Preliminary attempts at ultrastructural sulfhydryl demonstration

Summary Trials to visualize tissue-bound sulfhydryl (SH) groups were made by means of incubating thin slices of mammalian liver and pancreas specimens at pH 7.4 with the specific SH-reagents p-chloromercuribenzoate or mercury orange, the former followed by brief fixation in glutaraldehyde and/or inert dehydration, the latter used after dehydration of unfixed tissue. Uncontrasted thin sections showed no obvious increase in electron density over that of non-incubated controls. After contrasting with uranyl acetate, however, some lysosomes showed small, comma-like precipitates with both reagents.Trials to obtain a better visualization of precipitated mercaptides by subsequent application on the incubated sections of the sulfide-silver procedure for heavy metals gave, so far, only equivocal results.Read in part (Pihl, Gustafson and Falkmer, 1967) at the Annual Meeting of the Scandinavian Society for Electron Microscopy in Åbo, Finland, June 1–2, 1967.This work was supported by grants from the Swedish Medical Research Council (Project No. K68-12X-718-03).     

Histochemical evidence for lysosomal uptake of lead in tissue cultured fibroblasts

Summary Lysosomes of embryonic rat fibroblasts cultivated in vitro normally contain heavy metals, as shown with a modified sulfide-silver method (SSM). Cultures which received lead added to the cultivation medium showed an enhanced SSM-positivity. However, since the SSM demonstrates several different heavy metals the sulfides of which are weakly soluble in water, it was not possible to distinguish between naturally occurring heavy metals—largely iron—and added lead (supposed to have been taken up by the cells).By treating the cells with a 0.2 M TCA solution after an initial exposure to HS^– it was possible to dissolve FeS without affecting the PbS to any noticeable extent. When subsequently the development process of the SSM was applied to the cells, no metals could be demonstrated in the control cells whereas those exposed to lead showed presence of granules, most of which were identical with lysosomes as visualized with a Gomori type reaction. A lysosomal uptake of lead could thus be demonstrated with the modified SSM when combined with a simple dissolving process.Supported by the Swedish Medical Research Council grants nos. K70-12X-2037-05A and B71-12X-2037-06B.     

Heavy metal localization and age related accumulation in the rat nervous system

Summary A modified sulfide-silver method was used to demonstrate tissue bound heavy metals in the rat brain at various ages. An accumulation of sulfide-silver positive material was found to accompany aging, indicating heavy metal accumulation. This was verified by quantitative analysis using atomic absorption spectrophotometry. Iron appears to be the most important heavy metal. Besides differences between various ages, regional variations in heavy metal contents could constantly be shown. The heavy metals appear, at least in part, to be located in lysosomes. A heavy metal influence on the lysosomal membrane permeability is discussed.Supported by the Swedish Medical Research Council grant No. 12X-2037.     

Staining of fresh epiphyseal cartilage with toluidine blue

Summary Staining of fresh, unfrozen sections of rat tibia in 0.01% toluidine blue at pH 4.5 revealed the presence of large -metachromatic granules in the cells of the proliferative zone, small -metachromatic granules in the cells of the hypertrophic zone, and both types in cells of intermediate size in the maturing zone. The matrix of the reserve zone stained with toluidine blue; the matrix of the proliferative and upper hypertrophic zone was not stained, but staining appeared again in the lower hypertrophic (calcifying) zone and in the cartilage cores of the metaphyseal trabeculae. Similar but more erratic results were obtained with frozen sections of Carnoy-fixed, gelatin-embedded specimens. These results could not be obtained with simple frozen section techniques or ordinary histological processing, which disrupt the intracellular granules and alter the state of combination of the protein-polysaccharides of the matrix. The results support the concept that a change in the nature of the mucopolysaccharide-containing components is one of the factors involved in the change from non-calcifiable to calcifiable cartilage matrix.     

