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.     

Ultrastructural localization of intracellular calcium stores in Xenopus ovarian follicles as revealed by cytochemistry and X-ray microanalysis

The ultrastructural localization of calcium in full-grown ovarian follicles of Xenopus laevis was demonstrated after fixation in the presence of fluoride ions and by means of energy dispersive X-ray microanalysis. In hormonally untreated follicles (prophase I-arrested oocytes), two calcium sites were detected: follicle cells and oocyte pigment granules. In follicle cells, calcium containing deposits were preferentially associated with macrovilli, which ended by gap junctions. In human chorionic gonadotropin treated follicles (meiotically reinitiated oocytes), deposits were only seen in follicle cells. This is the first report of the cytochemical detection of intracellular Ca²⁺ in follicle cells of amphibians. The possible involvements of these Ca²⁺ stores in mediating the hormonal control of meiotic maturation are discussed.     

Carbonic anhydrase activity in a calcium-mobilizing epithelium of the crustacean Orchestia cavimana during molting

We investigated the involvement of the enzyme, carbonic anhydrase, in the calcification-decalcification processes occurring in the posterior caeca of the midgut of the terrestrial crustacean, Orchestia cavimana, before and after exuviation. This enzyme was ultrahistochemically localized throughout the membranes of the caecal epithelium as well as extracellularly, i.e., within pre-exuvial calcareous concretions and postexuvial calcified spherules. During the molt cycle, the pattern of carbonic anhydrase activity in the posterior caeca was correlated with the calcium content at this level. Acetazolamide treatment in vivo inhibited about 50% of the calcium uptake during both pre-exuvial secretion and postexuvial reabsorption. The role of carbonic anhydrase in this mineralizing-demineralizing epithelium is discussed and compared with that of other mechanisms involved in this calcium turnover.     

Carbonic anhydrase activity in a calcium-mobilizing epithelium of the crustacean Orchestia cavimana during molting

Summary We investigated the involvement of the enzyme, carbonic anhydrase, in the calcification-decalcification processes occurring in the posterior caeca of the midgut of the terrestrial crustacean, Orchestia cavimana, before and after exuviation. This enzyme was ultrahistochemically localized throughout the membranes of the caecal epithelium as well as extracellularly, i.e., within pre-exuvial calcareous concretions and postexuvial calcified spherules. During the molt cycle, the pattern of carbonic anhydrase activity in the posterior caeca was correlated with the calcium content at this level. Acetazolamide treatment in vivo inhibited about 50% of the calcium uptake during both pre-exuvial secretion and postexuvial reabsorption. The role of carbonic anhydrase in this mineralizing-demineralizing epithelium is discussed and compared with that of other mechanisms involved in this calcium turnover.     

Ultrastructural localization of potassium and calcium in an insect ommatidium as demonstrated by X-ray microanalysis

Summary Ultrastructural localization of potassium and calcium in the ommatidium of the house-cricketGryllus domeslicus L. was studied by X-ray microprobe analysis using samples prepared as thin sections (2 or 5 m) of freeze-dried and embedded tissue. Real resolution was limited by the size of ice crystals (Fig. 2) and estimated as about 1 m.Average values for potassium, calcium, sodium and phosphorus in different cells of the compound eye are given in Table 1.Striking non-uniformity in distribution of these elements over the cells and their compartments was found by probe scanning (Figs. 3, 4, 5). The highest potassium and calcium concentrations were measured in the pigmented zones of photoreceptors and pigment cells. The pigment granules are thought to be the ionic depots of the eye.Potassium and sodium are fully accessible to water in sections of embedded tissue, whereas all the calcium and half of the phosphorus are not.The functional significance of the non-uniformity discovered is briefly discussed.     

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.     

