Histological and histochemical observations on the foot glands in some byssus-bearing bivalves of the Waltair coast

Summary The glands responsible for the formation of the byssus threads inArca symmetrica, Barbatia obliquata andSeptifer bilocularis are the white gland, phenol gland and enzyme gland. Besides these, mucous glands are also present in the sub-epithelia. The size and shape of the cells of these glands vary in one and the same species. From histochemical investigations it has been revealed that these glands contain 1,2-glycol groups in addition to disulphides and sulphhydryls. The white gland secretes a protein material and the phenol gland is rich in phenols. These two combine to form a phenolic protein which is acted upon by a polyphenol oxidase secreted by the enzyme gland and leads to the formation of a byssus thread. The mucous gland cells secrete acid mucopolysaccharides, neutral mucins and glycoproteins.     

Ultrastructural and cytochemical study of a mucous gland of the foot of Mytilus galloprovincialis

The ultrastructural study of the ventral pair mucous gland (VPMG) of the foot of Mytilus galloprovincialis shows the presence of two cell types (type I and II cells) characterized by the cytoarchitecture typical of mucous secreting cells and distinct for the different structure of their secretory granules. The cytochemical tests performed on semithin (1 micron) and ultrathin sections show that type I secretory granules are made up of proteinaceous nucleoids and of a microfilamentous matrix containing both carboxylated and sulphated glycosaminoglycans. Type II secretory granules are mainly formed by glycoproteins. The ultrastructural and cytochemical studies do not support the hypothesis that VPMG secretions directly contribute to the formation of byssus threads. It is more probable that such secretions provide a protective and lubricating blanket during the multistep process of secretion, moulding and extrusion of byssus threads.     

Foot glands in Perna indica and Perna viridis (Pelecypoda: mytilidae) histology and histochemistry

The foot of Perna viridis is found to contain three main types of glands, the white gland, phenol gland and the enzyme gland. But in Perna indica there are only two glands, the phenol gland and the enzyme gland. Besides these, mucous glands are found in both of the species. The shape and size of the cells of these glands vary from species to species. Glycogen and 1 : 2 glycol groups are found in these gland cells. Proteins rich in disulfides and sulfhydryls are present in the phenol glands of both the species and in the white gland of p. viridis but they vary in the intensity of staining. The presence of phenols is confirmed in the phenol gland cells. Phospholipids and lipoproteins are intense in the white and phenol gland cells. They are absent in the enzyme gland. Alkaline and acid phosphatases activity in the enzyme gland cells could be demonstrated. The secretions of these glands help in the formation of the byssus threads. The mucous gland cells are subepithelial localised they secrete acid and neutral mucopolysaccharides together with glycoproteins. Which participate in the attachment of the byssus disc.     

SYNTHESIS, MIGRATION, AND RELEASE OF PRECURSOR COLLAGEN BY ODONTOBLASTS AS VISUALIZED BY RADIOAUTOGRAPHY AFTER [3H]PROLINE ADMINISTRATION

The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30–40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline. At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains. At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads. The parallel threads measured 280–350 nm in length and stained with the low pH-phosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils). The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules. Between 20 and 30 min, prosecretory and secretory granules respectively became labeled. These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules. Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process. At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin. This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process. It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus. Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis. In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils.     

Location and analysis of byssal structural proteins of Mytilus edulis

The acellular attachment organ (byssus) of the marine mussel Mytilus edulis L. is composed of threads that emanate from the body of the mussel to adhesive discs that anchor the threads to rocks, sand and other mussels. Three proteins have been purified by immunohistological methods and located to specific regions of the byssus. A collagenous protein with subunit molecular weights of 53,000, 55,000 and 65,000 is found in the matrix of the elastic thread region. Its 73,000-MW precursor was extracted from foot glands in the area proximal to the animal body and was identified by immune cross-reactivity. A cystine-rich, acidic protein was found in all regions of the byssus associated with a third protein, the polyphenolic protein. The L-dopa-containing polyphenolic protein appears in the cortex of the entire thread and adhesive plaque and at the substrate-plaque interface. Antiserum to this protein stains spherical vesicles in the phenol gland of the foot. Using immuno-electrophoretic methods, the polyphenolic protein and the cystine-rich protein were shown to form high molecular weight aggregates with aging of the byssus.     

