Cytochemical study of intracellular calcium in hamster spermatozoa during the acrosome reaction

Calcium was localized by a pyroantimonate technique in hamster spermatozoa during the acrosome reaction and pyroantimonate precipitates were observed in the anterior region of the acrosome. The calcium was also localized in the postacrosomal lamina of spermatozoa undergoing the acrosome reaction. Spermatozoa, incubated in capacitating medium containing verapamil, showed denser precipitates with an increase in concentration of this drug. Ionophore A23187 enhanced binding of calcium to the acrosomal region. The sodium channel inhibitor amiloride inhibited the acrosome reaction and the pyroantimonate precipitates were absent in these spermatozoa, whereas ionophore monensin enhanced the acrosome reaction. This suggests that the Na⁺/Ca⁺⁺ antiporter may be responsible for intracellular Ca⁺⁺ regulation during the acrosome reaction in hamster spermatozoa.     

ASSOCIATION OF CALCIUM WITH MEMBRANES OF SQUID GIANT AXON : Ultrastructure and Microprobe Analysis

Giant axons from the squid, Loligo pealei, were fixed in glutaraldehyde and postfixed in osmium tetroxide. Calcium chloride (5 mM/liter) was added to all aqueous solutions used for tissue processing. Electron-opaque deposits were found along the axonal plasma membranes, within mitochondria, and along the basal plasma membranes of Schwann cells. X-ray microprobe analysis (EMMA-4) yielded signals for calcium and phosphorus when deposits were probed, whereas these elements were not detected in the axoplasm.     

陆地棉雌蕊的花粉管生长途径中钙分布的超微细胞化学定位

用焦锑酸盐沉淀法对陆地棉(Gossypium hirsutum L.)授粉前后的柱头、花柱、珠孔与珠心组织中的钙进行了超微细胞化学定位。X射线波谱与能谱分析证明所定位的沉淀确系焦锑酸钙。观察结果表明:在整个花粉管生长途径中的雌蕊组织,钙分布均较其它相邻组织密集;钙主要分布在细胞壁与胞间基质等质外体系统中。在雌蕊中生长的花粉管,其尖端细胞器区也有丰富的钙。     

Ca++- and Na+, K+-ATPase activities in the fungiform papilla of the tongue of Rana Esculenta (Anura Ranidae)

In the fungiform papilla of Rana esculenta (Anura Ranidae), the Ca⁺⁺-ATPase is mainly distributed on the basolateral membrane of the sensory area cells (i.e., neuroepithelial, supporting, and mucous cells). Apical membranes of all cells facing the surface present a slight enzymatic activity. Lateral wall cells have a strong Ca⁺⁺-ATPase activity on basolateral and apical membranes. Strong Na⁺, K⁺-ATPase activity occurs on the apical surface of neuroepithelial cells. Ca⁺⁺-ATPase activity is absent on the surface of endothelial cells of the capillaries located under the sensory area. These observations lead us to conclude that the sensory area of fungiform papilla is the selective way for calcium influx. Furthermore the absence of ATPase activity on the surface of the endothelial cells indicates that there is no functional barrier to calcium influx into capillary, and that calcium can be removed by vessels from the sensory area.     

THE SUBCELLULAR LOCALIZATION OF CALCIUM ION IN MAMMALIAN MYOCARDIUM

This study was designed to investigate the proposition that subcellular calcium is sequestered in specific sites in mammalian myocardium. 29 functioning dog papillary muscles were fixed through the intact vascular supply by means of osmium tetroxide containing a 2% concentration of potassium pyroantimonate (K₂H₂Sb₂O₇·₄H₂O). Tissue examined in the electron microscope showed a consistent and reproducible localization of the electron-opaque pyroantimonate salts of sodium and calcium to distinct sites in the tissue. Sodium pyroantimonate was found exclusively in the extracellular space and clustered at the sarcolemmal membrane. Calcium pyroantimonate, on the other hand, identified primarily by its susceptibility to removal by chelation with EGTA and EDTA, was consistently found densely concentrated in the lateral sacs of the sarcoplasmic reticulum and over the sarcomeric I bands. M zones were virtually free of precipitate. The implications of these findings with respect to various parameters of muscle function are discussed.     