The ultrastructural basis for the identification of cell types in the pancreatic islets I. Guinea pig

Summary The pancreatic islets of the guinea pig have been studied by light and electron microscopy. The B granules in glutaraldehyde-fixed tissue often are cup-shaped with an indentation visible on one side of the granule. Phosphotungstic acid hematoxylin (PTAH) positive cells have been characterized by electron microscopy as three subtypes based on the size of the secretory granules. A_a cells are the most common and have secretory granules around 200 m in diameter. A_b cells have large secretory granules around 300 m in diameter and are relatively infrequent. A_c cells are the least common and have small (160 m) granules. Characteristic D cells are identifiable by electron microscopy and, on the basis of the subsequent study (Munger, Caramia, and Lacy, 1965), are identified as the silver positive cells observed by light microscopy.This investigation was supported in part by United States Public Health Service research grants GM-10102 and GM-03784 from the Institute of General Medical Sciences, and AM-01226 from the Institute of Arthritis and Metabolic Diseases.-The authors wish to acknowledge the valuable technical assistance of Mrs. Aileen Sevier and Mrs. Lidia Donohue.     

Electron microscopic demonstration of metals in rat mast cells

Summary This paper describes a modification of a cytochemical method for the demonstration of heavy metals. The well localized precipitate in the mast cell granules, which is also present in granules that have been separated from the cell, suggests that the metals are localized in the granules. It is demonstrated that mast cell grown cultures do not contain precipitate. The chelating and histamine inhibiting agent 8-hydroxyquinoline produced no changes in the histochemical pattern of the mast cell granules before nor after treatment with the histamine liberator 48/80 which provokes a release of granules from the cells. These observations suggest either that the metal (zinc) is bound to the granules in such a manner that the chelating agent cannot chemically, or based on the configurations of the metal-containing molecule, reach the metal and theraby prevent its transformation to a metal suphide.     

The pituitary polypetide “7B2” is associated with LH/FSH and TSH cells and is localized within secretory vesicles

Summary A highly conserved polypeptide termed 7B2, isolated from human and porcine pituitaries, has been reported by immunoreactivity to be distributed in various organs. However, the highest concentration has been found in the pituitary as demonstrated by a specific radioimmunoassay. In order to determine the type of cells within the pituitary that contain 7B2 and to analyse its intracellular localization, specific immunocytochemistry techniques (unlabeled antibody, peroxidase-antiperoxidase) were used both for light and electron microscopy. Immunocytochemistry of both expiants and monolayer-cell cultures of the adenohypophysis was studied.Immunoreactivity to 7B2 has been found in 21.9% of the total number of cells. After simultaneous staining of serial sections with appropriate antibodies, 7B2 was found to be colocalized with -LH/-FSH in gonadotrophs and with -TSH in thyrotrophs. In situ immunocytochemistry at the electron-microscopic level showed that immunoreactive 7B2 is compartmentalized within secretory granules. The small (130 to 250 nm) but not the large granules (400 to 700 nm) were labeled in gonadotroph-like cells and small granules (90 to 150 nm) were also labeled in thyrothrophlike cells. Study of the gonadotrophs in cell culture after Zamboni's fixation revealed weak to moderate immunoreaction in rough endoplasmic reticulum. The current findings as well as previous data indicate that 7B2 is synthesized, stored and possibly released from the adenohypophysis similarly to many other secretory products.     

Collagen formation — observations on its intracellular packaging and transport

Summary Odontoblasts, osteoblasts and fibroblasts of young rats were examined in the electron microscope after staining thin sections either with lead citrate alone or with uranyl acetate prior to lead citrate.With lead citrate alone, collagen fibrils in the extracellular matrix stand out as lucent structures against a moderately electron dense background. Within the cells, lucency is restricted to certain dilated portions of the Golgi saccules as well as to the secretory granules located nearby and in the secretory pole of the cells. The lucency present in these compartments may be attributed to fibrils that are similar to the lucent collagen fibrils in the extracellular matrix. Other cellular compartments, e.g. the rough ER, do not display lucency.When preparations are stained with uranyl acetate prior to lead citrate, lucency is observed neither in the matrix nor in the cells. In the matrix, collagen fibrils are easily identifiable by their cross banded pattern. In the odontoblasts, dilated portions of Golgi saccules between the outer and inner face contain filaments aligned in parallel that are approximately 3 000 Å in length. In saccules on the inner face filament aggregates are present, some of them exhibiting a cross banding pattern. In secretory granules, however, the contents appear rather homogeneous.It is suggested that filament aggregates of collagen can assemble in the Golgi apparatus from filamentous units. These are transported through the cell by way of secretion granules and are discharged to the extracellular matrix by exocytosis.This investigation was supported by grants of the Medical Research Council of Canada. The author wishes to express appreciation to Dr. C. P. Leblond for his guidance in the course of this work.     

