monosaccharides into glycogen and glycoprotein

Incorporation of [ 3H]galactose and [ 3H]glucose into the parenchyma, tegument, testis, and muscle of Fasciola hepatica slices was studied by lightand electron-microscope autoradiography. “Accumulation” labeling periods of up to 60 min were used. Both monosaccharides were found to be readily incorporated into glycogen in the parenchymal cells and muscle and [ 3H]glucose entered the glycogen stores of spermatozoa. No evidence was found for the involvement of any particular cell organelle in glycogenesis, but the demonstration of high synthetic activity in parenchymal evaginations to the base of the surface syncytial tegument supports physiological evidence that glucose enters the fluke mainly across the tegument. Ethylene glycol-dehydrated preparations showed that [ 3H]galactose was incorporated into glycoprotein by Type I tegumental cells, and perhaps also by sperm morulae. The carbohydrate component seems to be added to the tegumental secretions in the vesicular-lamellar region of the Golgi complex. Following the longest periods of incubation, labeling was observed in the tubules connecting the tegumental cells and syncytium, but not in the surface syncytium itself.

The localization of (3H) thymidine incorporation in the DNA of replicating spinach chloroplasts by electron-microscope autoradiography.

Electron-microscope autoradiography has been used to obtain information on the localization of DNA labelled with [3H]thymidine in chloroplasts known to be replicating and concomitantly synthesizing and segregating DNA, in cultured leaf disks. The studies were made using both Microdol-X developer and a 'compact' developer which gave a smaller grain size. About 80% of the grains were associated with the granal membranes and with presumptive DNA regions (3-nm fibril material in clear areas). Few grains occurred in association with the chloroplast envelope. We suggest that the DNA of chloroplasts is associated with the grana lamellae and extends into the stroma. Some light-microscope autoradiographs of whole chloroplasts show spiral or helical-like labelling patterns. We interpret these patterns as demonstration of the possibility that DNA occurs along the length of a continuous lamellar membrane system. Chloroplast fractionation experiments provided data consistent with the electron-microscope autoradiographic studies as most of the label was associated with chlorophyll-containing lamellae. We consider an association of chloroplast DNA molecules along the length of a continuous lamellar system would ensure an orderly segregation of DNA to daughter chloroplasts, during the binary fission of spinach chloroplasts by constriction division.

Fasciola hepatica: An electron microscope autoradiographic study of protein synthesis and secretion by gut cells in tissue slices

A loop technique for electron microscope autoradiographic studies on slices of Fasciola hepatica is described, and used to study the synthesis of protein-containing bodies by gut cells of the fluke. Slices were pulse labeled with tritiated tyrosine, methionine, leucine, and phenylalanine, and, after appropriate chase periods in unlabeled medium, were fixed with formaldehyde and prepared for electron microscopy by a procedure involving ethylene glycol dehydration. Labeled amino acids were found to be incorporated into protein, or glyoprotein, in the gut cells of the slices by a GER-Golgi complex mediated mechanism similar to that which occurs in vertebrate tissues. The precursors appeared to enter the cells across their basal and lateral plasma membranes and mature secretory bodies were discharged at the cell apex.

Lymphocyte capping and lymphocyte migration as associated events in the in vivo antigen trapping process. An electron-microscopic autoradiographic study in the spleen of mice.

The fate of 125I labeled antigen-antibody complexes in the first two hours after intravenous injection was followed in the spleen of mice by light and electron microscopic autoradiography. It appeared that AAC were phagocytized by granulocytes and by macrophages in the marginal zone and were present over the surface of lymphocytes in that area. By rinsing the spleen before fixation it could be shown that AAC were indeed bound to the lymphocyte membrane and not merely present in the blood plasma between the cells. The label was present in a spotty fashion or over a so-called uropod. The majority of labeled uropods (13 out of 17) pointed away from the follicle. From this it was inferred that these lymphocytes moved to and most probably into, the follicles. Inside the follicles, at 1 hour post injectionem, most of the label was associated with interfaces between lymphocytes. At 2 hours post injectionem there was a preferential localization over interfaces between lymphocytes and dendritic reticulum cells. It is conceivable that antigen that is introduced into the circulation is ultimately presented to dendritic reticulum cells in a complexed form with antibody, probably with complement, and with the B-cell receptor, since receptor shedding is a normal event following capping.

