- Email: [email protected]
Lectin histochemical study of bovine lingual glands
Archs oral Bid. Vol. 38, No. 10, pp. 881-884, 1993
Printed in Great Britain. All
LECTIN
0+03-9969/93 $6.00 + 0.00 Copyright
rights reserved
HISTOCHEMICAL
0
1993 Pergamon
Press Ltd
STUDY OF BOVINE LINGUAL GLANDS
ANNA MARIA GARGIULO, VERA PEDINI and PIERO CECCARELLI Istituto di Anatomia Normale degli Animali Domestici, Universita degli Studi di Perugia.
06126 Perugia, Italy (Accepted 24 May 1993) Summary-Bovine lingual glands consist of mucous acini capped by demilunes. Information on the chemical structure of their secretory glycoconjugates was obtained by means of a battery of peroxidaseconjugated lectins with affinity for specific terminal sugars. Sialidase procedures followed by lectin staining were also used to visualize the sugar sequences. Stored secretions in mucous acinar cells contained fucose, N-acetylglucosamine, c( and /I-N-acetylgalactosamine as terminal sugar residues and /?-galactose as penultimate sugar in a heterogeneous distribution. Demilunar cells failed to react with any of the lectins examined except that of Dolichos biJlorus. Key words: bovine, lingual glands, lectins.
immediately after slaughter were fixed for 6 h at room temperature in 6% mercuric chloride in 1% sodium acetate solution containing 0.1% glutaraldehyde. Tissues were dehydrated through graded ethanols, cleared in xylene and embedded in paraffin. Sections from tissues fixed in buffered glutaraldehyde-HgCl, were treated with Lugol’s solution before staining.
INTRODUCTION Mammalian tongues have two distinct sets of glands: lingual glands (glanduzae radicis linguae) at the base and Von Ebner’s glands (glandulae gustatoriae). In
some species [man (Sicher and Du Brul, 1975); bat (Navalade and Varute, 1972)] anterior lingual glands (glandula lingualis apicalis) are also present in the tongue tip (Barone, 1981). Lingual glands are located submucosally at the base and along the lateral margins of the tongue among muscle bundles. They consist of collections of large clusters of tubuloacinar aggregates forming multiple lobules. Histologically, the lingual glands of large ruminants and horses are mixed, containing mucous cells surrounded by demilunar cells. Generally, the mucous acini greatly outnumber the demilunes (Pedini, Gargiulo and Ceccarelli, 1992a). The nature and distribution of mucosubstances from lingual glands have been studied in several mammals (Nogueira and Carvalho, 1973; Tsuzuki, 1978; Poddar and Jacob, 1979; Imai, Shibata and Higashi, 1982; Jarrar and Taib, 1989; Sanford and Josephson, 1989) but we have found no previous studies on bovine lingual glands. Thus we have now investigated the histochemical composition of bovine lingual gland secretions with the aid of lectins.
Lectin histochemistry
Tissue sections, cut at about 5 pm, were incubated in a 10-20 pg/ml of lectin-HRP conjugate solution in 0.1 M PBS, pH 7.2, containing 0.1 mM CaCl,, MnCl, and MgCl, for 1 h at room temperature. After washing in PBS, lectin-binding sites were revealed using diaminobenzidine-hydrogen peroxide medium for 15 min at room temperature (Schulte and Spicer, 1983a,b). The lectins used were: DBA (Dolichos bifiorus), PNA (Arachis hypogea), WGA (Triticvm vulgaris), UEA-I (Ulex europaeus), SBA (Glycine max). All these HRP-labelled lectins were purchased from Sigma Chemical Co. Controls for lectin labelhng were run by incubating sections in HRPconjugated lectins with the addition of 0.2 M hapten sugars. Enzyme digestion
Enzymatic treatment was done on adjacent sections. Before lectin histochemistry, some sections were incubated at 37°C for 16 h in an 0.8 IU/ml solution of neuraminidase (sialidase) from Clostridium perfringens in 0.1 M sodium acetate buffer, pH 5.5, containing 10 mM CaCl,. Removal of sialic acid was confirmed on adjacent sections by the lack of staining with Alcian blue at pH 2.5 (Spicer and Warren, 1960). Controls for enzyme digestion entailed comparable exposure to the buffer in which the enzyme was dissolved. Sialic acid residues with 0-acetyl substituents at C-4 resisted sialidase (Moschera and Pigman, 1975)
MATERIALS AND METHODS Tissue preparation
Ten clinically healthy adult bovines (Bos taurus) of both sexes, killed at the local slaughterhouse, were used. Specimens of lingual glands obtained Abbreviations: DBA, Dolichos biJlorus agglutinin; HRP, horseradish peroxidase; PAS, periodic acid-Schiff; PBS, phosphate-buffered saline; PNA, peanut agglutinin; SBA, soybean agglutinin; UEA, Ulex europaeus agglutinin; WGA, wheatgerm agglutinin. 881
ANNA MARIA GARWLJLO EI al.