Amplified ribosomal DNA in meiotic prophase oocyte nuclei of acipenserid fishes

Summary In situ hybridization has been performed in sections through ovaries ofAcipenser ruthenus andAcipenser güldenstädti in order to detect the rDNA sequences. Hybridization resulted in specific labelling of the caps of extrachromosomal DNA present in pachytene oocyte nuclei and of the chromatin granules distributed beneath the nuclear envelope in early diplotene nuclei. In the same sections, the nuclei of all ovarian cells in both species (oogonia, leptotene, and zygotene stage oocytes, follicular cells, connective tissue cells) showed a very low, but similar labelling.Amplification of genes for rRNA thus occurs at the pachytene stage in early oogenesis ofAcipenseridae. No rDNA amplification could be detected in the previous stages.     

Immunocytochemistry of gonadotropic cells and identification of cell types in ultrathin cryosections of the pituitary of the rainbow trout,Salmo gairdneri

Summary The most frequently occurring cell types in the pars distalis of the pituitary gland of the rainbow trout, (i) the lactotropic, (ii) the gonadotropic, and (iii) the somatotropic cells, were identified in cryosections. Their morphological characteristics were compared with those of Epon-embedded material. Cell location, cell form, position of the nucleus, arrangement of rough endoplasmic reticulum and sizes of secretory granules proved to be useful parameters for identification. The size distribution of secretory granules of corresponding cells in cryosections and Epon sections proved to be similar. Additionally, both the immunoferritin and the unlabeled antibody enzyme method were applied for the immunocytochemical labeling of gonadotropic hormone-producing cells in cryosections. Anti-salmon-GTH as well as anti-carp-GTH serum showed the presence of GTH in both the smaller and the larger granules of the classical GTH cells, but also produced a reaction in TSH cells. Labelling of TSH cells was absent when using anti--carp-GTH. Specificity of the reaction depended upon the degree of dilution of the anti-GTH serum. Results with dilutions of 14,000 and 18,000 in the unlabeled antibody enzyme method, and of 18,000 up to 132,000 in the immunoferritin technique were optimal. Acid phosphatase activity in the smaller granules was demonstrated by enzyme cytochemistry in Epon sections. The relationship of the presence of hormone in these granules is discussed. The high sensitivity of the immunocytochemical labeling procedure is discussed with respect to cryo-ultramicrotomy.     

Localization of corticotropin- and endorphin-related peptides in the intermediate lobe of the rat pituitary

Summary The question is examined whether -melanocyte stimulating hormone (-MSH), adrenocorticotropic hormone (ACTH), met-enkephalin and -endorphin are detectable by enzyme immunocytochemistry in the cells of the intermediate lobe (PI) of the rat pituitary. By applying antibodies against MSH, ACTH and -endorphin on light microscopic sections, intense immunostaining was found in all PI-cells. At the ultrastructural level, after treatment of consecutive serial sections with these three antibodies the immunoreactivity was localized in the same secretory granules. No specific metenkephalin immunoreactivity could be detected in the cells of the intermediate lobe.Supported by Deutsche Forschungsgemeinschaft SFB 87/B2     

On the occurrence of argyrophil cells in salivary glands

Summary Salivary glands (parotid, submandibular and sublingual glands) of nine mammalian species were investigated with respect to presence and localization of argyrophil and argentaffin cells. With the exception of the parotid gland of the rat, no positive staining was observed within the examined glands. In the rat parotid distinctly argyrophil cells could be demonstrated in the intercalated ducts. Histochemical studies of the cells, ultrastructural analysis of their cytoplasmic granules as well as their reactions to certain drugs indicate that these cells are of exocrine rather than of endocrine nature. After a subcutaneous injection of pilocarpine, the intensity of the argyrophil staining was markedly reduced. No specific catecholamine fluorescence could be detected within the cells, not even after pretreatment of the animals with high doses of L-DOPA. The membrane-bounded cytoplasmic granules of the intercalated duct cells furthermore displayed a strong positive staining reaction after treatment of ultrathin Vestopal sections with the periodic acid-chromic acid-silver technique of Rambourg et al. (1969).Supported by grants from the Swedish Medical Research Council (Project No. 12X-718), and the Medical Faculty of the University of Umeå. The skilful technical assistance of Miss Siw Domeij is gratefully acknowledged     

