Calcium localization in the shell-forming tissue of the freshwater snail,Biomphalaria glabrata: a comparative study of various methods for localizing calcium

Summary The routes calcium might take across the mantle to the shell have been investigated with various electron-microscopical techniques in the freshwater snailBiomphalaria glabrata (Planorbidae, Basommatophora).In chemically-fixed tissue, calcium was precipitated with a tannic acid-antimonate technique in predominantly the intercellular spaces of the outer mantle epithelium and the interstitium below it. Some vacuoles of the outer mantle epithelium and one type of mucus cell in the inner mantle epithelium also contained precipitate. The presence of calcium in the precipitates was proved by electron energy loss spectroscopy combined with electron spectroscopic imaging. Incubation with lead acetate and uranyl acetate revealed binding-sites for calcium in the intercellular spaces of the epithelia interstitium and the mucus cells of the inner mantle epithelium. Precipitates were also seen after all incubations in the calcium spherites of the connective tissue.The concentrations of calcium and other elements were analysed in freeze-dried ultrathin sections of cryofixed mantle tissue by means of energy-dispersive X-ray microanalysis. Only in mitochondria of the musculature could high amounts of calcium and phosphorous be detected.     

X-ray microanalysis of freeze-dried digestive mucus in Anguilla anguilla L: modifications of ion content during the early steps of secretion

Summary— Digestive mucus of sea-water adapted eels has been observed and analyzed by the scanning electron microscope (SEM) after rapid freezing at liquid nitrogen temperature followed by freeze-drying. No chemical procedures were used in this technique. This allowed the maintenance of the mucous coating. Preliminary X-ray microanalysis carried out on freeze-fractured and freeze-dried samples of the oesophagus showed a decrease of K⁺ and an increase of Ca²⁺ and Cl^? from the basal part of the mucous cell towards its the apical part. This technique has proven to be satisfactory for it prevents translocation and loss of diffusible elements in situ and allows X-ray microanalysis in the SEM.     

Is mucus involved in biocrystallization?

Summary Freeze-dried intestinal mucus of sea-water-adapted eels was analysed by scanning electron microscopy (SEM) and X-ray microanalysis. Calcite crystals were observed in the mucus fibres; their concentration increased along the hindgut. Random SEM observations made in situ indicated that mucus fibres were involved in the genesis of these crystals. Calcium-rich mucus globules were found fused inside crystal matrices. Single typical rhombohedric crystals of various complexity appeared within the mucus framework. The steps of crystal biogenesis were reconstituted in in-vitro conditions.     

Inorganic polyphosphates are stored in spherites within the midgut of Anticarsia gemmatalis and play a role in copper detoxification

Inorganic polyphosphates (PolyP) are widespread molecules that have been shown to play a role in metal detoxification and heavy-metal tolerance. In the present report, we investigated the functional role of spherites as PolyP-metal binding stores in epithelial cells of the midgut of Anticarsia gemmatalis, a lepidopteran pest of soybean. PolyP stores were detected by DAPI staining and indirect immunohistochemistry as vesicles distributed in columnar cells and around goblet cell cavities. These PolyP vesicles were identified as spherites by their elemental profile in cell lysates that were partially modulated by P- or V-ATPases. PolyP levels along the midgut were detected using a recombinant exopolyphosphatase assay. When copper was added in the diet of larva, copper detection in spherites by X-ray microanalysis correlated with an increase in the relative phosphorous X-ray signal and with an increase in PolyP levels in epithelia cell lysate. Transmission electron microscopy of chemically fixed or cryofixed and freeze substituted tissues confirmed a preferential localization of spherites around the goblet cell cavity. Taken together, these results suggest that spherites store high levels of PolyP that are modulated during metal uptake and detoxification. The similarity between PolyP granules and spherites herein described also suggest that PolyP is one of the main phosphorous source of spherites found in different biological models. This suggests physiological roles played by spherites in the midgut of arthropods and mechanisms involved in heavy metal resistance among different insect genera.     

Ultrastructural localization of calcium, by electron-probe X-ray microanalysis, in the small intestine of suckling rats

The subcellular distribution of calcium has been investigated in samples, from the intestinal mucosa of 10-day rats, prepared for X-ray microanalysis by various techniques designed to minimize the loss of this element. Calcium retention and its threshold of detection was most satisfactory in freeze-dried frozen thin sections. In resin-embedded samples the best retention of calcium was found in specimens fixed in absolute ethanol, embedded without osmication, and sectioned onto glycerol. The results of this investigation indicate the presence of calcium in the supranuclear vacuole of enterocytes in the distal intestine of the neonatal rat. This calcium is probably taken up during the endocytosis of material from the intestinal lumen. The same mechanism may also be important in the uptake of other metals by suckling animals.     