882 Table
I. General pattern of lectin staining with and without neuraminidase Mucous
WGA UEA PNA NEU-PNA DBA NEU-DBA SBA
pretreatment
acini
Demilunes
Ducts
2 24* omI 23
0 0 0 0
0 0 2t 3t
3: 4: o-2
31 41 0
3t
2t
2t
Numbers indicate staining intensity on a subjectively estimated scale from 0, unreactive, to 4, most reactive. *The number of mucous cells stained with UEA I varies from 20 to 60%. tstaining was observed at the apical surface. $The number of cells stained with DBA comprises about 30% of the total cell population.
but were cleaved after removal of acetyl groups by saponification. This was done by immersing the sections in a 1% solution of potassium hydroxide in 70% ethanol for I5 min at room temperature.
1
Fig. 2. UEA-HRP
i
staining: acinar cells show UEA affinity. x 50
a variable
staining to the luminal border of ducts and to all mucous cells (Fig. 4). Saponification before sialidase digestion failed to increase the affinity for PNA imparted by this enzymatic degradation. Dolichos b$?orus (DBA)
RESULTS
Lectin binding to different components of bovine lingual glands without and with neuraminidase digestion is summarized in Table 1. Triticum vulgaris (WGA) Stored secretion in all the mucous acinar cells showed moderate staining with WGA (Fig. 1). Demilunes and ductal cells had no affinity for this lectin. Ulex europaeus (UEA -I) UEA-I staining was seen in 2040% of the mucous acinar cells. The intensity of cellular reaction was variable, ranging from moderate to strong (Fig. 2 and Table 1). No reactivity was seen in the demilunes and ducts.
The cytoplasm of about 30% of both the mucous cells and the demilunar cells showed a diffuse positive reaction with DBA (Fig. 5). The apical cytoplasm in cells lining ducts stained moderately with this lectin. Sialidase digestion increased the intensity of reaction in all binding sites (Fig. 6). KOH treatment before enzymatic digestion did not affect the DBA reactivity normally imparted by neuraminidase degradation. Glycine max (SBA) SBA stained the mucous cells variably, ranging from 0 to 2 (Table 1). The apical cytoplasm of ductal cells exhibited a moderate staining with this lectin. Demilunar cells failed to bind SBA. DISCUSSION
The apical surface of cells lining the ducts was moderately stained. A very faint reaction was seen in some mucous acinar cells (Fig. 3). Prior removal of sialic acid with neuraminidase imparted increased
Bovine lingual glands are structurally similar to bovine major salivary glands (Bloom and Carls66, 1974; Shackleford and Wilborn, 1970; Suzuki, Nishinakagawa and Otsuka, 1981), containing two types of secretory cells, the mucous acinar cells and the demilunar serous cells. Mucous acinar cells are far
Fig. 1. WGA-HRP staining: and diffusely
Fig. 3. PNA-HRP staining: acinar cells exhibit but always weak reaction. x 200
Arachis hypogea (PNA)
mucous stained.
acini are moderately x 50
a variable
Bovine lingual
Fig. 4. Sialidase of sialic acid,
PNA-HRP staining: acinar cells appear x 200
after the removal strongly stained.