Sclerotization of the perisarc of the calyptoblastic hydroid, Laomedea flexuosa 2. Histochemical demonstration of phenol oxidase and attempted demonstration of peroxidase

Histoehemical evidence is presented that cells, described as 'tanning cells', in the hydroid Laomedea flexuosa contain an orthodihydroxyphenol oxidase and transfer this enzyme to the perisarc. The detection of copper in the tanning cells provides further evidence for the presence of phenol oxidase, a copper-containing enzyme. Evidence is presented that this enzyme and the orthodihydroxyphenol, dopamine, are stored together in the same 1mu diameter spherical inclusions. The enzyme and orthodihydroxyphenolic substrate appear to be transferred together to the perisarc where it is thought that the dopamine is oxidized by the phenol oxidase to produce a quinone. The quinone is thought to cross-link the structural proteins to form a strong, inert exoskeleton, the perisarc. Attempts to demonstrate peroxidase using benzidine methods are also described. Both the spherical inclusions of the tanning cells and the perisarc of the hydrothecae give a reaction with benzidine in the absence of hydrogen peroxide. This suggests the presence of an oxidizing agent which is not a peroxidase but is possibly a quinone.     

Secretion and stabilization of the layers of the egg capsule of the dogfish Scyuorhinus canicula

The egg capsule of the dogfish is a unique, collagcnous, layered structure secreted by the nidamental gland which has nine, remarkably discrete, transverse zones of tubular glands. The present paper traces the origin of the four layers of the capsule to particular zones within the gland. Evidence is presented for the existence of DOPA, DOPA oxidase. protein(s) rich in tyrosyl residues and a peroxidase within the same storage granules within the secretory cells of the C and E zones. It is suggested that these interact when secreted to cross-link the inner and outer surfaces of the egg capsule. Evidence is presented that the middle layer which forms the bulk of the thickness of the egg capsule and has the highest collagen content may be partly stabilized by the peroxidation of tyrosyl residues. The mechanical significance of crosslinking in this system and the possible cytological mechanisms involved in the secretion of the tanning agents and enzymes are discussed.     

An ultrastructural study of byssus stem formation in Mytilus californianus

In Mytilus californianus, root lamellae of the byssus stem are formed by two morphologically distinct exocrine cell types. Type 1 cells contain large ellipsoid granules which are ultrastructurally identical to those of the collagen gland associated with byssus thread formation: these granules are secreted only at the base of the stem generator. Type 2 cells contain small cylindroid granules which are secreted only from the lateral surfaces of generator septa. The resultant matrix is biphasic because the two secretions are incompletely mixed. Lamellar sheets of matrix are propelled outward by the action of cilia and are molded into a cylinder at the neck region of the stem. However, the stem retains a lamellar pattern. Byssus threads are attached to the stem by flattened rings formed from thread material which is secreted into the cervical crevice surrounding the neck. The microanatomy of the stem forming region is described and a new term, “stem generator,” is proposed for this organ.     

Presence of Active Hatching Enzyme in the Secretory Granule of Prehatching Medaka Embryos

Secretory granules of hatching gland were isolated from a 0.3 M sucrose homogenate of whole medaka embryos at prehatching stage by differential centrifugation, followed by a Percoll density gradient centrifugation. The obtained preparation was almost free of melanosomes and composed exclusively of the secretory granules of hatching gland (hatching enzyme granules), as judged by morphological as well as enzymological criteria.
The aqueous extracts of the purified secretory granules showed a specific choriolytic activity as high as about 40 times that of a partially purified secretory granule preparation, P_1,000, and represented a single protein band with molecular weight of about 21,000 on SDS-polyacrylamide gel electrophoresis. It was also revealed that a major component of the hatching enzyme preparation (P II–0.3 enzyme, 13) purified from the hatching liquid was identical with the 21,000 molecular weight band.
These results suggest that the hatching enzyme is present in the secretory granules of prehatching embryos in an active molecular form.     

