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PubMed1978

Synthesis and migration of glycoproteins in cells of the rat thymus, as shown by radioautography after 3H-fucose injection.

Young male rats received a single intravenous injection of 3H-fucose and were killed after various time-intervals. Light- and electron-microscopic radioautographic studies of the thymus in animals killed shortly after injection showed that all of the different cell types present incorporated 3H-fucose label. The heaviest uptake occurred in macrophages and in hypertrophic epithelial cells located near the cortico-medullary border. Somewhat lighter incorporation was observed in medullary and cortical stellate epithelial cells and in cells designated as special cells, while the lightest reaction appeared over lymphocytes. In all cells the label was localized initially to the Golgi apparatus, where, presumably, it was incorporated into glycoproteins. With time, some of the labeled putative glycoproteins in all cell types migrated to the plasma membrane. In macrophages, much of the label migrated to lysosomal bodies, while in the special cells the label migrated to dense bodies which may also be of lysosomal nature. In stellate and hypertrophic epithelial cells much of the label migrated to characteristic vacuoles. The possible relationship between the observed glycoprotein synthesis in these cells and hormone production is discussed.

Animals↗
PubMed1979

Migration and turnover of entero-endocrine and caveolated cells in the epithelium of the descending colon, as shown by radioautography after continuous infusion of 3H-thymidine into mice.

Adult male mice were given a continuous infusion of about 0.5 muCi of 3H-thymidine per gram body weight per day for periods varying from 1 to 60 days. Semithin sections of descending colon were cut from/plastic-embedded blocks and stained by a method combining silver impregnation and iron hematoxylin, by which argentaffin entero-endocrine cells and caveolated cells could be identified. From radioautographs, the labeling index of these cells was determined. One to three days after the beginning of 3H-thymidine infusion, label is observed in some of the stained entero-endocrine cells in the bottom of the crypts; the apices of these cells reach the crypt lumen and are joined to neighboring cells by terminal bars (junctional complexes). After five to seven days, labeled entero-endocrine cells are seen on the sides of the crypts, where their base stretches along the basement membrane and their apex has lost its terminal bar connections to neighboring cells. Finally, by 13 and 24 days, labeled cells are observed within the epithelium at the mucosal surface. The turnover time, which is taken to be equal to the mean time required for migration from site of origin to site of loss on the mucosal surface, has been estimated at 23.3 days. This is much longer than the 4.6 days required by the two main cell types of the epithelium -- vacuolated-columnar and mucous cells -- to travel the same route. It is likely that, after entero-endocrine cells lose their terminal bar attachment to other epithelial cells, they migrate independently and very slowly. Labeled caveolated cells are first seen in the crypt bottom one day after the beginning of 3H-thymidine infusion. By three to five days, they are on the sides of the crypts; their base is stretched along the basement membrane, but their apex retains its attachment to neighboring cells by terminal bars. By seven days, labeled caveolated cells are on the mucosal surface. Their turnover time has been assessed at 8.2 days. This is, again, longer than for the two main types to which they are bound by terminal bars throughout migration. The discrepancy is explained by the caveolated cells arising deeper in the crypts than most vacuolated-columnar and mucous cells.

Animals↗
PubMed1984

Genesis of B lymphocytes in the bone marrow: extravascular and intravascular localization of surface IgM-bearing cells in mouse bone marrow detected by electron-microscope radioautography after in vivo perfusion of 125I anti-IgM antibody.