more numerous and constitute a considerably larger portion of the gland parenchyma than demilunar cells (Pedini et al., 1992a). Pure mucous acini, described in the ruminant mandibular glands (Shackleford and Wilborn, 1970; Suzuki et al., 1981) are not found in bovine lingual glands. Our previous histochemical investigations (Ceccarelli, Gargiulo and Pedini, 1992) demonstrated that mucous acinar cells reacted strongly to PAS and Alcian blue, whereas only a few cells showed a strong reactivity for high iron diamine. After the enzymatic removal of sialic acid, positivity for Alcian blue diminished. Demilunar cells were stained by PAS and very weakly by Alcian blue. With regard to the problem of classifying demilunar cells as serous or seromucous, we agree with Schulte and Spicer (1983a) when they say that ‘. . . the term serous should be applied to all epithelial cells possessing moderate to abundant rough endoplasmic reticulum and a uniform population of discrete, often separated secretory granules which contain a lower level of either acidic or neutral complex carbohydrate . . ’ . Therefore, on the basis of our histochemical (Ceccarelh et al., 1992) and ultrastructural study (unpublished data), we consider demilunes serous even if their secretory products contain carbohydrates.
Fig. 5. DBA-HRP staining: approx. 30% of the mucous acinar cells are diffusely stained; demilunes also react with DBA (arrows). x 130
glands
883
Fig. 6. Section similar to that in Fig. 9 but treated with sialidase before DBA-HRP conjugate; enzymatic treatment increases staining. x 130
Our results from lectin histochemistry demonstrate that the most reactive secretory cells are the mucous cells. In these cells, there was binding for UEA-I, WGA and SBA, indicating the presence of glycoconjugates with fucose, N-acetylglucosamine, c1 and /IN-acetylgalactosamine, respectively, as terminal sugar residues. The distribution of these saccharides in the stored glycoprotein secretion of mucous cells was not uniform. Removal of sialic acid imparted strong brown staining to all mucous cells with PNA, indicating the uniform presence of the terminal sequence siahc acid- (a -+ 3,6) galactosyl (fi 1 + 3) N-acetylgalactosamine. The same treatment imparted strong affinity for DBA in only 2&30% of the total cell population, demonstrating a variable content of sialic acid-o:-N-acetylgalactosamine residues. These findings are in agreement with previous biochemical (Bertolini and Pigman, 1970; Tsuji and Osawa, 1986) and histochemical (Menghi et al., 1992) studies on bovine major salivary glands. Saponification before sialidase digestion failed to increase the affinity of stored mucins for the lectins, indicating the absence or low levels of siahc acid residues containing 0 -acetyl substituents at C-4 (Schulte, Spicer and Miller, 1985). Demilunar cells failed to react with any of the lectins except DBA, as already shown in bovine mandibular gland by Menghi et al. (1992). Unlike other species (Poddar and Jacob, 1979; Pedini, Ceccarelli and Gargiulo, 1992b), there are no striated ducts in bovine lingual glands (Pedini et a[., 1992a) and the excretory system is made up of intraand interlobular ducts. The luminal border of these ducts reacted with PNA, SBA and DBA, and the reactivity was intensified after sialidase digestion. In the ovine mandibular gland there was also evidence of sialoglycoconjugates in striated and interlobular ducts (Schulte et al., 1985). Hence, our previous and present histochemical studies suggest the presence of acidic glycoconjugates with terminal sialic acid residues. Acidic glycoconjugates are generally present in the mucous cells of major salivary glands of some ruminants (El-Shafey, Al-Shaikhly and Al-Lawand, 1980; Schuhe et al., 1985; Menghi et al., 1992) and are probably responsible for the viscosity of saliva (Gottschalk, 1960;
884
ANNA MARIA GARGIULO et al
Shackleford and Klapper, 1962). Histochemical differences were present between bovine lingual glands and ferret lingual glands (Poddar and Jacob, 1979). This should be of no surprise, given the different diet of the two species. However, considerable differences also exist between the lingual glands of bovines and horses (Pedini et al., 1992b), indicating that in these species the chemical composition of the secretion may be influenced by the morphofunctional features of the alimentary tract rather than by the diet. Acknowledgement-This work was supported from the Italian M.U.R.S.T. (60%).