Cellular compartmentation of lysozyme and alpha-amylase in the mouse salivary glands. Immunogold approaches at light and electron microscopy level

The research was planned to study the subcellular distribution of enzymatic secretory products within the secretory structures of the mouse major salivary glands at light and electron microscopy level by immunogold silver stain (IGSS) technique and double-sided post-embedding immunogold binding and silver amplification in order to speculate about their compartmentation. In particular, we experimented the above immunogold labeling approaches to localize the lysozyme and to verify its distribution patterns in relation to another secretion enzyme, alpha-amylase. Co-presence of lysozyme and alpha-amylase was observed in the convoluted granular tubule cells of the submandibular gland and in the demilunar cells of the sublingual gland as well as in the electron-dense regions of the mottled secretory granules in the parotid gland. Exclusive binding patterns of lysozyme were observed in the acinar cells of the submandibular and sublingual glands where alpha-amylase did not occur.     

Electron microscopic identification of the histamine-storing argyrophil (enterochromaffin-like) cells in the rat stomach

Summary In the oxyntic gland area of the rat stomach the histamine-containing epithelial cells (also referred to as enterochromaffin-like cells because of their morphologic similarity with the 5-hydroxytryptamine-storing enterochromaffin cells) constitute the system of argyrophil cells in this area as previously shown by the combined use of fluorescence and light microscopic techniques. By performing the argyrophil staining reaction directly on ultra-thin sections it could be demonstrated in the electron microscope that the argyrophil cells have features suggesting that they are endocrine. Based on the ultrastructure of their secretory granules at least two such endocrine cell systems—both argyrophil—could be recognized in the oxyntic glands. The silver deposits were accumulated over the secretory granules of both these cell systems.It is well known that after injection of 1-3,4-dihydroxyphenylalanine, the histamine-storing (enterochromaffin-like) cells of the oxyntic glands store also dopamine. Under these conditions the enterochromaffin-like cells stain argentaffin, which has been shown at the light microscopic level. Also this reaction could be performed directly on ultra-thin sections. By electron microscopy it was then established that the two endocrine cell systems of the oxyntic gland area stained argentaffin upon treatment with 1-3,4-dihydroxyphenylalanine, and that the staining was confined to the secretory granules.The results clearly show that the enterochromaffin-like cells of the rat oxyntic gland area (which is devoid of 5-hydroxytryptamine-containing enterochromaffin cells) are identical with cells characterized as endocrine by ultrastructural criteria, and that gastric non-mast-cell histamine occurs in at least two separate systems of enterochromaffin-like cells.     

Localization of some enzymes in the main venom glands of viperid snakes

Several secretory and nonsecretory enzymes were localized histochemically in the main venom gland of 13 viperid snakes. All secretory cells show the intracellular oxidative enzymes succinate dehydrogenase and monoamine oxidase. The granular reactions obtained for both enzymes resemble mitochondria in distribution. Distinctive cells with a very high succinate dehydrogenase activity are dispersed among the secretory cells of all species except Atractaspis. Nonspecific acid phosphatase activity is found in the supranuclear region of the secretory cells in species that do not secrete this enzyme and throughout the cytoplasm in snakes that secrete the enzyme. Nonspecific alkaline phosphatase activity occurs in the secretory cells of those snakes whose venom shows this activity. Leucine amino peptidase (aryl amidase) activity is found in the venom and in the secretory cells of all the species. In Vipera palaestinae both the venom and the secretory cells of the main venom gland contain nonspecific esterase, L-amino acid oxidase and phosphodiesterase activities. The localization of phosphodiesterase and L-amino acid oxidase do not show major differences between glands at different intervals from an initial milking. Adenosine-monophosphate phosphatase activity is localized in the supranuclear region of the secretory cells in the glands of Vipera palaestinae and Aspis cerastes. Its activity is found in the venom of Aspis only.     