The role of mammalian bone marrow in generating surface IgM (sIgM)-bearing B lymphocytes is reviewed. Precursor cells in the marrow give rise to large, rapidly dividing cells bearing free cytoplasmic mu chains (c mu). The progeny of the large c mu+ cells form a population of small, nondividing c mu+ cells that mature into small lymphocytes, progressively expressing sIgM and other B-cell surface membrane components. Newly formed sIgM+ cells soon migrate through the bloodstream to the spleen and other lymphoid tissues, where they may die after a short lifespan or be activated to produce antibody molecules. The large-scale lymphocytopoiesis in the bone marrow thus maintains a population of rapidly renewed virgin B lymphocytes in the peripheral lymphoid tissues. This process continuously creates and selects B cell clones with the wide range of antibody specificities necessary to mediate primary humoral immune responses through postnatal life. A technique for perfusing radiolabeled anti-IgM antibodies in young mice has now permitted sIgM+ cells to be detected radioautographically in histological preparations of bone marrow under the electron microscope. Small sIgM+ lymphocytes are situated either singly or in small groups throughout the extravascular hemopoietic compartment of the bone marrow, often near sinusoid walls adjacent to late erythroblasts and reticular cells. Some regional concentrations of sIgM+ cells are apparent. sIgM+ cells also appear in transit through the sinusoidal endothelium and are markedly concentrated in the lumen of some sinusoids. Intrasinusoidal sIgM+ small lymphocytes have high densities of sIgM and long microvilli, on which sIgM molecules are concentrated. These studies reveal the localization and cell associations of specifically identified sIgM+ small lymphocytes in the extravascular marrow compartment and suggest that these cells may also undergo a transient intravascular storage and maturation phase. Use of this in vivo immunolabeling technique to detect other cell-surface markers may further elucidate the microenvironmental basis of B lymphocyte genesis in the bone marrow.

Animals↗
PubMed1991

Subcellular distribution of [3H]-dexamethasone mesylate binding sites in Leydig cells using electron microscope radioautography.

The present view is that glucocorticoid hormones bind to their cytoplasmic receptors before reaching their nuclear target sites, which include specific DNA sequences. Although it is believed that cytoplasmic sequestration of steroid receptors and other transcription factors (such as NFKB) may regulate the overall activity of these factors, there is little information on the exact subcellular sites of steroid receptors or even of any other transcription factors. Tritiated (3H)-dexamethasone 21-mesylate (DM) is an affinity label that binds covalently to the glucocorticoid receptor (GR), thereby allowing morphological localization of the receptor at the light and electron microscope levels as well as for quantitative radioautographic (RAG) analysis. After injection of 3H-DM into the testis, a specific radioautographic signal was observed in Leydig cells, which correlated with a high level of immunocytochemically demonstrable GR in these cells at the light-microscope level. To localize the 3H-DM binding sites at the electron microscope (EM) level, the testes of 5 experimental and 3 control adrenalectomized rats were injected directly with 20 microCi 3H-DM; control rats received simultaneously a 25-fold excess of unlabeled dexamethasone; 15 min later, rats were fixed with glutaraldehyde and the tissue was processed for EM RAG analysis combined with quantitative morphometry. The radioautographs showed that the cytosol, nucleus, smooth endoplasmic reticulum (sER), and mitochondria were labeled. Since the cytosol was always adjacent to tubules of the sER, the term sER-rich cytosol was used to represent label over sER networks, which may also represent cytosol labeling due to the limited resolution of the radioautographic technique. Labeling was highest in sER-rich cytosol and mitochondria, at 53% and 31% of the total, respectively. Cytosol (exclusively of all organelles) and nucleus showed comparatively weak labeling, at 9% and 7%, respectively. This study thus clearly establishes with the electron microscope, the localization of glucocorticoid binding sites using DM. It remains to be determined whether or not these DM binding sites represent bona fide glucocorticoid receptors or nonreceptor proteins that bind DM. Whereas the functional significance of the subcellular distribution of DM is not known, the labeling of the cytosol may represent localization of the steroid and GR in their traditional compartment. The steroid antagonistic properties of DM may have prevented the DM-GR complexes from translocating to the nucleus. However, the significant labeling of the sER-rich cytosol and mitochondria was unexpected and raises intriguing questions that are being addressed in current studies.

Animals↗
PubMed1975

Cellular renewal in the enamel organ and the odontoblast layer of the rat incisor as followed by radioautography using 3H-thymidine.

Renewal of the cell populations of the incisor was studied in 100 gm male rats injected with a single dose of 3H-thymidine and sacrificed at various times from one hour to 32 days after injection. Radioautographs showed that a cohort of labeled cells within the enamel organ, odontoblast layer, and pulp was carried passively with the erupting incisor from the apical end towards the gingival margin where the life cycle of these cells was terminated. Labeled cells in the upper and lower incisor, although traversing different absolute lengths, were found in approximately the same functional stage of their life cycle at similar times after the injection. Thus, by one and on-half days labeled ameloblasts began inner enamel secretion and, by eight days (upper) or nine days (lower), complement outer enamel secretion. By 32 days labeled ameloblasts had traversed the entire enamel maturation zone and were located at the gingival margin. Labeled odontoblasts followed closely the movement of labeled ameloblasts. The mean rate of ameloblast migration was 567 mum/day on the upper incisor and 651 mim/day on the lower. For the odontoblasts this rate was 55 mum/day (upper) and 631 mum/day (lower). Finally, it was found that as the rat age, the duration of the life cycle for epithelial and pulp cell populations of the incisor increased because of growth within the lonitudinal axis of the tooth. It was concluded that the apical end of the incisor literally "grows backward" in the bony socket, and hence, the duration of the life cycle becomes greater simply because it takes cells longer to physically reach the gingival margin.

