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.     

Evolutionary regularities of somatic polyploidy in salivary glands of the Gastropod molluscs. II. Subclass Pectinibranchia: order Anisorbranchia

Salivary glands of 5 species of gastropod molluscs of the order Anisobranchia, the most ancient order within the subclass Pectinibranchia, have been investigated by histological and cytochemical methods, including DNA cytophotometry. Glandular cells of the following types were recognized: granular cells (with glycoproteid granular inclusions), mucocytes-I (including sulfatic acid mucopolysaccharides), mucocytes-II (including neutral and acid polysaccharides and proteins), and epithelial ciliated cells. All the described cell types are considered to be independent and their morphofunctional characteristics coincide with those of salivary gland cells of the gastropod molluscs of subclasses Cyclobranchia and Scutibranchia. It has been shown that somatic polyploidy in salivary glands in the Anisobranchia molluscs, likely as in those of other Archaeogastropoda (Cyclobranchia and Scutibranchia), is actually absent.     

Evolutionary regularities of the somatic polyploidy manifestation in salivary glands of gastropod molluscs. IV. A subclass of Pectinibranchia: Orders Hamiglossa and Toxoglossa (Neogastropoda)

Salivary glands of 12 species of carnivorous gastropod molluscs, making the Neogastropoda group have been investigated by histochemical methods and DNA cytophotometry. We studied the anatomical and histological structure of ordinary (acinous) and accessory (tubular) salivary glands, and of unpaired glands (Leiblen's, "framboisée", and poisonous). Cells of three types were distinguished: granular cells (with glycoproteid granular inclusions), mucocytes-I (with sulfatic acid mucopolysaccharides), and epithelial ciliated cells. In ordinary and accessory glands of the studied Neogastropods, polyploid cells with polyploidy levels from 4c to 16c were revealed together with diploid cells. The functional significance and phylogenetic tendencies of polyploidy manifestation in salivary glands of prosobranch gastropods are discussed.     

Evolutionary regularities of somatic polyploidy expansion in salivary glands of gastropod mollusks. V. Subclasses Opisthobranchia and Pulmonata

Salivary glands of 25 species of euthyneural gastropod mollusks (Opisthobranchia and Pulmonata) have been investigated by means of histochemical methods and DNA cytophotometry in nuclei of cells. The cells of three basic types are distinguished in glandular epithelim: granular cells (with glicoproteid granular inclusions), mucocytes-I (with sulfatic acid mucopolysaccharides) and mucocytes-II (with neutral and acid nonsulfatic polysaccharides and proteins) and so the epithelial ciliated cells and cells of the ducts. It was shown that glandular cells of salivary glands of all discovered mollusks' species are polyploid in different degree. The highest ploidy level estimated by means of DNA content in most of species is 64-128c. The giant polyploidy, attained to 4096c, is discovered in cells of salivary glands of Tritonia diomedea. The functional conditionality connected with features of feeding of different mollusk species and phylogenetic tendencies of expansion of somatic polyploidy in class Gastropoda are discussed. In comparison with allogenic, facultative and small polyploidy manifestation in Prosobranchia the obligatory polyploidization of high degree revealed in cells of salivary glands of Opisthobranchia and Pulmonata is consider to be the original cytological arogenesis. The probable causes of such differences are conneted with euthyneural type of organization of central nervous system and giant polyploidy of neurons in Opisthobranchia and Pulmonata mollusks. The causes, mechanisms and significance of such correlations are unclear for the present.     

Morphology of the salivary glands of three Squamata species: Podarcis sicula sicula, Tarentola mauritanica and Coluber viridiflavus

