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PubMed1985

Dynamic histology of the antral epithelium in the mouse stomach: IV. Ultrastructure and renewal of gland cells.

The renewal of gland cells was investigated by three-dimensional reconstruction of typical mucous units of the pyloric antrum using electron microscopy and 3H-thymidine radioautography in 3 to 4 month-old CD1 mice. Based on analysis of 42 units, the average gland measured 31 micron in length and was composed of 37 (mucous) gland cells with eight enteroendocrine cells scattered among them. The gland neck cells located close to the isthmus showed the cytoplasmic and nuclear features characteristic of differentiating cells. The mid-gland cells occupying the central portion of the gland appeared to be at a more advanced stage of development and completing differentiation. The gland base cells comprising the blunt end of the gland were fully mature. To quantify the renewal process, the percent of gland cell nuclei carrying label was determined at several times following 3H-thymidine administration. The rate of proliferation was found to be greatest in the gland neck, lower in the mid-gland, and even lower within the gland base. Furthermore, the isthmus contributed to gland-cell renewal by providing an estimated 12.4 cells per day. Labeled cells migrated toward the blunt end of the gland. The migration rate became progressively slower with their descent, and many cells were lost along the migration pathway, mainly in the gland neck. The loss took place without being preceded by gradual cell degeneration, but occurred as a result of rapid extrusion to the lumen or, less frequently by pyknosis, which could be followed by phagocytosis. It is concluded that the rapid rate of mitosis within the isthmus and gland neck generates a pressure causing downward migration of the cells toward the blunt end of the gland. The rate of migration, however, gradually diminishes as cells descend into the gland, presumably owing both to decreasing proliferation rate and to cell loss. Thus, while cells migrate down toward the gland base, many are lost before reaching it. This sequence is described as "the cascade pattern" of renewal.

Animals↗
PubMed1986

Synthesis and migration of 3H-fucose-labeled glycoproteins in the ciliary epithelium of the eye: effects of microtubule-disrupting drugs.

3H-fucose was injected intravenously or intravitreously into albino rats. After time intervals of 10, 40, and 50 min, 1, 1.5, and 4 hr, 1, 3, and 7 days, and 1, 2, and 4 weeks after injection, the animals were sacrificed by intracardiac perfusion with glutaraldehyde. Samples of the ciliary body were prepared for light and electron microscope radioautography. Light microscope autoradiographs showed that the cells of both the inner and outer layers of ciliary epithelium actively incorporated 3H-fucose label in a reaction that peaked in intensity at 4 hr after injection, and then progressively declined. Electron microscope radioautographs revealed that, at early time intervals, most of the label was localized to the Golgi apparatus. With time, the plasma membrane of both cell types became increasingly labeled, and accounted for 60-70% of the total silver grains at 4 hr after injection. Adjacent to the basal cell surface of the inner layer cells, the fibers of the zonula became increasingly labeled from 1.5 hr onwards, providing strong evidence that these cells secrete glycoproteins to the zonula. When vinblastine was administered 30 min before 3H-fucose injection, followed by sacrifice 1.5 hr later, a much larger proportion of label remained localized to the Golgi apparatus than in controls, and the plasma membrane and zonula were much less labeled. These results suggest that, as documented in other cell types, microtubules may play a role in the intracellular transport of membrane and secretory glycoproteins in these cells.

Animals↗
PubMed1987

Radioautographic tracing of 3H-proline in the endodermal cells of the parietal yolk sac as an indicator of the biogenesis of basement membrane components.

The biogenesis of basement-membrane components was investigated in the endodermal cells of the rat parietal yolk sac in 12.5-day pregnant rats; 3H-proline was injected into conceptuses. After various time intervals, the parietal yolk sac, including endodermal cells and the associated Reichert's membrane, was removed and processed for electron-microscopic radioautography. Silver grains were counted over endodermal cell organelles and Reichert's membrane. At 2 and 5 min after 3H-proline injection, endodermal cells showed heavy labeling in rough endoplasmic reticulum (rER). Silver grain density over the rER decreased from 2 to 20 min and then remained at a plateau. Grain density was moderate over the Golgi apparatus initially but rose to a peak at 2 hr and decreased by 4 hr and later. Grain density was negligible over secretory granules at 2 and 5 min and increased moderately with time to reach a maximum at 8 hr. Thus, radioautographic peaks occurred sequentially in rER, Golgi apparatus, and secretory granules. By 4 hr and later, silver grains accumulated over Reichert's membrane. These results indicated that endodermal cells incorporated labeled proline into substances which were processed from the rER through the Golgi apparatus, transported from there to the cell surface by secretory granules, and released for export to Reichert's membrane. To clarify the nature of the exported substances, the amount of label present in proline and hydroxyproline residues after 3H-proline injection was measured in Reichert's membrane with or without the associated endodermal cells. Within the cells, 61.8% of the labeled proteins were classified as "sedentary" and 38.2% as "exportable." Of the label exported to Reichert's membrane, 66.3% consisted of type IV collagen and the rest of other basement-membrane components. The results obtained with this model suggest that basement-membrane proteins, including type IV collagen, are elaborated by the associated cells through the classical pathway: rER-Golgi apparatus-secretory granules.

