Evolutionary regularities of the appearance of polyploidy in salivary glands of gastropod mollusks. III. A subclass of Pectinibranchia: Orders Discopoda, Echinospirida, Aspidophora and Entomostoma (Mesogastropoda)

By means of histological methods and DNA cytophotometry, a study was made of the salivary glands of 16 species of gastropod molluscs belonging to the subclass Pectinibranchia and making a group of Mesogastropoda. Four cell types of salivary glands were distinguished: granular cells (with glycoproteid granular inclusions), mucocytes-I (with sulfatic acid mucopolysaccharides), mucocytes-II (with neutral and acid polysaccharides and proteins), and also the epithelial ciliated cells and mucous duct cells. Data of experiments on starvation and synchronous feeding of molluscs have testified that all described cell types are independent. In some species differentiation on protein and mucous departments within the glandular epithelium was shown. In some marine representatives of the orders Discopoda and Aspidophora polyploid cells with the ploidy levels from 8c to 32c were revealed along with diploid cells. The ecological and phylogenetic regularities of somatic polyploidy manifestation in Mesogastropoda are discussed.     

Anatomical and histochemical features of the digestive system of Octopus vulgaris Cuvier, 1797 with a special focus on secretory cells

This study reports a detailed anatomical and histological study of the digestive system of Octopus vulgaris. Emphasis was placed on characterising the glands and glandular cells and their distribution throughout the digestive tract. The use of classic histological and histochemical techniques revealed two morphological types of glandular cells: granular and mucous. Moreover, the histochemical analysis indicated specialisation of mucous glandular cells in the buccal mass, the submandibular gland and the caecum for secreting acid and neutral glycoconjugates. The cells of the anterior salivary glands are specialised for secreting neutral glycoproteins, and those of the posterior salivary glands are specialised for granular and mucous secretion. The oesophagus, crop and stomach lack glandular cells, but both granular and mucous glandular cells are found in the intestine. An unusual structure resembling the typhlosole of bivalves is described for the first time in the intestine of O. vulgaris. The highly ciliated epithelium and location of the structure in the anterior part of the intestine suggest a possible role in bypassing the caecum, stomach and intestine. We discuss how these cells and organs contribute to the process of digestion in the light of the present histological and histochemical data and of previously published information on the morphology and physiology of digestion in the octopus.     

Cellular types in the gill arch of the teleost Cyprinus carpio (Cyprinidae, Pisces). A histochemical and ultrastructural study

On adult specimens of the common carp (Cyprinus carpio), we have carried out a histochemical and ultrastructural study of the epithelia which form the gill arch. Secondary lamellae have two cellular types, granular and mucous, which produce neutral carbohydrates and proteins rich in tryptophane, and another mucous cell type which contains glycosaminoglycans. In gill rakers, three cellular types show different histochemical behaviour: 1) granular cells elaborate neutral mucosubstances; 2) a second cell type contains glycosaminoglycans,, and 3) a third cell type secretes neutral and acid carbohydrates. We discuss the possible role of these cells according to their secretion. We describe pillar and chloride cells in secondary lamellae, and chloride and neuroendocrine-like cells in gill rakers.     

Ultrastructural and histochemical study of the salivary glands of Aplysia depilans (Mollusca, Opisthobranchia)

The digestive system of the sea hare, Aplysia depilans , includes a pair of ribbon-shaped salivary glands. A central duct and a large blood vessel run close to each other along the length of these glands and both are surrounded by a layer of muscle cells. Three cell types form the glandular epithelium: granular cells, vacuolated cells and mucocytes. The granular cells possess cilia and spherical secretion granules, located primarily in the apical region. The granules of immature cells have a low electron density and are mainly formed by neutral polysaccharides with small amounts of proteins. The granules of mature cells are larger, have a high electron density and are mainly formed by proteins with lower amounts of neutral polysaccharides. Transition stages between immature and mature granular cells are observed. The vacuolated cells are large and frequently pyramidal in shape, but after the application of histochemical techniques almost all vacuoles remain uncoloured. The numerous vacuoles contain flocculent material in a clear background and the mitochondria possess large crystalline structures in the matrix. A pyramidal shape is also typical of the mucocytes, which are filled with vesicles containing granular masses surrounded by a network of secretion material. These large cells are strongly stained by Alcian blue, revealing the presence of acidic mucopolysaccharides. This is the first ultrastructural study of the salivary glands in opisthobranch gastropods.     

