Differential location and structural specificities of sialic acid-beta-D-Gal sequences belonging to sialoderivatives of rabbit oviduct under hormonal treatment.

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(alpha2,3)Gal and Sia(alpha2,6)Gal groups to be discriminated. Sialic acid residues linked to Gal(beta1,3)-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(alpha2,6) groups were restricted to the luminal surface of the lining epithelium while Sia(alpha2,3) groups were specifically located in the secretory, non-ciliated cells. In the ampullary epithelium, non-acetylated sialic acids alpha2,3-linked to Gal(beta1,3)-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.

Confocal evaluation of native and induced lectin binding contributes to discriminate between lingual gland glycocomponents in quail.

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 rostral portion of the anterior lingual gland was found to only secrete neutral glycocomponents, characterized by terminal beta-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-beta-galactose-N-acetylgalactosamine, sialic acid-beta-galactose-N-acetylglucosamine, and sialic acid-alpha-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 beta-galactose-N-acetylgalactosamine or alpha-N-acetylgalactosamine often located in distinct secretory elements.

Double-sided staining with a gold probe and silver enhancement to detect alpha-amylase and sugar moieties in the mouse salivary glands.

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 alpha-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 HRP-conjugated 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 alpha-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 alpha-amylase 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 beta-galactose, was found to be located.

Sex-related expression of sialic acid acceptor sugars in the mouse submandibular gland. Simultaneous visualization by confocal laser scanning microscopy.

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-alpha-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-alpha-N-acetylgalactosamine and sialic acid-beta-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 beta-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.

Confocal and electron microscopy to characterize sialoglycoconjugates in mouse sublingual gland acinar cells.

Double lectin labeling for confocal microscopy and lectin-protein A-gold binding for electron microscopy were applied to the mouse sublingual gland in order to study surface and cytoplasmic sialoglycoconjugates. For this purpose, serially cut sections were submitted to sialidase followed by incubation with lectins recognizing usually acceptor sugars for terminal sialic acids. At the electron microscope level, the residues subtended to sialic acid were individually identified on adjacent sections by an indirect technique of labeling, whereas with confocal microscopy the above sugars were simultaneously visualized on the same section by a double staining method using fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated lectins. Acinar cells were found to contain the terminal sequence sialic acid-beta-galactose in abundance while the sequence sialic acid-alpha-N-acetylgalactosamine appeared to be present in modest amounts. Both sialoglycoconjugates were homogeneously codistributed inside acinar cells. The combination with a saponification method also allowed the occurrence of C4 acetylated sialic acids linked to beta-galactose to be discovered, at the electron microscope level, on acinar cell secretory products.

Sialoglycoconjugate dimorphism of the mouse submandibular gland acinar cells. Ultrastructural evidence by lectin-protein A-gold probes and sialidase digestion.

An ultrastructural analysis of lectin receptors on the submandibular glands from mice of both sexes was performed utilizing horseradish peroxidase-labelled lectins in conjunction with antiperoxidase antibody and protein A-gold. Both qualitative and quantitative sex-related differences in terminal sugar expression within secretory granules were detected. Following sialidase digestion, also subterminal acceptor sugars for terminal sialic acids, proved to be differentially expressed in the submandibular glands of males and females. Heterogeneous distribution of sialoglycoconjugates characterized by the terminal disaccharide sialic acid-beta-galactose was found to occur in female acinar cells. Also DBA reactive sites indicating the presence of terminal alpha-N-acetylgalactosamine discriminated between male and female acinar secretory glycoconjugates. This difference was emphasized by sialidase pretreatment that evidenced a marked occurrence of sialic acid subtended to alpha-N-acetylgalactosamine in males in contrast to a modest presence in females. The different sialylation patterns of acinar cell secretory products, probably related to a different expression of O- and N-linked sialoglycoconjugates, give insight into the sexual dimorphism of the mouse submandibular gland known until recently for the convoluted granular tubules.