The kidney of a teleost, Spinachia spinachia II. Histochemical identification of sialic acid-containing glycoprotein and fine structure of mucus secreting cells

Kidney cells of the marine stickleback Spinachia have been studied with histochemical methods for the demonstration of glycoconjugates. The fine structure of epithelial cells is described. Mucus threads in the nephronic tubule of sexually mature males consist of neutral glycoprotein which corresponds with the secretory granules in proximal tubule segment II cells. Large lysosome-like inclusions, which also react with PAS, are present in many P II cells. All cells of the collecting duct epithelium differentiate into mucous cells in male Spinachia. The nature of their secretory products, which are well preserved by freeze-drying, is discussed. Sialylated glycoprotein is present in mucus granules and sulphated glycoprotein can be demonstrated at the apex of collecting duct cells. Collecting duct cell mucus can be digested with testicular hyaluronidase indicating that proteoglycans may be involved in the structure of macromolecules. The observations are compared with studies of mucus production in the urinary apparatus of several other vertebrates.     

Relationship between the golgi apparatus, gerl, and secretory granules in acinar cells of the rat exorbital lacrimal gland

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.     

Fine structural localization of peroxidase activity in the epithelium and the gland of the rat larynx

Summary Endogenous peroxidase activity was demonstrated in ciliated cells and secretory cells of the laryngeal epithelium and gland of rats, using the diaminobenzidine method for cytochemical demonstration of peroxidase activity. The intensity of peroxidase activity was greatly varied from cell to cell, but the fine structural localization of the activity was similar in various cell types. The activity was localized in cisternae of rough-surfaced endoplasmic reticulum including nuclear envelope, some vesicles and saccules of the Golgi complex, large membrane-limited granules, multivesicular bodies and probable lysosomes. In secretory cells, the activity was also found in secretory granules.The significance of peroxidase activity is not unclear, while the activity, at least a part of it, seems to be secreted into the cavity of the larynx. The possibility that peroxidase participates bactericidal mechanism, deserves further investigation.     

Collagen biogenesis and assembly into fibrils as shown by ultrastructural and 3H-proline radioautographic studies on the fibroblasts of the rat food pad

To examine whether collagen is assembled into fibrils within or outside fibroblasts, the connective tissue of the rat foot pad was investigated by electron microscopy and by radioautography at times varying from 4 min to 3 days after an intravenous injection of 3H-proline. The fibroblasts of the rat food pad are long polarized cells with the nucleus at one end, the Golgi apparatus in the center, and a region with long processes at the other end. This region contains secretory granules and is considered to be the secretory pole of the cell. In the Golgi apparatus the stacks of saccules are separated from rough endoplasmic reticulum (rER) by groups of "intermediate vesicles" including similarly structured tubules which may be over 300 nm long and are referred to as "intermediate tubules." The Golgi saccules exhibit distended portions which differ at the various levels of the stack. On the cis side, the distentions tend to be spherical and contain fine looping threads; in the middle of the stack, they are cylindrical and present distinct straight threads; whereas on the trans side, they are again cylindrical, but the straight threads are grouped in parallel aggregates. Between these cylindrical distentions and the secretory granules, there are transitional forms within which thread aggregates are packaged more and more tightly. Finally, the fibroblasts are associated with two types of collagen fibrils: extracellular ones arranged into large groups between the cells and intracellular ones located within long intracytoplasmic channels. Quantitative radioautography after 3H-proline injection reveals that the number of silver grains per unit area reaches a peak over the rER at 4-10 min, Golgi apparatus at 40 min, secretory granules at 60 min, and extracellular collagen fibrils at 3 h. At no time are intracellular collagen fibrils labeled. Qualitative observations further indicate that spherical Golgi distentions are mainly labeled at 40 min, and cylindrical distentions, at 60 min. In addition, from 20 min to 3 hr, some lysosomal elements are labeled. The biogenetic pathway leading to the formation of collagen fibrils is interpreted as follows.(ABSTRACT TRUNCATED AT 400 WORDS)     

The amylase-secreting cells of the stomach of the scallop, Pecten maximus: ultrastructural, immunohistochemical and immunocytochemical characterizations