The histology, histochemistry and ultrastructure of the salivary glands of three species of squamata, Podarcis sicula sicula (mandibular glands), Tarentola mauritanica (sublingual gland) and Coluber viridiflavus (supra- and infralabial glands) were studied. Each gland contained acidic cells, positive for both periodic acid Schiff and Alcian blue reactions. These cells can be distinguished as seromucous and mucous types based on the different electron density of their granules. α-Amylase, until now detected only in mammalian salivary glands, was not found in any of the salivary glands examined. The ultrastructural study revealed that the salivary glandular cells of T. mauritanica lack intercellular canaliculi, which by contrast, are present between the seromucous cells in the salivary glands of C. viridiflavus and P. s. sicula . Comparable variation is also seen when the ultrastructural features of the secretory granules in salivary glands of the three Squamata species are compared. The salivary granules of T. mauritanica and C. viridiflavus are more or less dense but structureless, while the mucous granules of P. s. sicula have a distinctive and characteristic substructure. Therefore, this study, designed to obtain comparative data on the histology, histochemistry and ultrastructure of the salivary glands of three representative, but hitherto unstudied, species of Squamata, reveals great variation in the structure of these glands within the Squamata lineage, even when compared to previously documented species.     

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.     

Morphological and Histochemical Study of Cephalic Glands of Bothrops jararaca (Ophidia,Viperidae)

Morphological and histochemical studies of the cell types in the cephalic glands of Bothrops jararaca have been performed. It is concluded: 1) mucous cells are found in the salivary labial, accessory glands; mucous-serous cells are found in the salivary labial, accessory and Harderian glands; serous-mucous cells are found only in the venom gland; 2) neutral mucosubstances and protein were found in the salivary labial, venom, accessory and Harderian glands; 3) hyaluronic acid was detected in the Harderian gland; 4) of the to sulfated acid mucosubstances, only chondroitin sulfate B was detected in the salivary labial and accessory glands; 5) sialic acid was detected in the salivary labial, accessory and Harderian glands.     

An ultrastructural study of the submandibular glands of the squirrel monkey, Saimiri sciureus

In squirrel monkey (Saimiri sciureus) the position of submandibular glands in the neck, on either side of the trachea, more closely resembles that of rodents than that of other primates. The glands exhibit seromucous acini and mucous tubules with seromucous demilunes. Electron microscopy shows basal cytoplasmic folds and well-developed intercellular tissue spaces and canaliculi only in relation to seromucous cells. Greatly dilated cisternae of the granular endoplasmic reticulum and prominent Golgi membranes are characteristic of the mucous cells. The secretory granules of seromucous and mucous cells are morphologically distinct and indicate chemically different products for the two cell types. Histochemically, the seromucous cell shows the presence of acid mucosubstance as indicated by the PAS and Alcian blue techniques. Preliminary studies showed no appreciable quantity of amylase in submandibular glands. The intercalated duct cell is juxtaposed with the acinar cell or mucous tubule cell. Short luminal microvilli, prominent Golgi complexes and scant apical granules are notable features of intercalated duct cells. Four cell types compose the striated ducts, viz., granular light cells, agranular dark cells, vesiculated dark cells, and basal cells. Peripheral nerves are found in five different locations: in the connective tissue (interstitial), between adjacent myoepithelial and mucous-secreting cells, in the intercellular space between adjacent secretory cells, and between basal plications of striated ducts and between adjacent myoepithelial and intercalated duct cells.     

Electron microscopic immunogold localization of salivary mucins MG1 and MG2 in human submandibular and sublingual glands.

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.     

Morpho-functional parameters of nucleoli in polyploid mucous and albumen cells of salivary gland in the snail Succinea lauta