Animals↗
PubMed1988

Synthesis of lens capsule and plasma membrane glycoproteins by lens epithelial cells and fibers in the rat.

The lens of the eye possesses a capsule which is a greatly hypertrophied basement membrane. To investigate the synthesis of glycoproteins destined for this capsule, 3H-fucose was injected into the vitreous body of intact rats weighing approximately 200 gm. The animals were killed from 10 min to 14.5 months later, and their lenses were processed for electron microscope radioautography. At 10 min after injection, more than 58% of the silver grains were localized to the Golgi apparatus of the lens epithelial cells. By day 1, the heaviest sites of reaction were the plasma membrane (more than 50% of total label), the basal cytoplasm, and the adjacent lens capsule, where a heavy band of reaction was seen. The remainder of the capsule exhibited a lighter diffuse reaction. In the lens fibers, the label was at first localized to clusters of vesicles but then migrated to the plasma membrane and to the region of the capsule adjacent to the basal surface of these fibers. Light microscope radioautographs of the lens capsule at later time intervals revealed that by 1 month after injection the diffuse reaction had disappeared, and only the strongly labeled band remained. By 14.5 months after injection, this band had migrated partially across the lens capsule, but the capsule itself had increased considerably in thickness. On the other hand, the distance between the labeled band and the free edge of the capsule had decreased from that seen at the time of injection.

Animals↗
PubMed1990

Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose.

The effects of short- and long-term stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of 3H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled 1 hr after labeling. The percentage of heavily labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after administration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.

Animals↗
PubMed1983

Toxic effect of phenytoin on developing cortical neurons in culture.

Studies were undertaken to determine the effect of chronic phenytoin exposure on developing neurons. Cerebral cortex from 16-day fetal mice was utilized to prepare primary dissociated cell cultures. Phenytoin was added to the cultures 10 days after plating and the cultures were harvested on day 17. Cortical cultures were assayed for neuronal cell number by phase microscopy and for high-affinity uptake of 3H-labeled gamma-aminobutyric acid (GABA) by both radioautography and scintillation spectrometry. Neuronal cell counts demonstrated a highly significant decrement in the number of neurons in cultures exposed to phenytoin at 15, 25, and 50 micrograms/ml. 3H-GABA-labeled neurons constituted 13% of the neurons present in both control and phenytoin-exposed cultures. These data indicate that phenytoin is toxic to cortical neurons in culture and that GABAergic neurons are affected to the same extent as the total neuronal population.

Animals↗
PubMed1975

Structure of rat ultimobranchial bodies after birth.

The evolution of ultimobranchial bodies in Holtzman rats during the first 64 weeks after birth was studied by reconstructing three-dimensional models from serial sections stained by the periodic acid-Schiff technique.Radio-autography with 125I was made to see if ultimobranchial cells and/or follicular cells lining the lumen of mixed follicles were able to iodinate proteins. The term ultimobranchial body designates herein an embryonic vesicular structure (derived from the third pharyngeal pouch) whose wall is made of stratified squamous epithelium. During the first week after birth, the vesicular ultimobranchial body elongates rapidly and becomes a canal or a duct. During the second week, cell desquamation brings about local dilatations in the lumen of these ducts; with further enlargement ultimobranchial follicles will appear. In one-day-old rats, mixed follicles are present. Only the follicular component of mixed follicles iodinates proteins as is shown by radioautography. The reconstructed models enlarge rapidly up to the 56th day after birth at which time their weight has increased nineteenfold. These same models show that the three morphological components of ultimobranchial parenchyma, namely ducts, follicles and mixed follicles, are in continuity within the thyroid parenchyma. The formation of new thyroid follicles after birth and the possiblility that the ultimobranchial parenchyma may function as an endocrine gland of holocrine type are discussed.