The binding of epidermolytic toxin from Staphylococcus aureus to mouse epidermal tissue

Summary Fluorescein-labelled epidermolytic toxin (FTC-toxin) ofStaphylococcus aureus and ferritin—toxin conjugate have been prepared and purified. FTC-toxin bound selectively to cryostat and resin-impregnated sections of neonatal mouse skin. Binding was localized at the keratohyalin granules and in the stratum corneum. In an epidermal cell (granular, spinous and basal) preparation, only keratohyalin granules of the granular cells bound FTC-toxin. Ferritin—toxin conjugate bound to skin sections at the same two sites as FTC-toxin and was competitive with the binding of free toxin. Keratohyalin granules in unstained sections had a novel patched appearance under the electron microscope, and the ferritin—toxin conjugate bound preferentially to the electron-lucent areas. In the stratum corneum it was shown by quantitative estimation that the target density decreased as the surface of the tissue was approached.     

Calcium and sulphur in neurosecretory granules and calcium in mitochondria as determined by electron microscope X-ray microanalysis

Summary Sections of neurosecretory cells fixed in glutaraldehyde and osmium tetroxide were studied by means of an EMMA-4 analytical microscope. Secretory granules in neurosecretory cells of the corpus cardiacum and of the brain, both in the desert locust Schistocerca and in the blowfly Calliphora, as well as neurosecretory granules in posterior pituitaries of the frog Rana and of the albino rat all contain a high concentration of calcium. A distinct sulphur peak was also a constant feature.In neurosecretory cells of the corpus cardiacum of Schistocerca the chromatin contained a high concentration of calcium. The mitochondria also contained much calcium, but part of this disappeared during preparation except when fixative and wash contained calcium chloride. By block staining with uranyl acetate most calcium is displaced from the mitochondria, whereas most of the calcium remains in the neurosecretory granules. Since the calcium peaks in spectra from neurosecretory granules appear of similar size, regardless of variations in the preparative procedure, this calcium must be firmly bound. The possible role of the calcium bound to the neurosecretory substance is discussed.The presence of sulphur in insect neurosecretory granules indicates the presence of a protein besides the hormone, i.e., an insect neurophysin.We wish to thank Dennis Greer for the operation of the analytical microscope. This work was supported by a Wellcome-Carlsberg Travelling Research Fellowship awarded to T.N. The EMMA-4 facility is supported by a grant from the British Science Research Council     

Granulated folliculo-stellate cells and growth hormone cells immunostained with anti-S 100 protein serum in the pituitary glands of the goat

Summary Goat pituitary glands were immunohistochemically studied with antisera for bovine S-100 protein, rat LH, FSH, TSH, prolactin, ovine GH, and porcine ACTH^1–39 by use of the superimposition technique on adjacent sections. Folliculo-stellate (F-S) cells were divided into two categories on the basis of ultrastructural properties: One consisted of a mass of agranular cells in which the pseudolumina were equipped with microvilli and cilia. Elongate gap junctions were often observed among these cells. The other was a group of granulated cells with or without pseudolumina. In this group the gap junctions were shown to be disintegrated. The dense granules 150–250 nm in diameter began to accumulate in the cells. However, neither type of these F-S cells was immunostained for S-100 protein. On the other hand, numerous polygonal, elongate, irregular or stellate cells containing S-100 protein were distributed throughout the gland. Most of them were immunohistochemically identical with the GH cells laden with the secretory granules 250–450 nm in diameter, but some of them were identical to TSH and prolactin cells which immunostained faintly for S-100 protein. This appears to be the first demonstration of GH cells intensely immunostained for S-100 protein.     

A dialysis system with phytoplankton for monitoring chemical pollution in freshwater ecosystems by alkaline phosphatase assay

The effects of three groups of chemicals, heavy metals, pesticides and phenolics, on alkaline phosphatase activity of intact Chlorella vulgaris cells were investigated. There was a marked inhibitory effect of heavy metals and a slight one due to phenolics, but the pesticides tested showed no effect. In order to detect heavy metals in freshwater ecosystems, we propose a dialysis system, which can be placed in the field and then provide early warning signals of toxicity. This phosphatase inhibition test is recommended the first stage of an enzymatic screening system for chemical pollution in water.     

