Scanning electron microscopy: Identification of mast cells by indirect cathodoluminescence

Summary The epithelial tissues of the rabbit gall bladder reacted for acid mucosaccharides were studied with the electron microscope. A series of acid mucosaccharide-containing ultrastructures of the gall bladder epithelium were observed in specimens treated with dialyzed iron, colloidal thorium and ruthenium red. In the epithelium stained with dialyzed iron, reactive ultrastructures are not only extra- but intracellular; the surface coat of the plasma membrane, pinocytotic vesicles, granules of secretion and certain elements of the Golgi apparatus. In the epithelial tissues stained by colloidal thorium or ruthenium red, the surface coat of the plasma membrane is the only ultrastructure which is reacted positively for the acid mucosaccharide stains. The present images of ultrastructural elements containing acid mucosaccharides are taken to indicate a multiple function of the substances in rabbit gall bladder epithelium and are well correlated with the results of previous light and electron microscopic studies on the gall bladder epithelium of various vertebrate species.     

Preparation of plasma membranes from fertilized sea urchin eggs

A new method is presented for preparation of highly purified plasma membranes from fertilized sea urchin eggs. The purified plasma membranes are in vesicle form and are highly enriched in ouabain inhibitable, Na+/K+ ATPase activity. Analysis of membrane proteins by sodium dodecyl sulfate-gel electrophoresis indicates that several high-molecular-weight proteins characteristic of plasma membranes from unfertilized eggs are absent in plasma membranes from fertilized eggs.     

Electron microscopic observations of the surface of L-cells in culture

Summary Techniques are described for the preparation of preshadowed replicas of both the upper and lower surfaces of L-cells in culture, and of cross sections of L-cells growing on a cellophane substrate. These revealed long slender microvilli, 800 to 1,100 A in diameter, projecting from both upper and lower surfaces of the cells. These microvilli were frequently observed to contact other cells and substrate, and to leave material behind on the substrate. The plasma membrane of the lower surface was separated from the substrate by an electron-lucent gap 200 to 300 A wide. The surface coat of the L-cell was visualized by staining with colloidal iron and ruthenium. Staining with colloidal iron was most intense on the surface of the microvilli. The gap between cell and substrate was intensely stained with ruthenium red. Enzymatic digestion of living cells revealed that both trypsin and neuraminidase reduced the staining of the cell coat by colloidal iron, whereas only trypsin altered its staining with ruthenium red. After trypsin treatment, fragments of an amorphous material with the staining characteristics of the cell coat were observed between the denuded cells. Treatment with ribonuclease, chymotrypsin or hyaluronidase did not affect the staining of the cell coat.     

ENZYMO-CYTOCHEMICAL OBSERVATIONS ON THE CORTICAL CHANGE IN THE EGGS OF CYPRINUS CARPIO AND CARASSIUS AURATUS

Acid phosphatase (AcPase) and cholinesterase (ChE) activities were examined by ultracytochemical techniques in the mature unfertilized and the fertilized eggs of Cyprinus carpio and Carassitus auratus , to reveal the differences among three kinds of structures, cortical alveoli, CA- and CB-granules, which discharge their contents on fertilization into the perivitelline space. Deposits of the reaction product for AcPase activity are localized on the plasmalemma of unfertilized eggs, in the cortical alveoli, cytoplasmic matrix, lamellae of the Golgi apparatus and sometimes in multivesicular bodies but not in CA- and CB-granules, mitochondria, rough endoplasmic reticulum or on the plasmalemma of fertilized eggs. Deposits of the reaction product for ChE activity are localized on the inner surface of the plasmalemma, in the cytoplasmic matrix, in mitochondria and on a small number of tubular or cisternal membranes of the endoplasmic reticulum in mature unfertilized eggs, and on the outer surface of the limiting membrane of CB-granules and on membranous structures (possibly Golgi lamellae) associated with their formation, in fertilized eggs. The deposits on the plasmalemma rapidly disappear almost completely, with discharge of the cortical alveoli soon after fertilization, but they are again seen on the inner surface of the plasmalemma when emiocytotic discharge of the CB-granules begins about 10 min after fertilization.     