Cytomorphological changes in the rabbit oviductal epithelium after human chorionic gonadotropin treatment.

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".

Contribution of confocal laser scanning microscopy to glycochemistry of mouse and rat submandibular glands by single and double lectin staining.

The localization of individual glycosidic residues in the mouse and rat submandibular glands was examined using confocal laser scanning microscopy (CLSM). For these organs we tested some procedures of fixation and embedding to better understand the distribution of some lectin-probes inside well preserved secretory cells and observed that fixation and inclusion steps did not influence appreciably the location and intensity of the reactive sites. The fixation mixture of 4% paraformaldehyde, 1% glutaraldehyde and 0.2% picric acid produced the most satisfactory results. In specimens labeled with PNA-, Con A-, LTA-FITC and WGA-, DBA-TRITC lectins, the convoluted granular tubules (CGT) proved to be composed of secretory granules with high-density lectin labeling. The complex organization of secretory glycocomponents within the granule matrix was further resolved by double labeling and dual scanning experiments. Some lectins exhibited colocalization while others displayed differential localization providing information about the occurrence of O- and N-linked glycoconjugates. The CLSM technique applied to fluorochrome-conjugated lectins also revealed a more marked dimorphism in the rat rather than in the mouse submandibular gland. In particular, the male rat submandibular gland was found to consist of CGT heterogeneous cell populations, while the mouse submandibular gland did not show glycochemical differences between cells. Female rats exhibited a lectin profile very different from that of female mouse.

Sialylation patterns of the mouse parotid secretory granules. Combined deacetylation, enzymatic degradation and lectin-gold binding.

We investigated the lectin cytochemistry of control and sialidase-digested sections of the mouse parotid gland by postembedding techniques. PNA and DBA lectins were used and their affinity sites were localized by employing conjugates with horseradish peroxidase that then reacted with anti-horseradish peroxidase antibody and protein A-gold. Potassium hydroxide pretreatment also was used before sialidase/PNA and DBA binding to investigate sialic acid acetylation. Ultrathin sections were obtained from specimens embedded in the acrylic hydrophilic resin, Bioacryl. The acini of mouse parotid gland contained polymorphous secretory granules differentially stained by the two lectins; the use of sialidase digestion and KOH deacetylation revealed that the sialic acids linked to beta-galactose are restricted to the electron-dense concentric areas of target granules, whereas the sialic acids linked to alpha-N-acetylgalactosamine contain C4-acetylated groups and are preferentially located in the electron-lucent regions of bizonal granules.

Glycocytochemistry of the mouse parotid gland. Investigation by lectin-gold techniques on Bioacryl and Lowicryl K4M embedded specimens.

In this study colloidal gold methodologies that allow the lectin affinity patterns in the mouse parotid gland to be studied by postembedding techniques were addressed. DBA, Con A, PNA, WGA and LTA lectins conjugated with gold particles or horseradish peroxidase for a direct or indirect technique of binding were used. Thin sections were obtained from tissue embedded in the acrylic hydrophilic resins Bioacryl and Lowicryl K4M. The most satisfactory results originated from sections infiltrated in Bioacryl and submitted to the indirect technique of binding. The mouse parotid gland appeared to be composed of polymorphous secretory granules differentially stained by lectins. The glycosylated components of acinar cells reacted intensely, and gold particles specified the ultrastructural distribution of lectin affinity sites reflecting the location of beta-galactose, alpha-N-acetylgalactosamine, beta-N-acetylglucosamine, alpha-D-mannose, and alpha-L-fucose in the electron-dense or electron-lucent areas of bizonal, mottled, and target granules.

Calpain inhibitor in rabbit skeletal muscle: an immunochemical and histochemical study.