A histological and histochemical study of the oesophagus and oesogaster of the Senegal sole, Solea senegalensis.

A histological and histochemical study was performed in the buccal cavity and papillae, which were around the teeth, as well as in the oesophagus and oesogaster of the Senegal sole, Solea senegalensis adult specimens. The oesophagus and oesogaster were made up of four distinct layers: mucosa, submucosa, muscular and serous. Two morphological types of epithelial cells were distinguishable in the oesophageal mucosa: the more numerous type cells possessed an electron-dense cytoplasm, whereas the cytoplasm was electron-clear in the other cells. Mucus-secreting cells were the dominant feature of the epithelium throughout the oesophagus. These goblet cells were filled with numerous mucous droplets of low electron-density. The oesophagus was devoid of taste buds. In the oesogaster mucosa, three types of cells were distinguished: dark, rodlet and light epithelial cells. Dark epithelial cells showed different characteristics from that in the oesophagus: the nucleus was irregular with an electron-dense hyaloplasm, the cytoplasm had a scarce smooth and granular endoplasmic reticulum; a Golgi apparatus consisted of four parallel cisternae, dense granules without membrane, lysosomes and numerous mitochondria. The rodlet cells were elongated, contained rod-like structures and were surrounded by an electron-dense capsule-like structure. The bulk of the rodlet cell was composed of up to 20 extended rodlet units. Light epithelial cells of the oesogaster had the same characteristics as those observed in the oesophagus and contained numerous mitochondria with a dense matrix, abundant smooth endoplasmic reticulum and numerous vesicles. In the goblet cells of the papillae, sulfomucin was recognised, since they showed alcianophilia (alcian blue pH 1.0 and 0.5). These cells were negative to protein reaction (bromophenol blue) and contained -S-S- and SH groups. Enzymatic activities (alkaline phosphatase, acid phosphatase, ATPase (pH 7.2 and 9.4) and lipid reactions were negative in the goblet cells of the buccal cavity. Epithelial cells of oesophagus contained a weak presence of acid and neutral mucopolysaccharides. Oesophageal goblet cells contained carboxylated, sulphated (weakly and strongly ionised) mucosubstances and sialic acid. Most goblet cells did not contain proteins and presented disulphide (-S-S-) and sulphydril (-SH) groups. Proteins in general, and in particular those rich in lysine, tyrosine and arginine were present in the epithelium, lamina propria, submucosa and muscular layer of the oesophagus. Lipids in general and phospholipids were observed in the oesophageal epithelium while unsaturated, acid and neutral lipids were not observed. The lamina propria and submucosa contained a weak presence of phospholipids and unsaturated lipids. Acid phosphatase and ATPase (pH 7.2) activities were observed in the lamina propria, submucosa and muscular regions, while ATPase (pH 9.2) activity was weak in these areas. ATPase activity (pH 7.2 and 9.5) was very weak in the epithelium. Oesophageal goblet cells were negative to lipid and enzymatic reactions.     

Synthesis and secretion of mucous glycoprotein by the gill of Mytilus edulis. I. Histochemical and chromatographic analysis of [14C]glucosamine bioincorporation

The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.     

仿刺参消化系统的组织学和组织化学研究

用组织学和组织化学方法研究了仿刺参的消化系统。消化道管壁由粘膜层、粘膜下层、肌层和外膜组成。粘膜层为假复层或单层的柱状细胞或立方细胞与粘液细胞。粘液细胞分布于前肠的前段和排泄腔。前肠和中肠上皮具蛋白酶、脂酶和非特异性酯酶活性。中肠上皮细胞游离端有密集微绒毛,游离端质膜呈碱性磷酸酶活性,上皮下有丰富的血窦,表明具吸收作用。     

幼龄皱纹盘鲍唾液腺和消化腺的超微结构与组织化学

以透射电镜观察和组织化学方法研究了45日龄皱纹盘鲍的唾液腺和消化腺。唾液腺由粘液细胞和纤毛细胞组成,粘液细胞含发达的粗面内质网和大量的粘原颗粒,分泌中性和酸性混合粘多糖。消化腺由消化细胞和嗜碱性细胞组成,消化细胞呈现活跃的内吞和细胞内消化,并具蛋白酶和非特异性酯酶活性。嗜碱性细胞含发达的粗面内质网和大量含铁的折光小体,折光小体的电子密度较低。     

Ultrastructural study of the epithelial mucous cells in lizards (Lacertilia)

Summary An ultrastructural study of the mucous cells of the intestinal epithelium of four lacertilian species (Lacerta lepida, Lacerta hispanica, Psammodromus algirus and Acanthodactylus erythrurus) is here reported. Two types of mucous cells have been found in these species: common mucous cells and granular mucous cells. Immature and mature forms of both types have been observed. The common mucous cells or typical goblet cells have the same characteristics in all four species. The granules of the granular mucous cells of the two species of Lacerta are similar but differ from those of the other two species.     