(1) alpha-amylase was extracted and purified from the stomach/digestive gland complex of the scallop Pecten maximus and an anti-serum was induced against the purified amylase by rabbit immunization. (2) The anti scallop amylase was used to localize the amylase-secreting cells in the stomach of Pecten maximus by immunofluorescence and immunogold labelling. The amylase-secreting cells are glandular cells particularly numerous in the main sorting area of the stomach. Their secretory granules were found strongly positive for anti-amylase. Three types of glandular cells were observed, actually corresponding to the three stages of the glandular-cell activity, synthesis, secretion and excretion. (3) The synthesizing cell shows the characteristic features of a protein-synthesizing cell: a conspicuous nucleolus and abundant granular endoplasmic reticulum. In the secretory cell, the secretory granules are formed by the Golgi apparatus and accumulate in the apical part of the cell. The secretory cell is filled with two types of secretory granules which are released in the stomach lumen by apocrine excretion. (4) The present study brings the first demonstration of the synthesis and extracellular release of amylase by glandular cells of the stomach epithelium of a bivalve.     

A pair of rosette glands in the embryo and zoeal larva of an estuarine crab Sesarma haematocheir, and classification of the tegumental glands in the embryos of other crabs

A pair of rosette glands (one of the tegumental glands in crustaceans) is present at the root of the dorsal spine of the thorax in mature embryos of the estuarine crab Sesarma haematocheir. Each rosette gland is spherical, 45-50 microm in diameter. This gland consists of three types of cells: 18-20 secretory cells, one central cell, and one canal cell. The secretory cells are further classified into two types on the basis of the morphology of secretory granules. There are 17-19 a cells, and only one b cell per rosette gland. An a cell contains spherical secretory granules of 2-3 microm in diameter. The granules are filled with highly electron-dense materials near the nucleus but have lower electron-density near the central cell. The secretory granules contained in the b cell have an irregular shape and are 1-1.5 microm in diameter. The density of the materials in the granules is uniform throughout the cytoplasm. The secretory granules contained in both the a and b cells are produced by the rough endoplasmic reticulum. Materials in the granules are exocytotically discharged into the secretory apparatus inside the secretory cell, sent to the extracellular channels in the central cell, and secreted through the canal cell. The rosette gland can be distinguished from the epidermal cells 2 weeks after egg-laying and the gland matures just before hatching. Materials produced by this gland are secreted after hatching and secretion continues through five stages of zoeal larvae. These rosette glands were never found in the megalopal larva. Rosette glands are found in the embryos of Sesarma spp. and Uca spp. In other crabs, tegumental glands are also found at the same position as in the embryo of S. haematocheir, but the fine structure of their glands is largely different from that of the rosette gland. On the basis of the morphology of secretory cells (a-g cell types), the tegumental glands of a variety of crab embryos can be classified into four types, including rosette glands (type I-IV). The function of these tegumental glands is not yet known, but different types of the gland seem to reflect the phylogeny of the crabs rather than differences of habitat.     

On the identity of the thyrotropic cell in the red-spotted newt

Summary Although the pars distalis of the red-spotted newt has previously undergone extensive cytological examination, the identity of its thyrotropic cells has remained uncertain. From the present ultrastructural study, cells of type 3 (Masur, 1969) containing granules 150–180 nm in diameter are concluded to be the thyrotropes. Such cells were found to be present in the regions of the pars distalis where thyroidectomy cells arise after ablation of the thyroid gland. Cells of type 3 are probably identical with a population of cells containing granules which stain with silver methenamine indicating the presence of a glycoprotein such as thyroid stimulating hormone (TSH). Thyroidectomy cells containing a few residual granules 150–180 nm in diameter were occasionally found in partes distales from newts killed 3 or 7 days after ablation of the thyroid gland, and were abundant in pituitaries 21 days after thyroidectomy. Only cells of type 3 responded (by vacuolation of granules) when animals were immersed in water containing 10 g/l of thyroxine. No cells of the pars distalis showed cytological change after administration of synthetic thyrotropic releasing hormone (TRH) giving additional support to the view that this hormone has no stimulatory role in amphibians.Work supported in part by research grant 1 R01 AM 16731-01 from the Institute of Arthritis, Metabolism and Metabolic Diseases and in part by grant PCM 75-17637 from the National Science Foundation