Electron cytochemical demonstration of sulfhydryl groups in keratohyalin granules and in the peripheral envelope of cornified cells

Summary Although it is widely accepted that sulfhydryl and disulfide groups are important in keratinization, it has generally not proved possible to demonstrate these in the keratohyalin granules.A type of nuclear and cytoplasmic keratohyalin granule and the peripheral envelope adjacent to the plasma membrane in cornified cells show considerable Cytochemical similarity (Jessen, 1970). These sites have now been shown to react highly specifically with silver-methenamine. Blocking experiments indicate that sulfhydryl groups are responsible for this reaction.The findings in the present paper confirm the validity of the previous suggestion that there are several types of keratohyalin granules, of which one type is of widespread occurrence in keratinizing tissue and may be involved in the deposition of the peripheral envelope in cornified cells.The author wishes to thank Dr. H. Moe and Dr. D. P. Knight for valuable discussion. This work was supported by grants from The Danish Medical Research Council (512-149/69 and 512-1008/71) and The Danish Science Research Council (512-1009/71).     

Occurrence and neonatal development of gastrin immunoreactivity in the digestive tract of the rat

Summary The distribution of gastrin immunoreactivity in the rat gut was examined by immunochemical and immunohistochemical techniques. Gastrin occurs predominantly in the antrum proper, but gastrin is found also in the adjacent part of the oxyntic mucosa and in the duodenum. In the remainder of the gut the gastrin concentration is very low. No gastrin cells and very low gastrin concentrations are observed in the antrum at birth. The gastrin concentration as well as the number of gastrin cells increases progressively with age. The antral gastrin concentration reaches adult or near-adult values 30–40 days after birth.This study was supported by the Swedish Medical Research Council (04 X-1007), by Riksföreningen mot Cancer (660-0 IX), Landsforeningen till Kraeftens Bekampelse, Danish Medical Research Council (512-6) and Fonden for Storkobenhavn, Faeroerne og Gronland.     

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

Summary 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 resorption 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.This report was supported by the Danish Research Council, Grant no. 512-4044We wish to thank Mrs. Ruth Nielsen for technical assistance during the work. The parathyroid extract was kindly supplied by Eli Lilly Sweden AB     

Uptake of peroxidase by calcitonin inhibited osteoclasts

Summary The present electron microscopic histochemical study demonstrates that osteoclasts from calcitonin treated bone are able to take up organic macromolecules even though the ruffled border has disappeared. The absorption occurs around the entire periphery of the osteoclast, but the amount of absorbed peroxidase seems to be reduced in comparison with that of untreated cells. It is concluded that the effect of calcitonin on osteoclasts is primarily a cessation of the exocytosis and the concomitant disappearance of the ruffled border.This research was supported by the Danish Medical Research Council, grant No. 512-7184     

Structural and histochemical aspects of epidermis development of fetal porcine skin

Epidermal development of fetal porcine skin was studied in fetuses from 41 days of gestation until birth with scanning and electron microscopy techniques as well as histochemical methods, including immunohistochemistry. The porcine fetus develops a relatively thick and solid multilayered cover of epidermal cells, which is not lost before birth. It consists of tightly packed cells of the periderm and the stratum intermedium. The periderm cells are totally filled with filamentous proteins; in the intermediate cells, the filamentous proteins are concentrated in the cell periphery, forming a thick marginal zone. Immunohistochemically, the cytofilaments could be identified as cytokeratins of lower and higher molecular weights. The first thin stratum corneum lamellae are formed below the stratum intermedium at about 80-85 days of gestation.     