Immunocytological localization of endogenous calcitonin-like molecules in the crustacean Orchestia

Summary Immunocytological mapping of calcitonin-like molecules (human form) performed in the terrestrial crustacean Orchestia, using PAP procedure on paraffin sections and immunogold method on ultrathin cryosections, reveals two reactive organs: central nervous system and posterior caeca of the midgut. Immunoreactivity within the nervous system is mainly located throughout perikarya and nerve fibers from both deuto- and tritocerebron and ventral nervous chain. Immunolabeling in the posterior caeca is detected on both cell components of the epithelium, with significant quantitative differences between molt and intermolt periods. The role of calcitonin-like substances in these organs is then discussed: at the nervous system level, a neuro transmitter function is suggested; the direct participation of these peptides in the regulation of calcium shifts through the caecal epithelium is hypothesized.     

X-ray microanalysis of calcium binding sites in Paramecium

Summary In Paramecium cells Ca⁺⁺-stimulated triggering of the exocytosis of secretory vesicles (trichocysts) was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some resting trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the inner lamellar sheath from where deposits seemed to radiate into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those resting trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca⁺⁺-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possibly in combination with the sudden activation of an ATPase systemlocalized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca⁺⁺ and then fixed with OsO₄ plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including ciliary granule plaques) also contained Ca, P and S. Cells contain osmiophilic calcium-storing vacuoles which were selectively rich in Ca and S but devoid of P.     

Action of 1,25-dihydroxyvitamin d3 on calcium metabolism in the crustacean Orchestia cavimana during the molt cycle

• 1.1. The possible involvement of vitamin D₃ in the calcium metabolism of the terrestrial crustacean Orchestia during the molt cycle was further investigated by measuring the effects of administration of exogenous 1,25 (OH)₂D₃ on three parameters of the calcium balance in two different compartments of the body.
• 2.2. At the hemolymph level, a strong hypocalcémie effect was observed in intermolt and early premolt. Within the posterior caeca of the midgut, stimulation of calcium storage and calcium release were noticed during a short period surrounding the time of exuviation, with concomitant variations of the epithelial carbonic anhydrase activity.
• 3.3. These results, together with other data, are discussed to determine the possible functions of vitamin D₃, or related molecules, in the calcium turnover within the different compartments of the body, according to the successive stages of the molt cycle.

     

Immunocytological localization of endogenous calcitonin-like molecules in the crustacean Orchestia.

Immunocytological mapping of calcitonin-like molecules (human form) performed in the terrestrial crustacean Orchestia, using PAP procedure on paraffin sections and immunogold method on ultrathin cryosections, reveals two reactive organs: central nervous system and posterior caeca of the midgut. Immunoreactivity within the nervous system is mainly located throughout perikarya and nerve fibers from both dueto- and tritocerebron and ventral nervous chain. Immunolabeling in the posterior caeca is detected on both cell components of the epithelium, with significant quantitative differences between molt and intermolt periods. The role of calcitonin-like substances in these organs is then discussed: at the nervous system level, a neuro transmitter function is suggested; the direct participation of these peptides in the regulation of calcium shifts through the caecal epithelium is hypothesized.     

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.     

X-ray microanalysis and chlorotetracycline staining of calcium vesicles in the green alga Mougeotia

Calcium ions have been proposed to play a key role in the sensory transduction of phytochrome-governed chloroplast movement in the green alga Mougeotia. To test this hypothesis, the intracellular pattern of calcium distribution was studied in this alga by two independent techniques, namely, X-ray microanalysis of fixed and of unfixed frozen-hydrated cells, as well as in vivo fluorescence by chlorotetracycline. Both methods of detection reveal a significant compartmentation of calcium in vesicles close to the chloroplast edge and, less frequently, in the cortical cytoplasm. Microfilaments, presumably actin, which could function in driving chloroplast movement, have been observed running between the chloroplast edge and the cortical cytoplasm (Wagner, G., Klein, K. (1978) Photochem. Photobiol. 27, 137). The vesicular calcium concentration is stable and decays only slowly in the absence of extracellular calcium much in the same way as the ability of the chloroplast to perform movements decreases. A functional relationship between vesicular calcium compartmentation and phytochrome-governed chloroplast movement in the green alga Mougeotia seems indicated.Abbreviation CTC chlorotetracycline     