Selective localization of histamine to electron dense granules in antigen-challenged sensitized rat mast cells and to similar granules isolated from sonicated mast cells. An electron microscope autoradiographic study.

The subcellular localization of histamine was studied in sensitized rat mast cells following antigen challenge and in granules obtained from sonicated cells, using an electron microscope autoradiographic technique. The mast cells were furnished with labelled histamine by incubation in 3-H-histidine. The silver grain distribution (reflecting the radioactive histamine) was highly non-random. The highest silver grain densities occured over homogeneous, electron dense (normal) granules and moderately electron dense granules. Swollen, less electron dense ("changed") granules with a reticular appearance and devoid of a limiting membrane had the lowest density of all subcellular structures studied and were therefore probably almost free of histamine. There was a good correlation between the percentage of electron dense granules, the histamine content and the silver grain density insaline-washed granule fractions isolated after sonication of mast cells for different times. These results support the hypothesis that histamine release occurs during the sequential exocytosis of storage granules, and during the sonication of mast cells, probably as a cation exchange between the amine, which is ionically bound to the heparin-protein complex of the granule matrix, and cations from the extracellular fluid. The exchanges will occur as soon as the perigranular membrane becomes premeable to water and cations.

Electron microscope autoradiographic study of intestinal absorption of decanoic and octanoic acids in the rat.

Intestinal absorption of [3H]octanoic acid and [3H]decanoic acid was investigated in the rat by electron microscope autoradiography. The common duct (bile and pancreatic common duct) of the rats was diverted and a loop of the duodenum was cannulated 24 h later. The lipid mixture to be investigated was introduced into each experimental loop, and after 15 min or less the loop was removed. One part of each loop was used to determine the distribution of radioactivity in different lipid fractions, and an autoradiographic study was performed on the other part of the loop. Radioactivity distribution studies confirmed that medium chain fatty acids are absorbed in their nonesterified form and established that these fatty acids are absorbed much more rapidly than oleic acid. Autoradiographic studies indicated that the medium chain fatty acids are taken up in a molecular or aggregate molecular form, leave the epithelial cells by way of the lateral plasma membrane, and are next found in the blood capillaries. Our results suggest that the Golgi complex does not play an important role in the absorption of unesterified fatty acids.

RNA synthetic activities of sertoli cells in the mouse testis.

Electron microscope autoradiographic studies of RNA synthesis in Sertoli cells of the adult mouse, using [‘H]uridine as a precursor, show a random distribution of grains in the nucleoplasmic area and a localized product on the nucleolar fibrillar component at early labeling times (15 mm). The condensed chromatin bodies associated with the nucleolus seem to have a nucleolar organizing function. At later labeling times (3 h) certain interchromatmn granules occurring in clusters in the nucleoplasm become partially labeled. After 7-8 days, the disappearance of label from Sertoli cells, contrasting with the longer persistence in spermatocytes, suggests a rapid turnover of RNA in Sertoli cells. Differential labeling patterns and structural changes in Sertoli cells cytoplasm are related to variations in functional activities presumably required for normal spermatogenesis and/or spermiogenesis. Sertoli cells are found in the seminiferous epithelium of several mammalian species. Though various functions have been attributed to these cells, it is generally accepted that Sertoli cells provide mechanical support for germinal cells. Moreover, they participate in the release of sperm into the lumen of the seminiferous tubules (VitaleCalpe and Burgos, 1970), dispose of cell debris by a phagocytic mechanism (Clegg and Macmillan, 1965; Kierszenbaum, 1970), and play an important role in the transfer of nutrients and metabolites between the intertubular space and the lumen of the tubules, acting as “bridge” cells (Vilar et al., 1962). It is also known that Sertoli cells constitute a unique stable cell population in the seminiferous epithelium. Whereas spermatogonia, spermatocytes, and sper