Effects of parathyroid hormone on osteoclasts in vivo

Summary Young rats were treated with high doses of parathyroid hormone (PTH). Osteoclasts from these animals revealed characteristic alterations in comparison to control cells: a) The cytoplasm contained large vacuoles with phagocytosed cells, some of which resembled osteoblasts or osteocytes. The vacuoles were interpreted as lysosomes because the engulfed cells often appeared partly digested and the vacuoles contained acid phosphatase as demonstrated histochemically, b) lipid droplets were present in the cytoplasm and usually located close to the endoplasmic reticulum and/or in regions with many free ribosomes, c) the Golgi complex was more frequently separated from the nuclei than in control cells, d) small coated cytoplasmic bodies were numerous in the peripheral cytoplasm, e) the membranes of the endoplasmic reticulum were fused in some places, f) cytoplasmic regions with numerous free ribosomes were frequent, g) large ring-shaped granules occurred in some mitochondria. Energy dispersive X-ray analysis of these granules provided evidence that they contained calcium and probably phosphorus, h) in some osteoclasts the mitochondria were enlarged. — The findings are consistent with an increased activity of osteoclasts and in particular a stimulation of the lysosomal system in these cells.This research was supported by grants no. 512-819, 512-1545 and 512-1912 from the Danish Research Council. The observations were reported in part at the annual meeting of the Scandinavian Society for Electron Microscopy in Aarhus 1972 (Lucht, 1973). — The authors thank Mr. K. Ibe for cooperation in the energy dispersive X-ray analysis, which was carried out at the JEOL (Europe) S. A. Application Center, Paris.     

Ultrastructure of the human enamel organ

Summary The fine structure of external enamel epithelium, stellate reticulum and stratum intermedium in primary tooth germs (bell stage) from four human foetuses was investigated.Characteristically, the cells of the differentiated external enamel epithelium, stellate reticulum and stratum intermedium exhibit many free ribosomes, few rough endoplasmic reticulum cisterns, well-developed Golgi complexes, many coated and smooth vesicles, often in relation to the cell membranes, and many bundles of tonofilaments. The cells are connected by numerous desmosomes and gap junctions.A parallel differentiation of stratum intermedium — external enamel epithelium, and the ameloblast layer is demonstrated.The morphology of the cells of the three layers indicates that these have secretory, transport and supporting functions.     

The interstitial cells in the renal medulla of rat,rabbit, and gerbil in different states of diuresis

Summary The inner zone of the renal medulla of rats, gerbils, and rabbits was investigated to determine whether or not there are any characteristic ultrastructural differences between the interstitial cells of these species. The effects on the interstitial cells of water deprivation and water loading were also investigated.In all three species, the Type 1 interstitial cells, the lipid containing cells, were abundant and their distribution and topographical relations as well as their general ultrastructure were similar. The previously reported significantly higher frequency in desert rats could not be confirmed. Although the lipid droplets of the interstitial cells were smaller in gerbils and rabbits when compared to rats, their fine structure was similar. Their electron dense outer zone was sometimes associated with a granular material and/or a lamellar material with a periodicity of about 40 Å resembling phospholipid myelin figures.Water-loaded rats showed a considerable increase in the number of lipid droplets when compared to dehydrated or untreated animals. In contrast, the interstitial cells of waterloaded gerbils and rabbits were depleted of lipid droplets.We are indebted to Professor A.B. Maunsbach for valuable discussions and criticism and to Mrss. Hanne Weiling and Birthe Overgaard for competent technical assistance. The gerbils used in this study were a gift from Leo AB, Helsingborg, Sweden. This study was supported by the Danish Medical Research Council (J. nos. 512-1067, 512-1545, 512-3633)     

Tooth formation and the 28,000-dalton vitamin D-dependent calcium-binding protein: an immunocytochemical study

Antiserum to the 28-kilodalton vitamin D-dependent calcium-binding protein (CaBP) was used to localize CaBP in histologic sections of the continuously erupting incisor in mandibles obtained from normal rats. With the peroxidase--anti-peroxidase technique, no CaBP was detected in undifferentiated ameloblasts or in those which had become columnar and were facing pulp. Calcium-binding protein was first noted in the cytoplasm of random ameloblasts facing dentin in the presecretion zone. As the ameloblasts became more mature in the zone of enamel secretion, CaBP was uniformly present in their cytoplasm. Ameloblasts with Tome's processes clearly contained CaBP in these processes as well as in the cell-body cytoplasm. Near the later developmental stages of the zone of enamel secretion, some of the adjacent underlying cells of the stratum intermedium also contained CaBP in their cytoplasm. In some stratum intermedium cells and papillary cells, CaBP extended into the zone of enamel maturation, but not to the end of that zone. Cytoplasmic CaBP continued to be present in ameloblasts as they progressed through the zone of enamel maturation to the final, shortened cells at the gingival margin of the erupting incisor. No CaBP was detected in odontoblasts, pulpal cells, the stellate reticulum, or the outer dental epithelium.     