The localization of calcium by X-ray microanalysis in myopathic muscle fibers

Summary An electron histochemical study was undertaken to localize calcium with ammonium oxalate precipitation technique in soleus muscle of rat in normal cases and in myopathy induced experimentally by a prolonged treatment of 2,4-dichlorophenoxyacetate (2,4-D). The calcium content of precipitates was detected by energy-dispersive X-ray microanalysis. In normal cases, the electron dense precipitates containing calcium were mainly found in the vesicles of sarcoplasmic reticulum, whereas in 2,4-D induced myopathy the deposits were shifted near the Z line into the myofibrils. Calcium, because the uptake into sarcoplasmic vesicles was inhibited by 2,4-D, could attach to other binding sites, such as to the troponin-C.A long-lasting binding of calcium might lead to a prolonged activation of the actin-myosin system.Fellow of the Max-Planck Society at the Abteilung Neurobiologie, Neuroanatomie, Max-Planck-Institut für biophysikalische Chemie, Göttingen. Federal Republic of Germany     

The localization of calcium by X-ray microanalysis in myopathic muscle fibers

An electron histochemical study was undertaken to localize calcium with ammonium oxalate precipitation technique in soleus muscle of rat in normal cases and in myopathy induced experimentally by a prolonged treatment of 2,4-dichlorophenoxyacetate (2,4-D). The calcium content of precipitates was detected by energy-dispersive X-ray microanalysis. In normal cases, the electron dense precipitates containing calcium were mainly found in the vesicles of sarcoplasmic reticulum, whereas in 2,4-D induced myopathy the deposits were shifted near the Z line into the myofibrils. Calcium, because the uptake into sarcoplasmic vesicles was inhibited by 2,4-D, could attach to other binding sites, such as to the troponin-C.A long-lasting binding of calcium might lead to a prolonged activation of the actin-myosin system.     

Ultrastructural location of calcium and magnesium during mineralisation of the cuticle of the shore crab,as determined by the K-pyroantimonate method and X-ray microanalysis

We have investigated the distribution of Ca²⁺ and Mg²⁺ in the new cuticle of moulting shore crabs (Carcinus maenas), using the K-pyroantimonate method in combination with X-ray microanalysis in order to identify antimony precipitates. During the premoult period, Ca²⁺ and Mg²⁺ accumulate in well-defined sites of the new pigmented layer. After moulting, mineralisation appears to begin preferntially at these sites. These form a honeycomb-like structure that quickly increases the rigidity of the new cuticle, with a small recruitment of material from extraneous sources. Mineralisation of the principal layer, on the other hand, immediately follows deposition of the organic matrix. Our experiments also provide evidence that the epidermal cell extensions associated with the pore canals are the means by which Ca²⁺ and Mg²⁺ are transferred from the epidermis into the mineralising cuticular layers. The plasma membrane of these cell extensions appears densely lined by particles of antimony precipitate that probably mark the location of the transporting sites. Shortly after moulting, the distribution of mineral deposits is such that the cell extensions cross the mineralised lamellae of the principal layer and constitute preferential access routes to the pigmented layer, where mineralisation is still in progress.     

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution

Summary Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2±0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3±1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of ⁴⁵Ca from prelabelled eggs. Exchangeable ⁴⁵Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.     

Localization of iron and sulfur in the lysosomes of thyroid epithelium using electron probe microanalysis and X-ray spectrometry

Thyroid lysosomes have been studied by means of EPMA combined with an electron microscope. In 4 months old mice, lysosomes are a few number and contain very low iodine concentrations and sulfur load is not significant. In 10 months old mice, iodine only or associated with sulfur is observed in homogeneous lysosomes varying in size and density. Those two types of localizations reflect two eventual steps in hormonal metabolism pathway of iodine.