An investigation by electron microscopy of the nucleoside phosphatase activity of amphibian and mammalian erythrocytes

Amphibian and mammalian blood was washed in isotonic saline, fixed in glutaraldehyde, and then stained in the ATPase medium of Wachstein and Meisel. The blood cells were subsequently postfixed in osmium tetroxide, embedded in epoxy resins, and studied by electron microscopy. The plasma membranes of amphibian erythrocytes, from the newt Triturus cristatus and the frog Rana esculenta, were stained after incubation in media containing ATP or ADP as substrates, but were unstained after incubation in media containing AMP or sodium β-glycerophosphate. The addition of 0.001 M ouabain to ATP-containing media did not inhibit the staining of the plasma membranes, but the omission of Mg⁺⁺ ions from the medium inhibited staining. The plasma membranes of rat and rabbit erythrocytes were never stained after incubation in any of the media used.     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) was found not to inhibit active sodium transport across the isolated frog skin when added in 10^–3 m concentration to the basal-lateral surface. The same Cd⁺⁺ concentration similarly had no effect on Na⁺ transport across the isolated epithelial cell layer from the frog skin, although this dose of Cd⁺⁺ did inhibit Na⁺ transport across the frog urinary bladder and large intestine. When 10^–3 m Cd⁺⁺ was added to the apical surface of the isolated frog skin or to the isolated epithelial cells from the frog skin, sodium transport was reversibly stimulated, in contrast to the irreversible inhibition noted above. If equimolar cysteine was added with Cd⁺⁺ to the apical surface of the skin, however, active Na⁺ transport was irreversibly inhibited. In conjunction with the inhibition produced by equimolar Cd⁺⁺ and cysteine, isotopic Cd⁺⁺ permeation into the tissue was three times higher when added with cysteine than in the absence of cysteine. Thus, the effects of Cd⁺⁺ on epithelial Na⁺ transport is variable according to the epithelium studied and the presence of potential carrier molecules.     

Tritrichomonas foetus: Ultrastructural localization of calcium in the plasma membrane and in the hydrogenosome

The osmium tetroxide-potassium pyroantimonate technique was used to localize Ca²⁺-containing sites in the protozoan Tritrichomonas foetus. Reaction product was seen in association with the plasma membrane and with a membrane-bound organelle, the hydrogenosome. Reaction product was also seen in some cytoplasmic vesicles and in lysosomes. Treatment of the ultrathin sections with EGTA resulted in removal of the pyroantimonate precipitate. These results suggest that the hydrogenosome may be involved in the control of the intracellular concentration of Ca²⁺ in T. foetus.     

Cytochemistry and X-ray microprobe analysis of the midgut of Tomocerus minor lubbock (Insecta,Collembola) with special reference to the physiological significance of the mineral concretions

Summary Histochemical and cytochemical analyses have been made on the mineral concretions within the midgut cells of Tomocerus minor. The classical histochemical methods are not specific and precise enough and have been supplemented with cytochemical techniques on ultrathin sections. The most interesting of these was the K-pyroantimonate technique combined with glutaraldehyde-osmium fixation. This technique shows the distribution of cations such as Ca⁺⁺, K⁺, Mg⁺⁺ and Na⁺ on the concentric layers of the concretions. Chloride ions can be detected by means of the silver lactate technique. The action of calcium chelators such as E.D.T.A. shows an important distribution of calcium ions in the concretions. The spectra obtained by electron probe microanalysis from areas of fresh, dried and carbon coated midguts as well as from carbon coated semithin or ultrathin sections reveal the presence of Ca, K, Mg, S, Cl and P principally. Other elements such as aluminium, silicon and manganese have also been detected. Iron is not always present. The chemical and X-ray analytical investigations indicate that the midgut concretions are mainly built up of calcium, potassium, magnesium and sodium phosphates, perhaps associated with chlorides and carbonates. An organic matrix formed by polysaccharides seems to join the different mineral layers. These concretions may be formed within the vesicles of rough endoplasmic reticulum. The midgut cells are highly differentiated and very active in transport. Extensive basal infoldings and apical microvilli as well as lateral membranes are a site of small cationic deposits. The possible pathway of ion transport in the cell and the physiological significance of the concretions are discussed. The principal function of these concretions seems to be the maintenance of the mineral balance and to trap foreign and excess ions.