Histochemical demonstration of succinic dehydrogenase activity during sequential molar development in the Swiss albino mouse

Summary Applying the ditetrazolium salt (Nitro-BT) method for succinic dehydrogenase, murine molar teeth were studied sequentially from the cap stage of development through the appositional stages of odontogenesis. Reaction-distribution and intensity varied relative to the developmental stage as well as the zone of maturation within a given stage. The peripheral cells of the parent dental lamina exhibited some activity, as did the outer enamel epithelium of the bell stage. During the period of matrix apposition, components of the stratum intermedium, ameloblastic zone and odontoblastic layer region of the dental papilla demonstrated intense enzymatic activity. Cells actively engaged in enamel matrix production demonstrated activity in the basal and distal cell segments. High activity continued in the papillary layer of the enamel organ, as well as in the cells of the dental sac during the postamelogenic period.Supported by PHS Grant No. 2800-02, National Institute of Dental Research, National Institutes of Health.     

Stratum intermedium lineage diverges from ameloblast lineage via Notch signaling

The stratum intermedium develops as flattened cell layers on the proximal side of the ameloblast layer during tooth development. However, little information is available regarding the origin and the role. In this study, we indicate that some stratum intermedium cells originate from the inner enamel epithelium (IEE) in rat incisor organ cultures using DiI as a tracer. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells express the Notch1 protein and Hes1 mRNAs, while the IEE and ameloblasts express the Jagged1. Further, we examined the role of Notch signaling using the dental epithelial cell line HAT-7. Recombinant Jagged1 protein enhanced the appearance of stratum intermedium cells in HAT-7 cultures and neutralization with an anti-Jagged1 antibody inhibited these effects. Additionally, overexpression of the Notch1 internal domain increased the number of stratum intermedium cells. We hypothesize that the stratum intermedium lineage differentiates from the ameloblast lineage via Notch signaling.     

Interstitial deletion in the “critical region” of the long arm of the X chromosome in a mentally retarded boy and his normal mother

Summary A family in which an interstitial deletion of the X chromosome, del(X)(q13q21.3), is segregating was ascertained through a boy with cleft lip and palate, agenesis of the corpus callosum, and severe mental retardation. The possible causal relationship to his chromosome abnormality is discussed. Although the deletion occurred within the critical region, the mother showed no signs of gonadal dysgenesis. A phenotypically normal daughter was, as her mother, monosomic for this region of the X, and both showed random inactivation of the X chromosome.Supported in part by grants to E.N. from the Carl Petersen's Foundation (B 995) and the Danish Medical Research Council (512-4276)     

Osteoclasts and their relationship to bone as studied by electron microscopy

Summary Osteoclasts in metaphyses from young rats were systematically sectioned at different levels. Two types of osteoclasts were recognized. One type had no ruffled border while the other, and predominant type contained a ruffled border in a part of its length; some of the latter contained two ruffled borders. The closest contact between osteoclast and bone occurred at the level of the ruffled border and this bone under the border showed characteristic changes indicative of resorption. In some osteoclasts the ruffled border consisted of numerous slender cytoplasmic projections separated by very narrow spaces or channels while in other osteoclasts it was more open. The ruffled border was commonly surrounded by a transitional zone containing numerous thin filaments. The osteoclast usually had its greatest dimension at the level of the ruffled border and the cytoplasm here contained many bodies and vacuoles but a sparse endoplasmic reticulum. Away from the level of the ruffled border the cytoplasmic vacuoles and bodies were fewer while the endoplasmic reticulum was often more pronounced. Parts of the osteoclasts were usually situated close to a vessel. It is suggested that there is a correlation between the development of the ruffled border and the degree of bone resorption and that osteoclasts without a ruffled border are, at least temporarily, inactive with respect to bone resorption. The numerous cytoplasmic bodies, interpreted as lysosomes, are presumed to be important in the resorption process. The closely adjacent positioning of osteoclasts and vessels may facilitate the transport of resorption products to the blood.This research was supported by the Danish Research Council. Grant no. 512–727, 512–819 and 512–1545.I wish to thank Professor Arvid B. Maunsbach for valuable discussions.     

