Enzymatic,analytical and structural aspects of electron-dense granules in cells of <Emphasis Type="Italic">Ucides cordatus</Emphasis> (Crustacea,Decapoda) hepatopancreas

Electron-dense granules (EDGs) are singular structures found in the tissues of several vertebrate and invertebrate organisms. Two types of EDGs were observed in hepatopancreatic cells of the crab Ucides cordatus: (1) a non-mineralized EDG, found mainly inside vacuoles, which reacted positively to acid phosphatase and D-amino acid oxidase, possibly formed by degradation of lipid membranes, and (2) a mineralized EDG surrounded by endoplasmic reticulum membranes that gave a positive reaction for glucose-6-phosphatase. In this study we show the fine structure and composition of the mineralized EDGs using cytochemistry, analytical transmission electron microscopy and field-emission scanning electron microscopy. They are formed of microvesicle-like structures that are arranged in concentric spherical layers in the most mineralized portions of the granule. Analytical microscopy of mineralized EDGs indicated that they are composed of amorphous calcium-magnesium phosphate. Isolated EDGs treated with NaOCl lose several elements, including P, when compared with EDGs treated with deionized water. Removal of the organic matrix by NaOCl induced marked changes in the mineralized EDGs, showing that the organic matrix plays an important role in its elemental composition and structure.     

Electron microscopical demonstration of glucose-6-phosphatase in native cryostat sections fixed with glutaraldehyde through semipermeable membranes.

The cytochemical demonstration of glucose-6-phosphatase (G6Pase) activity in native cryostat sections fixed with glutaraldehyde through semipermeable membranes is superior to conventional methods with regard to exact localization and lack of inactivation and diffusion of the enzyme, together with simultaneous excellent preservation of the tissue fine structure. In rat liver not only hepatocytes but also many bile duct epithelia and endothelia of arterioles and venules show a marked G6Pase activity in the membranes of the endoplasmic reticulum including the nuclear envelope.     

Electron microscopical demonstration of glucose-6-phosphatase in native cryostat sections fixed with glutaraldehyde through semipermeable membranes

Summary The cytochemical demonstration of glucose-6-phosphatase (G6Pase) activity in native cryostat sections fixed with glutaraldehyde through semipermeable membranes is superior to conventional methods with regard to exact localization and lack of inactivation and diffusion of the enzyme, together with simultaneous excellent preservation of the tissue fine structure. In rat liver not only hepatocytes but also many bile duct epithelia and endothelia of arterioles and venules show a marked G6Pase activity in the membranes of the endoplasmic reticulum including the nuclear envelope. This work was kindly supported by the Deutsche Forschungsgemeinschaft An erratum to this article is available at .     

Cytochemical reaction for cationic proteins as a marker of primary granules during development in chick heterophilis.

The ammoniacal silver reaction (ASR) for cationic proteins was used as a cytochemical marker for the primary or A granules in the cytoplasm of developing heterophils of chick bone marrow. The presence of the electron-dense particulate reaction product of silver, which is localized in the fully formed rod-shaped A granules, provides a marker by which the A granules could be distinguished from the B granules of similar size and by which the formation and maturation of both granule types could be followed through the developmental stages. Progressive developmental stages were ascertained on the basis of decreasing cell size, increasing condensation and margination of the chromatin, and the number and morphology of the granules; the stages were divided into promyelocyte, myelocyte, metamyelocyte and heterophil. During the promyelocyte stage, the first appearance of the electron-dense, membrane-bound, spherical granules (0.3--1.0 micrometer in diameter) is observed in the vicinity of an extensive Golgi complex. They occur in a cytoplasm containing rough-surfaced endoplasmic reticulum, ribosomal clusters, centrioles, mitochondria, microtubules, as well as the membranes, saccules, vesicles and vacuoles of the Golgi complex. These granules are considered as primary but their presence as the only granule type appears very brief. The ASR reaction product is first detected on the surface of these primary granules in late promyelocytes or myelocytes. The secondary or B granule, devoid of reaction for cationic protein at all stages, appears as a condensing vacuole in promyelocytes, but after some A granules are already present. The vacuole contents condense to form the B granules which are 0.1--0.6 micrometer in diameter, often oval-shaped, and contain a loose filamentous material surrounded by a membrane. Tertiary C granules or lysosomes appear during the myelocyte stage as dense core vesicles (0.1--0.2 micrometer in diameter) negative for cationic protein.     