Uptake of 35S-sulfate by morphologically differentiated replicating chondrocytes in Vivo: A double isotope electron microscope autoradiographic study

Developing cartilage in the 27-day regenerating forelimb of the newt Triturus viridescens was labeled with both 3H-thymidine and 35S-sulfate. The nuclear and cytoplasmic label was assessed quantitatively by EM autoradiography. It was found that chondrocytes incorporated 3H-thymidine into their nuclei while simultaneously incorporating 35S-sulfate in their cytoplasm. The incorporation of 35S-sulfate occurred primarily in the Golgi complex of morphologically differentiated chondrocytes at the base of the developing cartilage, and the 35S uptake by the DNA replicating chondrocytes was quantitatively equivalent per unit area of Golgi to that by the nonreplicating cells.

Pinocytotic uptake and intralysosomal crystal formation of coxsackievirus A9 in monkey kidney cells. An electron microscopic autoradiographic study.

Infection of monkey kidney cells by Coxsackievirus A 9 labelled with3H-uridine was studied. The penetration process and subsequent localization of viral RNA was examined by electron microscopic autoradiography: Single virions were pinocytozed and aggregated to groups within membrane bound vesicles. The virions were then rearranged to large crystals within these vesicles. At 3 hours after infection most of the crystal-containing vesicles displayed acid phosphatase activity and are therefore considered to be lysosomes.

Electron microscopic autoradiographic study of cholesteral passage across arterial and capillary endothelium.

The objective of this study was to compare for the first time, by means of electron microscopic autoradiography, the movements of orally administered labelled cholesterol across (a) normal arterial and capillary endothelium, and (b) normal and pathological arterial endothelium. For this purpose, 15 mCi/kg 3H-Cholesterol, spread in 5 daily doses, was given by stomach tube to (a) normal, (b) chronically hyperlipemic-atherosclerotic, (c) serum sick, and (d) desoxycorticosterone (DOCA) treated rabbits. The animals were killed 6 hours after the last 3H-Cholesterol feeding and thick unstained sections from their glutaraldehyde-digitonin fixed aortae, coronaries, myocardium and liver were then coated with a photographic emulsion, exposed for 2 or 6 months and examined electron-microscopically. It was found that in normal animals only very few labelled cholesterol molecules crossed the arterial endothelium, whereas large numbers of them crossed the capillary endothelium to enter the muscle cells of the heart and the hepatocytes of the liver. In contrast to the normal arterial endothelium, however, the arterial endothelium of hyperlipemic-atherosclerotic, serum sick and DOCA treated rabbits proved highly permeable to the labelled cholesterol molecules, allowing them to pour in great numbers into the arterial wall. The labelled lipid crossed the endothelium mainly trans-cytoplasmically, but in animals exposed to protracted hyperlipemia it also entered the arterial wall through opened interendothelial junctions that were occasionally observed in this condition. These results show that the normal arterial endothelium, unlike that of normal capillaries, has a very low permeability for physiologically presented cholesterol (or lipoprotein) molecules, but that certain pathological conditions such as protracted hyperlipemia, hypertension and immune insults change it and make it highly permeable to these molecules.

An electron microscopic autoradiographic study of proline incorporation by mouse lingual epithelium.

Mouse lingual epithelium incorporates significant amounts of L-proline-2, 3-H3 one hour after intraperitoneal injection of the tritiated amino acid. All viable cell strata incorporated approximately equal amounts of proline as assessed by autoradiographic techniques. Grain counts at 30 minutes, 1 hour, 4 hours and 24 hours, the four time periods studied, indicated a progressive incorporation of proline up to 4 hours following injection. Preferential incorporation of proline into any one cell structure or group of structures was not observed. Keratohyalin granules (KHG's) demonstrated incorporated proline; however, usually only one silver grain appeared over each granule, and, based on grain counts, the amount of proline incorporated by KHG's appeared slightly less than the general labeling observed in KHG-containing cells. This finding supports recent biochemical studies which have indicated a considerably lower proline content of keratohyalin than had previously been reported. Significant proline incorporation into the epithelial basal lamina was not observed during the 24 hours of this study. Thus, while recent recombination experiments have conclusively demonstrated that epithelial basal cells synthesize considerable quantities of basal lamina in a 24 hour period; it would appear that epithelial basal cells contribute little to a formed, intact basal lamina. This finding lends credence to the concept of a long basal lamina turnover time.