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Résumé L'analyse chimique des sphérocristaux de l'intestin moyen de Tomocerus minor a été réalisée. Les méthodes histochimiques courantes manquant souvent de spécificité et de sensibilité ont été complétées avec des méthodes cytochimiques sur coupes ultrafines. La plus intéressante a été la technique du pyroantimonate de K montrant la distribution des cations Ca⁺⁺, K⁺, Mg⁺⁺, Na⁺ sur les couches concentriques des sphérocristaux. La technique au lactate d'argent permet de déceler les ions Cl^-. L'action d'agents chélateurs du Ca tels l'E.D.TA. montre une importante distribution du calcium dans les sphérocristaux. L'analyse spectrographique d'étalements de mésentérons séches, carbonés et de coupes semi-fines ou ultrafines carbonées montre la présence de Ca, K, Mg, S, Cl, P, Na. D'autres éléments tels l'Al et le Si ont pu être détectés. Le Fe n'est pas toujours présent. Les sphérocristaux semblent formés essentiellement de phosphates de calcium, de potassium, de magnésium, de sodium associés peut-être à des chlorures ou des carbonates. Une matrice organique constituée essentiellement par des polysaccharides semble lier les différentes couches minérales. Ces sphérocristaux prennent naissance à l'intérieur des vésicules de l'ergastoplasme. Les cellules de l'intestin moyen sont très différenciées et sont le siège de nombreux transports actifs. Les replis basaux de la membrane plasmique, les microvillosités apicales, de même que les membranes latérales sont le siège de dépôts de cations. Le transport des ions dans les cellules ainsi que le rôle physiologique des sphérocristaux sont discutés. Le maintien de la balance hydrique ainsi que le piégeage d'ions étrangers ou en surplus semblent être la principale fonction des sphérocristaux.

     

N Lines in Striated Muscle: a Site of Intracellular Ca<Superscript>2+</Superscript>

PYROANTIMONATE in osmium penetrates intact cell membranes and produces electron dense precipitates by combining with several intracellular cations^1,2. The pyroantimonate technique provides a useful, method for ultrastructural studies on the N lines of muscle.     

Calcium pathway through a mineralizing epithelium in the crustacean Orchestia in pre-molt: Ultrastructural cytochemistry and X-ray microanalysis

During the pre-exuvial period of the terrestrial crustacean Orchestia, the calcium of the old cuticle is almost entirely reabsorbed and stored as calcareous concretions in the lumen of the midgut posterior caeca. The elaboration of these concretions is due to transport by the caecal epithelium. With ultrastructural cytochemistry controlled by X-ray microanalysis, it can be demonstrated that the main sites of ionized or ionizable calcium are the apical microvilli and an extracellular (lateral and basal) network of channels. Direct precipitating cytochemical methods, using potassium pyroantimonate or pyrophosphate, potassium oxalate or oxalic acid, sodium fluoride, sodium tungstate, and indirect substitution methods, using lead acetate or nitrate and cobalt nitrate were comparatively used. The results are interpreted in favour of the hypothesis of an extracellular transport pathway for calcium through the lateral smooth septate junctions, in conjunction with a more classical apical transport through the microvilli.     

Studies on Calcium and Sodium in Uterine Smooth Muscle Excitation under Current-Clamp and Voltage-Clamp Conditions

The objective of these studies was to define the roles of calcium and sodium in uterine smooth muscle excitation. The double sucrose-gap technique was used for current-clamp and voltage-clamp experiments. It was shown that neither sodium nor calcium alone is capable of supporting excitation in estrogen-dominated uterine smooth muscle. Calcium dependence was explained in part by increased membrane "leakage" current in calcium-free solution and calcium control of the voltage dependence of the early transient conductance. High concentrations of TTX did not affect the magnitude of the peak transient current while La⁺⁺⁺, Mn⁺⁺, and Co⁺⁺ greatly reduced or abolished it and decreased the steady-state current. From these and other data it was concluded that the regenerative mechanism in uterine smooth muscle has the functional characteristics of a single transient conductance channel whose activation requires the presence of both sodium and calcium. Insensitivity to TTX indicates that the molecular structure of the channel is unlike that in certain sodium-dependent systems, while the effects of La⁺⁺⁺, Mn⁺⁺, Co⁺⁺, and Ca⁺⁺ reveal a similar dependence of conductances on extracellular polyvalent cations.     