Cytoplasmic vacuoles and bodies of the osteoclast

Summary Cytoplasmic vacuoles and bodies in the osteoclast (rat) were studied by electron microscopy. The vacuole-like structures (0.03–5 in diameter) may be classed as a) vacuoles b) coated vacuoles and c) invaginations. The cytoplasmic bodies vary in size from 0.02–3 in diameter and these may similarly be classed as a) light cytoplasmic bodies, b) dense cytoplasmic bodies, c) coated cytoplasmic bodies and d) cytoplasmic bodies containing inclusions. Both the cytoplasmic vacuoles and the bodies are limited by a triple layered membrane of about 91 Å in thickness. Their relationship to the lysosomal system and the role of this system in the osteoclast is discussed.This research was supported by the Danish Research Council. Grant no. 512–727 and 512–819.     

Identification of inbred strains of mice,Mus musculus. I. Genetic control of inbred strains of mice using starch gel electrophoresis

Fifteen biochemical markers were tested in 30 inbred strains of mice to control the genetic constitution of each strain. Discrepancies in pattern from Standardized Nomenclature for Inbred Strains of Mice are reported and discussed.This work was supported by Grant No. 512–2532 from the Danish Medical Research Council.     

Electron microscopic localization of 5''-nucleotidase in the stratum intermedium and ameloblasts

Synopsis 5-nucleotidase was demonstrated at the fine structural level in the stratum intermedium and ameloblasts of the first mandibular molars of CD-1 mice. The enzyme was localized with the Wachstein & Meisel (1957) method along the plasma membranes of the cells of the stratum intermedium and ameloblasts. While 5-nucleotidase was present throughout the stratum intermedium, only the proximal region of the plasma membranes of ameloblasts was demonstrably active for this enzyme. 5-Nucleotidase has been implicated in transport of metabolites across cell membranes, and its localization in the present study supports this implication as well as the transport functions of the stratum intermedium and the stratum intermedium-ameloblastic interface.     

Effect of superior cervical ganglionectomy on monoamine content in the epithalamic area of the Mongolian gerbil (Meriones unguiculatus): A fluorescence histochemical study

Summary Extirpation of the superior cervical ganglion was performed in a series of Mongolian gerbils. One or two weeks after the ganglionectomy the animals were injected with a monoamine oxidase inhibitor. Subsequently perfusion fixation was performed using the glyoxylic acid-paraformal-dehydemagnesium method (Lorén et al., 1976) for fluorescence histochemical investigation of the monoamines of the pineal complex. In the ganglionectomized animals all of the blue-fluorescent sympathetic fibers in the pineal complex (superficial pineal gland, deep pineal gland and the pineal stalk) completely disappeared. The yellow indolamine fluorescence of the cells in the superficial pineal and the deep pineal, as well as in the pineal stalk, was markedly reduced after ganglionectomy. No change in the morphology or number of sympathetic fibers in the medial habenular nucleus was observed. These results indicate that the presence of sympathetic nerve fibers with perikarya in the superior cervical ganglion is necessary for maintaining a high indolamine content in all three parts of the pineal complex. In addition, the results also indicate that the deep pineal gland is a functional part of the pineal complex. The presence of a functionally active deep pineal, bordering the pineal recess, suggests that part of the pineal hormones might be secreted into the cerebrospinal fluid.This work was supported by the Carlsberg Foundation, the Swedish Natural Science Research Council, grant no. 2126-100, and the Danish Medical Research Council, grant no. 512-7134     

Quantitative analysis of epithelial changes during wound healing in palatal mucosa of guinea pigs