Regulation of secretory granule formation in chronically hypersecretory melanotrophs in the rat pituitary

The formation of secretory granules in chronically hypersecretory melanotrophs in the rat pituitary was studied. Hypersecretion was induced by treatment with the dopamine antagonist haloperidol (1.5 mg/kg daily for 7 days), which releases the normal neural dopaminergic inhibition of secretion from the melanotroph. Morphometric analysis showed a 100% increase in the volume fraction of granular endoplasmic reticulum after haloperidol treatment, while the volume fractions of electron-dense granules, electron-lucent granules and the Golgi apparatus were unaltered. The mean diameter of the mature secretory granules was increased by 10%, indicating a 30% increase in mean granule volume. A similar increase in diameter was observed in condensing granules within the Golgi area. With earlier results on the effect of chronic inhibition the study shows that a main adaptive response of the melanotroph to altered secretory conditions is a change in the volume of the secretory granules, regulated by a mechanism that operates at an early stage of granule formation.     

The formation and cytochemical characterization of cortical granules in ovarian oocytes of the golden hamster (Mesocricetus auratus).

The formation and cytochemical characterization of cortical granules in the ovarian oocytes of the golden hamster have been investigated by use of light and electron microscopical techniques. Particular emphasis is given to the changing population of organelles associated with cortical granule formation. Our observations indicate that cortical granules are produced by the participation of both the Golgi complex and the rough endoplasmic reticulum. Ultrastructural cytochemistry reveals that the cortical granules are composed of glycoprotein. The cortical granules are released at fertilization by a merocrine-type of secretory process.     

Immuno-electron microscopy of formation,degradation and exocytosis of the ovulation neurohormone in Lymnaea stagnalis

Summary The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis are involved in the control of egg laying and associated behaviour by releasing various peptides. One of these is the ovulation hormone (CDCH). The cellular dynamics of this peptide have been studied using an antiserum raised to a synthetic portion of CDCH comprising the 20–36 amino acid sequence. With the secondary antibody-immunogold technique, specific immunoreactivity was found in all CDC. Rough endoplasmic reticulum and Golgi apparatus showed very little reactivity as did secretory granules that were in the process of being budded off from the Golgi apparatus. However, secretory granules that were being discharged from the Golgi apparatus, were strongly reactive. Secretory granules within lysosomal structures revealed various degrees of immunoreactivity, indicating their graded breakdown. Large electrondense granules, formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were only slightly reactive. In the axon terminals secretory granules released their contents into the haemolymph by the process of exocytosis. The exteriorized contents were in most cases clearly immunopositive.The possibility has been discussed that CDCH is cleaved from its polypeptide precursor within secretory granules during granule discharge from the Golgi apparatus; subsequently, the mature secretory granules would be transported towards the neurohaemal axon terminals where they release CDCH into the haemolymph. Superfluous secretory material would be degraded by the lysosomal system including the large electron-dense granules.     

Ultrastructural and cytochemical aspects of oogenesis in Castrada viridis (Platyhelminthes, Rhabdocoela).