The coating of mouse myocardial cells. A cytochemical electron microscopical study.

The coating of mouse myocardial cells has been investigated with a variety of cytochemical methods. The coating of the surface membrane gives a positive reaction with ruthenium red, colloidal thorium, phosphotungstic acid (PTA) at low pH, silver methenamine after periodic oxidation (PA-silver technique) and with silver proteinate after periodic oxidation and thiocarbohydrazide treatment (PA-TCH-silver technique). The coating of the T system gives almost similar results. The nexuses do not react with PTA nor with the PA-silver and PA-TCH-silver techniques, but they are strongly stained with ruthenium red which reveals periodic structures in their gaps. The specificities of the colloidal thorium technique and PAT staining have been tested by chemical treatments (methylation, acetylation, saponification), enzymatic digestions (pronase, trypsin, hyaluronidase, neuraminidase) and carbohydrate extractions (with 0.1 N NaOH and 0.05 M H2SO4). These cytochemical data indicate, considering the specificity of the reactions, that the coating of the membrane surface and the T system contains polyanionic groups. A part of them, at least, would belong to a carbohydrate-containing material (glycoproteins), whereas at the level of nexuses the sugar residues would probably be absent.     

Histochemical staining of the complex carbohydrates of the midgut of the mosquito,Culex tarsalis coquillett

Histochemical staining of the midgut epithelial cell surface complex carbohydrates of the mosquito Culex tarsalis was examined electron microscopically. The microvillar surface is composed primarily of neutral vic-glycoconjugates; positively stained by silver methenamine and silver protein. Lanthanum and alcian blue staining indicate that the microvilli contain a minimal anionic component; possibly phosphoglycoconjugates. Similarly, the intercellular junctions contain a predominance of neutral vic-glycoconjugates. In addition, the intercellular junctions contain fixed positive charges, based on en bloc phosphotungstic acid staining. The midgut basolateral membrane system and the basal lamina are both highly anionic; stained by ruthenium red, tannic acid, alcian blue and periodic acid-chromic acid-phosphotungstic acid. The basolateral plasma membrane also contains some vic-glycoconjugates. Selective staining indicates that the anionic component of the basolateral plasma membrane and the basal lamina is predominantly carboxyl groups; no specific staining for sulfo- or phosphoglycoconjugates was observed.     

Localization of tropomyosin in sea urchin eggs

Localization of tropomyosin in sea urchin eggs was investigated immunohistochemically. A rabbit antiserum against tropomyosin prepared from lantern muscle of the sea urchin was used for the indirect immunofluorescence staining of unfertilized and fertilized eggs. The tropomyosin-specific fluorescence was observed at the peripheral region beneath the plasma membrane, mitotic apparatus and contractile ring. The mitotic apparatus isolated from sea urchin eggs was also stained with the anti-tropomyosin serum.     

Change in Membrane Fluidity of Sand Dollar Egg Cortices Caused by Ca2+-Induced Exocytosis: Microscopic Analysis with Fluorescence Anisotropy

The surface membrane fluidity of sand dollar eggs after exocytosis was investigated by using a specially designed video-microscope system to measure the fluorescence depolarization of isolated cortices stained with hexadecanoylaminofluorescein. When unfertilized eggs were stained before isolation, the plasma membranes became labeled with fluorescent dye, but cortical vesicles did not. The fluorescence anisotropy of the isolated cortices increased from 0.256 to 0.285 during exocytosis induced by Ca²⁺. The increased anisotropy was not changed by lowering the Ca²⁺concentration after exocytosis. Dislodging of cortical vesicles by shearing with a stream of solution had no affect on the anisotropy. These results suggest that the fluidity of the plasma membrane decreases after exocytosis. When cortices were stained after isolation, both plasma membranes and cortical membrane organelles became labeled. These cortices possessed an anisotropy of 0.215. After dislogding the cortical organelles the anisotropy increased to 0.232. These results indicate that the fluidity of the cytoplasmic membrane leaflets of cortical organelles is higher than that of the plasma membrane. Therefore, it was suggested that only the outer leaflet of the plasma membrane becomes less fluid after exocytosis.     