Ultrastructural and cytochemical studies in the mantle of Anodonta cygnea

The large central region of the mantle of Anodonta cygnea was studied using transmission and scanning electron microscopy (TEM and SEM) for ultrastructural analysis and light microscopy (LM) and TEM for cytochemical analysis. X-ray diffraction studies of the organic matrix pellicle deposited on the inner surface of the shell were also carried out. Two groups of columnar cells presenting desmosomes on the apical region were observed in the outer epithelium. One group secretes a structural and neutral mucopolysaccharide (MPS) identified with chitin, normally excreted to form the organic matrix of the shell. Another group presents numerous cytoplasmic vesicles and constitutes the predominant cells of this epithelium. Two different types of mucous cells were found in the inner epithelium. One type secretes a faintly acid mucopolysaccharide (MPS) with sulphate groups and another type secretes an association of this polysaccharide with a neutral MPS. Staining for sulfhydryl groups (probably cysteine) was also positive, suggesting the presence of proteoglycans in these secretions. A third type of cells was also observed presenting a very different ultrastructural aspect (columnar form) without large secretion masses. They may correspond to the replacing cells in this highly secretory epithelium. Elastic fibers were found on the base of the outer epithelium and amoebocytes were observed in the interepithelial tissue.     

Microscopic anatomy of the integument in the ribbon worm Micrura bella (Stimpson, 1857) (Pilidiophora,Nemertea)

The integument of ribbon worms in the order Heteronemertea is distinct from the integuments in the other taxa of nemerteans due to the presence of a special subepidermal glandular layer, the cutis. Among heteronemerteans, the ultrastructure of the cutis has been studied only in the Lineus ruber species complex. In the current study, ultrastructural (transmission electron microscopy) and histochemical studies of the epidermis and the cutis of Micrura bella from the basal Lineage A of the family Lineidae were performed. The epidermis consisted of ciliated and serous gland cells and is separated from the cutis by a layer of the subepidermal extracellular matrix; the basal lamina was not detected. The cutis comprised musculature, two types of mucous and four types of granular gland cells, and pigment cells with four types of granules. In the cutis of juvenile worms, type II granular gland cells and type II mucous cells were not observed. The integument of the caudal cirrus consisted of ciliated and serous gland cells and two intraepidermal lateral nerve cords; the cutis was absent. The compositions of the integument glands of M. bella and the L. ruber species complex are similar, except for the presence of type IV granular gland cells with narrow rod-shaped and lamellated granules exhibiting an alternating dark and light transverse layers and type II mucous cells found only in M. bella.     

The morphology of the mucous gland and its responses to prolactin in the skin of the red-spotted newt

The mucous gland of the red-spotted newt, Notophthalamus viridescens viridescens, Rafinesque was examined by histochemical and ultrastructural techniques and its cytological responses to various hormonal conditions were studied. Its secretory epithelial cells produce and release in merocrine fashion a neutral, unsulphated mucosubstance. The secretory epithelium is bounded peripherally by a thin, but apparent non-functional, myo-epithelium. The duct of this mucous gland consists of a single keratinized tubular cell that extends from the neck region of the gland to the surface of the epidermis. Mucous secretion is absent or greatly reduced on the skins of newts maintained under laboratory conditions for a few weeks but reappears after injection of ovine prolactin. Mucous glands in laboratory conditioned animals show a 4-fold increase in volume brought about by the engorgement of their epithelial cells with secretory granules. Ovine prolactin reduces the volume of the glands to unconditioned levels with a corresponding reduction in granular content, suggesting that prolactin functions in the release of the granules. This view is reinforced by the findings that autotransplantation of the pituitary gland prevents the conditioning effect and that glandular volume increases in auto-transplanted animals given ergocornine. Granular accumulation begins also in hypophysectomized newts but ceases after a week, indicating the need for some hypophyseal factor in the synthesis as well as the release of the granules. Ovine prolactin restores mucous glands of hypophysectomized newts to the unconditioned state. Contrary to earlier findings, ovine prolactin induces a reduction in the volume of the mucous gland in thyroidectomized newts.     