The heterocellular female gonad of the typhloplanoid Castrada viridis consists of a single germarium and two rows of vitellaria. The germarium, composed of a germinative zone and a growth zone, is surrounded externally by a layer of accessory cells the function of which is hypothesized in this study. The main feature of oocyte differentiation is the synthesis of small electron-dense inclusions produced by the rough endoplasmic reticulum (R.E.R.) and Golgi complex. The electron-dense content of the egg inclusions reacts positively to the cytochemical test used to detect polyphenols and is only partially extracted following incubation in protease. The genesis, composition, and peripheral location of egg inclusions in mature oocytes suggest that they could represent residual eggshell granules. The presumed function of eggshell granules is discussed and their fine morphology is compared with that observed in other neoophoran Platyhelminthes.     

Isolated pancreatic islets of the rat: an ultrastructural study.

Pancreatic islets from adult Wistar rats were isolated by an improved collagenase digestion technique. Examination of the preparations showed that they contained B cells possessing secretory granules, each having an eccentric electron-dense core surrounded by an electron-lucent halo; the Golgi bodies with their characteristic features were located in a juxtanuclear position. Roughly surfaced endoplasmic reticulum and mitochondria were present and were mostly normal in appearance. The cell possessed all the ultrastructural attributes indicating that they were fully functional and structurally intact.     

The Golgi complex in the esophageal mucous glands of the newly hatched chick

The general morphology of the mucous gland cell and the nature of the secretory granule in esophageal glands of the newly hatched chick have been described. Lightly basophilic supporting cells, attached to secretory cells by desmosomes and containing tonofilaments, are located on the basal lamina. Electron microscopic studies showed a morphological polarity of the Golgi complex which suggests that mucous precursors are transported from other sites within the cell to the Golgi complex for further packaging into secretory granules. Finally, acid mucopolysaccharides (AMPS) were specifically stained using the Thorotrast technique and not detected in the rough endoplasmic reticulum, the transitional elements, or in the lamellae at the forming face of the Golgi complex. Conversely, AMPS are found in the vicinity of the mature face of the Golgi complex, and in the secretory granules. The acquisition of cytochemical reactivity for AMPS within the Golgi complex is discussed.     

Cytochemistry of Ca(++)-dependent adenosine triphosphatase (Ca-ATPase) in rat anterior pituitary cells

In the present study, we demonstrate the localization of Ca(++)-ATPase in the anterior pituitary of the male rat. Ca(++)-ATPase was mainly distributed on the membrane system of the granular cells, which included the plasma membrane, the outer mitochondrial membrane, the enveloping membrane of secretory granules, the smooth endoplasmic reticulum and some components of the Golgi complex. No reaction product was detected on the membrane of the rough endoplasmic reticulum or that surrounding the lysosomes. A positive reaction was clearly observed on the membranes surrounding 'large' secretory granules, while that present on the membranes of the 'small' granules was comparatively weak. The cells which contained the 'large' granules were interpreted as growth hormone-secreting cells and those in which the 'small' granules were located as gonadotrophs. There were either no reaction or one that was barely detectable on the plasma membrane of the folliculo-stellate cells. These data along with our previous findings (Soji, 1982, 1984) suggest that the membranous enzymes are not uniformly distributed over all pituitary cells but rather are specific for a given cell population(s).     

Fine structure of the oxynticopeptic cells in the gastric glands of the ruin lizard, Podarcis sicula campestris De Betta, 1857

The results of an ultrastructural investigation of the gastric glands of the ruin lizard are reported. In this reptile the stomach can be divided into a larger fundus and a smaller pars pilorica. Fundic glands are characterized by three main kinds of cells: mucous, endocrine, and oxynticopeptic; the latter were not observed in the pyloric glands. The morphological features of the oxynticopeptic cells change from the proximal to the distal region of the fundic mucosa. In the proximal region, numerous electron-dense secretory granules, a well-developed granular endoplasmic reticulum, an evident Golgi complex, and a reduced system of smooth-surfaced vesicles and tubules in the apical cytoplasm characterize these cells. In the distal fundic region, oxynticopeptic cells possessed numerous mitochondria and a well-developed smooth-surfaced endoplasmic reticulum, but secretory granules were rare. These data suggest the existence of a gradient in the production of proteolytic enzymes, and perhaps also of hydrochloric acid, along the oral-aboral axis of the stomach. The results are discussed with regard to the evolution of the gastric glands and of the digestive mechanism in vertebrates.     