Ameloblasts↗
PubMed1982

Analysis of temporal and spatial patterns of rat vestibular hair cell differentiation by tritiated thymidine radioautography.

The location in time and space of the terminal mitoses of type I and type II sensory hair cells (HCI and HCII) of the developing crista ampullaris in rat lateral semicircular canal and macula utriculi was determined by radioautographs of specimens exposed to tritiated thymidine from the 13th to the 20th day of gestation. Qualitative analysis and statistical treatment of the percentages of labeled HCI and HCII show that the terminal mitoses occur first in the macula utriculi with a maximum percentage of the 14th day of gestation, for the HCI, and on the 15th day of gestation, for the HCII. In the lateral crista, the maximum percentage of labeled HCI occurs on the 17th and 18th day of gestation and on the 19th day of gestation for the HCII. A spatial distribution of this labeling activity is also described: the older cells are located at the top of the crista and at the level of the striola of the macula utriculi while the younger cells are found at the bottom of the crista and on the sides of the utricle. A study of the vestibular receptors in the fetuses shows that synaptic contacts already exist on the 18th day of gestation in the macula utriculi at the level of the striola and on the 19th day at the top of the crista; the cells situated on the periphery are still immature. The first hair cells to undergo their terminal mitoses are, therefore, connected first. These results also suggest that the two types of cells are genetically programmed and that the HCI start functioning first during the development of the labyrinth.

Acoustic Maculae↗
PubMed1981

Lectin-binding patterns of small lymphocytes in bone marrow, thymus and spleen: demonstration of lymphocyte subsets by quantitative radioautography.

Cells from mouse bone marrow, thymus and spleen were exposed to 125I-labeled concanavalin A (Con A). Lens culinaris lectin (LCL), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), phytohemagglutinin-P (PHA-P), peanut agglutinin (PNA), or wheat germ agglutinin (WGA) in a range of concentrations and examined radioautographically. Small lymphocytes in the three organs differed in the minimal concentration of each lectin which gave detectable surface labeling, while at optimal lectin concentrations, their labeling intensity profiles differed markedly. Inhibition by sugars demonstrated the labeling specificity. Major populations of bone marrow small lymphocytes bound WGA strongly, while Con A, SBA, HPA, PHA-P and LCL were bound only weakly, and PNA binding was lacking. Most thymus cells bound Con A, SBA, HPA, PHA-P and PNA strongly, WGA and LCL weakly. Subsets of bone marrow and thymus small lymphocytes differed from the major populations in their lectin-binding intensities. Spleen small lymphocytes were heterogeneous in the binding of each lectin. However, a major population bound LCL exceptionally strongly, while few cells bound PNA. Using a panel of lectins under standardized conditions, these studies show distinctive lectin-binding patterns for small lymphocytes in the bone marrow, thymus and spleen, respectively. Major and minor cell populations are distinguishable in each organ, providing an approach to discriminating lymphocyte lineages, subtypes and differentiation stages.

Agglutinins↗
PubMed1990

Preparation of bone for high-voltage electron microscopic radioautography.

This paper presents a reliable technique for the preparation of radioautographic specimens for study by high-voltage electron microscopy. Tissues are fixed by ventricular perfusion of aldehydes, sectioned, collected on slides, and coated with nuclear track emulsion using a coating machine. Sections are developed after 17 weeks' exposure in either undiluted Microdol-X or diluted D-19 developer, fixed in 25% sodium thiosulfate, and stained through the emulsion with uranyl acetate in absolute methanol and aqueous lead citrate. The technique produces sections of even thickness and staining density with negligible numbers of background grains and non-specific labeling. In this report, the efficacy of this technique for the study of the periosteal surface of demineralized alveolar bone specimens is demonstrated.

Animals↗