Morphological evidence for calcium stores at Sertoli-Sertoli and Sertoli-spermatid interrelations

Potassium pyroantimonate technique has been employed to localize calcium ultrastructurally at Sertoli-Sertoli and Sertoli-spermatid junctional specializations. Identification of Ca++ as the major cation precipitated was performed by EGTA sensitivity and X-ray microprobe analysis. Ca++ deposits have been demonstrated in the endoplasmic reticulum cisternae underlying junctional complexes and along the plasma membranes of both Sertoli and germ cells.     

Electron-microscopic demonstration of the distribution of calcium deposits in the exocrine pancreas of the rat after application of carbachol,atropine, cholecystokinin,and procaine

Summary In an attempt to identify a cellular Ca²⁺-pool, from which calcium is released when secretagogues are applied, tissue fragments of the rat exocrine pancreas were incubated and fixed with glutaraldehyde in the presence of calcium. By means of this procedure electron-dense deposits were found on plasma membranes. X-ray microanalysis showed that these deposits contain calcium. Stimulation of tissue fragments with the use of the secretagogues carbachol or cholecystokinin reduced the number of deposits by about 80%. When the antagonist atropine was applied after carbachol stimulation, deposits reappeared on cell membranes, which then disappeared again after a second stimulation with cholecystokinin. In the presence of procaine, carbachol was inhibited and only slightly reduced the Ca²⁺-deposits on the plasma membranes.These results suggest that a calcium pool, from which calcium is released to induce enzyme secretion on stimulation, is located in the cell membrane     

Cytohistochemical techniques for calcium localization and their application to diseased plants

Lesion delimitation and resistance of old bean (Phaselous vulgaris L., cv. Red Kidney) plants to Rhizoctonia solani Kühn have been suggested to result from increased calcium pectate formation in walls. Ultrastructural histochemistry was used to determine the site of calcium in tissues adjacent to lesions and in older bean hypocotyls. Hypocotyl lesion tissue and uninoculated control tissue were treated with ammonium oxalate or potassium pyroantimonate during fixation. Treatment with potassium pyroantimonate, but not with oxalate, resulted in granular deposits in cell walls of healthy and lesion tissue. Granules also occurred on the plasma membrane of cells adjacent to lesions and in organelles of damaged cells, but wall granule density was not increased. Cell walls from healthy 24-day-old plants had a greater granule density than those for 8-day-old plants. Wall granules were removed from thin sections with ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. Energy dispersive analysis of x-rays also suggested that potassium pyroantimonate localized calcium. Chemical analyses showed that some calcium was retained in tissues after fixation. The results suggest that there are different mechanisms for lesion delimitation and age-induced resistance.     

Fine Structure and Cytochemistry of the Hydrogenosome of Tritrichomonas foetus1

Fine structural studies of the hydrogenosomes of Tritrichomonas foetus using an improved fixative reveal that they are enclosed by two closely apposed 6 nm membranes, which separate at some regions forming a large intramembranous vacuole where Ca⁺⁺-binding sites are located. Fixation of the cells in a glutaraldehyde solution containing 5 mM CaCl₂ and postfixation in an osmium tetroxide-potassium ferrocyanide solution led to the appearance of a reaction product associated with certain regions of the membrane of the hydrogenosomes and in the cisternae of the endoplasmic reticulum, in the recurrent flagellum, and in the plasma membrane. Treatment of ultrathin sections with EGTA removed the reaction product. These results, in association with others previously described, indicate the existence of several similarities between the hydrogenosomes and the mitochondria.     