by a grant
REFERENCES Barone R. (1981) Anutomia Comparata dei MammtJeri Domestici. Edagricole, Bologna. Bertolini M. and Pigman W. (1970) The existence of ohgosaccharides in bovine and ovine submaxillary mucins. Carbohydr. Res. 14, 53363. Bloom G. B. and Carlso B. (1974) Fine structure and peroxidase activity of the bovine mandibular gland cells. Anat. Histol. Embriol. 3, 308-323. Ceccarelli P., Gargiulo A. M. and Pedini V. (1992) Histochemical studies on glycoconjugates of bovine lingual salivary glands. 19th Congr. EAVA, 15 p. Ghent, Antwerp. El-Shafey M. S., Al-Shaikhly A. K. J. and Al-Lawand S. (1980) Micromorphology and histochemistry of polysaccharides in the goat mandibular salivary gland. Anat. Am. 147, 3341. Gottschalk A. (1960) Correlation between composition, structure shape and function of salivary mucoprotein. Nature 186, 9499951. Imai M., Shibata T. and Higashi N. (1982) Structure and carbohydrate histochemistry of the minor salivary glands of the Japanese Macaque (Macaca fuscata yukui). Okajimas Folia Anat. Jap. J8, 485-500.‘ Jarrar B. M. and Taib N. T. (1989) Histochemical characterization and distribution of mucosubstances and enzyme activity in the lingual salivary glands of the one-humped camel (Camelus dromedarius). Rev. Elev. Med. Vet. Pays trop. 42(l), 63371. Menghi G., Accih D., Scocco P. and Materazzi G. (1992) Sialoglycoderivatives of bovine submandibular gland identified in situ by histochemical techniques combined with lectins. Histochemie 97, 397403. Moschera J. and Pigman W. (1975) The isolation and characterization of rat sublingual mucus glycoprotein. Carbohydr. Res. 40, 5367.
Navalade M. N. and Varute A. T. (1972) Histochemical studies on the mucins of the vertebrate tongues: IVHistochemical analysis of mucosubstances in lingual mucous glands of four varieties of bats differing in their feeding habits. Ann. Hisiochim. 17, 273-287. Nogueira J. C. and Carvalho A. D. V. (1973) Histochemistry of the mucins in the posterior lingual salivary glands of some mammals. Rev. bras. Pesquisas med. biol. 6, 267-274. Pedini V., Gargiulo A. M. and Ceccarelli P. (1992a) Le ghiandole linguali de1 bovino e de1 cavallo. Atti SISVet XLVI, 293-296. Pedini V., Ceccarelli P. and Gargiulo A. M. (1992b) Lectin histochemistry of glycoconjugates in horse hnguaiglands. Acta Med. Veter. 38, 311-316. Poddar S. and Jacob S. (1979), Histoloav__ and mucosubstance histochemistry of ferret lingual glands. Acta Anat. 105, 65-74. Sanford S. E. and Josephson G. K. A. (1989) Identification of lingual glands and ducts ventrally in pigs tongues. J. Anat. 163, 2755278. Schulte B. A. and Spicer S. S. (1983a) Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse. Histochem. J. 15, 1217-1238. Schulte B. A. and Spicer S. S. (1983b) Light microscopic histochemical detection of terminal galactose and Nacetylgalactosamine residues in rodent complex carbohydrates using a galactose oxidase_Schiff sequence and peanut lectin-horseradish peroxidase conjugate. J. Histothem. Cytochem. 31, 19-24. Schulte B. A., Spicer S. S. and Miller R. L. (1985) Lectin histochemistry of secretory and cell-surface glycoconjugates in the ovine submandibular gland. Cell. Tiss. Res. 240, 5766. Shackleford J. M. and Klapper C. E. (1962) Structure and carbohydrate histochemistry of mammalian salivary glands. Am. J. Anat. 111, 25-33. Shackleford J. M. and Wilborn W. H. (1970) Ultrastructural aspects of calf submandibular glands. Am. J. Anat. 127, 2599280. Sicher H. and DuBrul E. L. (1975) Oral Anatomy, 6th edn. C. V. Mosby, St Louis, MO. Spicer S. S. and Warren L. (1960) The histochemistry of sialic acid containing mucoproteins. J. Histochem. Cytothem. 8, 135-137. Suzuki S., Nishinakagawa H. and Otsuka J. (1981) Fine structure of the bovine mandibular gland. Mem. Fat. Agr. Kagoshima Univ. 17, 1477155. Tsuji T. and Osawa T. (1986) Carbohydrate structures of bovine submaxillary mucin. Cat-b. Res. 151, 391402. Tsuzuki M. (1978) Pathomorphological study on the tongue. IIIb, Histochemical study on carbohydrate in rabbit lingual gland. Nihon Univ. dent. J. 52, 472479.