Age Factors↗
PubMed1976

DNA synthesis and cell proliferation in the simple liver acinus of 10 to 20-day-old rats: evidence for cell fusion.

Radioautography after 3H-thymidine injection, mitotic arrest by colchicine and camera lucida drawings were used to study DNA synthesis, mitosis, formation of binucleated cells and morphogenesis in the simple liver acinus of Rappaport in 10-20-day old rats. By ten days the arrangement of hepatic cell plates had already attained the adult configuration - irregular and thick in acinar zone 1 (periportal), straight and thin in acinar zone 3 (pericentral). The DNA synthetic index of parenchymal and bile duct cells slowly decreased during the observation period. Zonal labeling remained steady in the relationship: zone 1 greater than zone 2 greater than zone 3. Mitosis of parenchymal and bile duct cells reached a peak at 12 days, decreasing slowly thereafter. Mitosis also exhibited the relationship zone 1 greater than zone 2 greater than zone 3. The number of binucleated cells remained constant until after day 14 when it increased rapidly. Zonal distribution of binucleated cells was just the reverse of that for DNA synthesis and mitosis, that is zone 3 greater than zone 2 greater than zone 1. Radioautographic studies of binucleated cells labeled with 3H-thymidine indicated that a small percentage of them were formed by fusion of mononucleated cells. Conclusions are: (1) the 10-20-day old rat liver is expanding its cell population primarily in acinar zones 1 and 2 while overt differentiation is occurring in acinar zone 3, (2) ingestion of solid food around day 16 may be related to binucleated cell production due in part to altered portal venous blood changing the cellular microenvironments, (3) binucleated cells arise by suppression of cytokinesis, cell fusion and/or other non-mitotic routes.

Animals↗
PubMed1976

Kinetics of intraepithelial lymphocytes in the small intestine of thymus-deprived mice and antigen-deprived mice.

The kinetics of lymphoid cells within the epithelium of the small gut has been studied in various thymus-deprived mice and in antigen-deprived mice by the use of 3H-thymidine injections and radioautography. In thymus-deprived mice--including adult thymectomized, thymectomized and irradiated, neonatally thymectomized, and nude mice - and in germ-free mice decreased numbers of intraepithelial lymphocytes (IL) were found. On the other hand, the radioautographic results indicated that the remaining IL populations included both newly formed and long-lived lymphoid cells in the same percentages as found in sham-operated controls and normal mice. It is concluded that although the presence of the thymus and the antigen content of the gut is of importance to the maintenance of the numbers of cells in the lymphoid populations of the intestinal wall, the basic kinetics of these cell populations are preserved in deprived mice.

Animals↗
PubMed1976

Cellular composition of the bone marrow in the chicken. I. Identification of cells.

Incorporation of Fe55 in vivo was used for verifying on radioautographs the identity of chicken bone marrow cells that are in the process of hemesyntheses. Under the experimental conditions all labeled cells may be considered to be linked with erythroid differentiation. They were classified into five maturational stages according to their morphology and capacity for DNA synthesis. Granulocytes were identified by the presence of specific granules. All mononuclear cells were classified as lymphocytes which had a pachychromatic nucleus, a high nucleocytoplasmic ratio, lacked the capacity for DNA synthesis, and resembled small lymphocytes of the bursa, spleen and bone marrow that bound, in vitro, anti-chicken gamma globulins labeled with I125. Radioautography with H3TdR was used to identify proliferating and non-proliferating members of each cell population.

Animals↗
PubMed1979

Localization of a highly specific neuronal protein, serotonin binding protein, in thyroid parafollicular cells.

A highly specific serotonin binding protein (SBP) has been found in serotonergic neurons in both brain and gut. This protein has an extremely high affinity for serotonin and may be a storage protein. Serotonin is found in many endocrine cells, including parafollicular cells of the sheep thyroid, as well as in neurons. SBP is also present in sheep thyroid. The present study was done to localize the protein in the gland. Thyroid glands were divided into five segments. Concentrations of serotonin and SBP, as well as parafollicular cell volume were measured in each. Serotonin was assayed by enzymatic conversion to melatonin using tritiated S-adenosylmethionine. SBP was assayed by molecular sieve chromatography on sephadex G-50. The relative volume of parafollicular cells was obtained by stereological analysis of electron micrographs. Experiments were also done to demonstrate these cells by histofluorescence and radioautography following incubation with tritiated 5-hydroxytryptophan. Good correlations were found between serotonin and SBP concentrations, and parafollicular cell volume. These peaked in the rostro-central portion of the gland and were minimal at the poles. We conclude that thyroid SBP is probably localized in parafollicular cells.