Variation of some characteristics of nucleoli of polyploid mucous and albumen cells was examined in salivary glands of the snail Succinea lauta. The number, total area and Ag-protein content of nucleoli, and DNA content in each nucleus were estimated on squashed preparations incubated with AgNO3, decolorized and then Feulgen stained. The ultrastructure of nucleoli was studied by electron microscopy. Differentiated mucous cells had 4c-8c-16c-32c nuclei; albumen cells had 8c-16c-32c-64c-128c nuclei. The ultrastructure of nucleoli of the two cell types was essentially the same. Normally, a large fibrous to granular zone was observed in the nucleoli, without a clear distinction between fibrous and granular components. At the same time, aggregations of granular matter could be discerned at the periphery of nucleoli. No fibrous centers were observed. Occassionally, nucleolonema-like structures occurred. Normally each nucleolus contacted several chromosomes. On squashed preparations, the least size of nucleoli was 2-3 microm, and the largest size amounted to 14 microm in mucous cells, and to 50-80 microm in albumen cells. The number of nucleoli rose from 1-2 in tetraploid nuclei to 2-3 in 32c-nuclei, and to 5-7 in 128c-nuclei. The disparity between the ploidy levels of nuclei and the numbers of nucleoli may be due, presumably, to aggregation of chromosome NORs. The Ag-protein content in the nucleoli, and the total nucleolar area displayed a strong mutual correlation. Both parameters differed significantly by 1.5-2.2 times in mucous and albumen cells of the same ploidy level. Thus, in albumen and mucous cells the total Ag-protein content in octaploid nuclei was 3.3 and 2.2 relative units (r. u.), respectively. In 16c- and 32c-nuclei of albumen cells, it was 7.6 and 15.1 r. u.; and in the same nuclei of mucous cells--3.8 and 6.8 r. u., respectively. On the whole, in albumen cells, in the course of 4 endocycles (4c-128c), the total Ag-protein content increased by 17 times. Therefore, the mean multiplication factor for this parameter was found to be 2.05 per endocycle. In mucous cells, in the course of 3 endocycles (4c-32c), the total Ag-protein content increased by 5.2 times against 8 times expected, with the mean multiplication factor equal to 1.75 per endocycle. Thus, in the course of polyploidization of albumen and mucous cell nuclei, the gene dosage effect was fully pronounced in the former, and only partly in the latter. This differtence is due obviously to peculiarities of differentiation of the two cell types, in particular, to differences in the number of activated ribosomal genes.     

Morphology of the exocrine glands of the frog skin

Frog skin contains three distinct types of exocrine glands: granular (poison), mucous, and seromucous. The granular gland forms a syncytial secretory compartment within the acinus, which is surrounded by smooth muscle cells. The mucous and seromucous glands are easily identifiable as distinct glands. The serous and mucous secretory cells are arranged in a semilunar configuration opposite the ductal end and are filled with granules. Within the acinus, located at the ductal pole of the gland, are distinct groups of cells with few or no granules in the cytoplasm. In both the mucous and seromucous gland there is a cell type with abundant mitochondria; the one in the mucous gland is located in the region adjacent to the secretory cells. The duct of these glands is two-layered, with the individual cells appearing morphologically similar to the layers of the skin epithelium as the duct traverses the skin. The duct appears to be patent throughout its length. The morphological heterogeneity and distinct distribution of the cell types within the gland acinus may be indicative of a functional heterogeneity that allows the production of distinctly different types of secretion from the same gland type, depending on the type of stimulus.     

A reassessment of argasid tick salivary gland ultrastructure from an immuno-cytochemical perspective

Previous morphological and histochemical studies of argasid tick salivary glands indicated that they were less complex than ixodid salivary glands, with only three granular cell types. The present study shows that there exist at least four different granular cell types in the salivary glands of the argasid tick Ornithodoros savignyi, based on immuno-localization of the anti-hemostatic factors, apyrase and savignygrin. Both anti-hemostatic factors were localized to dense core granule type 'a' and to granule type 'b', that shares a similar homogenous morphology with non-labeled granule type 'd'. Furthermore, the major tick salivary gland proteins (TSGPs), previously implicated in granule biogenesis, were localized to all the granular cell types. This indicates that granular cell types with different morphologies can express the same proteins, while cell types that show similar morphologies may not express the same proteins. Argasid tick salivary glands seem to be more complex than previously thought and might not be amenable to morphological classification alone. Alternative classification methodologies that rely on physical expression patterns of the salivary gland proteome might be more reliable as markers for a specific granular cell type.     