Histological description of the ink gland of the tropical sea hare Aplysia dactylomela Rang, 1828

This work describes for the first time the ink gland of Aplysia dactylomela Rang, 1828 using light microscopy and histochemical tests. The results reveal that this organ is covered by a layer of simple epithelium and that there are some differences between the epithelium facing the mantle shelf and that facing the mantle cavity. The former consists of columnar cells, which may have a secretory function, whereas the latter is a cuboidal epithelium. Underneath the epithelium and along the whole gland there are fibres of smooth muscle and collagen, organized in groups of parallel bundles. There are vesicles of different diameters, apparently with similar morphology. Some are filled with ink, whereas others are either granular or clear. The ink is released through a duct formed by invaginations of the cuboidal epithelium. Tests with bromophenol blue and periodic acid Schiff indicated that the ink and granulated vesicles contain proteins and carbohydrates or maybe glycoproteins. Between the fibre bundles and the vesicles there are dispersed cells. A diagram is presented emphasizing the covering epithelium, the distribution of muscle and collagen fibres, the dispersed cells, vesicles and ducts. This organization is similar to that of the other Aplysia species studied to date, A. californica.     

Organogenesis of the digestive tract in the white seabream,Diplodus sargus. Histological and histochemical approaches

The ontogeny of the digestive tract of the white seabream, Diplodus sargus during the larval development up to day 45 post-hatching (dph) has been studied using histological and histochemical techniques. The oesophageal goblet cells appeared around 6 dph and contained neutral and acid mucosubstances (PAS/diastase-PAS and Alcian Blue pH 2.5 positive reactions). An incipient stomach can be distinguished from 2 dph but the first sign of gastric gland development was detected around 13-15 dph, increasing in number and size by 22-23 dph. Gastric glands were concentrated in the cardiac stomach region and they had a high content of protein rich in tyrosine, arginine and tryptophan. Acidophilic supranuclear inclusions related to pynocitosis of proteins, were already observed in the intestinal cells of the posterior intestine around 4-6 dph (exogenous feeding) and they were present until 25 dph. The intestinal mucous cells appeared between 15-18 dph and contained a mixture of neutral and acid mucosubstances/glycoconjugates, carboxylated ones being more abundant than the sulphated ones. The stomach and gastric glands were fully developed by the first month of life marking the beginning of digestive features characteristic of the juvenile stage. Around 4-6 dph, glycogen, proteins and neutral lipids were observed in the granular cytoplasm of hepatocytes. Strongly acidophilic zymogen granules were also present, at this time, in the basophilic cytoplasm of the exocrine pancreatic acinar cells and contained abundant proteins, especially rich in arginine, tyrosine and tryptophan.     

Evolutionary regularities of somatic polyploidy manifestation in salivary glands of gastropod molluscs. I. Subclasses Cyclobranchia and Scutibranchia

By means of histological and cytochemical methods, including DNA cytophotometry, the salivary glands of 11 species of molluscs of two old gastropod subclasses--Cyclobranchia and Scutibranchia (limpets)--have been investigated. In spite of some anatomical differences, the glandular epithelium of investigated molluscs includes functionally similar cell types: granular cells (with glycoproteid granular inclusions), mucocytes-I (that include sulfatic acid mucopolysaccharides), mucocytes-II (that include neutral and acid polysaccharides and proteins) and also the epithelial ciliated cells. Data of experiments on starvation and synchronous feeding of molluscs testify that all described cell types are independent. According to DNA cytophotometry data, the glandular cell nuclei are diploid in the main; only small part of the nuclei, varying in different species from 0.5 to 5.0%, displayed tetraploid DNA mass. A conclusion is made that in the oldest subclasses of gastropods (Cyclobranchia and Scutibranchia) somatic polyploidy, as a factor of tissue growth in salivary glands, is actually absent.     

Histochemical demonstration of proteins, lipids and polysaccharides in Japanese monkey oocytes

A histochemical study of proteins, lipids and polysaccharides was carried out in the oocytes of Japanese monkey (Macaca fuscata) during development of follicles. There were a small number of protein granules reactive to acrolein-Schiff in the cytoplasm of oocytes from primordial, secondary and vesicular follicles, while there were no lipid droplets, granules of neutral polysaccharides or acid polysaccharides in the cytoplasm. Proteins reactive to acrolein-Schiff, neutral polysaccharides reactive to periodic acid-Schiff and acid polysaccharides stainable with alcian blue were observed in the zona pellucida of the oocytes of secondary and vesicular follicles. The zona pellucida contained sudanophilic lipids composed of neutral fats and lipoids, besides the proteins and polysaccharides.     