Animals↗
PubMed1980

Proliferation of epithelial cells in the adult primate choroid plexus.

An adult rhesus monkey was injected intraperitoneally with [H3] thymidine (2.3 microCi/gram body weight) and perfused 90 minutes later with a mixture of aldehydes. One and a half micrometer plastic sections were then cut and dipped into liquid emulsion for radioautography. Labeled cells were observed in the choroid plexus of the anterior lateral ventricle; cell identification was evaluated using electron micrographs taken from serial thin sections of re-embedded. radioautographic 1.5-micron sections. The ultrastructure and location of both mitotic figures and labeled cells confirmed the presence of undifferentiated basal choroid plexus epithelial cells in the adult primate central nervous system.

Animals↗
PubMed1981

An electron microscopic radioautographic study of collagen secretion in periodontal ligament fibroblasts of the mouse: II. Colchicine-treated fibroblasts.

Colchicine administered intravenously depolymerized microtubules and disrupted the normal organization of the Golgi apparatus in periodontal ligament fibroblasts. Radioautography with 3H-proline indicated that collagen secretion was completely inhibited during a period of approximately 4 hours following the onset of the colchicine effect. During this period of secretory inhibition, labeled collagen precursors were present within a variety of dense bodies, primarily located in a juxtanuclear location replacing the normal Golgi complex. The time course of 3H-proline labeling from 2 to 8 hours suggested that small, newly formed dense bodies fused to form larger dense bodies and pleomorphic structures (zebra bodies), within which collagen precursors appeared to undergo partial polymerization. Autophagosomes, many labeled with 3H-proline, also increased in number after colchicine administration. A gradual decline in 3H-proline label occurred from 4 to 24 hours, presumably due to exocytosis of dense bodies or by the digestion of labeled collagen precursors within autophagosomes. These results support the concept that an intact microtubular network is essential for the organized transport of collagen precursors, from the rough endoplasmic reticulum to the Golgi apparatus, and the eventual transport and exocytosis of collagen secretory granules.

Animals↗
PubMed1982

Formation of the outer dense fibers during spermiogenesis in the rat.

The morphogenesis of the outer dense fibers (ODF) in rat spermatids has been studied by electron microscopy, and the synthesis and incorporation of proteins into the ODF during this process have been followed by radioautography using 3H-proline and 3H-cystine as precursors for ODF proteins. In the first phase (step 8-14), nine very fine fibers termed anlagen of the ODF develop in association with the microtubule doublets. These first appear along the most proximal portion of the axoneme in step 8 of spermiogenesis; during steps 9-14 they gradually increased in length in a proximal-to-distal direction, being first observable along the forming midpiece and later along the principal piece as well. In the second phase (steps 15-16), the rudimentary fibers suddenly increase in diameter, with the most dramatic growth occurring in step 16, and assume a close resemblance to the mature ODF. This striking transformation, which appears to result from simultaneous deposition of electron-dense material along the length of anlagen of the ODF, coincides with a period of rapid incorporation of 3H-proline-and 3H-cystine-containing proteins, which become permanent structural components of the ODF. These proteins, which comprise the bulk of the ODF, are synthesized in the cytoplasm of spermatids during the acrosome and early maturation phases. In the final phase (steps 17-19) the fibers continue to enlarge very slowly, assuming their definitive form in step 19 of spermiogenesis. Thus formation of the ODF in the rat is a lengthy multistep procedure, requiring from step 8-19 of spermiogenesis and utilizing proteins synthesized throughout most of this period.

Animals↗
PubMed1983

Two anatomical pathways for the renewal of surface glycoproteins in chloride cells of fish gills.