Summary The epithelium of wounded guinea pig palate was subjected to stereologic analysis. A total of 18 biopsies (animals) were used. Biopsies were taken at 18, 48, 96 and 120 h after wounding. Point counting procedures were employed to analyse electron micrographs sampled from one (18 h) or two epithelial strata (48, 96 and 120 h). The essential modulations in epithelial structure as wound healing proceeds were as follows: During the early phases characterized by formation and advancement of epithelial lips (18 and 48 h), migrating cells converged towards a cell type which structurally was less differentiated than normal basal cells. This alteration was expressed by a decrease in volume density of cytoplasmic organelles, mainly mitochondria, free ribosomes and tonofilament bundles, coupled with an increase in volume density of lysosomal bodies. Concomitantly, the volume density of cytoplasmic ground substance rose markedly. Subsequent to fusion of contralateral migratory lips (96 and 120 h) reversion to normal epithelial structure was indicated by the increment in magnitude of basal cell parameters. Further structural density gradients from basal towards upper cell layers appeared. This pattern was mainly displayed by mitochondria, free ribosomes, and tonofilament bundles. The magnitude and gradation of most tissue and cell parameters were not yet re-established at 120 h. The density of tonofilament bundles and the density level of cytoplasmic ground substance in particular deviated.This investigation was supported in part by grants No. 512-5958 and No. 512-5151 from the Danish State Medical Research Council     

Acid phosphatase of osteoclasts demonstrated by electron microscopic histochemistry

Summary Location of acid phosphatase outside and inside the osteoclast was studied by electron microscopic histochemistry. Osteoclasts with a ruffled border apposed to the bone showed enzyme activity in a) membrane-limited cytoplasmic bodies of different dimensions, b) some Golgi vesicles and inner Golgi cisternae, c) vacuoles and vacuole-like profiles, d) extracellular channels and channel expansions in the ruffled border, e) cell-bone interspace. The possibility of bone degradation by lysosomal enzymes both in the cytoplasmic vacuoles and in the cell-bone interspace is discussed.This research was supported by the Danish Medical Research Council. Grant. no. 512-819.I am indebted to Professor Arvid B. Maunsbach for valuable discussions and suggestions and to Mrs. Ruth Nielsen for technical assistance.     

Epithelial cytodifferentiation and extracellular matrix formation in enamel-free areas of the occlusal cusp during development of mouse molars: light and electron microscopic studies

Mandibular first molars in mice ranging in age from 18 days prenatal to 5 days postnatal were used for light and electron microscopic examinations of the enamel-free area (EFA) during development of the occlusal cusp (mesiobuccal cusp). Notable morphological changes in the inner enamel epithelium and the cells of the stratum intermedium were observed. At prenatal age of 18 days, the inner enamel epithelium of the EFA (EFA epithelium) was composed of a layer of columnar cells and covered by the cells of the stratum intermedium. Two days after birth, the EFA epithelium was made up largely of preameloblasts, with mitochondria located in the proximal side of the cells toward the stratum intermedium. The cells of the stratum intermedium were irregularly shaped, with wide intercellular spaces between them. At a postnatal age of 3 days, most of the EFA epithelial cells resembled maturation-stage ameloblasts, being short and columnar in shape and having nuclei located in their proximal side. Distal cell membranes were folded, and mitochondria were scattered throughout the cytoplasm. In 4-day-old mice, the EFA epithelium was found to be formed of short columnar or cuboidal cells with distinct intercellular spaces. The cells of the stratum intermedium could no longer be detected, and cells of the EFA epithelium could not be distinguished from those of the stellate reticulum. Odontoblasts of the EFA were arranged and polarized parallel to the basal lamina, and odontoblastic processes extended toward the cusp tip. The orientation of thin and thick collagen fibers within predentin and dentin was also parallel to the basal lamina. Even after dentin mineralization, disrupted basal lamina and long, aperiodic, fine fibrils were found between the epithelium and the dentin. Following the disappearance of the basal lamina and fine fibrils, stippled material and crystals appeared on the dentin surface. The mineralized matrix, which x-ray microanalytical energy peaks identified as containing calcium and phosphorus, was continuous with enamel in the distal slope of the cusp at the cusp tip. Thus, the inner enamel epithelium of the EFA differentiated into secretory cells capable of enamel-like matrix formation.(ABSTRACT TRUNCATED AT 400 WORDS)