Morphological changes during conoid extrusion in Toxoplasma gondii tachyzoites treated with calcium ionophore

Treatment of tachyzoites of Toxoplasma gondii with the calcium ionophore A23187 induced dramatic ultrastructural changes that were observed by light and electron microscopy. Light microscopy showed a higher percentage (22%) of tachyzoites with the conoid extruded when compared to control parasites. Electron microscopy confirmed the conoid extrusion by both transmission and scanning electron microscopy. Freeze-fracture replicas showed that the plasma membrane adjacent to cytoplasmic dense granules appeared devoid of intramembranous particles. Membrane-limited vesicles and filopodium-like structures at the cell surface were observed in treated cells. 3-D reconstruction from serial sections confirmed the data and showed a heterogeneity in dense granule shape not reported in control cells.     

Effect of monoamines on the taste buds in the mouse

Summary Mouse taste buds were investigated following administration of monoamines and their precursors by fluorescence and electron microscopy. The appearance of fluorescent cells within the taste bud and the ultrastructural changes of vesicles in the gustatory cells were due to the treatment of 5-hydroxytryptophan. Small dense-cored vesicles (30–60 nm in diameter) appeared throughout the cytoplasm and accumulated especially at the presynaptic membranes of afferent synapses. Large dense-cored vesicles (80–100 nm) increased twice in number, and electron densities of their cores became more dense as compared with untreated mice. Fluorescent cells appeared in the taste bud of l-DOPA treated mice, whereas no ultrastructural changes were observed. These results suggest that the gustatory cells of the taste bud are capable of taking up and storing monoamines, which might act as neurotransmitters from the gustatory cells to the nerves.     

Morphological study of cell organelles during development. I - The nuclear sac and the endoplasmic reticulum on the rat nephron

The maturation of the endoplasmic reticulum (ER) of rat kidney tubule cells was studied with an osmium impregnation technique. Thick sections (0.3-0.6 micron) of kidney tissue were made after a five-day impregnation with osmium tetroxide and examined by standard transmission electron microscopy at 80-100 kV. Studies were performed on rat foetuses from 18-21 days of gestation, on newborns, and on 2-20 day old animals. At the undifferentiated stage, only a small percentage of the tubule cells were impregnated; in these, the perinuclear sac was stained and a few nuclear pores were already seen. Rudimentary, but thick canalicular projections seemed to originate from the perinuclear sac and become more extensive with maturity. Flattened saccules appeared later and fenestrations were seen in proximal tubule cells only when they seemed to have reached their functional specialization. In some cells, only the Golgi apparatus was stained. In the distal tubule cells, there was also progressive formation of a network consisting first of canaliculi and later of saccules which were rarely fenestrated. The osmium impregnation technique appears to be useful as an index of the ER organization development.     

Morphologic and cytochemical characteristics of amine-containing globule leukocytes in rat tracheal epithelium

Amine-containing cells in the tracheal epithelium are typically of the small-granule type (diameter approximately 100 nm). However, in the rat, another amine-containing cell type has been identified that possesses the amine-handling features of the APUD-series of cells (amine precursor uptake and decarboxylation) but not the ultrastructural characteristics. It has been postulated that these cells may be related to cutaneous melanocytes. In this study, fluorescent cells were present in the laryngeal and tracheal epithelial lining of adult Sprague-Dawley rats following freeze-drying and exposure to formaldehyde vapor (FIF or formaldehyde-induced fluorescence). Microspectrofluorimetry revealed an emission maximum at 493 nm. The excitation maximum could not be calculated but appeared to be around or below 350 nm (to record spectra below requires the use of quartz optics). Yellow fluorescence also emanated from serotonin-containing mast cells (excitation and emission maxima: 401/515 nm). Tracheal segments processed according to the aqueous formaldehyde ( AFIF ) technique, for the demonstration of 5- hydroxytryptophan (5-HTP) or serotonin (5-HT), failed to identify fluorescent cells in the epithelial lining even though connective-tissue mast cells were evident. Subsequent treatment of AFIF -fixed sections with formaldehyde and HCl vapors ( AFIF -HCl) resulted in the formation of a fluorogenic compound within numerous cells in the tracheal lining (455/537 nm). This spectral shift and increase in intensity of fluorescence following acidification are characteristic for standards and/or cells that contain tryptamine, tryptophan, or peptides with NH2-terminal tryptophan and are markedly different from microspectrofluorimetric data reported for the phenylethylamines or serotonin. It is therefore postulated that these cells contain a closely related beta-(3-indolyl) ethylamine-like compound, serotonin excluded. The morphology of the fluorescent cells was similar when prepared according to the FIF or AFIF -HCl techniques. Conjunctive staining, the examination of a single section first by fluorescence microscopy and subsequently by other histochemical and cytochemical methods, demonstrated that the fluorescent granules were also methylene blue, alcian blue, periodic-acid Schiff, and ferric- fericyanide positive. Subsequent correlative electron microscopic examination of Epon-embedded AFIF -HCl-treated tracheal sections demonstrated that these amine-containing cells were globule leukocytes.     

