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Morphological and histoenzymological differences have been observed between intercalated and principal cells of the quail Coturnix coturnix japonica collecting ducts. The present study was designed to shed light on the lectin affinity of the collecting duct cells within cortex and medulla by the use of HRP-labelled lectins combined with glycosidase degradation. Binding of PNA and RCA-1 lectins consequent to enzymatic release of sialic acid revealed abundant sialylated carbohydrate moieties within the principal cell cytoplasm. This characteristic binding pattern differed considerably from the staining observed in the intercalated cells. Interesting information also emerged about the presence of sialoglycoconjugates having the terminal disaccharide sialic acid-B-N-acetylgalactosamine originating from the increased SBA binding and the unmodified DBA labelling after removal of sialic acid. Sequential degradation by sialidase1B-galactosidase followed by incubation with DBA offered the possibility to suspect that the receptor sugar for the penultimate Bgalactose may be N-acetylgalactosarnine. Conversely, we were not able to define the acceptor sugar for penultimate B-GalNAc owing to the lack of availability of B-N-acetylgalactosaminidase enzyme. When although further studies are clearly needed to elucidate the physiological role of the cellular sialoglycoconjugates detected, the present results already provide valuable insight into the carbohydrate composition of intercalated and principal cells in the quail collecting ducts.

Sialoderivatives expressed in the rabbit oviduct under hormonal treatment have been investigated in situ by lectin histochemistry with a view to specifying further regional and temporal specializations which enable ampulla and isthmus to play distinct roles in the reproductive events. Application of MAL II and SNA lectins allowed sialoglycoconjugates containing Sia(a2,3)Gal and Sia(a2,6)Gal groups to be discriminated. Sialic acid residues linked to Gal(ß1,3)-D-GalNAc sequences were identified using PNA combined with sialidase digestion. Information on structural features of sialic acids were acquired by deacetylation and differential oxidation pretreatments. In both oviductal portions, Sia(a2,6) groups were restricted to the luminal surface of the lining epithelium while Sia(a2,3) groups were specifically located in the secretory, non-ciliated cells. In the ampullary epithelium, non-acetylated sialic acids a2,3-linked to Gal(ß1,3)-D-GalNAc sequences were largely present. Only at ovulation time were sialic acid residues containing acetyl substituents on C4 also found. A great variety of sialic acids were found in the isthmic epithelium which showed the highest expression of acetylated forms at the first hours after the hormonal treatment. The heterogeneity of sialoderivatives differently expressed in the ampulla and isthmus as well as their distinct cycle-dependent modulation suggest that sialylated components may contribute to the molecular and functional specificities within the oviductal epithelium.

The intracellular distribution of lectin receptor sites was studied in the rat Pyla osteoblasts using immunofluorescence at the confocal microscopy level. This immortalized cell line was found to represent a satisfactory model to study the occurrence and distribution of sugar moieties. Our data showed distinct affinity patterns of lectins recognizing different terminal or internal sugar residues. For some lectins, the binding patterns appeared to be cell cycle-independent, whereas for PNA the cell cycle greatly influenced the nuclear binding. By combining lectin affinity with sialidase degradation and alcoholic saponification the sialic acid acceptor sugars and derivatives were also visualized. In particular, glycoconjugates with sialic acids linked to Bgalactose, and mainly C4 acetylated, were located in the cytoplasm, while glycoconjugates characterized by sialic acids linked to a-N-acetylgalactosamine, and devoid of acetyl groups at C4, were almost exclusively found in the nucleus. The comparison of lectin affinities, with and without prior glycosidase digestions, allowed us to gain further insight into the chemical composition of glycoconjugates that act as the lectin receptor sites that appeared to belong to 0- and N-linked glycoconjugates. The use of additional enzymatic treatments were useful to better establish the localization of nuclear receptor sites and results were compared with previous studies about endogenous and exogenous lectins in an attempt to reconcile the association of lectins and sugars within the nucleus and their possible involvement in modulation of cell proliferation andlor response to chemical signals. The above digestions also provided information about the cytoplasmic binding patterns.

A confocal analysis was performed on the quail (Coturnix coturnix japonica) lingual salivary glands where the carbohydrate chains were studied by lectin histochemistry. For this purpose, appropriate FITC- and TRITC-conjugates were used for double binding also accomplished with sialidase digestion. The glycosidic components of the quail lingual salivary glands were found to be heterogeneously distributed on the different secretory structures as well as on the single secretory elements of each adenomere. The rostra1 portion of the anterior lingual gland was found to only secrete neutral glycocomponents, characterized by terminal B-galactose, N-acetylgalactosamine and fucose residues in contrast to the caudal portion that was shown to be extremely heterogeneous and to produce sialylated glycoconjugates characterized by the terminal sequences sialic acid-B-galactose-N-acetylgalactosamine, sialic acid-13-galactose-N-acetylglucosamine,a nd sialic acida- N-acetylgalactosamine partly codistributed within secretory adenomeres. The posterior lingual gland was observed to be the major contributor to the secretion of salivary mucins containing sialoglycoconjugates with terminal sialic acid residues linked to B-galactose-Nacetylgalactosamine or a-N-acetylgalactosamine often located in distinct secretory elements.

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 doublesided 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, a-amylase. Co-presence of lysozyme and a-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 a-amylase did not occur.