Printed in Great Britain. All
LECTIN
0+03-9969/93 $6.00 + 0.00 Copyright
rights reserved
HISTOCHEMICAL
0
1993 Pergamon
Press Ltd
STUDY OF BOVINE LINGUAL GLANDS
ANNA MARIA GARGIULO, VERA PEDINI and PIERO CECCARELLI Istituto di Anatomia Normale degli Animali Domestici, Universita degli Studi di Perugia.
06126 Perugia, Italy (Accepted 24 May 1993) Summary-Bovine lingual glands consist of mucous acini capped by demilunes. Information on the chemical structure of their secretory glycoconjugates was obtained by means of a battery of peroxidaseconjugated lectins with affinity for specific terminal sugars. Sialidase procedures followed by lectin staining were also used to visualize the sugar sequences. Stored secretions in mucous acinar cells contained fucose, N-acetylglucosamine, c( and /I-N-acetylgalactosamine as terminal sugar residues and /?-galactose as penultimate sugar in a heterogeneous distribution. Demilunar cells failed to react with any of the lectins examined except that of Dolichos biJlorus. Key words: bovine, lingual glands, lectins.
immediately after slaughter were fixed for 6 h at room temperature in 6% mercuric chloride in 1% sodium acetate solution containing 0.1% glutaraldehyde. Tissues were dehydrated through graded ethanols, cleared in xylene and embedded in paraffin. Sections from tissues fixed in buffered glutaraldehyde-HgCl, were treated with Lugol’s solution before staining.
INTRODUCTION Mammalian tongues have two distinct sets of glands: lingual glands (glanduzae radicis linguae) at the base and Von Ebner’s glands (glandulae gustatoriae). In
some species [man (Sicher and Du Brul, 1975); bat (Navalade and Varute, 1972)] anterior lingual glands (glandula lingualis apicalis) are also present in the tongue tip (Barone, 1981). Lingual glands are located submucosally at the base and along the lateral margins of the tongue among muscle bundles. They consist of collections of large clusters of tubuloacinar aggregates forming multiple lobules. Histologically, the lingual glands of large ruminants and horses are mixed, containing mucous cells surrounded by demilunar cells. Generally, the mucous acini greatly outnumber the demilunes (Pedini, Gargiulo and Ceccarelli, 1992a). The nature and distribution of mucosubstances from lingual glands have been studied in several mammals (Nogueira and Carvalho, 1973; Tsuzuki, 1978; Poddar and Jacob, 1979; Imai, Shibata and Higashi, 1982; Jarrar and Taib, 1989; Sanford and Josephson, 1989) but we have found no previous studies on bovine lingual glands. Thus we have now investigated the histochemical composition of bovine lingual gland secretions with the aid of lectins.
Lectin histochemistry
Tissue sections, cut at about 5 pm, were incubated in a 10-20 pg/ml of lectin-HRP conjugate solution in 0.1 M PBS, pH 7.2, containing 0.1 mM CaCl,, MnCl, and MgCl, for 1 h at room temperature. After washing in PBS, lectin-binding sites were revealed using diaminobenzidine-hydrogen peroxide medium for 15 min at room temperature (Schulte and Spicer, 1983a,b). The lectins used were: DBA (Dolichos bifiorus), PNA (Arachis hypogea), WGA (Triticvm vulgaris), UEA-I (Ulex europaeus), SBA (Glycine max). All these HRP-labelled lectins were purchased from Sigma Chemical Co. Controls for lectin labelhng were run by incubating sections in HRPconjugated lectins with the addition of 0.2 M hapten sugars. Enzyme digestion
Enzymatic treatment was done on adjacent sections. Before lectin histochemistry, some sections were incubated at 37°C for 16 h in an 0.8 IU/ml solution of neuraminidase (sialidase) from Clostridium perfringens in 0.1 M sodium acetate buffer, pH 5.5, containing 10 mM CaCl,. Removal of sialic acid was confirmed on adjacent sections by the lack of staining with Alcian blue at pH 2.5 (Spicer and Warren, 1960). Controls for enzyme digestion entailed comparable exposure to the buffer in which the enzyme was dissolved. Sialic acid residues with 0-acetyl substituents at C-4 resisted sialidase (Moschera and Pigman, 1975)
MATERIALS AND METHODS Tissue preparation
Ten clinically healthy adult bovines (Bos taurus) of both sexes, killed at the local slaughterhouse, were used. Specimens of lingual glands obtained Abbreviations: DBA, Dolichos biJlorus agglutinin; HRP, horseradish peroxidase; PAS, periodic acid-Schiff; PBS, phosphate-buffered saline; PNA, peanut agglutinin; SBA, soybean agglutinin; UEA, Ulex europaeus agglutinin; WGA, wheatgerm agglutinin. 881
ANNA MARIA GARWLJLO EI al.