The cytoplasm of chloride cells found in the epithelium lining the gills of guppies (Lebistes reticulatus) contains, in addition to the Golgi apparatus and cisternae of endoplasmic reticulum, two distinct membranous components, the vesiculotubular and the tubular systems. While the latter is connected to the laterobasal plasma membrane, the former, made up of small vesicles and short membranous tubules, is seen mainly between the Golgi apparatus and the apical cavity which invaginates the apex of the cell. The role of these two systems in the transport of glycoproteins from the Golgi apparatus to the cell surface was investigated in fishes maintained in fresh and salt water, injected with 3H-fucose, and sacrificed at various intervals thereafter (10 and 30 min; 2.5, 8, 15.5, 24, and 48 hours). The distribution of the label was analyzed by quantitative radioautography in sections examined with the light and electron microscopes. The light microscopic data suggested that the label incorporated in the supranuclear region, where the Golgi apparatus is located, migrated toward the apical and the laterobasal regions of the chloride cells. The relative concentration of the tracer over the various components of the cytoplasm of these cells was calculated from data collected on electron microscope radioautographs at various intervals after 3H-fucose injection. The curves obtained supported the view that glycoproteins synthesized in the Golgi apparatus were transported to the apical surface via the vesiculotubular system, and to the laterobasal membrane via the tubular system.

Animals↗
PubMed1984

The effect of beta-aminoproprionitrile on the periodontal ligament: II. Radioautographic study of collagen secretion from fibroblasts.

Fibroblasts are distributed evenly throughout the periodontal ligament (PDL) of normal mice. In mice fed beta-aminoproprionitrile (beta-APN) the fibroblasts undergo aggregation to form palisades of closely juxtaposed cells abutting pools of acellular collagenous matrix. Individual fibroblasts within these aggregates retain their polarized cytoplasmic organization and continue to synthesize and secrete collagen. However, unlike normal PDL fibroblasts, the beta-APN-treated cells appear immobilized by well-developed cell-to-cell adherens-type junctions along their lateral surfaces. We studied collagen secretion from beta-APN-treated fibroblasts by light and electron microscopic radioautography after injection of 3H-proline. Newly synthesized collagen was secreted from the distal ends of the beta-APN-aggregated fibroblasts as a distinct band of labeled material, resembling the pattern of matrix deposition seen in osteogenesis and dentinogenesis. The radioactive band of collagenous matrix was displaced further away from the fibroblasts at 2 and 4 days after 3H-proline injection as more collagen was secreted. This pattern of radiolabeled collagen secretion confirmed previous observations that PDL fibroblasts are highly polarized and that collagen secretory granules are extruded from the distal or secretory pole of the cell. In normal PDL the even distribution of fibroblasts and the complex interrelationship of their distal cell processes leads to a diffuse pattern of silver grain deposition, masking the oriented flow of new collagen from the distal ends of individual fibroblasts. Analysis of electron microscopic radioautographs revealed that newly synthesized collagen was packaged and secreted from beta-APN-treated fibroblasts via the normal cytoplasmic pathways but at a slower rate.

Aminopropionitrile↗
PubMed1985

Effects of L-azetidine-2-carboxylic acid on matrix secretion and Golgi structure in fibroblasts and osteoblasts of the mouse.

The effect of a proline analogue, L-azetidine-2-carboxylic acid (LACA), on collagenous matrix secretion by periodontal ligament fibroblasts and alveolar bone osteoblasts was studied by light and electron microscopic radioautography after injection of 3H-glycine; 3H-glycine labeled material accumulated in the cisternae of the rough endoplasmic reticulum and was not secreted for over 4 hours. The Golgi complex of both fibroblasts and osteoblasts showed a marked alteration of its composition after LACA administration. All Golgi saccules and collagen secretion granules disappeared within 2 hours. Flattened Golgi cisternae were still present and appeared to be unaffected by the administration of LACA. These observations indicate that Golgi saccules in collagen-secreting cells are the initial staging areas for the formation of collagen secretory granules and that there is a close correlation between form and function in the Golgi apparatus of collagen-secreting cells.

Alveolar Process↗
PubMed1986

Root-analogue versus crown-analogue dentin: a radioautographic and ultrastructural investigation of the mouse incisor.

The present paper reports on differences between the root- and crown-analogue dentin portions of the continuously growing mouse incisor. Conventional light microscopy and radioautography were used to study dentin formation and the uptake of [3H]-proline and [3H]-serine. It was found that, although the dentin apposition rate along the crown-analogue part (covered by enamel) equalled or slightly exceeded that along the root-analogue part (covered by cementum), the processing of predentin into dentin was considerably faster in the root aspect. Comparison of the two dentin portions at the ultrastructural level revealed that differences occurred in the morphology of the secretory granules of the odontoblast layer. Two types of granules were observed: those that were and those that were not loaded with electron-dense particles of 30 nm diameter. While the former type was most frequent along the crown-analogue aspect of the incisor, the latter type was particularly found along its root-analogue aspect. This difference may reflect differences between the two dentin portions in the composition of the noncollagenous matrix.

Animals↗