Calpastatin, the endogenous inhibitor of calcium-activated neutral proteases (calpains; EC 3.4.22.17), was studied in the rabbit vastus lateralis muscle by means of immunochemical and immunohistochemical techniques. Immunoaffinity chromatography using an antibody raised against the 34-kDa monomer of the 68-kDa dimeric inhibitor allowed us to isolate three main proteins (130-, 100- and 80-kDa). These proteins strongly inhibited calpain activity in muscle homogenate (I50 at about 50 micrograms/ml). Immunohistochemical experiments showed that calpastatin-related immunoreactivity was present in all fibre types (oxidative, glycolytic, oxidative-glycolytic) at both surface and cytoplasmic level. However, a few (20%) of the slow-twitch, oxidative fibres (5% of the total fibres), did not contain the cytoplasmic inhibitor. Specific immunoreactivity for calpastatin was also associated with the interstitial connective tissue. These results suggest that (i) calpastatin in skeletal muscle, as in other tissues, is present as a mixture of proteins of various molecular weights and (ii) the inhibitor may act not only in the cytoplasm but also at the surface or extracellular level.

Glycoconjugate composition of mammalian parotid glands elucidated in situ by lectins and glycosidases.

Sugar specific lectins (PNA, RCA I, LPA, SBA, DBA, GSA IB4, GSA II, WGA, LTA, UEA I, Con A, LCA) with and without prior selective glycosidase digestion (sialidase, alpha-fucosidase, alpha-mannosidase, beta-N-acetylglucosaminidase, alpha- and beta-galactosidase, beta-glucosidase) were used in order to investigate the distribution of native accessible carbohydrates and obtain information dealing with the composition of terminal disaccharides within glycoconjugates present in acinar compartments and ductal segments of mammalian (mouse, rat, hare, and rabbit) parotid glands. Glycoconjugates containing variable amounts of mannose, glucose, N-acetylgalactosamine and N-acetylglucosamine were present in the parotid glands of all species. However, these carbohydrate chains exhibited a different composition of terminal sequences within each type of gland. For example, sialylated components having the terminal dimers sialic acid-galactose and sialic acid-N-acetylgalactosamine were found in all acinar cells, whereas fucoglycoconjugates with terminal disaccharide fucose-galactose were localized in the rat striated ducts and hare acinar cells. The terminal sequence alpha-galactose-beta-galactose was demonstrated in the mouse acinar cells. Finally, glycoconjugates characterized by the terminal dimer beta-galactose-N-acetylgalactosamine were demonstrated in the mouse acinar and ductal cells and the rat ductal ones. Thus, present findings outlined and further confirmed the possibility to elucidate the oligosaccharide structure in situ using lectin histochemistry combined with enzymatic degradation.

On the fine structure and complex carbohydrate cytochemistry of the rabbit parotid gland.

The parotid gland of the rabbit, a lagomorph species, was studied by ultrastructural techniques and carbohydrate ultracytochemical stainings. The rabbit parotid gland is a peculiar mixed gland consisting of serous and mucous secretory cells due to their histochemical properties supported by biochemical findings. Acinar cells exhibit heterogeneous features of secretory granules with different electrondensity and occasional presence of subunits. Intercalated duct cells show nuclei with deep indentation and apical granules partly similar to acinar secretory products. Striated ducts are characterized by three different cell populations, namely "light cells" with small secretory granules, "dark cells" rich of scattered mitochondria and typical striated cells. The presence of differentiated cell types within striated duct segments lends credence to the idea that, in addition to the role in electrolyte transport, some ductal cells may be involved in secretion and/or absorption of glycosylated products.

The hare parotid gland: ultrastructure and histochemistry of acinar and ductal cells.

The parotid gland of the hare Lepus europaeus was studied by electron microscopy. The parenchyma consists of secretory areas (acini) and ductal segments (intercalated, striated and excretory ducts). Acinar cells showed a marked heterogeneity due to the presence of secretory granules characterized by different size, electron-density and degree of membrane merging in addition to the occurrence of flattened and dilated rough endoplasmic reticulum. Acinar cells had variable affinity for PAS and PA-TCH-SP staining but were HID- and LIT-TCH-SP negative. Intercalated duct cells lacked secretory granules. Striated ducts were lined by four cell types namely light cells, dark cells, vacuolated cells, and typical striated cells. Excretory ducts showed some scalloping on the apical region of the light cells.