882 Table
I. General pattern of lectin staining with and without neuraminidase Mucous
WGA UEA PNA NEU-PNA DBA NEU-DBA SBA
pretreatment
acini
Demilunes
Ducts
2 24* omI 23
0 0 0 0
0 0 2t 3t
3: 4: o-2
31 41 0
3t
2t
2t
Numbers indicate staining intensity on a subjectively estimated scale from 0, unreactive, to 4, most reactive. *The number of mucous cells stained with UEA I varies from 20 to 60%. tstaining was observed at the apical surface. $The number of cells stained with DBA comprises about 30% of the total cell population.
but were cleaved after removal of acetyl groups by saponification. This was done by immersing the sections in a 1% solution of potassium hydroxide in 70% ethanol for I5 min at room temperature.
1
Fig. 2. UEA-HRP
i
staining: acinar cells show UEA affinity. x 50
a variable
staining to the luminal border of ducts and to all mucous cells (Fig. 4). Saponification before sialidase digestion failed to increase the affinity for PNA imparted by this enzymatic degradation. Dolichos b$?orus (DBA)
RESULTS
Lectin binding to different components of bovine lingual glands without and with neuraminidase digestion is summarized in Table 1. Triticum vulgaris (WGA) Stored secretion in all the mucous acinar cells showed moderate staining with WGA (Fig. 1). Demilunes and ductal cells had no affinity for this lectin. Ulex europaeus (UEA -I) UEA-I staining was seen in 2040% of the mucous acinar cells. The intensity of cellular reaction was variable, ranging from moderate to strong (Fig. 2 and Table 1). No reactivity was seen in the demilunes and ducts.
The cytoplasm of about 30% of both the mucous cells and the demilunar cells showed a diffuse positive reaction with DBA (Fig. 5). The apical cytoplasm in cells lining ducts stained moderately with this lectin. Sialidase digestion increased the intensity of reaction in all binding sites (Fig. 6). KOH treatment before enzymatic digestion did not affect the DBA reactivity normally imparted by neuraminidase degradation. Glycine max (SBA) SBA stained the mucous cells variably, ranging from 0 to 2 (Table 1). The apical cytoplasm of ductal cells exhibited a moderate staining with this lectin. Demilunar cells failed to bind SBA. DISCUSSION
The apical surface of cells lining the ducts was moderately stained. A very faint reaction was seen in some mucous acinar cells (Fig. 3). Prior removal of sialic acid with neuraminidase imparted increased
Bovine lingual glands are structurally similar to bovine major salivary glands (Bloom and Carls66, 1974; Shackleford and Wilborn, 1970; Suzuki, Nishinakagawa and Otsuka, 1981), containing two types of secretory cells, the mucous acinar cells and the demilunar serous cells. Mucous acinar cells are far
Fig. 1. WGA-HRP staining: and diffusely
Fig. 3. PNA-HRP staining: acinar cells exhibit but always weak reaction. x 200
Arachis hypogea (PNA)
mucous stained.
acini are moderately x 50
a variable
Bovine lingual
Fig. 4. Sialidase of sialic acid,
PNA-HRP staining: acinar cells appear x 200
after the removal strongly stained.