Ultrastructural studies on the surface membrane of the mouse egg.

Fertilized and unfertilized mouse eggs were examined by scanning and transmission electron microscopy for evidence of mosaicism in the organization and concanavalin A-binding properties of their surface membranes. No obvious quantitative mosaicism in concanavalin A binding was noted. The egg membrane was microvillous over most of its surface, but was smooth in the region overlying the 2nd metaphase spindle of the unfertilized egg and on the polar body of the fertilized egg.     

An electron microscopic study of surface polysaccharides in Bacteroides

The electron microscopic appearance of the cell surface of Bacteroides strains and Klebsiella pneumoniae stained with ruthenium red or colloidal iron is described. The effect of polymyxin B (PMB) was also registered. It was found that all Bacteroides strains have a polysaccharide lined 'micro-capsule' external to the outer membrane which could aggregate and form blebs. The blebs so formed were distinct from other types of bleb formed in Klebsiella involving the outer membrane and induced by PMB. Such types of PMB alterations were not induced in Bacteroides.     

Perivitelline space of mammalian oocytes: extracellular matrix of unfertilized oocytes and formation of a cortical granule envelope following fertilization.

Extracellular matrices (ECM) present around unfertilized and fertilized mammalian oocytes were studied ultrastructurally in samples prepared in the presence of ruthenium red to facilitate stabilization of extracellular materials. Unfertilized mouse, hamster, and human oocytes have an ECM comprising granules and filaments in their perivitelline spaces (PVS). This matrix is more abundant in the human than in hamsters and mice. The granule/filament matrix appears identical to the matrix seen between cumulus and corona radiata cells following ruthenium red processing and previously shown to comprise protein and hyaluronic acid. By including ruthenium red during fixation, it is possible to demonstrate the existence of cortical granule exudate in the PVS of fertilized oocytes from hamsters, mice, and humans. Much of the cortical granule exudate is trapped in the PVS and forms a new coat around the fertilized oocyte. This material is particulate when stained with ruthenium red and appears to be uniformly dispersed around the entire oocyte surface. We refer to this new coat as the cortical granule envelope. This envelope is observed in the PVS of all developmental stages up to and including blastocysts in all three species. Following hatching of mouse and hamster blastocysts, the cortical granule envelope is no longer present. Possible functions of this envelope are discussed.     

Cytochemistry and morphology of synaptic structures in the cerebral cortex of rat.

Studies have been carried out on the synapses in the cerebral cortex of rat by using impregnation with ethanolic solution of phosphotungstic acid, contrast staining with ruthenium red and impregnation with bismuth iodide, with or without subsequent uranyl acetate and lead citrate staining. It has been established that dense projections are adequately visualized with methods demonstrating basic chemical groups (phosphotungstic acid and bismuth iodide), whereas the synaptic vesicles are stained by techniques demonstrating acid chemical groups (ruthenium red and uranyl acetate and lead citrate). On the basis of these observations a hypothesis is forwarded concerning the mechanisms of migration of synaptic vesicles towards the presynaptic membrane. Measurements of the parameters of the dense projections suggest that the configuration of the presynaptic vesicular grid is not uniform along the presynaptic areas.     