Structural and cytochemical observations in the submandibular gland of unweaned cats.

An investigation by electron microscope was performed on the submandibular gland of unweaned cats for studying its morphology and carbohydrate histochemistry. The cytoarchitecture was comparable with that of adult animals with the exception of the degree of membrane merging in both demilunar and acinar cell secretory granules. Conversely, the submandibular gland of unweaned cats showed a peculiar HID-TCH-SP reactivity at acinar cell level.

Characterization of glycoconjugates in an embryonic human epithelial line and changes consequent to adaptation to a hyperosmotic medium.

Cell surface and cytoplasmic glycoconjugates were characterized in embryonic human explant cells (a transformed heteroploid line) cultured in iso-osmotic medium (0.137 M NaCl) and in hyperosmotic medium (0.274 M NaCl) for 10 days in order to study the changes induced in these compounds by hyperosmoticity. Cytochemical and ultracytochemical staining selective for glycoconjugates was carried out. The following results were obtained: (1) glycoproteins, glycosaminoglycans and glycolipids are present on the cell surface and in the cytoplasm of the explant cells; (2) lectin histochemistry combined with glycosidase digestion demonstrated the presence of the disaccharides fucose-N-acetylglucosamine and sialic acid-beta-galactose as terminal sequences; (3) histophotometric evaluation of lectin labelling showed a noticeable decrease in histochemical reactivity of adapted cells; (4) plasma membrane cell coat decreased in adapted cells, which was emphasized by ultracytochemical reactions and a rearrangement of glycolipids in the cytoplasm.

Ultrastructure and complex carbohydrate ultracytochemistry of the cat parotid gland.

The structure and glycoconjugate content of the cat parotid gland were analyzed at electron microscopic level by applying morphological techniques and three ultrastructural histochemical methods - HID-TCH-SP, LID-TCH-SP and PA-TCH-SP. This gland appeared as a typical salivary gland composed of acinar secretory cells, intercalated ducts, striated ducts and excretory ducts. The most common configuration of secretory granules consisted of a dense core surrounded by a variable electron-lucent halo. All ductal segments were characterized by the presence of different cell populations and small apical granules greatly different from those localized in the acinar cells. By using HID-TCH-SP we were able to demonstrate that in a few acinar cells there are sulphated sites, whereas PA-TCH-SP staining revealed the presence of vic-glycol radicals in all acinar cells preferentially located on the halo of secretory granules.

Codistribution of lectin reactive glycoderivatives and PA-TCH-SP positive sites in rat oviduct.

Specimens from rat ampulla and isthmus were stained with a battery of 10 lectin-horseradish peroxidase conjugates and lectin binding patterns were correlated with the distribution of periodate-reactive vicinal diol groups as determined by the PA-TCH-SP (periodic acid-thiocarbohydrazide-silver proteinate) sequence. The free surface of ciliated and non-ciliated cells was stained moderately to intensely by all lectins and PA-TCH-SP sequence. Binding sites for WGA, UEA I, LTA, Con A were also detected on the tight junctional regions. Secretory granules reacted with all stainings, except for LPA. The localization of certain sugars (sialic acid, fucose) appeared useful for understanding the functional meaning of positive sites.

Attempt to visualize an endogenous lectin recognizing alpha-L-fucose immunohistochemically in the rabbit oviduct.

An immunohistochemical study was conducted in order to visualize an endogenous lectin, having specificity for alpha-L-fucose residues, in the ampullary and isthmic tracts of the rabbit oviduct. 2 different sequences (S1 and S2) were tested using antilectin UEA I antibody. Reactive sites towards S2 sequence were localized at ciliated cell level in the ampulla; in the isthmus, instead, the staining was present both in ciliated and secretory cells. A role in the sugar immobilization and gamete and zygote protection was postulated for the endogenous lectin evidenced.