Evolutionary regularities of development of somatic polyploidy in salivary glands of gastropod mollusks: V. Subclasses Opisthobranchia and Pulmonata

Salivary glands of 25 species of Opisthobranchia and Pulmonata gastropod mollusks were studied with the use of histochemical methods and cytophotometry of DNA in cellular nuclei. In the secretory epithelium, cells of three main types are identified: granular cells (with granular glycoprotein inclusions), mucocytes-I (containing sulphatized acid mucopolysaccharides), and mucocytes-II (containing neutral and acid nonsulphatized polysaccharides and protein), as well as epithelial ciliated cells and cells of ducts. It is shown that the salivary gland secretory cells of all studied mollusk species are polyploidized, but to different degree. In most species, the maximal degree of polyploidy estimated by the DNA content amounts to 64–128c. Giant polyploidy—up to 4096c—is revealed in salivary gland cells of Tritonia diomedea. The functional properties due to the peculiarities of nutrition of different molluscan species and phylogenetic tendencies of development of somatic polyploidy in the class of Gastropoda are discussed. The high degree of obligatory polyploidization revealed in salivary gland cells of the opisthobranchian and pulmonate mollusks is considered as a peculiar cytological arogenesis as compared with allogenic, facultative, and slight manifestations of polyploidy in prosobranchian gastropod mollusks. The probable causes of such differences are due to the euthyneural type of organization of the central nervous system and to the giant neuronal polyploidy in opisthobranchian and pulmonate mollusks. The causes, mechanisms, and significance of such correlations are so far unclear.     

Salivary gland ultrastructure of the unfed adult and feeding female of Haemaphysalis (Rhipistoma) leachi (Ixodoidea: Ixodidae)

The paired salivary glands of unfed adult Haemaphysalis (Rhipistoma) leachi contain one type of agranular and three types of granular alveoli connected to a salivary duct system. Type I agranular alveoli consist of one large, central cell surrounded by peripheral cells with numerous basal membrane infoldings indicative of epithelia involved in fluid transport. Glycogen particles, lipid-like droplets, and the parallel pattern of infolded membranes disappeared from the peripheral cells during feeding. Types II, III, and IV granular alveoli contain some agranular interstitial epithelial cells, cap cells, and fundus cells, but are predominantly composed of structurally different granular cell types a, b, c, d, e, and f. Agranular cells develop during the early stages of feeding. Granular a, c, e, and f cells release their granules directly after attachment to the host and possibly are involved in cement secretion required for firm attachment to it. The b cell granules are replaced by b₁ filamentous granules during feeding. Golgi bodies and rough endoplasmic reticulum (RER) participate in the formation of most types of granules. The d cells contain lamella-like structures and condensing vacuoles, probably responsible for lysosome formation. The main salivary duct and all types of alveoli are innervated by neurosecretory axons.     

The dorsal tail tubercle of Mertensiella caucasica and M. luschani (Amphibia: Salamandridae)

Males of the two species of Mertensiella (M. caucasica and M. luschani) possess a tubercle projecting from the skin of the dorsal tail base, the single morphological character that defines the genus. The dorsal tail tubercle functions during courtship, and its role is similar in both species. The tubercle is inserted into the cloaca of the female during ventral amplexus, shortly before the male deposits a spermatophore. Histological examination, however, revealed that the dorsal tubercles differ structurally between the two species. In M. caucasica, the tubercle consists primarily of elongate mucous glands, with granular glands occurring only at the base. Both mucous and granular glands of the tubercle are larger than those in typical skin. Unlike typical skin, however, mucous glands are larger than granular glands. In M. luschani, mucous glands and granular glands occur throughout the tubercle, and the granular glands are larger than the mucous glands, although both types are larger than those in typical skin. The dorsal tubercles of M. caucasica and M. luschani may not be homologous structures and may have resulted from convergent evolution. J Morphol 232:93–105, 1997. © 1997 Wiley-Liss, Inc.     