Alteration of lipid organization following fertilization of sea urchin eggs

The fluorescent probe merocyanine 540 was used to examine the organization of the lipids in the external leaflet of the plasma membrane after fertilization of sea urchin eggs. These lipids in unfertilized eggs are closely packed, as evidenced by their inability to bind the dye, whereas in fertilized eggs and cells of embryos up to at least the gastrula stage, the membrane becomes more loosely organized, and stains with bright ring fluorescence. Induction of late fertilization events with ammonia failed to induce this change in staining behavior. Sperm components are not required to induce this alteration since parthenogenetically activated eggs stained. However, treatment of eggs with procaine, which specifically inhibits the early event of cortical granule fusion, was effective in suppressing staining. These results indicate that cortical granule fusion after fertilization results in a change in the organization of the lipids of the plasma membrane of sea urchin eggs.     

Starfish sperm-oocyte jelly binding triggers functional changes in cortical granules. A study using acid phosphatase and ruthenium red ultrastructural histochemistry

Starfish oocytes were examined before fertilization, immediately after insemination, and during the cortical reaction by means of acid phosphatase and ruthenium red ultrastructural histochemistry. Oocyte cortical granules are composed of a lamellar body and a surrounding matrix which is subdivided into dense and light portions. In unfertilized oocytes cortical granules are not stained by ruthenium red but show a weak acid phosphatase activity in the light portion of the granule matrix. Immediately after the adhesion of the spermatozoon to the oocyte jelly coat, the light matrix portion of cortical granules appears stained by ruthenium red and shows a strong acid phosphatase activity. During the cortical reaction, cortical granules are released into the perivitelline space and the lamellar body, surrounded by the stained matrix, fuses with the fertilization envelope. Our data suggest that membrane permeability changes and enzyme activation occur in the egg when the spermatozoon binds to the oocyte jelly coat.     

Starfish sperm-oocyte jelly binding triggers functional changes in cortical granules

Summary Starfish oocytes were examined before fertilization, immediately after insemination, and during the cortical reaction by means of acid phosphatase and ruthenium red ultrastructural histochemistry. Oocyte cortical granules are composed of a lamellar body and a surrounding matrix which is subdivided into dense and light portions. In unfertilized oocytes cortical granules are not stained by ruthenium red but show a weak acid phosphatase activity in the light portion of the granule matrix. Immediately after the adhesion of the spermatozoon to the oocyte jelly coat, the light matrix portion of cortical granules appears stained by ruthenium red and shows a strong acid phosphatase activity. During the cortical reaction, cortical granules are released into the perivitelline space and the lamellar body, surrounded by the stained matrix, fuses with the fertilization envelope. Our data suggest that membrane permeability changes and enzyme activation occur in the egg when the spermatozoon binds to the oocyte jelly coat.     

Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate

Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from demonstration of the mobility of integral membrane components.     

Insulin receptor sites as membrane markers during embryonic development. I. Data obtained with unfertilized and fertilized sea urchin eggs.

Binding of insulin to sea urchin egg plasma membrane has been studied by biochemical and immunocytochemical methods. Unfertilized and fertilized eggs as well as embryos during the first cell division have been used. 1. Competition experiments between 125I-insulin (1 nM) and an excess of native insulin (30 muM) indicate a specific hormone fixation to membrane crude extracts from unfertilized and fertilized eggs. The magnitude of "specific binding" is comparable to values recorded for mammalian cells. 2. Inhibition of insulin fixation by concanavalin A (100 mug/ml) suggests the glycoprotein composition of plasma membrane receptors. 3. An 30-min incubation of unfertilized and fertilized eggs in the presence of insulin leads to a significant increase in cyclic AMP content. 4. An immunocytochemical method demonstrates that insulin is selectively and specifically bound to the plasma membrane of eggs incubated in the presence of insulin before fixation. It can be concluded that insulin receptor sites are components of sea urchin eggs plasma membrane. Insulin binding which leads to cyclic AMP accumulation is not deeply modified by fertilization and does not include visible morphological changes in the eggs.     