Mucous granule exocytosis and CFTR expression in gallbladder epithelium

A mechanistic model of mucous granule exocytosis by columnar epithelial cells must take into account the unique physical-chemical properties of mucin glycoproteins and the resultant mucus gel. In particular, any model must explain the intracellular packaging and the kinetics of release of these large, heavily charged species. We studied mucous granule exocytosis in gallbladder epithelium, a model system for mucus secretion by columnar epithelial cells. Mucous granules released mucus by merocrine exocytosis in mouse gallbladder epithelium when examined by transmission electron microscopy. Spherules of secreted mucus larger than intracellular granules were noted on scanning electron microscopy. Electron probe microanalysis demonstrated increased calcium concentrations within mucous granules. Immunofluorescence microscopic studies revealed intracellular colocalization of mucins and the cystic fibrosis transmembrane conductance regulator (CFTR). Confocal laser immunofluorescence microscopy confirmed colocalization. These observations suggest that calcium in mucous secretory granules provides cationic shielding to keep mucus tightly packed. The data also suggests CFTR chloride channels are present in granule membranes. These observations support a model in which influx of chloride ions into the granule disrupts cationic shielding, leading to rapid swelling, exocytosis and hydration of mucus. Such a model explains the physical-chemical mechanisms involved in mucous granule exocytosis.     

X-ray microanalysis of freeze-dried digestive mucus in Anguilla anguilla L: modifications of ion content during the early steps of secretion

Digestive mucus of sea-water adapted eels has been observed and analyzed by the scanning electron microscope (SEM) after rapid freezing at liquid nitrogen temperature followed by freeze-drying. No chemical procedures were used in this technique. This allowed the maintenance of the mucous coating.Preliminary X-ray microanalysis carried out on freeze-fractured and freeze-dried samples of the oesophagus showed a decrease of K⁺ and an increase of Ca²⁺ and Cl⁻ from the basal part of the mucous cell towards its the apical part. This technique has proven to be satisfactory for it prevents translocation and loss of diffusible elements in situ and allows X-ray microanalysis in the SEM.     

An electron microscopic histochemical and X-ray microprobe study of spherites in a mussel

Transmission electron microscopic, histochemical and X-ray analytical microprobe techniques were used to study the inorganic-organic relationship in the spherites (calcospherules) from the mantle, i.e. subadjacent to the outer mantle epithelium, of the fresh-water mussel Amblema. These structures were shown to contain calcium which could be chelated by the flotation of sections on solutions of either formic acid or ethylene glycol bis-(beta-amino ethyl ether)-N, N1-tetra-acetic acid (EGTA), Analysis of both non-chelated and sections revealed a significant sulfur peak. Chelated spherites were also intensely stained with acid phosphotungstic acid (PTA), Such data is indicative of the presence of an organic glycoprotein (proteoglycan) matrix which could serve to bind mineral ions, thus forming organic-inorganic aggregates for calcium transport and homeostasis. In this regard, the spherites are analogous to both calcium phosphate containing mitochondrial granules and the initial calcification sites in vertebrate mineralizing tissues.     

Calcium reabsorption in the posterior caeca of the midgut in a terrestrial crustacean,Orchestia cavimana

Summary During molting, the epithelium of the posterior caeca (PC) of the midgut in the terrestrial crustacean, Orchestia cavimana, is active in calcium turnover. In the preexuvial period, epithelial cells that progressively differentiate into cell-type III secrete ionic calcium (originating from the old cuticle) from the base to the apex of the cell within a typical extracellular network of channels; the calcium is then stored in the PC lumen as calcareous concretions. Immediately after exuviation, the epithelial cells rapidly differentiate into cell-type IV, reabsorbing calcium from the concretions through successive generations of spherites which quickly appear, grow, and then disappear from the apex to the base of the same extracellular network. The PC epithelium is thus alternatively calcium-loaded and unloaded. When the calcium-reabsorbing process is complete (average 48 h after exuviation), the epithelial cells again differentiate into two different regional cellular types (cell-type I in the distal segment and cell-type II in the proximal segment) characteristic of the intermolt period.The dynamic changes in the PC epithelium during the postexuvial period are discussed, including the characteristic features of cell-type IV and of the reabsorption spherites.     

Calcification in insects: The dwelling-tube and midgut of machaerotid larvae (Homoptera)

The dwelling-tubes of machaerotid larvae consist of a mineralized organic scaffolding of mucofibrils. The mineral component accounts for 85 per cent of the dry weight and is composed of calcium, ferrous iron, manganese, magnesium, potassium, sodium, phosphate, carbonate, and chloride and of these the major ions are calcium and carbonate. Ferric iron in the form of ferritin is probably also present.Calcium, manganese, magnesium, and phosphate are derived from spherites secreted by a specialized region of the midgut. Calcium and phosphate are present in the spherites, probably as amorphous tricalcium phosphate. Subsequent to secretion the spherites are slowly dissolved and the calcium is incorporated into the dwelling-tube as calcium carbonate. Thus it appears that within the dwelling-tube calcium phosphate is converted to calcium carbonate.Ferritin and ferrous iron are secreted by another specialized region of the midgut and are also incorporated into the dwelling-tube.     