Fluorescent labeling of resin-embedded sections for correlative electron microscopy using tomography-based contrast enhancement

Locating areas of interest by electron microscopy can be laborious. This is particularly true for electron tomography, where the use of thicker sections may obscure relevant details in the projection images. We evaluated the applicability of fluorescent probes to thin plastic sections, in combination with fluorescence microscopy, as an aid in selecting areas for subsequent electron microscopic analysis. We show that pre-embedding labeling of DNA and RNA with acridine orange yielded a predominant nuclear stain. The stain greatly reduced the time needed to scan sections for mitotic cells, or cells with characteristic nuclei such as neutrophils. Post-embedding labeling with SYTOX green yielded a nuclear stain comparable to acridine orange, and wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 labeled mucous granules and the Golgi area in intestinal goblet cells. The fluorescent labels were visualized directly on sections on electron microscope grids. It was therefore possible to establish a coordinate system based on the position of the grid bars, allowing for easy retrieval of selected areas. Because the fluorescent probes were incompatible with osmium tetroxide treatment, contrast in the sections was faint. We propose a simplified electron tomography procedure for the generation of 2D views with enhanced contrast and resolution.     

Maturation of the human hypothalamo-hypophyseal neurosecretory system in prenatal ontogeny. A light-optical and electron microscopic study

The hypothalamic-hypophysial neurosecretory system (HHNS) was studied in the human foetuses from the age of 8 weeks till birth. The hypothalamus of 8 weeks old foetuses is weakly differentiated, no individual cell groups, so-called nuclei, are identified. The supraoptic (SON) and paraventricular (PVN) nuclei are identified from the age of 12 weeks on. The size of cell nuclei increases with the age. The Homori-positive granules were first found in some SON and PVN cell and in neurohypophysis in the 18 weeks old foetuses. It was shown under the electron microscope that the neurohypophysis of 8 weeks old foetuses consisted mainly of pituicytes with axons among the cytoplasmic processes of the latter. After the age of 10 weeks, the area of parenchyma of the neurohypophysis occupied by axons increased and typical elementary neurosecretory granules appeared in them. The data obtained are discussed with respect to the participation of HHNS in the regulation of water metabolism in the human foetuses.     

The relationship between the folliculo-stellate network and the thyrotropic cells of the avian adenohypophysis

Summary The folliculo-stellate network of the avian adenohypophysis consists of stellate cells surrounding colloid-containing follicular cavities into which cilia and microvilli project. Other identifying criteria are agranularity, junctional complexes at the apical pole, presence of cytoplasmic processes ramifying between adjacent secretory cells, and close appositions of plasma membranes linking folliculo-stellate cells and presumptive thyrotropic cells.Transmission electron microscopy reveals that TRH and L-DOPA induce simultaneous ultrastructural changes in the folliculo-stellate network and in the thyrotropic cells. TRH transforms at cell of the cephalic lobe into a highly hypertrophic cell in which enlargement of cisterns of rough endoplasmic reticulum containing secretory granules, development of a large Golgi complex, presence of newly synthesized secretory granules, and granulation of the cytoplasm are the main features. In the meantime, the follicular cavities become dilated by large amounts of homogeneous colloid. The administration of L-DOPA also leads to the development of dilated cisterns in presumptive thyrotropic cells of the cephalic lobe. Intracisternal granules, immature secretory granules, and large Golgi complexes, however, are not observed. Degranulation of the cytoplasm is obvious. The follicular cavities of both cephalic and caudal lobes are enlarged and filled with colloid in which granular elements are noted.The ultrastructural changes observed in thyrotropic cells and in the folliculo-stellate network reflect functional changes induced by the experimental manipulation. These changes may be related, directly or indirectly, or completely independent.     

Endocrine-like cells of the pulmonary epithelium of the human adult lung

Summary The morphological and histochemical characteristics of endocrinelike cells of the pulmonary epithelium of the right lower lobe of 12 human adult lungs were studied.Few cells were reactive to the argyrophil silver method of Grimelius and of Sevier and Munger and cells with a similar morphology and distribution emitted a green or yellow fluorescence after treatment of the lung epithelium with the amine precursors L-DOPA or L-HTP, respectively. A greater number of cells seems to be demonstrated by electron microscopy. The cells were characterized by small, round secretory granules showing a central dense core and a very thin clear halo between the core and the surrounding membrane.The cells are thought to be related to the endocrine-like cells of the pulmonary epithelium of the human foetal lung and to cells of carcinoids of larger bronchi.     