Lectin binding sites in normal and phenobarbitale/halothane treated rat liver

Summary The content of carbohydrate residues of both normal and phenobarbitale-halothane-hypoxia exposed rat liver has been examined by means of lectin histochemistry. Eight biotinylated lectins specific to galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose and mannose were applied to paraffin sections of rat liver at light microscopic levels. The most distinct binding was observed at the structures of the perisinusoidal functional unit: Kupffer cells are bound by S-WGA, SBA and PNA. Bile canaliculi display binding sites for RCA I and WGA. Cytoplasm of hepatocytes appears lectin-negative, except for PSA. The enhanced reaction of S-WGA, PNA and SBA after the preincubation of the sections with neuraminidase indicates the occurance of sialic acid in Kupffer cells. The phenobarbitale-halothane-hypoxia exposed rat liver shows centrolobular degeneration of hepatocytes with a diminished amount of hepatocytes and Kupffer cells as well. The lectin binding pattern of sinusoidal walls, membranes of hepatocytes and bile canaliculi remains the same compared to that of normal rat liver. This finding suggests that at least the carbohydrate content of membranes in the liver resists severe destruction under phenobarbitale-halothane-hypoxia. It is assumed that there exists a connection between intact carbohydrate residues and the regeneration of liver parenchyma.     

Stage-specific localization of cytoskeletal actin mRNA in murine seminiferous tubules and intestinal epithelia as demonstrated by in-situ hybridization

Summary In-situ hybridization experiments have been performed using isoactin ( and )-specific riboprobes in various tissues of the rat and mouse. Distribution of the grains of actin mRNAs for both and types was similar throughout sections of the rat testis. Although both mRNAs were evenly distributed in the seminiferous tubule, extremely heavy labeling was observed in about 10% of the seminiferous tubules that could be identified as stage XII of spermatogenesis. At high magnification, grains of the mRNA were found in the cytoplasm of elongating spermatids and in the Sertoli cell cytoplasm at the adluminal side. Much higher density of the grains of mRNA was observed in the neck region of the spermatids at stage XII. Thus, the dense distribution of cytoskeletal actin mRNAs is stage-specific in the tubule during spermatogenesis in the rat. The high expression of both and actin mRNAs was also observed in the epithelial cells of the intestinal crypts.     

Establishment and characterization of a mid-kidney cell line derived from golden pompano <Emphasis Type="Italic">Trachinotus ovatus</Emphasis>, a new cell model for virus pathogenesis and toxicology studies

Golden pompano Trachinotus ovatus, a popularly cultured and commercially important marine fish worldwide, has been recognized as a promising candidate for mariculture. However, outbreaks of infectious bacterial or viral diseases and environmental deterioration have led to great economic losses in T. ovatus aquaculture recently. In our research, we established a new mid-kidney cell line, designated as TOK, from golden pompano, T. ovatus. The optimized growth temperature and working concentration of fetal bovine serum (FBS) were 28°C and 10–20%, respectively. Foreign genes could express well in TOK cells. The modal number of TOK cells was 54. The TOK cells were susceptive to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV), and the virus could propagate in cells. Propagation was verified by qRT-PCR, and virions were observed under electron microscopy. Cytotoxicity analysis revealed that TOK cells were sensitive to different concentrations of extracellular products (ECPs) from Vibrio alginolyticus and V. anguillarum. Moreover, heavy metals (Cd, Cu, and Hg) also showed dose-dependent cytotoxicity to the TOK cell line. We established a mid-kidney cell line from T. ovatus which could be applied to cytotoxicity assays of heavy metals. The newly established TOK cell line possesses great application potential in genetic manipulation, virus–host interaction studies, and toxicity assays of bacterial extracellular products and heavy metals.     

Evidence for FSH-like material in ACTH granules of certain corticotropic cells in the pituitary of the pig

Summary The ultrastructural immunocytochemical peroxidase-antiperoxidase technique used on serial sections reveals that certain corticotropic cells contain both ACTH and FSH, but not LH (ACTH/FSH cells). To determine the specificity of the anti-FSH staining in these cells, immunocytochemical absorption experiments were performed. The results indicate that (1) anti-FSH and -ACTH antisera are bound to different antigens in the corticotropic cell, and (2) anti-FSH staining is specific for a FSH-like antigen. In the ACTH/FSH cells both hormones are stored in the same secretory granules, distributed among other granules that contain only ACTH.