Mineral congregations, “spherites” in the midgut gland ofCoelotes terrestris (Araneae): Structure,composition and function

Summary Spherites in the digestive and secretory cells of the midgut gland of the agelenid spiderCoelotes terrestris were studied by electron microscopy and histochemical methods. Spherites measured 1–6 m in diameter and were characterized by alternating layers of electron dense and electron lucent material. The main-components of spherites were calcium phosphates and calcium carbonates. Guanine and barium, as well as aminopeptidase and alkaline phosphatase were also present. The matrix consisted of proteins and carbohydrates. Numerous spherites were found together with excretory products within the excretory vacuoles of the digestive cells.Spiders fed with food containing lead, showed deposition of the metall in the spherites. It is then proposed that spherites, aside from their role in storing calcium and other ions, may function in detoxification of heavy metals.     

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.     

X-RAY MICROANALYSIS IN CELL BIOLOGY AND CELL PATHOLOGY

Electron probe X-ray microanalysis has been used for the last 25 years by biologists to obtain information about the distribution of elements at the cell and tissue level. During this period, progress has mainly been made through the development of more adequate techniques for specimen preparation (mainly low temperature techniques) and quantitative analysis, so that accurate analysis of the physiologically important cellular ions can be carried out. Use ofin vitrosystems and cell cultures may further increase the number of problems to which X-ray microanalysis can be applied. Among the numerous applications of X-ray microanalysis in cell biology and cell pathology, applications in the areas of epithelial ion transport, the role of calcium in secretory and contractile cells, and the role of ions in cell proliferation and cancer are discussed in more detail.     

The distribution and function of mucous cells and their secretions in the alimentary tract of Arrhamphus sclerolepis krefftii

The mucosa of the mouth, pharynx, oesophagus and rectum of Arrhamphus sclerolepis krefftii contain saccular mucous cells and the lining of the intestinal mucosa contains goblet mucous cells. Saccular mucous cells in the buccal epithelium are present in relatively low densities and contain acidic and neutral glycoprotein-secreting cells in an approximately 1:1 ratio. The saccular mucous cells in the mucosa of the pharynx, oesophagus and rectum are abundant and contain acidic glycoprotein which consists principally of sialomucin with traces of sulphomucin distributed around the periphery of the mucous vacuoles. Goblet cells in the intestinal mucosa contain neutral glycoprotein. Mechanically digested plant material within the lumen of the gut is bound by a sheath of acidic glycoprotein which is in contact with the intestinal mucosa. From these observations and with information on the known properties of acidic glycoproteins, a novel mechanism for the involvement of mucus in the extraction of nutrients from plant material mechanically digested by fish is proposed.     

Electron microscope studies of the intracellular origin and formation of calcifying granules and calcium spherites in the hepatopancreas of the snail,Helix pomatia L.

Summary It has been previously demonstrated that the hepatopancreas of the snail, Helix pomatia, produce proteinaceous particles (termed b-granules), which are involved in the process of calcification. In calcium cells of this organ calcium spherites arise from these granules. The present study retraces the intracellular development of b-granules from Golgi vesicles and from Golgi saccules encircling the cytoplasmic areas up to the formation of calcium spherites. It is suggested that the mature b-granules are well-defined cytoplasmic units with calcifying capacity. The fine structure of b-granules and calcium spherites is described. The possible function of various cell organelles in the formation of the b-granules and spherites is discussed.This investigation was supported by grants from the Swedish Natural Science Research Council. The assistance of Mrs. I. Rehnberg is gratefully acknowledged.     

A study of the concentrically laminated concretions, 'spherites' in the regenerative cells of the midgut of Lepidopterous larvae

Concentrically laminated granules, spherites, are sometimes found in the regenerative cells of midgut of some species of lepidopterous larvae. The spherites are formed in cytoplasmic vesicles just before ecdysis and disappear during the differentiation of the regenerative cells to columnar and goblet cells. They function as intracellular stores of compounds used in the growth of the cell. Phosphates of magnesium and perhaps calcium are probable constituents. Spherites are sometimes also found in the degenerating columnar cells where they are excreted into the lumen with the exfoliating epithelium. The phenomenon of periodic precipitation which is the physical-chemical basis of the formation of spherites is discussed.     