more numerous and constitute a considerably larger portion of the gland parenchyma than demilunar cells (Pedini et al., 1992a). Pure mucous acini, described in the ruminant mandibular glands (Shackleford and Wilborn, 1970; Suzuki et al., 1981) are not found in bovine lingual glands. Our previous histochemical investigations (Ceccarelli, Gargiulo and Pedini, 1992) demonstrated that mucous acinar cells reacted strongly to PAS and Alcian blue, whereas only a few cells showed a strong reactivity for high iron diamine. After the enzymatic removal of sialic acid, positivity for Alcian blue diminished. Demilunar cells were stained by PAS and very weakly by Alcian blue. With regard to the problem of classifying demilunar cells as serous or seromucous, we agree with Schulte and Spicer (1983a) when they say that ‘. . . the term serous should be applied to all epithelial cells possessing moderate to abundant rough endoplasmic reticulum and a uniform population of discrete, often separated secretory granules which contain a lower level of either acidic or neutral complex carbohydrate . . ’ . Therefore, on the basis of our histochemical (Ceccarelh et al., 1992) and ultrastructural study (unpublished data), we consider demilunes serous even if their secretory products contain carbohydrates.
Fig. 5. DBA-HRP staining: approx. 30% of the mucous acinar cells are diffusely stained; demilunes also react with DBA (arrows). x 130
glands
883
Fig. 6. Section similar to that in Fig. 9 but treated with sialidase before DBA-HRP conjugate; enzymatic treatment increases staining. x 130
Our results from lectin histochemistry demonstrate that the most reactive secretory cells are the mucous cells. In these cells, there was binding for UEA-I, WGA and SBA, indicating the presence of glycoconjugates with fucose, N-acetylglucosamine, c1 and /IN-acetylgalactosamine, respectively, as terminal sugar residues. The distribution of these saccharides in the stored glycoprotein secretion of mucous cells was not uniform. Removal of sialic acid imparted strong brown staining to all mucous cells with PNA, indicating the uniform presence of the terminal sequence siahc acid- (a -+ 3,6) galactosyl (fi 1 + 3) N-acetylgalactosamine. The same treatment imparted strong affinity for DBA in only 2&30% of the total cell population, demonstrating a variable content of sialic acid-o:-N-acetylgalactosamine residues. These findings are in agreement with previous biochemical (Bertolini and Pigman, 1970; Tsuji and Osawa, 1986) and histochemical (Menghi et al., 1992) studies on bovine major salivary glands. Saponification before sialidase digestion failed to increase the affinity of stored mucins for the lectins, indicating the absence or low levels of siahc acid residues containing 0 -acetyl substituents at C-4 (Schulte, Spicer and Miller, 1985). Demilunar cells failed to react with any of the lectins except DBA, as already shown in bovine mandibular gland by Menghi et al. (1992). Unlike other species (Poddar and Jacob, 1979; Pedini, Ceccarelli and Gargiulo, 1992b), there are no striated ducts in bovine lingual glands (Pedini et a[., 1992a) and the excretory system is made up of intraand interlobular ducts. The luminal border of these ducts reacted with PNA, SBA and DBA, and the reactivity was intensified after sialidase digestion. In the ovine mandibular gland there was also evidence of sialoglycoconjugates in striated and interlobular ducts (Schulte et al., 1985). Hence, our previous and present histochemical studies suggest the presence of acidic glycoconjugates with terminal sialic acid residues. Acidic glycoconjugates are generally present in the mucous cells of major salivary glands of some ruminants (El-Shafey, Al-Shaikhly and Al-Lawand, 1980; Schuhe et al., 1985; Menghi et al., 1992) and are probably responsible for the viscosity of saliva (Gottschalk, 1960;
884
ANNA MARIA GARGIULO et al
Shackleford and Klapper, 1962). Histochemical differences were present between bovine lingual glands and ferret lingual glands (Poddar and Jacob, 1979). This should be of no surprise, given the different diet of the two species. However, considerable differences also exist between the lingual glands of bovines and horses (Pedini et al., 1992b), indicating that in these species the chemical composition of the secretion may be influenced by the morphofunctional features of the alimentary tract rather than by the diet. Acknowledgement-This work was supported from the Italian M.U.R.S.T. (60%).