Electron microscopic demonstration of rat liver glycogen phosphorylase activity with iron (Fe++)

Summary In this study a new electron microscopic method for the demonstration of liver glycogen phosphorylase activity has been presented.Prior to incubation the liver samples were shortly fixed in cold paraformaldehyde. Inorganic phosphate, liberated in the reaction catalyzed by the enzyme, were precipitated with iron (Fe⁺⁺) present in the incubating medium. Postfixation was performed in glutaraldehyde and osmium tetroxide.The ferrous phosphate precipitate was detected electron microscopically in unstained sections.The precipitate was mainly localized to endoplasmic membranes but also in glycogen particles. The method is imperfect in demonstrating phosphorylase activity bound to glycogen particles because of poor preservation of glycogen during treatment.     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) significantly increased potential difference (PD) and short-circuit current (SCC) across isolated frog skin when added to the outside Ringer's solution at 10^–4, 10^–3 and 5×10^–3 m concentration. Resistance was reduced by 10^–4 m Cd⁺⁺ but not significantly changed by the higher concentrations. When SCC was first stimulated by vasopressin, 10^–4 and 10^–3 m Cd⁺⁺ produced additive stimulation which was reversible by washing with Cd⁺⁺-free Ringer's. If SCC was first stimulated by Cd⁺⁺, further stimulation by vasopressin was additive with 10^–4 m Cd⁺⁺ but completely inhibited by 10^–3 m Cd⁺⁺. Elevating the calcium ion (Ca⁺⁺) concentration of the outer Ringer's from 10^–3 m to 5×10^–3 m or 10^–2 m prior to Cd⁺⁺ treatment did not reduce the magnitude of SCC stimulation by Cd⁺⁺. Removal of Ca⁺⁺ from the outside Ringer's with 2×10^–3 m EDTA increased SCC as predicted. Subsequent addition of 5×10^–3 m Cd⁺⁺ drastically reduced SCC below control levels while equimolar concentrations of Cd⁺⁺ and EDTA reduced SCC only to control levels. These results suggest that Cd⁺⁺ interacts with the components of the apical plasma membranes of epithelial cells which are associated with the stimulation of SCC by vasopressin and Ca⁺⁺ removal and may be a useful probe for elucidating these components.     

Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation

Summary Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the⁴⁵Ca-exchange method to determine free cellular Ca⁺⁺, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca⁺⁺). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca⁺⁺] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca⁺⁺ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca⁺⁺] results in a release of the fusion-block and an increase in cellular Ca⁺⁺ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca⁺⁺] must exist. The latter suggests further that cell surface Ca⁺⁺ was also critical.     

Cyto- and histochemical demonstration of lignins in plant cell walls: an evaluation of the chlorine water/ethanolamine-silver nitrate method of Coppick and Fowler

Summary The chlorine water/ethanolamine-silver nitrate method introduced by Coppick and Fowler for the detection of lignins was evaluated for cyto- and histochemical work using different reagents and fixatives for specimens embedded in epoxy resin. Fixation schedules tested included ethanol, glutaraldehyde, and glutaraldehyde followed by O_sO₄ as a post-fixative. Chlorine water, sodium hypochlorite, and calcium hypochlorite were the oxidising agents evaluated for their efficacy as part of the Coppick and Fowler procedure. The Coppick and Fowler method was tested against stem woody tissue ofLophomyrtus obcordata, and haustorial xylem tissue of the sucker of its attached dwarf mistletoeKorthalsella lindsayi. The presence of lignins in walls of these cells was indicated in thin sections for transmission electron microscopy by fine electron-dense deposits. Post-staining thin sections did not affect the lignin reaction, but tended to mask its effect due to increased wall contrast. In histological preparations lignified walls stained orange/brown. Counter-staining in methylene blue/azur B caused lignified walls to appear dark green/brown and non-lignified walls blue. Fixation in either ethanol or glutaraldehyde produced identical staining for lignins. Penetration by chlorine water was sometimes irregular, more so with glutaraldehyde fixation, with parts of tissues consequently not responding to the lignin reaction. Post-fixation in osmium tetroxide following primary fixation in glutaraldehyde slightly improved penetration of chlorine water. However, osmium caused greater amounts of extraneous stain deposits compared with other fixative regimes. Chlorine water was confirmed as the most effective oxidising agent for reacting with groups in lignins to produce reducing residues in the Coppick and Fowler method. Sodium hypochlorite caused no reaction. Calcium hypochlorite exhibited limited oxidative capacity resulting in slight staining for lignins. The Coppick and Fowler procedure was concluded to be a suitable method for demonstrating lignins in cyto- and histochemical preparations using material fixed in either ethanol or glutaraldehyde, and with embedding in epoxy resin.