Calcium localization in oat aleurone cells using chlorotetracycline and X-ray microanalysis

Calcium distribution was studied in oat caryopses. Using the chlorotetracycline method it was found that membrane-associated Ca²⁺ was present in the aleurone layer. X-ray microanalysis confirmed the presence of calcium in aleurone cells; it also demonstrated the presence of considerable amounts of calcium in the cell wall surrounding these cells.Abbreviation CTC chlorotetracycline     

A morphological and electron-microprobe study of the inorganic composition of the mineralized secretory products of the calciferous gland and chloragogenous tissue of the earthworm,Lumbricus terrestris L.

Summary The two pairs of lobes of the calciferous gland of the earthworm Lumbricus terrestris are specialized oesophageal diverticulae that secrete spherites ranging from 0.5–7.0 m in diameter. Correlative transmission and scanning electron microscopy indicated that the spherites (which are predominantly CaCO₃) are formed extracellularly in distinctive bays bounded by secretory-cell processes, and are mobilized anteriorly from the gland lumina to the lumen of a non-secretory pouch, where the majority coalesce and undergo phase transformation to concretions 0.5–1.0 mm in diameter consisting of a mass of cuboidal crystals with facets up to 40 m. The distribution of Sr (0.1 ml 5 % SrCl₂ injected into the posterior coelomic cavity) was monitored in the mineralized secretory products of the calciferous glands by X-ray microanalysis of 10 m — thick air-dried cryostat sections in a SEM. Strontium was not detected in chloragosomes at 2 h and 24 h post-injection. Strontium was transported anteriorly and specifically incorporated into gland spherites (detectable within 2h). This technique of Sr localization afforded sufficient structural and analytical resolutions to provide a confirmation of the sequence of extracellular changes in the gland/pouch system. In addition we were able to distinguish a population of growing spherites from the vast majority of mature spherites; size alone was a singularly poor indication of spherite growth.The major element constituents of the chloragosomes were P, Ca and Zn (Ca: P ranging from 0.4 to 1.0; Zn: P from about 0.05 to 0.45). Analysis of individual spherites showed that Ca was probably bound to P or P-containing matrix components, whilst Zn was probably linked to one or more different but unknown constituents.     

Ultracytochemical Localization of Calcium in the Stigma,Style and Micropyle of Sunflower

Calcium was localized ultracytochemically in the stigma, style and micropyle of sun-flower (Helianthus annuus L. ) by pyroantimonate precipitation technique. To identify the element constitution of the pyroantimonate deposits, wave-dispersive X-ray microanalysis (WDX) method was employed in addition to the energy-dispersive X-ray microanalysis (EDX) and the deposits observed were directly proved as calcium pyroantimonate. In the stigma, calcium was more abundant on the receptive surface, especially outside and inside the papillae, than on the noo-receptive surface. In the style, more calcium was seen in the transmitting tissue as compared with the adjacent parenchymatous tissue, and was concentrated at the intercellular matrix and the boundary between the cell wall and the intercellular matrix. In micropyle region, the transmitting tissue on the side proximal to the funicle contained large amount of calcium, where as more calcium was localized in the intercellular matrix. As for the pollen tubes growing along the gynoecium, calcium was mainly localized at the pectin layer of the tube wall after 1.5 hours of pollination.     

Crystalline calcium in littorinid mucus trails

Previous work has shown that the feet of terrestrial and freshwater snails are important in calcium regulation, often secreting granules of CaCO₃. This phenomenon has not, until now, been observed in marine snails. Here we report the presence of CaCO₃ granules in the trail mucus of Littorina littorea (L.), L. saxatilis (Olivi) and L. obtusata (L.) Fixed mucus trails on plastic coverslips were examined by X-ray microanalysis under the SEM. Of the single-metal granules observed in the mucus trails the most abundant were of calcium (means: L. littorea, 440 mm⁻²; L. saxatilis, 401 mm⁻²; L. obtusata, 348 mm⁻²) followed for each species by silicon (maximum mean density: L. saxatilis, 120 mm⁻²) and iron (maximum mean density: L. saxatilis, 65 mm⁻²) granules. Single-metal granules of Al, Ti, Mg and P were also found but only in the mucus trails of L. obtusata, perhaps reflecting its different collection site from the other two species. The mean size of the calcium granules showed significant interspecific variation (L. littorea, 1.32 μm diameter±0 08 μm, n = 143; L. saxatilis, 1.80 μm±0.12, n = 113; L. obtusata, 2.14 μm±0.09, n = 167). Most calcium granules (L. littorea, 80%, n = 35; L. saxatilis, 57%, n = 113; L. obtusata, 69%, n = 167) were attached to, or embedded within, microthreads of mucus which tended to run parallel to the direction of locomotion. The significance of this is unknown although it may imply that the CaCO₃ granules are secreted with the mucus. It is concluded that calcium losses via this route are too small for pedal mucus to function significantly in ionoregulation of calcium. The calcium in the trail may therefore perform other functions, for example indicating trail polarity.