Adrenergic innervation of the rat submandibular and parotid glands. An electron microscopic autoradiographic study of the uptake of tritiated norepinephrine.

AbstractThe adrenergic innervation of the rat submandibular and parotid glands was investigated by monitoring the uptake of H3‐NE by nerve terminals with electron microscopic autoradiographic techniques. Both glands contained nerve bundles in the stroma encompassed by a Schwann cell sheath. Silver grains due to H3‐NE were observed overlying several nerve terminals found in these nerve bundles, while other nerve terminals in the same bundle were devoid of silver grains. In the submandibular gland, adrenergic nerve terminals characterized by silver grains due to H3‐NE were observed in the stroma in close relationship to, but not in direct apposition to parenchymal cells. In the parotid gland, silver grains due to H3‐NE were observed overlying nerve terminals contained within the basement lamina and in close contact with acinar cells.

Post-injury DNA synthesis, mitosis and ultrastructural reorganization of adult frog cardiac myocytes. An electron microscopic-autoradiographic study.

From the 5th day up to the end of 3rd week following local crushing of the frog ventricle myocardium, ca. 13% of myocyte nuclei, in the vicinity of the damaged zone, were labelled after a single 3H-thymidine (3HTdr) injection, and 30–50% of these were labelled after repeated 3HTdr administration. The number of myocyte mitoses was maximal (ca. 1.3%) at the beginning of the 3rd week. The reactive proliferation of myocytes was accompanied by their “partial dedifferentiation”. This involved the nuclear euchromatic rearrangement, increase in size of nuclei and nucleoli, accumulation of the sarcoplasm rich in free ribosomes and rough endoplasmic reticulum, hyperplasia of the Golgi apparatus, and the appearance of 80–100 A in diameter cytofilaments. Electron microscope autoradiography has shown that all these changes may be more or less pronounced in myocytes incorporating 3HTdr. The myofibril ultrastructure was found to be unchanged during S phase. However, in the mitotically dividing myocytes, the majority of Z-disks were disintegrated resulting in progressive release of myofilament bundles. Both 3HTdr labelled and mitotic myocytes were anchored to the adjacent ones by desmosomes and intercalated disks. No free myoblasts were observed.

Electron microscope autoradiographic studies on the effect of chloroquine on the uptake of tritiated nucleosides and methionine by Plasmodium berghei.

Electron microscope autoradiographic studies on the effect of chloroquine on the uptake of tritiated nucleosides and methionine by Plasmodium berghei.

Nuclear and cytoplasmic DNA synthesis in cotton embryos: a correlated light and electron microscope autoradiographic study.

To investigate the possibility, implied by an earlier report, that large amounts of degradable DNA are probably present in the cytoplasm of young cotton embryos, an investigation was undertaken to establish the distribution, amount and metabolic stability of DNA in cotton embryos. Several sensitive cytochemical tests failed to detect any but small amounts of extranuclear DNA. Quantitative determination of the nucleic acid content of embryos during embryogenesis showed that the amounts of DNA and RNA remained fairly constant during embryogenesis, with a ratio of RNA to DNA of about 3.5 to 1. Quantitative autoradiography at both the light and electron microscope levels of sections from embryos pulse-labeled with 3H-thymidine showed that the grain density over the nucleus and cytoplasm did not change during a seven-hour period after labeling, nor did the distribution of label in the cytoplasm. Virtually all incorporation was eliminated by the inclusion of iododeoxy-uridine in the medium. Almost all of the nuclear label and at least 90% of the cytoplasmic label after 3H-thymidine incorporation was eliminated by deoxyribonuclease. It was concluded that there are no unusual features related to DNA distribution or metabolism in cotton embryo; i.e., that only small amounts of DNA are present in the cytoplasm and that all of the DNA is metabolically stable.