The cephalic glands of Brazilian reptilia and amphibia. IV. Histology of labial, premaxillary, and Duvernoy's glands in the colubrid snake Oxyrhopus trigeminus

A study of the morphology of the salivary glands of the colubrid snake Oxyrhopus trigeminus showed the following: The acini of supralabial, infralabial, and premaxillary glands are formed by mucous and mucoserous cells; the tubules of Duvernoy's gland are formed by seromucous cells; and mucous cells produce neutral and acid mucosubstances, mucoserous cells secrete neutral and acid mucosubstances and protein, and seromucous cells have neutral mucosubstance and protein secretions.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Modification of the structure of penial glands in males of the intertidal molluscs Littorina saxatilis and L. obtusata under the influence of the infestation by trematode parthenites

Sporocycts of the "pygmaeus" microphallides (Microphallus piriformes) are localized in hepatopancreas and gonads of Littorina molluscs causing total parasitic castration. A histological study of penial glands in Littorina saxatilis and L. obtusata males infested with trematodes M. piriformes has been made. Copulatory organs of noninfested molluscs, molluscs after recent contamination (with not completely formed daughter sporocysts), and molluscs containing mature metacercariae inside daughter sporocysts were examined. Based on the data obtained, probable dynamics of the histological structure of infested glandular apparatus was established. It was shown, that the trematode infestation have an influence on the muscular and secretory parts of penial glands. The wall of the penial gland muscular capsule becomes more fine in infested L. saxatilis. On the contrary, this wall is vastly thicker in infested L. obtusata, as compared with noninfested individuals. Glandular cells of the molluscs' penial glands decreases the amount of granular secret in both species. In L. obtusata the number of secretory cells is shown to be reduced up to their total disappearance. The above pathological changes probably prevent normal function of penial glands.     

Cyclic AMP-receptor proteins in human salivary glands

In mammalian species, cyclic AMP receptor proteins (cARP) are the regulatory (R) subunits of cyclic AMP-dependent protein kinase (PKA), the cellular effector of cyclic AMP-mediated signal transduction. An isoform of the PKA type II R subunit (RII), cARP, is a polyfunctional protein, present in most tissues and cells. It is expressed in salivary and other glands of rodents, and secreted into the saliva of rats and Man. The aim of the present study was to determine the expression of cARP in human salivary glands using immunoelectron microscopy. Thin sections of normal salivary glands embedded in LR Gold resin were labeled with anti-cARP primary antibody, then with gold-conjugated secondary antibody. Labeling was present in the secretory granules and cytoplasm of parotid, submandibular (SMG) and sublingual gland serous cells. Quantitative analysis showed considerable variability in granule labeling from sample to sample, indicating shifts in expression and cellular location of cARP. Unlike rodent salivary glands, the granules of intercalated and striated duct cells also were labeled. The cytoplasm and granules of mucous cells of the SMG and sublingual glands were unlabeled, while the Golgi complex and filamentous bodies in these cells showed moderate reactivity. Mitochondria and nuclei of both serous and mucous cells were unlabeled. Labeling also was present in the connective tissue adjacent to the epithelial cells. The results indicate that serous cells of the parotid and SMG are the major source of salivary cARP. They also reveal significant species differences in the glandular distribution of RII. RII binds to cytoskeletal and nuclear proteins, and may function to regulate extracellular cyclic AMP levels. Thus, the tissue and cellular distribution of RII may serve as an index of regulation of gene expression and cell differentiation.     

Specialized mucous glands and their possible adaptive role in the males of some species of Rana (Amphibia,Anura)

A structural and ultrastructural study was carried out on the cutaneous glands in some species of Rana (R. dalmatina, R. iberica, R. italica, R. "esculenta," and R. perezi), giving particular attention to the mucous secretory units. Two different types of mucous glands occur in R. dalmatina, R. iberica, and R. italica. Besides the ordinary mucous units, which are randomly distributed over the body surface in both males and females, a further population of mucous glands was observed on the male dorsal skin. The latter is recognizable by the peculiar morphology of the epithelial cells and some characteristics of the secretory product. Specialized mucous glands are absent in both sexes of R. "esculenta" and R. perezi. The possible adaptive role of the specialized mucous glands is discussed in light of the absence of vocal sacs in males of R. dalmatina, R. iberica, and R. italica. Chemosignals released by sexually dimorphic mucous units may replace vocal communication during the breeding season and so play an important role in female attraction and/or territorial announcement. The morphology and possible function of the specialized mucous glands in the three species of Rana are compared with the breeding glands of other frogs and with the hedonic glands of some urodeles (Salamandridae and Plethodontidae), which are known to produce pheromonal substances during courtship.