Immunocytochemical localization of pleurocidin to the cytoplasmic granules of eosinophilic granular cells from the winter flounder gill

The teleost gill is considered to be of significant immunological importance, as it is one of the first tissues exposed to environmental or pathogenic challenge and thus should be well equipped to mount an effective immune response. This study characterizes ultrastructurally and immunocytochemically a tissue granulocyte (eosinophilic granular cell) from the winter flounder gill that was previously determined to be involved in the gene expression and synthesis of a known antimicrobial peptide (pleurocidin). The cell is irregular in shape with a cytoplasm characterized by numerous large, electron-dense, membrane-bounded granules. The nucleus is euchromatic and closely associated with a prominent rough endoplasmic reticulum. The cytoplasm typically contains two to three mitochondria and a centralized Golgi apparatus surrounded by numerous electron-lucent vesicles. Immunogold staining of the cells with an anti-pleurocidin antibody shows large number of gold particles in direct association with the electron-dense granules. These data provide the first evidence definitively showing storage of an antimicrobial peptide in the cytoplasmic granules of an eosinophilic granule cell resident in gill tissue.     

Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study.

The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 microns in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereological study of endoplasmic reticulum, Golgi complex and secretory granules in the B-cells of normal and alloxan-treated mice.

Different B-cell organelles (lamellar and vesicular endoplasmic reticulum, Golgi complex, whole secretory granules and secretory granule cores) were studied stereologically in pancreatic islets from control mice and mice killed 10 or 60 min following alloxan injection. Ten min following alloxan a significant decrease was observed in the volume, surface and numerical densities of whole secretory granules and their cores, and a significant increase was found in the volume and surface densities of vesicular endoplasmic reticulum. At the 60 min observation time, a significant decrease was seen in the volume density of lamellar endoplasmic reticulum and Golgi complex, and in the volume, surface and numerical densities of whole secretory granules and their cores, and a significant increase was observed in the volume and surface densities of vesicular endoplasmic reticulum, and in the mean values for volume and surface of whole secretory granules and their cores. The stereological data indicate swelling of endoplasmic reticulum, decreased Golgi area, and decreased number and total volume and surface of secretory granules during the first hour after alloxan administration to mice. The observations may be consistent with inhibited insulin synthesis.     

Widespread association of annexin II with intracellular membrane systems and involvement of annexin II in granule--granule fusion in addition to granule--plasma membrane fusion in anterior pituitary cells

The subcellular localization in anterior pituitary secretory cells of annexin II, one of the Ca2+-dependent phospholipid-binding proteins, was examined by immunohistochemistry and immunoelectron microscopy. Annexin II was associated with the plasma membrane, the membranes of secretory granules and cytoplasmic organelles, such as rough endoplasmic reticulum, mitochondria and vesicles, and with the nuclear envelope. Annexin II was frequently detected at the contact sites of secretory granules with other granules and with the plasma membrane. The anterior pituitary and adrenal medulla were treated with Clostridium perfringens enterotoxin, which induces Ca2+ influx, and examined under an electron microscope. The anterior pituitary cells showed multigranular exocytosis, i.e. multiple fusions of secretory granules with each other and with the plasma membrane, but adrenal chromaffin cells, which lack annexin II on the granule membranes, never showed granule--granule fusion and only single granule exocytosis. From these results, we conclude that, in anterior pituitary secretory cells, annexin II is involved in granule--granule fusion in addition to granule--plasma membrane fusion. © 1998 Chapman & Hall     

Cytochemical evidence of the origin of the dense tubular system in the mouse platelet

Summary Glucose-6-phosphatase (G6Pase) was used as a marker enzyme for the endoplasmic reticulum in mouse megakaryocytes and platelets. G6Pase activity was localized in the dense tubular system of the platelets. Enzyme activity was also observed in the nuclear envelope, and in the rough endoplasmic reticulum of the megakaryocytes. However, the Golgi apparatus of the megakaryocyte was never involved. The present study has added new cytochemical evidence for the hypothesis that the dense tubular system of the platelet originates from the endoplasmic reticulum of the megakaryocyte.     