Changes in the topography of the sea urchin egg after fertilization

Changes in the topography of the sea urchin egg after fertilization were studied by scanning and transmission electron microscopy. Strongylocentrotus purpuratus eggs were treated with dithiothreitol to modify the vitelline layer and to prevent formation of a fertilization membrane. Dithiothreitol treatment caused the microvilli to become more irregular in shape, length, and diameter than those of untreated eggs. The microvilli were similarly modified by trypsin treatment. This effect did not appear to be due to disruption of cytoskeletal elements beneath the plasma membrane, for neither colchicine nor cytochalasin B altered microvillar morphology. Thus, it appears that the vitelline layer may act in the maintenance of surface form of unfertilized eggs. Since dithiothreitol-treated eggs did not elevate a fertilization membrane, scanning electron microscopy could be used to directly observe modifications in the egg plasma membrane after fertilization. The wave of cortical granule exocytosis initiated at the point of attachment of the fertilizing sperm was characterized by the appearance of pits that subsequently opened, releasing the cortical granule contents and leaving depressions upon the egg surface. The perigranular membranes inserted during exocytosis were seen as smooth patches between the microvillous patches remaining from the original egg surface. This produced a mosaic surface with more than double the amount of membrane of unfertilized eggs. The mosaic surface subsequently reorganized to accommodate the inserted membrane material by elongation of microvilli. Blebs and membranous whorls present before reorganization suggested the existence of an unstable intermediate state of plasma membrane reorganization. Exocytosis and mosaic membrane formation were not blocked by colchicine or cytochalasin B, but microvillar elongation was blocked by cytochalasin B treatment.     

Integration of sperm and egg plasma membrane components at fertilization

Studies examining the integration of the sperm and egg plasma membranes, subsequent to gamete fusion in the surf clam, Spisula solidissima, were carried out employing the concanavalin A-horseradish peroxidase-diaminobenzidine procedure (Con A-HRP-DAB). When unfertilized Spisula eggs were incubated in Con A, either prior to or after aldehyde fixation and reacted with HRP-DAB, enzymatic precipitate was found associated with the vitelline layer and plasmalemma. The plasma membranes of sperm treated in a similar manner failed to stain. The plasma membranes of fertilized eggs reacted with Con A-HRP-DAB and examined by 1 min postinsemination were associated uniformly with enzymatic precipitate except at sites of sperm incorporation. These portions of unstained plasma membrane were derived from the spermatozoon and delimited the contents of the fertilization cone. From 2 to 4 min postinsemination, HRP-DAB reaction product became associated with the plasma membrane delimiting the fertilization cone. By 4 min postinsemination no difference in staining of the plasma membranes derived from the egg or the sperm (plasmalemma delimiting the fertilization cone) was detected. Evidence is presented suggesting that the acquisition of HRP-DAB reaction product by the former sperm plasmalemma is due to the movement of Con A binding sites from the egg plasma membrane.     

ELECTRON MICROSCOPE STUDIES ON THE SURFACE COAT OF THE NEPHRON

Attempts to make visible the carbohydrate coat at the free cell surface of glomeruli as well as the tubules of rabbit kidney were undertaken. The ruthenium red procedure was performed, according to Luft, at various pH values. Moreover, the colloidal iron and the colloidal thorium methods were used. Neuraminidase digestion was also performed. In the ruthenium red procedure the luminal face of the epithelial cells of the nephron was coated distinctly with reaction product. The results obtained revealed that some of the differences at various levels of the nephron depended on the pH values. In glomeruli and proximal convoluted tubules the optimum pH value was 7.4; in the ascending limb of Henle loops and distal convoluted tubules the optimum pH value was 6.8. The ruthenium red-positive surface coat was either closely connected with, or appeared as a part of, the outer leaflet of the unit membrane. The slit pores of glomeruli were also covered by a coat continuous with the surface coat of the adjacent foot processes. The coat lining the microvilli of proximal convoluted tubules completely filled the intervillous spaces. Also, both the colloidal iron method and the colloidal thorium method evidenced the presence of surface coat. Pre-treatment with neuraminidase abolished the effect of the Hale reaction. These results may indicate that the surface coat of the epithelia of the nephron shows the presence of glycoproteins containing siliac acid residues.