by a grant
REFERENCES Barone R. (1981) Anutomia Comparata dei MammtJeri Domestici. Edagricole, Bologna. Bertolini M. and Pigman W. (1970) The existence of ohgosaccharides in bovine and ovine submaxillary mucins. Carbohydr. Res. 14, 53363. Bloom G. B. and Carlso B. (1974) Fine structure and peroxidase activity of the bovine mandibular gland cells. Anat. Histol. Embriol. 3, 308-323. Ceccarelli P., Gargiulo A. M. and Pedini V. (1992) Histochemical studies on glycoconjugates of bovine lingual salivary glands. 19th Congr. EAVA, 15 p. Ghent, Antwerp. El-Shafey M. S., Al-Shaikhly A. K. J. and Al-Lawand S. (1980) Micromorphology and histochemistry of polysaccharides in the goat mandibular salivary gland. Anat. Am. 147, 3341. Gottschalk A. (1960) Correlation between composition, structure shape and function of salivary mucoprotein. Nature 186, 9499951. Imai M., Shibata T. and Higashi N. (1982) Structure and carbohydrate histochemistry of the minor salivary glands of the Japanese Macaque (Macaca fuscata yukui). Okajimas Folia Anat. Jap. J8, 485-500.‘ Jarrar B. M. and Taib N. T. (1989) Histochemical characterization and distribution of mucosubstances and enzyme activity in the lingual salivary glands of the one-humped camel (Camelus dromedarius). Rev. Elev. Med. Vet. Pays trop. 42(l), 63371. Menghi G., Accih D., Scocco P. and Materazzi G. (1992) Sialoglycoderivatives of bovine submandibular gland identified in situ by histochemical techniques combined with lectins. Histochemie 97, 397403. Moschera J. and Pigman W. (1975) The isolation and characterization of rat sublingual mucus glycoprotein. Carbohydr. Res. 40, 5367.
Navalade M. N. and Varute A. T. (1972) Histochemical studies on the mucins of the vertebrate tongues: IVHistochemical analysis of mucosubstances in lingual mucous glands of four varieties of bats differing in their feeding habits. Ann. Hisiochim. 17, 273-287. Nogueira J. C. and Carvalho A. D. V. (1973) Histochemistry of the mucins in the posterior lingual salivary glands of some mammals. Rev. bras. Pesquisas med. biol. 6, 267-274. Pedini V., Gargiulo A. M. and Ceccarelli P. (1992a) Le ghiandole linguali de1 bovino e de1 cavallo. Atti SISVet XLVI, 293-296. Pedini V., Ceccarelli P. and Gargiulo A. M. (1992b) Lectin histochemistry of glycoconjugates in horse hnguaiglands. Acta Med. Veter. 38, 311-316. Poddar S. and Jacob S. (1979), Histoloav__ and mucosubstance histochemistry of ferret lingual glands. Acta Anat. 105, 65-74. Sanford S. E. and Josephson G. K. A. (1989) Identification of lingual glands and ducts ventrally in pigs tongues. J. Anat. 163, 2755278. Schulte B. A. and Spicer S. S. (1983a) Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse. Histochem. J. 15, 1217-1238. Schulte B. A. and Spicer S. S. (1983b) Light microscopic histochemical detection of terminal galactose and Nacetylgalactosamine residues in rodent complex carbohydrates using a galactose oxidase_Schiff sequence and peanut lectin-horseradish peroxidase conjugate. J. Histothem. Cytochem. 31, 19-24. Schulte B. A., Spicer S. S. and Miller R. L. (1985) Lectin histochemistry of secretory and cell-surface glycoconjugates in the ovine submandibular gland. Cell. Tiss. Res. 240, 5766. Shackleford J. M. and Klapper C. E. (1962) Structure and carbohydrate histochemistry of mammalian salivary glands. Am. J. Anat. 111, 25-33. Shackleford J. M. and Wilborn W. H. (1970) Ultrastructural aspects of calf submandibular glands. Am. J. Anat. 127, 2599280. Sicher H. and DuBrul E. L. (1975) Oral Anatomy, 6th edn. C. V. Mosby, St Louis, MO. Spicer S. S. and Warren L. (1960) The histochemistry of sialic acid containing mucoproteins. J. Histochem. Cytothem. 8, 135-137. Suzuki S., Nishinakagawa H. and Otsuka J. (1981) Fine structure of the bovine mandibular gland. Mem. Fat. Agr. Kagoshima Univ. 17, 1477155. Tsuji T. and Osawa T. (1986) Carbohydrate structures of bovine submaxillary mucin. Cat-b. Res. 151, 391402. Tsuzuki M. (1978) Pathomorphological study on the tongue. IIIb, Histochemical study on carbohydrate in rabbit lingual gland. Nihon Univ. dent. J. 52, 472479.