Schistosoma mansoni: cytochemistry and morphology of the gastrodermal Golgi Apparatus

The gastrodermal Golgi apparatus of adult Schistosoma mansoni displays two distinct morphologies. In one type, there is an identifiable cis (forming) face where vesicles from the endoplasmic reticulum fuse to form the cisternae. A morphological change occurs in the cisternae as the trans (emitting) face is approached with the cisternae becoming progressively flattened. The cisternae at the emitting face produce a membrane-bound secretory granule with moderately electron-dense contents and a vacuolar structure that may be analogous to a condensing vacuole as reported in several vertebrate secretory cells. In a second type, vesicles possessing a thicker membrane than those of the transfer vesicles are observed at the emitting face. They are not observed when the secretory granules are present. Several cytochemical markers were used to aid in studying the polarity of the Golgi apparatus. Enzymes studied were thiamine pyrophosphatase (TPPase) (EC 3.6.1.1), nucleoside diphosphatase (NDPase) (EC 3.6.1.6) using uridine diphosphate as a substrate, and nicotinamide adenine dinucleotide phosphatase (NADPase) (EC 3.1.3.2). Reaction products from all enzyme markers were observed in the cisternae and, to some extent, in the transfer vesicles. At times, NADPase and TPPase reaction products were observed in all cisternae and in the transfer vesicles of the Golgi. When this distribution was evident, the latter vesicles were observed in clusters occasionally fusing with lipid-like globules dispersed throughout the gastrodermis. Heterogeneity in cisternae was observed when NDPase, TPPase, and osmium reduction techniques were used. NDPase activity was limited to the middle cisternae while reduced osmium was observed in the outer two cisternae and in some transfer vesicles. TPPase reaction product was also observed in the secretory granules and in the condensing vacuoles. It is hypothesized that a functional bipolarity may be demonstrated by the Golgi. Under certain stress conditions, the forming face of the Golgi may package lysosomal enzymes while the emitting region of the Golgi appears to be responsible for the packaging of the secretory granules. The fusion of transfer vesicles and, at times, secretory granules with lipid-like globules is postulated to represent a mechanism by which enzymes may be transported to the lumen of the cecum.     

Developmental pattern of glucose-6-phosphatase activity in the small intestine of the mouse fetus.

The distribution of glucose-6-phosphatase (G6Pase) activity in the epithelium of the small intestine in mouse embryos (the last 4 days of gestation) was studied by electron microscope cytochemistry and by enzymatic assays. At 16 days, the lead phosphate deposited by the cytochemical reaction is localized on the rough endoplasmic reticulum (RER) and nuclear envelope of very few cells in the duodenum and jejunum. Positive cells are more frequently seen in the upper part of the developing villi. At 17 days of gestation, a tremendous burst in RER differentiation is noticed in all parts of the small intestine and concomitantly glycogen disappears. At 18 days of gestation all the principal cells of the intestinal mucosa show a well differentiated positive RER and the enzyme is also present in the smooth endoplasmic reticulum. Biochemically, G6Pase activity is detected in the proximal 2 thirds of the small intestine at 17 days of gestation and appears at 18 days in the last third. Afterwards the activity increases up until birth. These results suggest (1) that the endoplasmic reticulum differentiates very late in the intestinal mucosa of mouse embryos (2) that the differentiation with respect to G6Pase is asynchronous between the enterocytes, (3) that for a given cell all the cisternae of RER are involved in G6Pase synthesis at the same moment and (4) that the enterocytes of the duodenum differentiate sooner and faster that those of the jejunum and ileum.