In the present study we report the development of an ultrastructural electron microscopic double-sided staining technique that, using gold probes of 10 nm and enhancement of the gold signal by silver amplification, allows the demonstration of two antigenic sites on the same section. The labeling was carried out in the following manner: one face of uncoated floating grids was incubated with an antibody directed to a - amylase, followed by a secondary gold-labeled antibody, amplification of gold particles, drying and carbon coating; subsequently, the reverse face of the same grid, was processed for lectin cytochemistry, with and without sialidase digestion, and it was incubated with HRPconjugated lectins, anti-HRP antibody and protein-A gold. Also the reverse sequence of steps and amplification of gold signal after the first or second labeling were experimented. The resultant small and large particles revealed different distributional patterns of antigenic sites on the opposite faces of the same tissue section. The transparency of the resin-embedded ultrathin sections in the electron beam allowed the simultaneous visualization of the gold probes of different sizes present on the two faces. The analysis of immunolabeling revealed that the a-amylase is chiefly secreted by the parotid and submandibular glands. The application of this double-sided staining technique also indicated that, when present in glycosylated form, the aamylase enzyme does not contain sialic acid in the submandibular and sublingual glands; conversely, its location on the electron-dense areas of target granules in the parotid acinar cells seems to suggest that a sialylated isoenzymatic form can occur within these granule regions where sialic, acid linked to 0-galactose, was found to be located.

The novel combination of sialidase digestion with simultaneous PNA and DBA binding yielded marked differences on sialoglycoconjugate occurrence and distribution in the mouse submandibular gland acinar cells of the two sexes. Striking differences in the structure of terminal disaccharides within stored secretory sialoglycoconjugates were also found. High content of sialic acid, characterized by the terminal sequence sialic acid-a-N-acetylgalactosamine, was established to only occur in the male acini where secretory cells appeared to be differently stained; indeed, some cells exhibited codistribution of sialic acid-a-Nacetylgalactosamine and sialic acid-B-galactose terminal disaccharides, whereas other ones exclusively contained one of the two kinds of terminal sequences. In the female acinar cells, the secretory products were found to be almost exclusively composed by glycoconjugates having sialic acid subtended to B-galactose without appreciable differences between acinar cells. Our finding of such extensive differences in the acinar cells of male and female mice adds new insights into the submandibular gland sexual dimorphism, commonly attributed to the androgen responsiveness of the granular convoluted tubule portion of the gland.

An electron microscopic investigation was performed to examine the ultrastructural changes occurring in the rabbit oviductal epithelium after human chorionic gonadotropin (HCG) administration. Mainly, the non-ciliated secretory cells proved to be affected by the hormonal treatment which resulted in qualitative and quantitative modifications of the secretory patterns differently expressed in the ampulla and isthmus. Thus, morphological evidence of intense secretion was observed in both the oviduct regions at preovulatory stages. Following ovulation, timing of expression of active secretory patterns in the ampulla and isthmus correlated well with the rate of gamete transport and relative functional roles of the oviductal regions in the reproductive process. At present, HCG-induced changes concerning the ciliated cells seem to consist of the occurrence of secretory granules responsible for the appearance of "mixed cells".

Sections from the major salivary glands of rats and mice were used to locate, charactecize and compare sialoglycoconjugates by means of lectin histochemistry, sialidase digestion, periodate oxidation and potassium hydroxide deacetylation. The gland sialylated macromolecules contained the terminal dimers sialic acid-B-galactose and sialic acid-a-N-acetylgalactosamine but differed in the varieties of sialic acids and the linkages of sialic acids to penultimate sugars. Indeed, the submandibular and parotid glands exhibited a notable occurrence of periodate labile sialic acids with C7 andlor C8 andlor C9 acetyl groups in their polyhydroxyl chains. In particular, C9 acetylated sialic acids were mostly linked a2-6 to B-galactose. The sublingual glands, instead, were strongly characterized by a presence of C9 acetylated sialic acids bound a2-3 to B-galactose. Also, sialic acids with O-acetyl substituents at C4 were evident in the mouse parotid gland and in the rat submandibular and sublingual glands. The great variety of sialoderivatives expressed by the rodent salivary glands was correlated with the differential involvement of these compounds in lubricating and defensive processes. Sex-related differences regarding the sialic acid location, acetylation degree and linkage were shown in the submandibular glands of both species.

Sialoglycoconjugates were investigated in the bovine sublingual gland by direct visualization of sialic acid with specific lectins (LPA, SNA) and by histochemical procedures combined with sialidase digestion and lectins. The most reactive histological sructures were found to be acini which contained glycoconjugates with terminal disaccharides consisting of sialic acid linked to galactose or N-acetylgalactosamine. Resistance to periodate oxidation was interpreted as demonstrating a relevant presence of C7, C8 and Cg acetylated sialic acids. KOH-Sialidase-DBA and KOHAlcian blue sequences allowed the identification of C4 acetylated sialic acids.

This review was focused on the salivary gland oligosaccharide chains studied by lectin histochemistry combined with exoglycosidase digestion. Glycoconjugates play an important role in many biofunctions and, generally. salivary mucins, which consist of numerous oligosaccharide chains attached at closely spaced intervals to a peptide backbone, serve as lubricants and protective agents, but in many instances we are ignorant about the role of biochemically identified oligosaccharides. Lectin histochemistry represents the greatest analytic tool to study carbohydrates in situ; in addition, there is availability of selective enzymes, so glycosidase degradation is useful to both investigate the structure of a given oligosaccharide and verify the influence of neighbouring sugars on the affinity towards the respective specific lectins. Using stepwise digestion of samples, followed by lectin labelling, the structure of terminal short oligosaccharides with blood-group activity was also elucidated. Additional histochemical methodologies were developed to establish the presence of acetylated groups in sialic acid residues, and the position of the linkage to the underlying monosaccharide. Sequencing approaches by exoglycosidases and lectins were also seen to be particularly useful when substantial differences did not emerge in lectin affinity, glycoconjugate composition and complex carbohydrate cytochemistry.