Ultrastructural cytology of the cyclic corpus luteum of the cow

Corpora lutea (CL) from cows on day 12 of the oestrous cycle were studied by electron microscopy to investigate whether, and how, different subpopulations of luteal cells can be identified in tissue sections. Tissues from 6 CL were examined, and nucleated profiles of luteal cells were classified as large, medium or small on the basis of their areas in electron micrographs. Cut-off points for area categories for large, medium and small-sized cells were based on diameters of greater than 25, 20-25 and less than 20 microns, respectively, if the measured areas were converted to a circular shape after correction for shrinkage. The only qualitative features which distinguished cells of large size from those of small size were the presence of clusters of secretory granules, and of exocytosis of these granules, in large cells only. However, these features were observed in only 59% of large cells, probably primarily due to sampling limitations in single sections. Other qualitative features which have been regarded as diagnostic of large or small luteal cells were observed in cells in all size categories. It was concluded that large and small luteal cells in the cyclic CL of the cow are distinguishable by their ultrastructural features. However, these data do not support the recent suggestion that the mid-cycle CL of the cow contain two subpopulations of large luteal cells in approximately equal numbers.     

Pyronin Y as a fluorescent stain for paraffin sections

Pyronin Y has long been used, in combination with other dyes such as Methyl Green, as a differential stain for nucleic acids in paraffin tissue sections. It also forms fluorescent complexes with double-stranded nucleic acids, especially RNA, enabling semi-quantitative analysis of cellular RNA in flow cytometry. However, the possibility of using pyronin Y as a fluorescent stain for paraffin tissue sections has rarely been investigated. We herein report that in sections stained with Methyl Green–pyronin Y, red blood cells, elastic fibre of blood vessels, zymogen granules of pancreatic acinar cells, surface membrane of heptocytes and kidney tubular cells showed strikingly strong green and/or red fluorescence, while the nuclei of cells appeared non-fluorescent. The use of confocal laser-scanning microscope greatly improved the resolution and selectivity of the fluorescent images. Staining with pyronin Y alone gave similar results in terms of fluorescence properties of the specimens. Pretreatment of paraffin sections with RNase significantly reduced cytoplasmic pyronin Y staining as judged by transmission light microscopy, but it had little effect on the fluorescence intensity of red blood cells, elastic fibres and zymogenbreak granules.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin.

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

The ultrastructural identity of pancreatic polypeptide cells in chicks

A very similar ultrastructure has been attributed to pancreatic polypeptide and somatostatin cells in chickens. In order to characterize any possible differences between them, cells shown to be immunoreactive for these hormones in semi-thin sections of chick pancreas were identified in adjacent thin sections prepared for conventional electron microscopy. In this way the ultrastructural features of the immunoreactive cells could be determined. In general, in somatostatin-immunoreactive cells, granule profiles are almost exclusively round, whereas in pancreatic polypeptide cells there are elongate as well round profiles. Within cells of both types the electron density of the granule matrix varies from one granule to another, but the range of density is greater in pancreatic polypeptide granules. The latter are slightly smaller than somatostatin granules.     

Monoamine-containing granulated cells in the frog lung

Summary The epithelium of the primary bronchus of the frog lung has been studied by fluorescence and electron microscopy. Clusters of five to ten, ovoid, brilliantly yellow fluorescent cells were observed in the basal portion of the epithelium. These cells contained numerous electron-dense granules of variable shape and size. The granules gave a positive argentaffin reaction at the ultrastructural level, suggesting a possible existence of monoamines in the granules. In addition, synaptic contact between the intraepithelial nerves and the cells, which was characterized by the aggregation of the granules toward the presynaptic membrane thickening of the cell, was also noted. These data are discussed in relation to similar studies in birds and mammals, and a possible function of these cells suggested.     

Estimation of the copper-iron correlation in the liver under experimental conditions. A microscopic and microchemical study

Liver obtained from copper-intoxicated rats was studied by electron microscopy and electron probe microanalysis. Large ultrastructural disorders in liver cells appeared at the level of lysosomes and mitochondria. Diffuse and aggregate copper granules were observed in the entire cell, but mainly in pericentrolobular and periportal zones. Probe analysis revealed an increased copper deposit, particularly during the chronic copper intoxication, and a very high concentration of iron gradually accumulated.     