Morphology and glycoconjugate histochemistry of the palpebral glands of the adult newt, Notophthalmus viridescens.

The eyelids of the newt were studied in 10 microns serial paraffin and 1-2 microns plastic sections using standard histological stains and special stains for glycoconjugates. The eyelids contain four different glands. Simple acinar serous and simple acinar mucous glands occur in the skin; unicellular mucous glands occur in the conjunctiva; and convoluted tubular seromucous glands are present in connective tissue beneath the conjunctiva. The first two are identical to cutaneous glands found elsewhere on the head and body. The simple acinar serous glands are surrounded by myoepithelial cells and release their secretion, which is composed largely of proteins with minimal glycoconjugate content, by a holocrine mechanism. The secretory product of the simple acinar mucous glands is composed of neutral glycoconjugates with a minor content of acidic glycoconjugates; the mucin exhibits strong PAS and PAPD staining and weak staining by AB and PAPS methods. The unicellular conjunctival mucous glands secrete both neutral and acidic glycoconjugates as shown by positive reactions with PAS, PAPD, PAPS, and AB methods. Convoluted tubular seromucous glands in the ventral eyelid synthesize both proteins and neutral glycoconjugates. The mucous secretions of the conjunctival glands probably provide lubrication and protection for the cornea.     

Loss of traditional mucous glands and presence of a novel mucus-producing granular gland in the plethodontid salamander Ensatina eschscholtzii

All amphibians are thought to possess two distinct types of dermal gland: mucous and granular. Mucous glands typically produce an acidic, flocculent and carbohydrate positive secretion that keeps skin moist and helps maintain homeostasis. In this study, we use a battery of histochemical tests to identify these traditional mucous glands in the dermis of the plethodontid salamander Ensatina eschscholtzii . The periodic acid-Schiff, alcian blue, toluidine blue and carmine reactions reveal the absence of both acidic glycosaminoglycans and traditional mucous glands. Instead, E. eschscholtzii produces neutral mucus in specialized granular glands, which we name mucus-producing granular glands (MPGGs). The loss of traditional mucous glands is a derived trait for Ensatina .  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 469–477.     

Morphological and histochemical observations on the intestinal epithelium of Ascardia galli (Nematoda: Ascaridida).

The intestinal epithelium of Ascardia galli has been studied with various cytological and cytochemical techniques. It consists of large epithelial cells resting on a thick collagenous basal lamina. Their luminal surface is provided with microvilli. The intestinal cells store considerable amounts of glycogen and neutral lipids. Some intracellular granular inclusions, which stain for proteins, phospholipids and lipoproteins, are distributed throughout the cytoplasm. The brush border is composed of microvilli whereas the outer surface coat consists of saliva resistant PAS-positive material. The detailed histochemical analysis of surface material has revealed that it is composed of nonacetylated acid mucopolysaccharides rich in hyaluronic acid with carboxylate polyanions. The brush border shows intense activities of acid phosphatase and glucose-6-phosphatase, moderate of ATPase, and lipase, weak of 5'-nucleotidase. Acid phosphatase-positive intracellular structures are seen in the intestinal epithelium which form distinct aggregations.     

Description of epithelial granular cell in catshark spiral intestine: Immunohistochemistry and ultrastructure

We evaluated the histology of the spiral intestine of the blackmouth catshark (Galeus melastomus), a small shark distributed in the eastern Atlantic and Mediterranean Sea basin. Entire digestive tracts of 10 G. melastomus were studied using histochemical, immunohistochemical, and ultrastructural methods. Our studies identified a unique, large granular cell type in the intestinal epithelium. Transmission electron microscopy showed that the epithelial granular cell type made intimate contact, by means of junctional complexes, with adjacent epithelial and mucous cells. Several histochemical staining methods showed that the cytoplasmic granules were strongly eosinophilic. Immunostaining of intestinal sections revealed immunoreactivity of the granular cell to tumor necrosis factor-α (TNF-α) antibody. However, no reactivity to inducible-nitric oxide synthase (i-NOS), interleukin-6 (IL-6), interleukin IL-1β, lysozyme, serotonin 5-HT antibodies was detected.