Cutaneous mast cell depletion and recovery in murine graft-vs-host disease

Mast cells as studied by light microscopy with metachromatic staining, have been noted to "disappear" from the skin of mice with chronic graft-vs-host disease (GVHD) produced across minor histocompatibility barriers. This mast cell disappearance is accompanied by ultrastructural evidence of loss of granule contents. In this study, we followed cutaneous mast cells in chronic GVHD over 9 mo by three methods: Light microscopy of toluidine blue-stained sections showed that mast cells not seen at day 42 reappeared between days 94 and 125, were supramaximal at days 146 and 164, and returned to normal levels at days 195 and 280. Double immunofluorescent staining of mast cells for the presence of surface IgE receptors and cytoplasmic granules (avidin) revealed IgE receptor-bearing cells that lacked avidin-binding granules at the time when mast cells were not apparent on light microscopy. By electron microscopy, reappearing mast cells have the morphology of immature dermal mast cells. Ultrastructural abnormalities of mast cells persist some 150 days after GVHD induction. The possible relationship of these mast cell changes to the development of dermal fibrosis in chronic GVHD is discussed.     

The ultrastructural identity of pancreatic polypeptide cells in chicks

Summary A very similar ultrastructure has been attributed to pancreatic polypeptide and somatostatin cells in chickens. In order to characterize any possible differences between them, cells shown to be immunoreactive for these hormones in semi-thin sections of chick pancreas were identified in adjacent thin sections prepared for conventional electron microscopy. In this way the ultrastructural features of the immunoreactive cells could be determined. In general, in somatostatin-immunoreactive cells, granule profiles are almost exclusively round, whereas in pancreatic polypeptide cells there are elongate as well as round profiles. Within cells of both types the electron density of the granule matrix varies from one granule to another, but the range of density is greater in pancreatic polypeptide granules. The latter are slightly smaller than somatostatin granules.     

Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells

Adenomatous polyposis coli (APC) tumor suppressor protein has been shown to be localized near the distal ends of microtubules (MTs) at the edges of migrating cells. We expressed green fluorescent protein (GFP)-fusion proteins with full-length and deletion mutants of Xenopus APC in Xenopus epithelial cells, and observed their dynamic behavior in live cells. During cell spreading and wound healing, GFP-tagged full-length APC was concentrated as granules at the tip regions of cellular extensions. At higher magnification, APC appeared to move along MTs and concentrate as granules at the growing plus ends. When MTs began to shorten, the APC granules dropped off from the MT ends. Immunoelectron microscopy revealed that fuzzy structures surrounding MTs were the ultrastructural counterparts for these GFP signals. The COOH-terminal region of APC was targeted to the growing MT ends without forming granular aggregates, and abruptly disappeared when MTs began to shorten. The APC lacking the COOH-terminal region formed granular aggregates that moved along MTs toward their plus ends in an ATP-dependent manner. These findings indicated that APC is a unique MT-associated protein that moves along selected MTs and concentrates at their growing plus ends through their multiple functional domains.     

The smooth endoplasmic reticulum in neurohypophysial axons of the rat: Possible involvement in transport,storage and release of neurosecretory material

Summary The intra-axonal organization of the smooth endoplasmic reticulum was studied in the neurohypophysis of rats during and after water deprivation. Parallel to conventional electron microscopy, the material was treated with a double impregnation staining technique specifically designed to contrast the intracellular membranous system. In conventionally stained ultrathin sections from severely dehydrated rats most axons appeared to be free of membranous organelles, whereas corresponding axons treated with the double-impregnation technique generally exhibited a highly developed system of smooth endoplasmic reticulum. In axonal endings, both techniques revealed a profusion of microvesicles in intimate relationship with tubular elements of the smooth endoplasmic reticulum. In short-term (12 h) rehydrated rats, a similarly developed system of smooth endoplasmic reticulum was still observed at all axonal levels with both procedures. After 24 to 48 h of rehydration the tubules of the smooth endoplasmic reticulum exhibited, in double impregnated material, numerous dilatations which resembled the adjacent neurosecretory granules. In conventionally stained ultrathin sections, an accumulation of electron dense material occurred within tubules of the smooth endoplasmic reticulum in the more proximal axonal segments, while in the more terminal segments, which contained numerous elongated granules, membrane continuity was frequently observed between newly formed granules and the smooth endoplasmic reticulum. After 7 days of rehydration the general pattern of the axonal smooth endoplasmic reticulum was comparable to that in untreated rats. These results are discussed in the light of a suggested involvement of the axonal smooth endoplasmic reticulum in the non-granular transport of neurosecretory material in connection with (1) storage in distally formed granules, and (2) release via microvesicles. Acknowledgements: The authors wish to express their gratitude to Mrs. M. Balmefrézol for her skillful technical assistance